Sample records for based sulfotransferase assay

  1. A versatile polyacrylamide gel electrophoresis based sulfotransferase assay

    Directory of Open Access Journals (Sweden)

    Prather Brittany


    Full Text Available Abstract Background Sulfotransferases are a large group of enzymes that regulate the biological activity or availability of a wide spectrum of substrates through sulfation with the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS. These enzymes are known to be difficult to assay. A convenient assay is needed in order to better understand these enzymes. Results A universal sulfotransferase assay method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE is described. This assay has been successfully applied to substrates as small as α-naphthol and as big as proteoglycans. As examples, we present the assays for recombinant human CHST4, TPST1, CHST3 and HS6ST1. In order to assess whether a small molecule can be applicable to this type of assay, a method to estimate the relative mobility of a molecule to PAPS is also presented. The estimated relative mobilities of various sulfated small molecules generated by SULT1A1, SULT1E1, SULT2A1 and CHST4 are in the range of ± 0.2 of the actual relative mobilities. Conclusion The versatility of the current method comes from the ability that SDS-PAGE can separate proteins and small molecules according to different parameters. While mobilities of proteins during SDS-PAGE are inversely related to their sizes, mobilities of small molecules are positively related to their charge/mass ratios. The predicted relative mobility of a product to PAPS is a good indicator of whether a sulfotransferase can be assayed with SDS-PAGE. Because phosphorylation is most similar to sulfation in chemistry, the method is likely to be applicable to kinases as well.

  2. Nonradioactive glycosyltransferase and sulfotransferase assay to study glycosaminoglycan biosynthesis. (United States)

    Ethen, Cheryl M; Machacek, Miranda; Prather, Brittany; Tatge, Timothy; Yu, Haixiao; Wu, Zhengliang L


    Glycosaminoglycans (GAGs) are linear polysaccharides with repeating disaccharide units. GAGs include heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan. All GAGs, except for hyaluronan, are usually sulfated. GAGs are polymerized by mono- or dual-specific glycosyltransferases and sulfated by various sulfotransferases. To further our understanding of GAG chain length regulation and synthesis of specific sulfation motifs on GAG chains, it is imperative to understand the kinetics of GAG synthetic enzymes. Here, nonradioactive colorimetric enzymatic assays are described for these glycosyltransferases and sulfotransferases. In both cases, the leaving nucleotides or nucleosides are hydrolyzed using specific phosphatases, and the released phosphate is subsequently detected using malachite reagents.

  3. Assays for determining heparan sulfate and heparin O-sulfotransferase activity and specificity. (United States)

    Sterner, Eric; Li, Lingyun; Paul, Priscilla; Beaudet, Julie M; Liu, Jian; Linhardt, Robert J; Dordick, Jonathan S


    O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [(35)S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.

  4. Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays. (United States)

    Prather, Brittany; Ethen, Cheryl M; Machacek, Miranda; Wu, Zhengliang L


    Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.

  5. Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography-tandem mass spectrometry. (United States)

    Bansal, Sumit; Lau, Aik Jiang


    Sulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3→97.0) and cortisol (m/z 407.2→331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2pmol DHEA-S and the calibration curve was linear from 0.2 to 200pmol. The intra-day and inter-day accuracy and precision was sulfotransferase enzyme assays, and it is the first UPLC-MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.

  6. Engineering sulfotransferases to modify heparan sulfate

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    Xu, Ding; Moon, Andrea F.; Song, Danyin; Pedersen, Lars C.; Liu, Jian (NIH); (UNC)


    The biosynthesis of heparan sulfate (HS) involves an array of specialized sulfotransferases. Here, we present a study aimed at engineering the substrate specificity of different HS 3-O-sulfotransferase isoforms. Based on the crystal structures, we identified a pair of amino acid residues responsible for selecting the substrates. Mutations of these residues altered the substrate specificities. Our results demonstrate the feasibility of tailoring the specificity of sulfotransferases to modify HS with desired functions.

  7. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas


    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  8. Fluorescent peptide sensors for tyrosylprotein sulfotransferase activity. (United States)

    Zhou, Wenbo; Duckworth, Benjamin P; Geraghty, Robert J


    Tyrosine sulfurylation is a post-translational modification important for protein-protein interactions in the extracellular space that are instrumental in cell adhesion, cell signaling, immune responses, and pathogen recognition of host cells. Tyrosine sulfurylation is catalyzed by the tyrosylprotein sulfotransferases (TPSTs), and in humans there are two isoforms: hTPST1 and hTPST2. The study of hTPST function and the development of small molecule probes to examine the role of hTPSTs in cell biology have been delayed by the absence of a continuous direct assay for hTPST activity. We have developed a fluorescent peptide-based assay to directly monitor tyrosine sulfurylation in real time. TPST-mediated tyrosine sulfurylation of the peptides disrupts fluorophore quenching and results in increased fluorescence emission. The assay can be used to study TPST enzymatic activity, and we show that recombinant hTPSTs are active in the absence of divalent metal ions and that optimal activity is at pH 6.0. We further show that the assay can also be used to identify inhibitors of tyrosine sulfurylation. A clear understanding of hTPST function in normal cell biology and in disease states will require the identification of small molecule inhibitors or probes to modulate enzymatic activity, and our results will facilitate that process.

  9. Genotoxicity of three food processing contaminants in transgenic mice expressing human sulfotransferases 1A1 and 1A2 as assessed by the in vivo alkaline single cell gel electrophoresis assay. (United States)

    Høie, Anja Hortemo; Svendsen, Camilla; Brunborg, Gunnar; Glatt, Hansruedi; Alexander, Jan; Meinl, Walter; Husøy, Trine


    The food processing contaminants 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 5-hydroxymethylfurfural (HMF) and 2,5 dimethylfuran (DMF) are potentially both mutagenic and carcinogenic in vitro and/or in vivo, although data on DMF is lacking. The PHIP metabolite N-hydroxy-PhIP and HMF are bioactivated by sulfotransferases (SULTs). The substrate specificity and tissue distribution of SULTs differs between species. A single oral dose of PhIP, HMF or DMF was administered to wild-type (wt) mice and mice expressing human SULT1A1/1A2 (hSULT mice). DNA damage was studied using the in vivo alkaline single cell gel electrophoresis (SCGE) assay. No effects were detected in wt mice. In the hSULT mice, PhIP and HMF exposure increased the levels of DNA damage in the liver and kidney, respectively. DMF was not found to be genotoxic. The observation of increased DNA damage in hSULT mice compared with wt mice supports the role of human SULTs in the bioactivation of N-hydroxy-PhIP and HMF in vivo.

  10. Characterization of human iodothyronine sulfotransferases

    NARCIS (Netherlands)

    M.H.A. Kester (Monique); E. Kaptein (Ellen); T.J. Roest (Thirza); C.H. van Dijk (Caren); D. Tibboel (Dick); W. Meinl; H. Glatt; M.W. Coughtrie; T.J. Visser (Theo)


    textabstractSulfation is an important pathway of thyroid hormone metabolism that facilitates the degradation of the hormone by the type I iodothyronine deiodinase, but little is known about which human sulfotransferase isoenzymes are involved. We have investigated the s

  11. In vivo imaging of sulfotransferases (United States)

    Barrio, Jorge R; Kepe, Vladimir; Small, Gary W; Satyamurthy, Nagichettiar


    Radiolabeled tracers for sulfotransferases (SULTs), their synthesis, and their use are provided. Included are substituted phenols, naphthols, coumarins, and flavones radiolabeled with .sup.18F, .sup.123I, .sup.124I, .sup.125I, or .sup.11C. Also provided are in vivo techniques for using these and other tracers as analytical and diagnostic tools to study sulfotransferase distribution and activity, in health and disease, and to evaluate therapeutic interventions.

  12. Crystal structure of sulfotransferase STF9 from Mycobacterium avium. (United States)

    Hossain, Md Murad; Moriizumi, Yuuji; Tanaka, Shotaro; Kimura, Makoto; Kakuta, Yoshimitsu


    Sulfotransferases catalyze the sulfate conjugation of a wide variety of endogenous and exogenous molecules. Human pathogenic mycobacteria produce numerous sulfated molecules including sulfolipids which are well related to the virulence of several strains. The genome of Mycobacterium avium encodes eight putative sulfotransferases (stf1, stf4-stf10). Among them, STF9 shows higher similarity to human heparan sulfate 3-O-sulfotransferase isoforms than to the bacterial STs. Here, we determined the crystal structure of sulfotransferase STF9 in complex with a sulfate ion and palmitic acid at a resolution of 2.6 Å. STF9 has a spherical structure utilizing the classical sulfotransferase fold. STF9 exclusively possesses three N-terminal α-helices (α1, α2, α3) parallel to the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) binding motif. The sulfate ion binds to the PAPS binding structural motif and the palmitic acid molecule binds in the deep cleft of the predicted substrate binding site suggesting the nature of endogenous acceptor substrate of STF9 resembles palmitic acid. The substrate binding site is covered by a flexible loop which may have involvement in endogenous substrate recognition. Based on the mutational study (Hossain et al., Mol Cell Biochem 350:155-162; 2011) and structural resemblance of STF9-sulfate ion-palmitic acid complex to the hHS3OST3 complex with PAP (3'-phosphoadenosine-5'-phosphate) and an acceptor sugar chain, Glu170 and Arg96 are appeared to be catalytic residues in STF9 sulfuryl transfer mechanism.

  13. Molecular cloning and characterization of chondroitin-4-O-sulfotransferase-3. A novel member of the HNK-1 family of sulfotransferases. (United States)

    Kang, Hyung-Gyoo; Evers, Matthias R; Xia, Guoqing; Baenziger, Jacques U; Schachner, Melitta


    We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated chondroitin-4-sulfotransferase-3 (C4ST-3) (GenBank accession number AY120869) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 341 amino acids with a 12-amino acid cytoplasmic domain and a 311-amino acid luminal domain containing a single potential N-linked glycosylation site. C4ST-3 has the greatest amino acid sequence identity when aligned with chondroitin-4-O-sulfotransferase 1 (C4ST-1) (45%) but also shows significant amino acid identity with chondroitin-4-O-sulfotransferase 2 (C4ST-2) (27%), dermatan-4-O-sulfotransferase 1 (29%), HNK-1 ST (26%), N-acetylgalactosamine-4-O-sulfotransferase 1 (26%), and N-acetylgalactosamine-4-O-sulfotransferase 2 (23%). C4ST-3 transfers sulfate to the C-4 hydroxyl of beta1,4-linked GalNAc that is substituted with a beta-linked glucuronic acid at the C-3 hydroxyl. The open reading frame of C4ST-3 is encoded by three exons located on human chromosome 3q21.3. Northern blot analysis reveals a single 2.1-kilobase transcript. C4ST-3 message is expressed in adult liver and at lower levels in adult kidney, lymph nodes, and fetal liver. Although C4ST-3 and C4ST-1 have similar specificities, the highly restricted pattern of expression seen for C4ST-3 suggests that it has a different role than C4ST-1.

  14. Metabolic activation of N-hydroxy arylamines, N-hydroxy heterocyclic amines and ring-hydroxymethyl polycyclic aromatic hydrocarbons by human sulfotransferases

    Energy Technology Data Exchange (ETDEWEB)

    Chou, H.C.


    Arylamines and polycyclic aromatic hydrocarbons (PAHs) are two major classes of chemical carcinogens. N-Hydroxylation of arylamines is regarded to be a necessary process for their mutagenicity and carcinogenicity, while alkyl-hydroxylation is the major metabolic pathway for alkyl-substituted PAHs. Evidence has been presented that sulfation of several N-hydroxy arylamines and hydroxymethyl PAHs is an important pathway leading to the formation of ultimate carcinogens in experiment animals. Sulfation of these chemicals forms putative sulfuric acid ester intermediates that can rearrange to electrophilic nitrenium or carbenium ions capable of forming covalent adducts with important cellular macromolecules. In order to study the metabolic activation by sulfotransferase(s) in various human tissue preparations an in vitro enzymatic assay was established. A metabolic phenotyping method was also developed for thermostable phenolsulfotransferase (TS-PST) in platelet homogenates (correlated with TS-PST activity in other tissues) based on a simple colorimetric assay using 2-naphthol as substrate. By using a PAPS-regenerating system to supply the activated sulfate and calf thymus DNA to trap the reactive metabolites, we found that N-hydroxy derivatives of the carcinogens, 4-aminobiphenyl (4-ABP), 4,4[prime]-methylene-bis(2-chloroaniline) (MOCA), 2-acetylaminofluorene (2-AAF), 2-aminofluorene (2-AF), 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and 2-amino-6-methyldipyrido [1,2-1:3[prime],2[prime]-d]imidazole (Glu-P-1) were metabolically activated by human TS-PST. On the other hand, three methyl-hydroxylated derivatives (7-OH, 12-OH, and 7,12-diOH) of 7, 12-dimethylbenz[a]anthracene (DMBA) were metabolically activated by human steroid sulfotransferase. Human sulfotransferase(s)-mediated activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was not observed.

  15. Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene

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    Gaedigk, A.; Beatty, B.G.; Grant, D.M. [Hospital for Sick Children, Toronto, Ontario (Canada)


    Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p1.2) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family. 27 refs., 3 figs.

  16. Biochanin A induction of sulfotransferases in rats. (United States)

    Chen, Yue; Huang, Chaoqun; Zhou, Tianyan; Zhang, Shunfen; Chen, Guangping


    Biochanin A (BCA) is a dietary isoflavone present in red clover (Trifoliumn pretense) and many herbal products. BCA has been reported to have chemopreventive actions against various cancers including prostate, breast, colon cancer, and so on. Sulfotransferases are a family of phase II drug-metabolizing enzymes, which are important for xenobiotic detoxification and regulation of biological signaling molecule biological activities. Sulfotransferase gene expressions are regulated by different hormones and xenobiotics. Improper regulation of sulfotransferases leads to improper functions of biological signaling molecules, which in turn can cause cancer or other diseases. BCA inhibits the enzyme activities of the phase I drug-metabolizing enzymes CYP1A1 and CYP1B1 in Chinese hamster ovary cells and induces the phase II drug-metabolizing enzymes UDP-glucuronosyltransferases in human prostate cancer cells. BCA induction of sulfotransferases has not been studied. This investigation evaluates the in vivo regulation of sulfotransferases at protein and mRNA levels in the liver and intestine of Sprague-Dawley rats treated with BCA (0, 2, 10, and 50 mg/kg/day) for 7 days. Our experimental results demonstrate for the first time that chronic BCA treatment can significantly induce the expression of rat sulfotransferase 1A1 (rSULT1A1, AST-IV), sulfotransferase 2A1 (rSULT2A1, STa), and rat estrogen sulfotransferase (rSULT1E1, EST) in rat liver and intestine. Our Western blot results are in good agreement with real-time RT-PCR data, suggesting that BCA induction of sulfotransferases occurs at the transcriptional level.

  17. Interactions of cytosolic sulfotransferases with xenobiotics. (United States)

    James, Margaret O; Ambadapadi, Sriram


    Cytosolic sulfotransferases are a superfamily of enzymes that catalyze the transfer of the sulfonic group from 3'-phosphoadenosine-5'-phosphosulfate to hydroxy or amine groups in substrate molecules. The human cytosolic sulfotransferases that have been most studied, namely SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1, are expressed in different tissues of the body, including liver, intestine, adrenal, brain and skin. These sulfotransferases play important roles in the sulfonation of endogenous molecules such as steroid hormones and neurotransmitters, and in the elimination of xenobiotic molecules such as drugs, environmental chemicals and natural products. There is often overlapping substrate selectivity among the sulfotransferases, although one isoform may exhibit greater enzyme efficiency than other isoforms. Similarly, inhibitors or enhancers of one isoform often affect other isoforms, but typically with different potency. This means that if the activity of one form of sulfotransferase is altered (either inhibited or enhanced) by the presence of a xenobiotic, the sulfonation of endogenous and xenobiotic substrates for other isoforms may well be affected. There are more examples of inhibitors than enhancers of sulfonation. Modulators of sulfotransferase enzymes include natural products ingested as part of the human diet as well as environmental chemicals and drugs. This review will discuss recent work on such interactions.

  18. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten


    The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  19. Enzyme kinetics of conjugating enzymes: PAPS sulfotransferase. (United States)

    James, Margaret O


    The sulfotransferase (SULT) enzymes catalyze the formation of sulfate esters or sulfamates from substrates that contain hydroxy or amine groups, utilizing 3'-phosphoadenosyl-5'-phosphosulfate (PAPS) as the donor of the sulfonic group. The rate of product formation depends on the concentrations of PAPS and substrate as well as the sulfotransferase enzyme; thus, if PAPS is held constant while varying substrate concentration (or vice versa), the kinetic constants derived are apparent constants. When studied over a narrow range of substrate concentrations, classic Michaelis-Menten kinetics can be observed with many SULT enzymes and most substrates. Some SULT enzymes exhibit positive or negative cooperativity during conversion of substrate to product, and the kinetics fit the Hill plot. A characteristic feature of most sulfotransferase-catalyzed reactions is that, when studied over a wide range of substrate concentrations, the rate of product formation initially increases as substrate concentration increases, then decreases at high substrate concentrations, i.e., they exhibit substrate inhibition or partial substrate inhibition. This chapter gives an introduction to sulfotransferases, including a historical note, the nomenclature, a description of the function of SULTs with different types of substrates, presentation of examples of enzyme kinetics with SULTs, and a discussion of what is known about mechanisms of substrate inhibition in the sulfotransferases.

  20. Understanding the substrate specificity of the heparan sulfate sulfotransferases by an integrated biosynthetic and crystallographic approach. (United States)

    Liu, Jian; Moon, Andrea F; Sheng, Juzheng; Pedersen, Lars C


    Heparan sulfates (HSs) have potential therapeutic value as anti-inflammatory and antimetastasis drugs, in addition to their current use as anticoagulants. Recent advances in chemoenzymatic synthesis of HS provide a way to conveniently produce homogenous HS with different biological properties. Crystal structures of sulfotransferases involved in this process are providing atomic detail of their substrate binding clefts and interactions with their HS substrates. In theory, the flexibility of this method can be increased by modifying the specificities of the sulfotransferases based on the structures, thereby producing a new array of products.

  1. Phenol sulfotransferase. II. Inactivation by phenylglyoxal, N-ethylmaleimide and ribonucleotide 2',3'-dialdehydes. (United States)

    Borchardt, R T; Schasteen, C S; Wu, S E


    affinity labeling reagents for phenol sulfotransferase, causing enzyme inactivation by the possible formation of a Schiff base adduct with an active-site lysine residue.

  2. Phenol sulfotransferases: Candidate genes for Batten disease

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, T.P.; Probst, P.; Obermoeller, R.D. [M.D. Anderson Cancer Center, Houston, TX (United States)] [and others


    Batten disease (juvenile-onset neuronal ceroid lipofuscinosis; JNCL) is an autosomal recessive neurodegenerative disorder, characterized by the cytosomal accumulation of autofluorescent protolipopigments in neurons and other cell types. The Batten disease gene (CLN3) has not yet been identified, but has been mapped to a small region of human chromosome area 16p12.1-p11.2. We recently reported the fortuitous discovery that the cytosolic phenol sulfotransferase gene (STP) is located within this same interval of chromosome 16p. Since phenol sulfotransferase is expressed in neurons, can sulfate lipophilic phenolic compounds, and is mapped near CLN3, STP is considered as a candidate gene for Batten disease. YAC and cosmid cloning results have further substantiated the close proximity of STP and a highly related sulfotransferase (STM), encoding the catecholamine-preferring enzyme, to the CLN3 region of chromosome 16p. In this report, we summarize some of the recent progress in the identification of two phenol sulfotransferase genes (STP and STM) as positional candidate genes for Batten disease. 42 refs., 1 tab.

  3. Overproduction, purification and preliminary X-ray diffraction analysis of a sulfotransferase from Mycobacterium tuberculosis H37Rv

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    Tanaka, Shotaro; Moriizumi, Yuuji; Kimura, Makoto; Kakuta, Yoshimitsu, E-mail: [Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581 (Japan)


    A sulfotransferase from M. tuberculosis was crystallized and preliminarily analyzed using X-ray diffraction. Sulfotransferase STF1 from the Mycobacterium tuberculosis H37Rv genome was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 1.5 Å resolution using synchrotron radiation at SPring-8. The crystals are monoclinic and belong to space group P2{sub 1}, with unit-cell parameters a = 40.86, b = 95.76, c = 48.04 Å, β = 106.43°. The calculated Matthews coefficient is approximately 2.1 Å{sup 3} Da{sup −1} assuming the presence of one molecule of STF1 in the asymmetric unit. A substrate-binding assay using a PAP–agarose column suggests that STF1 exhibits sulfotransferase activity.

  4. Cell based assay for hypoglycemic drugs screening

    Institute of Scientific and Technical Information of China (English)

    LiZHANG; Juan-juanHU; Guan-huaDU


    OBJECTIVE: To establish a cell based assay for hypoglyc emicdrugs. METHODS: The five cell lines, BALB/c3T3, HepG2, NIH3T3, Be17402, and L929 were incubated with insulin (0-125n mol/L) for 48 h. Their sensitivities to insulin were studied by detecting glucose consumption. The dose-response and time-response relationship between the sensitive cell line (BALB/c 3T3)

  5. Mass-based readout for agglutination assays (United States)

    Chunara, Rumi; Godin, Michel; Knudsen, Scott M.; Manalis, Scott R.


    We present a mass-based readout for agglutination assays. The suspended microchannel resonator (SMR) is used to classify monomers and dimers that are formed during early stage aggregation, and to relate the total count to the analyte concentration. Using a model system of streptavidin functionalized microspheres and biotinylated antibody as the analyte, we obtain a dose-response curve over a concentration range of 0.63-630nM and show that the results are comparable to what has been previously achieved by image analysis and conventional flow cytometry.

  6. Characterization of iodothyronine sulfotransferase activity in rat liver

    NARCIS (Netherlands)

    E. Kaptein (Ellen); G.A.C. van Haasteren (Goedele); E. Linkels; W.J. de Greef; T.J. Visser (Theo)


    textabstractSulfation is an important pathway in the metabolism of thyroid hormone because it strongly facilitates the degradation of the hormone by the type I iodothyronine deiodinase. However, little is known about the properties and possible regulation of the sulfotransferase(s)

  7. The effect of ligands on the thermal stability of sulfotransferases: a molecular dynamics simulation study. (United States)

    Zhang, Pu-pu; Zhao, Li; Long, Shi-yang; Tian, Pu


    Human cytosolic sulfotransferases (hSULTs) are important phase II metabolic enzymes. They catalyze transfer of the sulfuryl-group (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyl or primary amine moieties of a large number of endogenous and xenobiotic substrates. Broad selectivity and specificity of binding and activity within the sulfortransferases family could be detected by thermal denaturation assays, which have been made more and more suitable for high throughput screening based on recent technical advances. Here molecular dynamics simulations were used to explore the effect of the cofactor (PAPS) and substrate (LCA) on the thermal stability of the enzyme. It was found that the apo-enzyme unfolded fastest upon heating. The holo-enzyme with bound substrate LCA unfolded slowest. This thermo-denaturation order is consistent with that observed in experiments. Further it was found that the cofactor and substrate will pronouncedly increase the thermal stability of the active pocket regions that interact directly with the ligands. In addition, cofactor and substrate show noticeable synergy effect on the thermal stability of the enzyme.

  8. Rat phenol-preferring sulfotransferase genes (Stp and Stp2): Localization to mouse chromosomes 7 and 17

    Energy Technology Data Exchange (ETDEWEB)

    Khan, A.S.; Taylor, B.R.; Ringer, D.P. [Oklahoma Medical Research Foundation, Oklahoma City, OK (United States)] [and others


    The phenol-preferring sulfotransferases aryl sulfo-transferase IV and N-hydroxyarylamine sulfotransferase catalyze sulfate conjugation of N-hydroxy-2-acetylaminofluorene, a metabolite capable of causing hepatocarcinogenesis in rats. We utilized published cDNA sequences of these sulfotransferases to type the progeny of two multilocus crosses and determined that the genes, aryl sulfotransferase (Stp) and N-hydroxyarylamine sulfotransferase (Stp2), map to positions on mouse chromosomes 7 and 17. 19 refs., 1 fig.

  9. Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis

    Directory of Open Access Journals (Sweden)

    King Roberta S


    Full Text Available Abstract Background Ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine. Results The two sulfotransferase genes were coded as Ixosc SULT 1 & 2 and corresponding proteins were referred as Ixosc Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that Ixosc SULT1 was statistically increased upon blood feeding while Ixosc SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins Ixosc Sult 1(R and 2(R showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in I. scapularis

  10. Inhibition of thyroid hormone sulfotransferase activity by brominated flame retardants and halogenated phenolics. (United States)

    Butt, Craig M; Stapleton, Heather M


    Many halogenated organic contaminants (HOCs) are considered endocrine disruptors and affect the hypothalamic-pituitary-thyroid axis, often by interfering with circulating levels of thyroid hormones (THs). We investigated one potential mechanism for TH disruption, inhibition of sulfotransferase activity. One of the primary roles of TH sulfation is to support the regulation of biologically active T3 through the formation of inactive THs. We investigated TH sulfotransferase inhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs), BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinated analogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). A new mass spectrometry-based method was also developed to measure the formation rates of 3,3'-T2 sulfate (3,3'-T2S). Using pooled human liver cytosol, we investigated the influence of these HOCs on the sulfation of 3,3'-T2, a major substrate for TH sulfation. For the formation of 3,3'-T2S, the Michaelis constant (Km) was 1070 ± 120 nM and the Vmax was 153 ± 6.6 pmol min(-1) (mg of protein)(-1). All chemicals investigated inhibited sulfotransferase activity with the exception of BDE 47. The 2,4,6-trihalogenated phenols were the most potent inhibitors followed by the OH BDEs and then halogenated BPAs. The IC50 values for the OH BDEs were primarily in the low nanomolar range, which may be environmentally relevant. In silico molecular modeling techniques were also used to simulate the binding of OH BDE to SULT1A1. This study suggests that some HOCs, including antimicrobial chemicals and metabolites of flame retardants, may interfere with TH regulation through inhibition of sulfotransferase activity.

  11. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis

    Energy Technology Data Exchange (ETDEWEB)

    Shi,R.; Lamb, S.; Bhat, S.; Sulea, T.; Wright, G.; Matte, A.; Cygler, M.


    Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.

  12. Downregulation of sulfotransferase expression and activity in diseased human livers. (United States)

    Yalcin, Emine B; More, Vijay; Neira, Karissa L; Lu, Zhenqiang James; Cherrington, Nathan J; Slitt, Angela L; King, Roberta S


    Sulfotransferase (SULT) function has been well studied in healthy human subjects by quantifying mRNA and protein expression and determining enzyme activity with probe substrates. However, it is not well known if sulfotransferase activity changes in metabolic and liver disease, such as diabetes, steatosis, or cirrhosis. Sulfotransferases have significant roles in the regulation of hormones and excretion of xenobiotics. In the present study of normal subjects with nonfatty livers and patients with steatosis, diabetic cirrhosis, and alcoholic cirrhosis, we sought to determine SULT1A1, SULT2A1, SULT1E1, and SULT1A3 activity and mRNA and protein expression in human liver tissue. In general, sulfotransferase activity decreased significantly with severity of liver disease from steatosis to cirrhosis. Specifically, SULT1A1 and SULT1A3 activities were lower in disease states relative to nonfatty tissues. Alcoholic cirrhotic tissues further contained lower SULT1A1 and 1A3 activities than those affected by either of the two other disease states. SULT2A1, on the other hand, was only reduced in alcoholic cirrhotic tissues. SULT1E1 was reduced both in diabetic cirrhosis and in alcoholic cirrhosis tissues, relative to nonfatty liver tissues. In conclusion, the reduced levels of sulfotransferase expression and activity in diseased versus nondiseased liver tissue may alter the metabolism and disposition of xenobiotics and affect homeostasis of endobiotic sulfotransferase substrates.

  13. A nano switch mechanism for the redox-responsive sulfotransferase. (United States)

    Lin, Chih-Heng; Lin, En-Shyh; Su, Tian-Mu; Hung, Kuo-Sheng; Yang, Yuh-Shyong


    Cellular redox signaling is important in diverse physiological and pathological processes. The activity of rat phenol sulfotransferase (rSULT1A1), which is important for the metabolism of hormone and drug, is subjected to redox regulation. Two cysteines, Cys232 and Cys66, nanometer away from each other and from the enzyme active site were proposed to form disulfide bond to regulate the activity of rSULT1A1. A nano switch, composed of a flexible loop from amino acid residues 59-70, explained how this long distance interaction between two cysteines can be achieved. The enzyme properties were investigated through site-directed muatagnesis, circular dichroism, enzyme kinetics and homologous modeling of the rSULT1A1 structures. We proposed that the formation of disulfide bond between Cys232 and Cys66 induced conformational changes of sulfotransferase, then in turn affected its nucleotide binding and enzyme activity. This discovery was extended to understand the possible redox regulation of other sulfotransferases from different organisms. The redox switch can be created in other redox-insensitive sulfotransferases, such as human phenol sulfotransferase (hSULT1A1) and human alcohol sulfotransferase (hSULT2A1), to produce mutant enzymes with redox regulation capacity. This study strongly suggested that redox regulation of drug and hormone metabolism can be significantly varied even though the sequence and structure of SULT1A1 of human and rat have a high degree of homology.

  14. Nematodes join the family of chondroitin sulfate-synthesizing organisms: Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans (United States)

    Dierker, Tabea; Shao, Chun; Haitina, Tatjana; Zaia, Joseph; Hinas, Andrea; Kjellén, Lena


    Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS. PMID:27703236

  15. Effect of folic acid on methotrexate induction of sulfotransferases in rats. (United States)

    Dutta, Sangita Maiti; Maiti, Smarajit; Chen, Guangping


    Our earlier investigation showed that MTX is an inducer of rat and human sulfotransferases. Here we report that folic acid treatment inhibited MTX induction of aryl sulfotransferase (AST-IV) in female rat liver and hydroxysteroid sulfotransferase (STa) in male rat liver. This is important for understanding the clinical mechanisms of MTX.

  16. Inflammatory cues modulate the expression of secretory product genes, Golgi sulfotransferases and sulfomucin production in LS174T cells. (United States)

    Croix, Jennifer A; Bhatia, Shikha; Gaskins, H Rex


    The signals that mediate goblet cell expression of specific mucin chemotypes are poorly defined. Animal and in vitro studies show that acidomucin chemotypes may be altered by inflammation and changes in intestinal microbiota. To examine factors that may elicit this response, human adenocarcinoma-derived LS174T cells, which have a goblet cell-like phenotype and produce both sulfo- and sialomucins, were used to examine the effects of selected microbial and host factors on expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production. Expression of genes encoding mucin 2 (MUC2), resistin-like molecule β (RETNLB), and trefoil factor 3 (TFF3) and Golgi sulfotransferases, carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), was measured by quantitative reverse transcriptase-polymerase chain reaction following treatment with bacterial flagellin, tumor necrosis factor α (TNF-α) or the mucogenic cytokine interleukin-13 (IL-13). Expression of the toll-like receptor 5 (TLR5) gene was also analysed. Sulfomucin expression was examined via high-iron diamide/alcian blue (HID/AB) histochemistry and immunofluorescent staining for the Sulfo Le(a) antigen, which is synthesized in part by GAL3ST2. Flagellin, IL-13 and TNF-α all significantly increased GAL3ST2, MUC2, TFF3 and TLR5 expression, while only IL-13 increased RETNLB and CHST5 expression. Based on HID/AB histochemistry, mucin sulfation was significantly increased in response to both flagellin and IL-13 but not TNF-α. Only treatment with flagellin increased the expression of the Sulfo Le(a) antigen. Collectively, these results indicate that bacterial flagellin, IL-13 and TNF-α differentially modulate the expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production.

  17. Identification and Characterization of Tyrosylprotein Sulfotransferase from Human Saliva

    Directory of Open Access Journals (Sweden)


    Full Text Available Tyrosylprotein sulfotransferase (TPST, the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96. Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.

  18. Computer-determined assay time based on preset precision

    Energy Technology Data Exchange (ETDEWEB)

    Foster, L.A.; Hagan, R.; Martin, E.R.; Wachter, J.R.; Bonner, C.A.; Malcom, J.E. [Los Alamos National Lab., NM (United States). Nuclear Materials Measurement and Accountability


    Most current assay systems for special nuclear materials (SNM) operate on the principle of a fixed assay time which provides acceptable measurement precision without sacrificing the required throughput of the instrument. Waste items to be assayed for SNM content can contain a wide range of nuclear material. Counting all items for the same preset assay time results in a wide range of measurement precision and wastes time at the upper end of the calibration range. A short time sample taken at the beginning of the assay could optimize the analysis time on the basis of the required measurement precision. To illustrate the technique of automatically determining the assay time, measurements were made with a segmented gamma scanner at the Plutonium Facility of Los Alamos National Laboratory with the assay time for each segment determined by counting statistics in that segment. Segments with very little SNM were quickly determined to be below the lower limit of the measurement range and the measurement was stopped. Segments with significant SNM were optimally assays to the preset precision. With this method the total assay time for each item is determined by the desired preset precision. This report describes the precision-based algorithm and presents the results of measurements made to test its validity.

  19. Cell-based Assays to Identify Inhibitors of Viral Disease (United States)

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong


    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  20. Oestrogen sulfotransferase ablation sensitizes mice to sepsis. (United States)

    Chai, Xiaojuan; Guo, Yan; Jiang, Mengxi; Hu, Bingfang; Li, Zhigang; Fan, Jie; Deng, Meihong; Billiar, Timothy R; Kucera, Heidi R; Gaikwad, Nilesh W; Xu, Meishu; Lu, Peipei; Yan, Jiong; Fu, Haiyan; Liu, Youhua; Yu, Lushan; Huang, Min; Zeng, Su; Xie, Wen


    Sepsis is the host's deleterious systemic inflammatory response to microbial infections. Here we report an essential role for the oestrogen sulfotransferase (EST or SULT1E1), a conjugating enzyme that sulfonates and deactivates estrogens, in sepsis response. Both the caecal ligation and puncture (CLP) and lipopolysaccharide models of sepsis induce the expression of EST and compromise the activity of oestrogen, an anti-inflammatory hormone. Surprisingly, EST ablation sensitizes mice to sepsis-induced death. Mechanistically, EST ablation attenuates sepsis-induced inflammatory responses due to compromised oestrogen deactivation, leading to increased sepsis lethality. In contrast, transgenic overexpression of EST promotes oestrogen deactivation and sensitizes mice to CLP-induced inflammatory response. The induction of EST by sepsis is NF-κB dependent and EST is a NF-κB-target gene. The reciprocal regulation of inflammation and EST may represent a yet-to-be-explored mechanism of endocrine regulation of inflammation, which has an impact on the clinical outcome of sepsis.

  1. A capillary electrophoresis method with dynamic pH junction stacking for the monitoring of cerebroside sulfotransferase. (United States)

    Li, Wenjin; Zech, Isabell; Gieselmann, Volkmar; Müller, Christa E


    Metachromatic leukodystrophy (MLD) is a rare and severe genetic disease. Inhibition of cerebroside sulfotransferase (CST) has been proposed as a promising new therapeutic strategy for the treatment of MLD. CST catalyzes the transfer of a sulfate group from the coenzyme 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to cerebroside yielding cerebroside sulfate and adenosine-3',5'-diphosphate (PAP). So far only a few weak CST inhibitors have been described. The goal of the present study was to establish a suitable assay for identifying and characterizing novel CST inhibitors. To this end, we developed and optimized a capillary electrophoresis (CE) based assay for monitoring the catalytic activity of CST by measuring the formation of PAP. A sample matrix consisting of 5mM phosphate buffer with about 0.0001% polybrene at pH 7.4 and a background electrolyte (BGE) containing 75 mM phosphate buffer with 0.002% polybrene at pH 5.6 were utilized to achieve a stacking effect for PAP by dynamic pH junction. This led to a limit of detection for the enzymatic product PAP of 66.6 nM. The CE method was sensitive, robust, and suitable for CST inhibitor screening, Ki value determination, and enzyme kinetic studies. Selected reference compounds were tested in order to validate the assay, including the substrates cerebroside and psychosine, and the inhibitor Congo Red. The newly developed CE method will be useful for the identification and development of novel CST inhibitors which are urgently needed for the treatment of MLD.

  2. A Cell-Based Assay to Assess Hemichannel Function (United States)

    Krishnan, Srinivasan; Fiori, Mariana C.; Cuello, Luis G.; Altenberg, Guillermo A.


    Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms.

  3. Traditional and Model Based Assay of Irregular Geometry Items

    Energy Technology Data Exchange (ETDEWEB)



    The Analytical Development Section (ADS) of SRNL was requested to perform a waste disposal assay of two heater boxes which had been used in the HB Line dissolvers. They had been sent to SRNL for study to make recommendations on how to prevent future failure of the units when they were replaced. The study having been completed, the units needed to be characterized prior to sending to Solid Waste for disposal. An assay station consisting of a turntable, HPGe detector, CANBERRA Inspector, transmission source and a portable computer was set up to do the required assays. The assays indicate the presence of U-235, Pu-239 and Cs-137. No measurable amounts of U-235 or Pu-239 were found. Therefore the Minimum Detectable Activities for U-235 and Pu-239 were calculated. For Heater Box 1, 0.23 grams of U-235 and 0.24 grams of Pu-239. For Heater Box 2, the results were 0.21 grams of U-235 and 0.21 grams of Pu-239. This paper describes and documents the assays employed to determine the amount of U, Pu and Cs contents of the heater boxes. The paper provides results of SNM assays using traditional calibration of the system and on one based on modeling. It also provides the scientific community with data that will assist the user in determining the method of choice for assaying items with irregular geometries.

  4. High accuracy in silico sulfotransferase models. (United States)

    Cook, Ian; Wang, Ting; Falany, Charles N; Leyh, Thomas S


    Predicting enzymatic behavior in silico is an integral part of our efforts to understand biology. Hundreds of millions of compounds lie in targeted in silico libraries waiting for their metabolic potential to be discovered. In silico "enzymes" capable of accurately determining whether compounds can inhibit or react is often the missing piece in this endeavor. This problem has now been solved for the cytosolic sulfotransferases (SULTs). SULTs regulate the bioactivities of thousands of compounds--endogenous metabolites, drugs and other xenobiotics--by transferring the sulfuryl moiety (SO3) from 3'-phosphoadenosine 5'-phosphosulfate to the hydroxyls and primary amines of these acceptors. SULT1A1 and 2A1 catalyze the majority of sulfation that occurs during human Phase II metabolism. Here, recent insights into the structure and dynamics of SULT binding and reactivity are incorporated into in silico models of 1A1 and 2A1 that are used to identify substrates and inhibitors in a structurally diverse set of 1,455 high value compounds: the FDA-approved small molecule drugs. The SULT1A1 models predict 76 substrates. Of these, 53 were known substrates. Of the remaining 23, 21 were tested, and all were sulfated. The SULT2A1 models predict 22 substrates, 14 of which are known substrates. Of the remaining 8, 4 were tested, and all are substrates. The models proved to be 100% accurate in identifying substrates and made no false predictions at Kd thresholds of 100 μM. In total, 23 "new" drug substrates were identified, and new linkages to drug inhibitors are predicted. It now appears to be possible to accurately predict Phase II sulfonation in silico.

  5. Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: II. The 6-O-sulfotransferase family. (United States)

    Cadwallader, Adam B; Yost, H Joseph


    Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 6-O-sulfotransferases (6-OST) catalyze the transfer of sulfate groups to the 6-O position of glucosamine residues of HS. We report here the characterization and developmental expression analysis of the 6-OST gene family in the zebrafish. The zebrafish 6-OST gene family consists of four conserved vertebrate orthologues, including a gene duplication specific to zebrafish. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin development. Members of the 6-OST gene family have spatially and temporally distinct restricted expression, suggesting in vivo functional differences exist between members of this family.

  6. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas


    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  7. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  8. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

    Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon;


    Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...... human EPO was evaluated as a capturing antibody in a label-free biolayer interferometry-based quantification assay. Human recombinant EPO can be specifically detected in Chinese hamster ovary cell supernatants in a sensitive and pH-dependent manner. This method enables rapid and robust quantification...... of EPO in a high-throughput setting....

  9. Heparan sulfate 6-O-sulfotransferase 3 is involved in bone marrow mesenchymal stromal cell osteogenic differentiation‍. (United States)

    Zhao, Shancheng; Deng, Chao; Wang, Zhen; Teng, Liping; Chen, Jinghua


    The roles of sugar chains such as heparan sulfate (HS) in stem cell self-renewal and differentiation are poorly understood. HS is a sugar chain with linear sulfated polyanionic disaccharide repeating structures that interact with many proteins, including structural proteins in the extracellular matrix and growth factors and their receptors. Thus, unraveling the role of HS in stem cell self-renewal and differentiation could provide new insights and technical routes in clinical stem cell applications. Here, we purified rat bone marrow mesenchymal stromal cells (BMMSCs) by density gradient centrifugation, analyzed mesenchymal stromal cell surface stemness marker expression by flow cytometry, and identified the sulfotransferases responsible for sulfation ester modification of HS. An osteogenic differentiation model was established by chemical induction reagents and confirmed via alkaline phosphatase (ALP) activity detection and the expression of the osteogenic differentiation markers Runx2 and Ocn. The expression profiles of HS sulfotransferases in rat BMMSCs before and after osteogenic induction were detected by RT-PCR and Western blot. Cell spheroids were formed in both control and osteogenic culture systems when BMMSCs were grown to high confluence. We determined that this type of cell spheroid was a highly calcified nodule by histochemical staining. Among all the sulfotransferases examined, heparan sulfate 6-O-sulfotransferase 3 (HS6ST3) mRNA and protein were upregulated in these calcified cell spheroids. HS6ST3 knockdown BMMSCs were established with RNA interference, and they had significantly lower ALP activity and decreased expression of the osteogenic differentiation markers Runx2 and Ocn. These findings suggest that HS6ST3 is involved in BMMSC differentiation, and new glycotherapeutic-based technologies could be developed in the future.

  10. Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate. (United States)

    Zhou, Xianxuan; Chandarajoti, Kasemsiri; Pham, Truong Quang; Liu, Renpeng; Liu, Jian


    Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

  11. Parabens inhibit human skin estrogen sulfotransferase activity: possible link to paraben estrogenic effects. (United States)

    Prusakiewicz, Jeffery J; Harville, Heather M; Zhang, Yanhua; Ackermann, Chrisita; Voorman, Richard L


    Parabens (p-hydroxybenzoate esters) are a group of widely used preservatives in topically applied cosmetic and pharmaceutical products. Parabens display weak associations with the estrogen receptors in vitro or in cell based models, but do exhibit estrogenic effects in animal models. It is our hypothesis that parabens exert their estrogenic effects, in part, by elevating levels of estrogens through inhibition of estrogen sulfotransferases (SULTs) in skin. We report here the results of a structure-activity-relationship of parabens as inhibitors of estrogen sulfation in human skin cytosolic fractions and normal human epidermal keratinocytes. Similar to reports of paraben estrogenicity and estrogen receptor affinity, the potency of SULT inhibition increased as the paraben ester chain length increased. Butylparaben was found to be the most potent of the parabens in skin cytosol, yielding an IC(50) value of 37+/-5 microM. Butylparaben blocked the skin cytosol sulfation of estradiol and estrone, but not the androgen dehydroepiandrosterone. The parabens were also tested as inhibitors of SULT activity in a cellular system, with normal human epidermal keratinocytes. The potency of butylparaben increased three-fold in these cells relative to the IC(50) value from skin cytosol. Overall, these results suggest chronic topical application of parabens may lead to prolonged estrogenic effects in skin as a result of inhibition of estrogen sulfotransferase activity. Accordingly, the skin anti-aging benefits of many topical cosmetics and pharmaceuticals could be derived, in part, from the estrogenicity of parabens.

  12. Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells. (United States)

    Chen, Yue; Zhang, Shunfen; Zhou, Tianyan; Huang, Chaoqun; McLaughlin, Alicia; Chen, Guangping


    Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT.

  13. Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: I. The 3-O-sulfotransferase family. (United States)

    Cadwallader, Adam B; Yost, H Joseph


    Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3-O-sulfotransferases (3-OST) catalyze the transfer of sulfate groups to the 3-O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3-OST gene family in a vertebrate. There are eight 3-OST genes in zebrafish: seven genes with homology to known 3-OST genes in mouse and human, as well as a novel, 3-OST-7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3-OST family can be subdivided into two distinct subgroups. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3-OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family.

  14. ApoHRP-based Assay to Measure Intracellular Regulatory Heme (United States)

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A.; Dhahbi, Joseph M.


    The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells. PMID:25525887

  15. Miniaturized Aptamer-Based Assays for Protein Detection

    Directory of Open Access Journals (Sweden)

    Alessandro Bosco


    Full Text Available The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM. We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (μL range assay conditions.

  16. A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay. (United States)

    Park, Jun Hee; Cho, Yea Seul; Kang, Sungmuk; Lee, Eun Jeong; Lee, Gwan-Ho; Hah, Sang Soo


    A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3',5'-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.

  17. Cell based assays for anti-Plasmodium activity evaluation. (United States)

    Mokgethi-Morule, Thabang; N'Da, David D


    Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed.

  18. ABAP: antibody-based assay for peptidylarginine deiminase activity. (United States)

    Zendman, Albert J W; Raijmakers, Reinout; Nijenhuis, Suzanne; Vossenaar, Erik R; Tillaart, Marloes van den; Chirivi, Renato G S; Raats, Jos M H; van Venrooij, Walther J; Drijfhout, Jan W; Pruijn, Ger J M


    Members of the family of peptidylarginine deiminases (PADs, EC catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.

  19. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.


    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  20. Caffeine induction of sulfotransferases in rat liver and intestine. (United States)

    Zhou, Tianyan; Chen, Yue; Huang, Chaoqun; Chen, Guangping


    Sulfotransferases (SULTs) are important phase II drug-metabolizing enzymes. Regulation of SULTs by hormones and other endogenous molecules is relatively well understood, while xenobiotic induction of SULTs is not well studied. Caffeine is one of the most widely consumed psychoactive substances. However, SULT regulation by caffeine has not been reported. In this report, male and female rats were treated with different oral doses of caffeine (2, 10, 50 mg kg⁻¹ per day) for 7 days. Western blot and real-time RT-PCR were used to investigate the changes in SULT protein and mRNA expression following the caffeine treatment. Caffeine induced both rat aryl sulfotransferase (rSULT1A1, AST-IV) and rat hydroxysteroid sulfotransferase (rSULT2A1, STa) in the liver and intestine of female rats in a dose-dependent manner. Caffeine induction of rSULT1A1 and rSULT2A1 in the female rat intestine was much stronger than that in the liver. Although caffeine induced rSULT1A1 significantly in the male rat liver, it did not significantly induce rSULT2A1. In male rat intestine, caffeine significantly induced rSULT2A1. The different SULTs induction patterns in male and female rats suggest that the regulation of rat SULTs by caffeine may be affected by different hormone secretion patterns and levels. Our results suggest that consumption of caffeine can induce drug metabolizing SULTs in drug detoxification tissues.

  1. Automated microfluidic screening assay platform based on DropLab. (United States)

    Du, Wen-Bin; Sun, Meng; Gu, Shu-Qing; Zhu, Ying; Fang, Qun


    This paper describes DropLab, an automated microfluidic platform for programming droplet-based reactions and screening in the nanoliter range. DropLab can meter liquids with picoliter-scale precision, mix multiple components sequentially to assemble composite droplets, and perform screening reactions and assays in linear or two-dimensional droplet array with extremely low sample and reagent consumptions. A novel droplet generation approach based on the droplet assembling strategy was developed to produce multicomponent droplets in the nanoliter to picoliter range with high controllability on the size and composition of each droplet. The DropLab system was built using a short capillary with a tapered tip, a syringe pump with picoliter precision, and an automated liquid presenting system. The tapered capillary was used for precise liquid metering and mixing, droplet assembling, and droplet array storage. Two different liquid presenting systems were developed based on the slotted-vial array design and multiwell plate design to automatically present various samples, reagents, and oil to the capillary. Using the tapered-tip capillary and the picoliter-scale precision syringe pump, the minimum unit of the droplet volume in the present system reached ~20 pL. Without the need of complex microchannel networks, various droplets with different size (20 pL-25 nL), composition, and sequence were automatically assembled, aiming to multiple screening targets by simply adjusting the types, volumes, and mixing ratios of aspirated liquids on demand. The utility of DropLab was demonstrated in enzyme inhibition assays, protein crystallization screening, and identification of trace reducible carbohydrates.

  2. A functional assay-based strategy for nanomaterial risk forecasting

    Energy Technology Data Exchange (ETDEWEB)

    Hendren, Christine Ogilvie, E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Lowry, Gregory V., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Carnegie Mellon University, 119 Porter Hall, Pittsburgh, PA 15213 (United States); Unrine, Jason M., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Plant and Soil Sciences, University of Kentucky, Agricultural Science Center, Lexington, KY 40546 (United States); Wiesner, Mark R., E-mail: [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Duke University, 121 Hudson Hall PO Box 90287, Durham, NC 27708 (United States)


    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical–chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical–chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. - Highlights: • Approaches to predict risk directly from nanomaterial (NM) properties are problematic. • We propose

  3. Development of a Drosophila cell-based error correction assay

    Directory of Open Access Journals (Sweden)

    Jeffrey D. Salemi


    Full Text Available Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT interactions before anaphase onset results in chromosomal instability (CIN, which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5. Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due to the activity of kinesin-14 (Ncd when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC. Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and

  4. High content cell-based assay for the inflammatory pathway (United States)

    Mukherjee, Abhishek; Song, Joon Myong


    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  5. Development of a Drosophila cell-based error correction assay. (United States)

    Salemi, Jeffrey D; McGilvray, Philip T; Maresca, Thomas J


    Accurate transmission of the genome through cell division requires microtubules from opposing spindle poles to interact with protein super-structures called kinetochores that assemble on each sister chromatid. Most kinetochores establish erroneous attachments that are destabilized through a process called error correction. Failure to correct improper kinetochore-microtubule (kt-MT) interactions before anaphase onset results in chromosomal instability (CIN), which has been implicated in tumorigenesis and tumor adaptation. Thus, it is important to characterize the molecular basis of error correction to better comprehend how CIN occurs and how it can be modulated. An error correction assay has been previously developed in cultured mammalian cells in which incorrect kt-MT attachments are created through the induction of monopolar spindle assembly via chemical inhibition of kinesin-5. Error correction is then monitored following inhibitor wash out. Implementing the error correction assay in Drosophila melanogaster S2 cells would be valuable because kt-MT attachments are easily visualized and the cells are highly amenable to RNAi and high-throughput screening. However, Drosophila kinesin-5 (Klp61F) is unaffected by available small molecule inhibitors. To overcome this limitation, we have rendered S2 cells susceptible to kinesin-5 inhibitors by functionally replacing Klp61F with human kinesin-5 (Eg5). Eg5 expression rescued the assembly of monopolar spindles typically caused by Klp61F depletion. Eg5-mediated bipoles collapsed into monopoles due, in part, to kinesin-14 (Ncd) activity when treated with the kinesin-5 inhibitor S-trityl-L-cysteine (STLC). Furthermore, bipolar spindles reassembled and error correction was observed after STLC wash out. Importantly, error correction in Eg5-expressing S2 cells was dependent on the well-established error correction kinase Aurora B. This system provides a powerful new cell-based platform for studying error correction and CIN.

  6. Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yue; Zhang, Shunfen [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Zhou, Tianyan [Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083 (China); Huang, Chaoqun; McLaughlin, Alicia [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Chen, Guangping, E-mail: [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States)


    Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.

  7. The 3'-terminal exon of the family of steroid and phenol sulfotransferase genes is spliced at the N-terminal glycine of the universally conserved GXXGXXK motif that forms the sulfonate donor binding site.


    Chiba, H; Komatsu, K.; Lee, Y.C.; Tomizuka, T; Strott, C A


    The guinea pig estrogen sulfotransferase gene has been cloned and compared to three other cloned steroid and phenol sulfotransferase genes (human estrogen sulfotransferase, human phenol sulfotransferase, and guinea pig 3 alpha-hydroxysteroid sulfotransferase). The four sulfotransferase genes demonstrate a common outstanding feature: the splice sites for their 3'-terminal exons are identically located. That is, the 3'-terminal exon splice sites involve a glycine that constitutes the N-terminal...

  8. Exposure-based validation list for developmental toxicity screening assays

    NARCIS (Netherlands)

    Daston, George P.; Beyer, Bruce K.; Carney, Edward W.; Chapin, Robert E.; Friedman, Jan M.; Piersma, Aldert H.; Rogers, John M.; Scialli, Anthony R.


    Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as "positive" or "negative" in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a

  9. Manganese-dependent Dopa/tyrosine sulfation in HepG2 human hepatoma cells: Novel Dopa/tyrosine sulfotransferase activities associated with the human monamine-form phenol sulfotransferase


    Sakakibara, Yoichi; Katafuchi, Junko; Takami, Yasunari; Nakayama, Tatsuno; Suiko, Masahito; Nakajima, Hiroshi; Liu, Ming-Cheh


    Human monoamine (M)-form phenol sulfotransferase (PST) was PCR-cloned and transiently expressed in COS-7 cells. The recombinant enzyme was demonstrated to display not only the previously reported sulfotransferase activity toward dopamine, but also novel manganese-dependent Dopa/tyrosine sulfotransferase activities. These results imply a new functional role of the human M-form PST in the homeostatic regulation of Dopa and tyrosine.

  10. Antibody-Based Assays for Phenotyping of Extracellular Vesicles

    Directory of Open Access Journals (Sweden)

    Lotte Hatting Pugholm


    Full Text Available Extracellular vesicles (EVs are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA. This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

  11. GTP-specific fab fragment-based GTPase activity assay. (United States)

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri


    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  12. Colloidal gold probe based rapid immunochromatographic strip assay for cortisol

    Energy Technology Data Exchange (ETDEWEB)

    Nara, Seema, E-mail: [Department of Applied Mechanics (Biotechnology), Motilal Nehru National Institute of Technology, Allahabad 211004 (India); Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Tripathi, Vinay [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India); Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Singh, Harpal [Center for BioMedical Engineering, Indian Institute of Technology, New Delhi 110016 (India); Shrivastav, Tulsidas G. [Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, Munirka, New Delhi 110067 (India)


    A rapid and semi-quantitative immunochromatographic strip (ICS) test for cortisol analysis in serum was developed. The test strip was based on a competitive assay format. Colloidal gold nanoparticles were synthesized and coupled with cortisol-3-carboxymethyloxime-adipic acid dihydrazide-bovine serum albumin (F-3-CMO-ADH-BSA) antigen to directly compete with cortisol in human serum samples. F-3-CMO-ADH-BSA-gold label and uncoupled colloidal gold nanoparticles were appropriately characterized using UV-vis spectroscopy, transmission electron microscopy and atomic force microscopy. Anticortisol antibody raised against F-3-CMO-BSA immunogen in New Zealand white rabbits was coated on the NC membrane as test line. Anti-BSA antibody was used as control line. The lower detection limit of the ICS test was 30 ng mL{sup -1} with visual detection and was completed in 10 min. About 30 human serum samples were also analyzed by the developed strip test and their range of cortisol concentration was established. The developed ICS test is rapid, economic and user friendly.

  13. Nanobeads-based assays. The case of gluten detection (United States)

    Venditti, Iole; Fratoddi, Ilaria; Vittoria Russo, Maria; Bellucci, Stefano; Crescenzo, Roberta; Iozzino, Luisa; Staiano, Maria; Aurilia, Vincenzo; Varriale, Antonio; Rossi, Mosè; D'Auria, Sabato


    In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

  14. A label-free, fluorescence based assay for microarray (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  15. Roles of GlcNAc-6-O-sulfotransferases in lymphoid and nonlymphoid tissues. (United States)

    Kawashima, Hiroto


    Recent studies using sulfotransferase-deficient mice have revealed various physiological functions of sulfated glycans. Studies using gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 showed that these sulfotransferases play critical roles in lymphocyte homing. Recent studies indicated that GlcNAc6ST-2 is expressed not only in lymph node high endothelial venules but also in the colonic epithelial cells in mice, and that this sulfotransferase plays a critical role in GlcNAc-6-O-sulfation of the colonic-mucins, as revealed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type (WT) and GlcNAc6ST-2-deficient mice. After induction of colitis by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of WT mice. These studies demonstrate that GlcNAc-6-O-sulfotransferases play important roles not only in lymphoid tissues but also in nonlymphoid tissues. This chapter describes experimental procedures for assessing the functions of GlcNAc-6-O-sulfotransferases using gene-targeted mice.

  16. The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

    Directory of Open Access Journals (Sweden)

    Livia Pica-Mattoccia


    Full Text Available Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

  17. Molecular cloning, expression, and functional analysis of a predicted sulfotransferase STF9 from Mycobacterium avium. (United States)

    Hossain, Md Murad; Moriizumi, Yuuji; Tanaka, Shotaro; Kimura, Makoto; Kakuta, Yoshimitsu


    Sulfotransferases catalyze the transfer of sulfate group from para-nitrophenyl sulfate (pNPS) or 3'-phosphoadenosine-5'-phosphosulfate (PAPS) onto acceptor molecules in the biosynthesis of sulfate esters. Human pathogenic mycobacteria are known to produce numerous sulfated molecules on their cell surface which have been implicated as important mediators in host-pathogen interactions. The open reading frame stf9, a predicted homologue of sulfotransferase in the Mycobacterium avium genomic data, was cloned and over expressed in Escherichia coli. The recombinant STF9 conserved the characteristic PAPS binding motif of sulfotransferase and was purified as a 44 kDa soluble protein which exhibited transfer of sulfate group from pNPS (K (m) 1.34 mM, V (max) 7.56 nmol/min/mg) onto 3'-phosphoadenosine-5'-phosphate (K (m) 0.24 mM, V (max) 10.36 nmol/min/mg). The recombinant STF9 protein was also capable of transferring sulfate group from PAPS onto certain acceptor substrates in E. coli, and showed binding affinity to the PAP-agarose resin, supporting the sulfotransferase activity of the recombinant STF9 protein. This is the first report of molecular evidence for sulfotransferase activity of a protein from M. avium. Mutation of Arg96 to Ala and Glu170 to Ala abolishes sulfotransferase activity, indicating the importance of Arg96 and Glu170 in STF9 activity catalysis.

  18. Identification of chondroitin/dermatan sulfotransferases in the protochordate, Ciona intestinalis. (United States)

    Tetsukawa, Akira; Nakamura, Jun; Fujiwara, Shigeki


    Sulfated glycosaminoglycans are important components of connective tissues. The pattern of sulfation is important for their biological functions. Ascidians, the closest relatives of vertebrates, have a simple chordate body plan. In the present study, we identified an almost complete set of genes encoding proteins homologous to chondroitin/dermatan sulfotransferases in the genome of the ascidian Ciona intestinalis. We found eight genes encoding 4-O-sulfotransferases, eight genes encoding 6-O-sulfotransferases, and three genes encoding uronyl 2-O-sulfotransferases. The number of sulfotransferase genes was unexpectedly large, considering that ascidians do not have a well-developed endoskeleton. In addition, most of the genes within each sub-family seemed to have arisen by gene duplication events that occurred in the ascidian lineage after divergence from the main chordate lineage. This suggests that a unique pattern of sulfation independently developed during ascidian evolution. Some of the genes identified in the present study showed tissue-specific expression in the epidermis, notochord, muscle, and central nervous system. Region-specific expression in the epidermis was also observed. The present study provides useful information for further comparative and functional analyses of sulfotransferases and proteoglycans in chordate embryos.

  19. Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: III. 2-O-sulfotransferase and C5-epimerases. (United States)

    Cadwallader, Adam B; Yost, H Joseph


    Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains, termed the HS fine structure, which give HS specific binding affinities for extracellular ligands. HS 2-O-sulfotransferase (2-OST) catalyzes the transfer of sulfate groups to the 2-O position of uronic acid residues of HS. We report here the characterization and developmental expression patterns of 2-OST in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin. The 2-OST gene has spatially and temporally distinct expression, which is a surprise given the essential role of 2-OST in HS fine structure formation. Furthermore, although 2-OST and C5-epimerase are predicted to be interdependent for protein translocation from the endoplasmic reticulum to the Golgi, their expression is not coordinately regulated during zebrafish development.

  20. Expanding the available assays: adapting and validating In-Cell Westerns in microfluidic devices for cell-based assays. (United States)

    Paguirigan, Amy L; Puccinelli, John P; Su, Xiaojing; Beebe, David J


    Microfluidic methods for cellular studies can significantly reduce costs due to reduced reagent and biological specimen requirements compared with many traditional culture techniques. However, current types of readouts are limited and this lack of suitable readouts for microfluidic cultures has significantly hindered the application of microfluidics for cell-based assays. The In-Cell Western (ICW) technique uses quantitative immunocytochemistry and a laser scanner to provide an in situ measure of protein quantities in cells grown in microfluidic channels of arbitrary geometries. The use of ICWs in microfluidic channels was validated by a detailed comparison with current macroscale methods and shown to have excellent correlation. Transforming growth factor-β-induced epithelial-to-mesenchymal transition of an epithelial cell line was used as an example for further validation of the technique as a readout for soluble-factor-based assays performed in high-throughput microfluidic channels. The use of passive pumping for sample delivery and laser scanning for analysis opens the door to high-throughput quantitative microfluidic cell-based assays that integrate seamlessly with existing high-throughput infrastructure.

  1. Differential expression and enzymatic properties of GalNAc-4-sulfotransferase-1 and GalNAc-4-sulfotransferase-2. (United States)

    Boregowda, Rajeev K; Mi, YiLing; Bu, Hongyin; Baenziger, Jacques U


    We have cloned two GalNAc-4-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that transfer sulfate to terminal beta1,4-linked GalNAc. In conjunction with the action of protein-specific beta1,4GalNAc-transferases, GalNAc-4-ST1 and GalNAc-4-ST2 account for the presence of terminal beta1,4-linked GalNAc-4-SO(4) on glycoproteins such as lutropin, thyrotropin (TSH), proopiomelanocortin (POMC), carbonic anhydratase-VI (CA-VI), and tenascin-R. GalNAc-4-ST1 and GalNAc-4-ST2 can be distinguished by their differing specificity for oligosaccharide acceptors and temperature lability. The differences in properties have been used to show that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity are proportionate to the levels of their respective transcripts. Furthermore, we have found that both transcript and activity levels of GalNAc-4-ST1 and GalNAc-4-ST2 vary widely among different tissues indicating that the regulation of their expression differs. Differences in specificity and the regulation of expression may account for existence of two GalNAc-4-sulfotransferases in vivo. The highest levels of both GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are present in the pituitary of the mouse with multiple cell types that produce glycoproteins terminating with GalNAc-4-SO(4). Genetic ablation of both GalNAc-4-ST1 and GalNAc-4-ST2 may be necessary to alter the pattern and/or extent of sulfate addition to terminal beta1,4GalNAc in tissues such as pituitary.

  2. Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays. (United States)

    Henriksen, Ulla; Fog, Jacob; Loechel, Frosty; Praestegaard, Morten


    Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.

  3. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique


    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  4. Pseudotype-based neutralization assays for influenza: a systematic analysis

    Directory of Open Access Journals (Sweden)

    George William Carnell


    Full Text Available The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI-competent antibodies that target the globular head of HA thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D towards the production of a universal vaccine has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1 to H16. The accurate and sensitive measurement of antibody responses elicited by these next-generation influenza vaccines is however hampered by the lack of sensitivity of the traditional influenza serological assays hemagglutinin inhibition (HI, single radial hemolysis (SRH and microneutralization (MN. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly-neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies.

  5. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay. (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G


    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

  6. The 2.7 Å resolution structure of the glycopeptide sulfotransferase Teg14

    Energy Technology Data Exchange (ETDEWEB)

    Bick, Matthew J. [Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Banik, Jacob J. [Howard Hughes Medical Institute, Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Darst, Seth A. [Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Brady, Sean F., E-mail: [Howard Hughes Medical Institute, Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Laboratory of Molecular Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States)


    The 2.7 Å resolution crystal structure of Teg14, a glycopeptide sulfotransferase cloned from an uncultured soil bacterium, is described. The relationship of Teg14 to other sulfotransferases is discussed. The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAP have recently been reported [Bick et al. (2010 ▶), Biochemistry, 49, 4159–4168]. Here, the 2.7 Å resolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3′-phosphoadenosine 5′-phosphosulfate-dependent sulfotransferases facilitating crystallographic experiments, especially in the field of microcrystallography.

  7. Crystal structure of human tyrosylprotein sulfotransferase-2 reveals the mechanism of protein tyrosine sulfation reaction. (United States)

    Teramoto, Takamasa; Fujikawa, Yukari; Kawaguchi, Yoshirou; Kurogi, Katsuhisa; Soejima, Masayuki; Adachi, Rumi; Nakanishi, Yuichi; Mishiro-Sato, Emi; Liu, Ming-Cheh; Sakakibara, Yoichi; Suiko, Masahito; Kimura, Makoto; Kakuta, Yoshimitsu


    Post-translational protein modification by tyrosine sulfation has an important role in extracellular protein-protein interactions. The protein tyrosine sulfation reaction is catalysed by the Golgi enzyme called the tyrosylprotein sulfotransferase. To date, no crystal structure is available for tyrosylprotein sulfotransferase. Detailed mechanism of protein tyrosine sulfation reaction has thus remained unclear. Here we present the first crystal structure of the human tyrosylprotein sulfotransferase isoform 2 complexed with a substrate peptide (C4P5Y3) derived from complement C4 and 3'-phosphoadenosine-5'-phosphate at 1.9 Å resolution. Structural and complementary mutational analyses revealed the molecular basis for catalysis being an SN2-like in-line displacement mechanism. Tyrosylprotein sulfotransferase isoform 2 appeared to recognize the C4 peptide in a deep cleft by using a short parallel β-sheet type interaction, and the bound C4P5Y3 forms an L-shaped structure. Surprisingly, the mode of substrate peptide recognition observed in the tyrosylprotein sulfotransferase isoform 2 structure resembles that observed for the receptor type tyrosine kinases.

  8. Receptor-based screening assays for the detection of antibiotics residues - A review. (United States)

    Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui


    Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics.

  9. Structure, dynamics and selectivity in the sulfotransferase family. (United States)

    Leyh, Thomas S; Cook, Ian; Wang, Ting


    Combined structure, function and molecular dynamics studies of human cytosolic sulfotransferases (SULT1A1 and 2A1) have revealed that these enzymes contain a ≈ 30-residue active-site cap whose structure responds to substrates and mediates their interactions. The binding of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) gates access to the active site by a remodeling of the cap that constricts the pore through which acceptors must pass to enter the active site. While the PAPS-bound enzyme spends the majority (≈ 95%) of its time in the constricted state, the pore isomerizes between the open and closed states when the nucleotide (PAPS) is bound. The dimensions of the open and closed pores place widely different steric constraints on substrate selectivity. Nature appears to have crafted these enzymes with two specificity settings - a closed-pore setting that admits a set of closely related structures, and an open setting that allows a far wider spectrum of acceptor geometries. The specificities of these settings seem well matched to the metabolic demands for homeostatic and defensive SULT functions. The departure of nucleotide requires that the cap open. This isomerization dependent release can explain both the product bursts and substrate inhibition seen in many SULTs. Here, the experimental underpinnings of the cap-mechanism are reviewed, and the advantages of such a mechanism are considered in the context of the cellular and metabolic environment in which these enzymes operate.

  10. Estrogen sulfotransferase/SULT1E1 promotes human adipogenesis. (United States)

    Ihunnah, Chibueze A; Wada, Taira; Philips, Brian J; Ravuri, Sudheer K; Gibbs, Robert B; Kirisci, Levent; Rubin, J Peter; Marra, Kacey G; Xie, Wen


    Estrogen sulfotransferase (EST/SULT1E1) is known to catalyze the sulfoconjugation and deactivation of estrogens. The goal of this study is to determine whether and how EST plays a role in human adipogenesis. By using human primary adipose-derived stem cells (ASCs) and whole-fat tissues from the abdominal subcutaneous fat of obese and nonobese subjects, we showed that the expression of EST was low in preadipocytes but increased upon differentiation. Overexpression and knockdown of EST in ASCs promoted and inhibited differentiation, respectively. The proadipogenic activity of EST in humans was opposite to the antiadipogenic effect of the same enzyme in rodents. Mechanistically, EST promoted adipogenesis by deactivating estrogens. The proadipogenic effect of EST can be recapitulated by using an estrogen receptor (ER) antagonist or ERα knockdown. In contrast, activation of ER in ASCs inhibited adipogenesis by decreasing the recruitment of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ) onto its target gene promoters, whereas ER antagonism increased the recruitment of PPARγ to its target gene promoters. Linear regression analysis revealed a positive correlation between the expression of EST and body mass index (BMI), as well as a negative correlation between ERα expression and BMI. We conclude that EST is a proadipogenic factor which may serve as a druggable target to inhibit the turnover and accumulation of adipocytes in obese patients.

  11. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others


    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

  12. Sulfation of ractopamine and salbutamol by the human cytosolic sulfotransferases. (United States)

    Ko, Kyounga; Kurogi, Katsuhisa; Davidson, Garrett; Liu, Ming-Yih; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh


    Feed additives such as ractopamine and salbutamol are pharmacologically active compounds, acting primarily as β-adrenergic agonists. This study was designed to investigate whether the sulfation of ractopamine and salbutamol may occur under the metabolic conditions and to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating two major feed additive compounds, ractopamine and salbutamol. A metabolic labelling study showed the generation and release of [(35)S]sulfated ractopamine and salbutamol by HepG2 human hepatoma cells labelled with [(35)S]sulfate in the presence of these two compounds. A systematic analysis using 11 purified human SULTs revealed SULT1A3 as the major SULT responsible for the sulfation of ractopamine and salbutamol. The pH dependence and kinetic parameters were analyzed. Moreover, the inhibitory effects of ractopamine and salbutamol on SULT1A3-mediated dopamine sulfation were investigated. Cytosol or S9 fractions of human lung, liver, kidney and small intestine were examined to verify the presence of ractopamine-/salbutamol-sulfating activity in vivo. Of the four human organs, the small intestine displayed the highest activity towards both compounds. Collectively, these results imply that the sulfation mediated by SULT1A3 may play an important role in the metabolism and detoxification of ractopamine and salbutamol.

  13. Cell-based assays in GPCR drug discovery. (United States)

    Siehler, Sandra


    G protein-coupled receptors (GPCRs) transmit extracellular signals into the intracellular space, and play key roles in the physiological regulation of virtually every cell and tissue. Characteristic for the GPCR superfamily of cell surface receptors are their seven transmembrane-spanning alpha-helices, an extracellular N terminus and intracellular C-terminal tail. Besides transmission of extracellular signals, their activity is modulated by cellular signals in an auto- or transregulatory fashion. The molecular complexity of GPCRs and their regulated signaling networks triggered the interest in academic research groups to explore them further, and their drugability and role in pathophysiology triggers pharmaceutical research towards small molecular weight ligands and therapeutic antibodies. About 30% of marketed drugs target GPCRs, which underlines the importance of this target class. This review describes current and emerging cellular assays for the ligand discovery of GPCRs.

  14. Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay). (United States)

    Schmid, Claudia; Arndt, Christian; Reifferscheid, Georg


    The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene. The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor. The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay. This test procedure enables screening for mutations within one working day. Incubation for 16 h reveals high sensitivity.

  15. Mechanistic insights into the specificity of human cytosolic sulfotransferase 2A1 (hSULT2A1) for hydroxylated polychlorinated biphenyls through the use of fluoro-tagged probes. (United States)

    Ekuase, E J; van 't Erve, T J; Rahaman, A; Robertson, L W; Duffel, M W; Luthe, G


    Determining the relationships between the structures of substrates and inhibitors and their interactions with drug-metabolizing enzymes is of prime importance in predicting the toxic potential of new and legacy xenobiotics. Traditionally, quantitative structure activity relationship (QSAR) studies are performed with many distinct compounds. Based on the chemical properties of the tested compounds, complex relationships can be established so that models can be developed to predict toxicity of novel compounds. In this study, the use of fluorinated analogues as supplemental QSAR compounds was investigated. Substituting fluorine induces changes in electronic and steric properties of the substrate without substantially changing the chemical backbone of the substrate. In vitro assays were performed using purified human cytosolic sulfotransferase hSULT2A1 as a model enzyme. A mono-hydroxylated polychlorinated biphenyl (4-OH PCB 14) and its four possible mono-fluoro analogues were used as test compounds. Remarkable similarities were found between this approach and previously published QSAR studies for hSULT2A1. Both studies implicate the importance of dipole moment and dihedral angle as being important to PCB structure in respect to being substrates for hSULT2A1. We conclude that mono-fluorinated analogues of a target substrate can be a useful tool to study the structure activity relationships for enzyme specificity.

  16. Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Andrea F.; Xu, Yongmei; Woody, Susan M.; Krahn, Joseph M.; Linhardt, Robert J.; Liu, Jian; Pedersen, Lars C. (NIH); (UNC); (Rensselaer)


    Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Site-directed mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production.

  17. Effects of solvents and dosing procedure on chemical toxicity in cell-based in vitro assays.

    NARCIS (Netherlands)

    Tanneberger, K.; Rico Rico, A.; Kramer, N.I.; Busser, F.J.M.; Hermens, J.L.M.; Schirmer, K.


    Due to the implementation of new legislation, such as REACh, a dramatic increase of animal use for toxicity testing is expected and the search for alternatives is timely. Cell-based in vitro assays are promising alternatives. However, the behavior of chemicals in these assays is still poorly underst

  18. Development of a lipase-based optical assay for detection of DNA

    DEFF Research Database (Denmark)

    Pinijsuwan, Suttiporn; Shipovskov, Stepan; Surareungchai, Werasak


    A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate...

  19. Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength.

    Directory of Open Access Journals (Sweden)

    Taylor R Paskin

    Full Text Available Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli, planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green, as well as ultraviolet (UV and infrared (IR which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV causing the most intense photophobic responses while longer wavelengths produce no effect (red or an apparent attraction (IR. In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment.

  20. Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification. (United States)

    Derda, Ratmir; Lockett, Matthew R; Tang, Sindy K Y; Fuller, Renee C; Maxwell, E Jane; Breiten, Benjamin; Cuddemi, Christine A; Ozdogan, Aysegul; Whitesides, George M


    This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

  1. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus. (United States)

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng


    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  2. Fluorescence Hybridization Assay Based On Chitosan-Linked Softarrays (United States)


    was incubated in the wells to reduce the Schiff base resulting from the reaction of aldehyde and amine groups. After this reaction, the yellowish...color representative of a Schiff base disappeared and the background fluorescence signal dropped to the initial ~8 to 12 fluorescence intensity (FI

  3. Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate. (United States)

    Dou, Wenfang; Xu, Yongmei; Pagadala, Vijayakanth; Pedersen, Lars C; Liu, Jian


    Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.

  4. Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays (United States)

    de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra


    Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

  5. Uronyl 2-O sulfotransferase potentiates Fgf2-induced cell migration. (United States)

    Nikolovska, Katerina; Spillmann, Dorothe; Seidler, Daniela G


    Fibroblast growth factor 2 (Fgf2) is involved in several biological functions. Fgf2 requires glycosaminoglycans, like chondroitin and dermatan sulfates (hereafter denoted CS/DS) as co-receptors. CS/DS are linear polysaccharides composed of repeating disaccharide units [-4GlcUAb1-3-GalNAc-b1-] and [-4IdoUAa1-3-GalNAc-b1-],which can be sulfated. Uronyl 2-O-sulfotransferase (Ust)introduces sulfation at the C2 of IdoUA and GlcUA resulting inover-sulfated units. Here, we investigated the role of Ust-mediated CS/DS 2-O sulfation in Fgf2-induced cell migration. We found that CHO-K1 cells overexpressing Ust contain significantly more CS/DS2-O sulfated units, whereas Ust knockdown abolished CS/DS 2-O sulfation. These structural differences in CS/DS resulted in altered Fgf2 binding and increased phosphorylation of ERK1/2 (also known as MAPK3 and MAPK1, respectively). As a functional consequence of CS/DS 2-O sulfation and altered Fgf2 binding, cell migration and paxillin activation were increased. Inhibition of sulfation, knockdown of Ust and inhibition of FgfR resulted in reduced migration. Similarly, in 3T3 cells Fgf2 treatment increased migration, which was abolished by Ust knockdown. The proteoglycan controlling the CHO migration was syndecan 1. Knockdown of Sdc1 in CHO-K1 cells overexpressing Ust abolished cell migration.We conclude that the presence of distinctly sulfated CS/DS can tune the Fgf2 effect on cell migration.

  6. A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay. (United States)

    Emter, Roger; Natsch, Andreas


    The KeratinoSens™ assay was the first cell-based in vitro test in the skin sensitisation adverse outcome pathway to be endorsed by an ECVAM statement. It includes a cell viability assessment, which serves two purposes: It forms part of the prediction model to exclude false-positive irritants and cytotoxicity provides some information on sensitizer potency of chemicals, which can feed into a multivariate potency model. In the KeratinoSens™ protocol, Nrf2-dependent luciferase induction and the MTT-viability assay are performed in parallel plates. Resazurin-based viability assays do not require cell lysis and are compatible with luciferase measurements in the same cells. Here, we performed detailed comparison of the tetrazolium-based MTT assay and the PrestoBlue® assay on 35 reference chemicals tested in the full KeratinoSens™ protocol. Log-transformed IC50 and IC30 values measured with both methods correlate with an R(2) of 0.97 and 0.95. A single chemical showed divergent results and analysis by four different viability assays indicated the PrestoBlue® read-out to be correct. The new more rapid and resource efficient approach has clear advantages: Dose-response curves show lower variability and the two endpoints are measured on the same cells. This approach is a valid addition to or replacement of the MTT-readout in the KeratinoSens™ assay and it is recommended as a general tool for luciferase-based reporter assays.

  7. Ethanol up-regulates phenol sulfotransferase (SULT1A1) and hydroxysteroid sulfotransferase (SULT2A1) in rat liver and intestine. (United States)

    Maiti, Smarajit; Chen, Guangping


    Ethanol-consumption impairs physiological-efficiency/endurance, expedites senescence. Impaired-regulations of steroids/biomolecules link these processes. Steroids are catabolized by cytosolic-sulfotransferases (SULTs). Ethanol-induction of eukaryotic-SULTs-expression is scanty. Plant (Brassica-napus) steroid-sulfotransferase; BNST3/BNST4 (gene/BNST) is highly ethanol-inducible (protein/mRNA). Resembling mammalian-SULTs catalytic-mechanism BNSTs show broad substrate-specificities (mammalian-steroids; estradiol/dehydroepiandrosterone/pregnanolone). Recently, ethanol-regulation of SULTs-expression is verified in rat liver/intestine/cultured human-hepatocarcinoma (Hep-G2) cells at enzyme-activity/protein-expression (Western-blot) level. Here, two week's ethanol ingestion by male rat significantly increased SULT2A1 in their liver/intestine (p sulfotransferase (SULT1A1) in intestine (p < 0.001) at enzyme-activity/protein levels. In human cells, ethanol significantly (2-fold) increased hSULT1A1/hSULT1E (2-3 fold) protein expressions paralleling their enzymatic-activities (p < 0.05-p < 0.01). The earlier finding of alcohol-association to the physiological impairment may be corroborated by our present findings. Inductions of SULT-expressions by ethanol have significant physiological/pharmacological consequences.

  8. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo


    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  9. Microchip-based ultrafast serodiagnostic assay for tuberculosis (United States)

    Mani, Vigneshwaran; Paleja, Bhairav; Larbi, Karima; Kumar, Pavanish; Tay, Jo Ann; Siew, Jie Yee; Inci, Fatih; Wang, ShuQi; Chee, Cynthia; Wang, Yee Tang; Demirci, Utkan; De Libero, Gennaro; Singhal, Amit


    Access to point-of-care (POC), rapid, inexpensive, sensitive, and instrument-free tests for the diagnosis of tuberculosis (TB) remains a major challenge. Here, we report a simple and low-cost microchip-based TB ELISA (MTBE) platform for the detection of anti-mycobacterial IgG in plasma samples in less than 15 minutes. The MTBE employs a flow-less, magnet-actuated, bead-based ELISA for simultaneous detection of IgG responses against multiple mycobacterial antigens. Anti-trehalose 6,6′-dimycolate (TDM) IgG responses were the strongest predictor for differentiating active tuberculosis (ATB) from healthy controls (HC) and latent tuberculosis infections (LTBI). The TDM-based MTBE demonstrated superior sensitivity compared to sputum microscopy (72% vs. 56%) with 80% and 63% positivity among smear-positive and smear-negative confirmed ATB samples, respectively. Receiver operating characteristic analysis indicated good accuracy for differentiating ATB from HC (AUC = 0.77). Thus, TDM-based MTBE can be potentially used as a screening device for rapid diagnosis of active TB at the POC. PMID:27775039

  10. A cost effective base-matching assay with low backgrounds.


    SU, X.; Mushinsky, G; Comeau, A.M.


    Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.

  11. Heparan sulfate 6-O-Sulfotransferase is essential for muscle development in zebrafish

    NARCIS (Netherlands)

    Bink, R.J.; Habuchi, H.; Lele, Z.; Dolk, E.; Joore, J.; Rauch, G.; Geisler, R.; Wilson, S.W.; Hertog, J. den; Kimata, K.; Zivkovic, D.


    Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding z

  12. Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost. (United States)

    Berry, Scott M; Pezzi, Hannah M; Williams, Eram D; Loeb, Jennifer M; Guckenberger, David J; Lavanway, Alex J; Puchalski, Alice A; Kityo, Cissy M; Mugyenyi, Peter N; Graziano, Franklin M; Beebe, David J


    Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R2 = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.

  13. Development of a heavy metals enzymatic-based assay using papain

    Energy Technology Data Exchange (ETDEWEB)

    Shukor, Yunus [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)]. E-mail:; Baharom, Nor Azlan [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Rahman, Fadhil Abd. [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Mohd. Puad [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Shamaan, Nor Aripin [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Syed, Mohd. Arif [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)


    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC{sub 50} (concentration of toxicant giving 50% inhibition) of Hg{sup 2+}, Ag{sup 2+}, Pb{sup 2}, Zn{sup 2+} is 0.39, 0.40, 2.16, 2.11 mg l{sup -1}, respectively. For Cu{sup 2+} and Cd{sup 2+} the LOQ (limits of quantitation) is 0.004 and 0.1 mg l{sup -1}, respectively. The IC{sub 50} and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox{sup TM}, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.

  14. Using Exclusion-Based Sample Preparation (ESP to Reduce Viral Load Assay Cost.

    Directory of Open Access Journals (Sweden)

    Scott M Berry

    Full Text Available Viral load (VL measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD, accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1 and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP.71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL and high accuracy (average difference between methods of 0.08 log, R2 = 0.97. Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.

  15. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki


    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  16. Digital magnetic tagging for multiplexed suspension-based biochemical assays (United States)

    Mitrelias, T.; Trypiniotis, T.; Palfreyman, J. J.; Hong, B.; Vyas, K.; Hayward, T. J.; Llandro, J.; Kopper, K. P.; Bland, J. A. C.; Robertson, P. A.; Barnes, C. H. W.


    Microarrays and suspension (or bead)-based technologies have attracted significant interest for their broad applications in high throughput molecular biology. However, the throughput of microarrays will always be limited by the array density and the slow diffusion of molecules to their binding sites. Suspension-based technologies, in which all the reactions take place directly on the surface of microcarriers functionalized with molecular probes, could offer true multiplexing due to the possibility of extending their detection capability by a straightforward expansion of the size of the chemical library of probes. To fully exploit their potential, the microcarriers must be tagged, but the number of distinct codes available from spectrometric/graphical/physical encoding methods is currently fairly limited. A digital magnetic tagging method based on magnetic microtags, which have been anisotropy engineered to provide stable magnetization directions which correspond to digital codes, is reported. The tags can be suspended in solution and functionalized with a variety of biological molecular probes. Magnetic tagging offers several benefits compared to the traditional optical encoding techniques currently employed. It offers minimal background signals, potential for a large number of distinct codes, miniaturization of devices, and the ability to write a code in situ. Experimental data showing the reading of individual magnetic microbars from samples comprising 50×20 μm2 Ni elements, as well as micromagnetic simulations that show the feasibility of stray field detection, are presented. The stray fields of the magnetic microbars spanning a range of 60 mOe were detected by a microfabricated fluxgate sensor scanned in a raster fashion over the sample that was placed about 70 μm away. Free floating tags have also been fabricated for use in microfluidic systems. A magnetic lab-on-a-chip device could be used for tagging biomolecular probes for applications in genome

  17. Smartphone based visual and quantitative assays on upconversional paper sensor. (United States)

    Mei, Qingsong; Jing, Huarong; Li, You; Yisibashaer, Wuerzha; Chen, Jian; Nan Li, Bing; Zhang, Yong


    The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform.

  18. Spectrophotometric total reducing sugars assay based on cupric reduction. (United States)

    Başkan, Kevser Sözgen; Tütem, Esma; Akyüz, Esin; Özen, Seda; Apak, Reşat


    As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method.

  19. In silico mechanistic profiling to probe small molecule binding to sulfotransferases. (United States)

    Martiny, Virginie Y; Carbonell, Pablo; Lagorce, David; Villoutreix, Bruno O; Moroy, Gautier; Miteva, Maria A


    Drug metabolizing enzymes play a key role in the metabolism, elimination and detoxification of xenobiotics, drugs and endogenous molecules. While their principal role is to detoxify organisms by modifying compounds, such as pollutants or drugs, for a rapid excretion, in some cases they render their substrates more toxic thereby inducing severe side effects and adverse drug reactions, or their inhibition can lead to drug-drug interactions. We focus on sulfotransferases (SULTs), a family of phase II metabolizing enzymes, acting on a large number of drugs and hormones and showing important structural flexibility. Here we report a novel in silico structure-based approach to probe ligand binding to SULTs. We explored the flexibility of SULTs by molecular dynamics (MD) simulations in order to identify the most suitable multiple receptor conformations for ligand binding prediction. Then, we employed structure-based docking-scoring approach to predict ligand binding and finally we combined the predicted interaction energies by using a QSAR methodology. The results showed that our protocol successfully prioritizes potent binders for the studied here SULT1 isoforms, and give new insights on specific molecular mechanisms for diverse ligands' binding related to their binding sites plasticity. Our best QSAR models, introducing predicted protein-ligand interaction energy by using docking, showed accuracy of 67.28%, 78.00% and 75.46%, for the isoforms SULT1A1, SULT1A3 and SULT1E1, respectively. To the best of our knowledge our protocol is the first in silico structure-based approach consisting of a protein-ligand interaction analysis at atomic level that considers both ligand and enzyme flexibility, along with a QSAR approach, to identify small molecules that can interact with II phase dug metabolizing enzymes.

  20. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays. (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M


    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  1. 3'-Phosphoadenosine-5'-phosphosulfate: Photoaffinity ligand for sulfotransferase enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Otterness, D.M.; Powers, S.P.; Miller, L.J.; Weinshilboum, R.M. (Mayo Clinic/Mayo Foundation, Rochester, MN (USA))


    Sulfation is an important pathway in the biotransformation of many drugs, xenobiotic compounds, neurotransmitters, and hormones. The sulfate donor for these reactions is 3'-phosphoadenosine-5'-phosphosulfate (PAPS). We set out to determine whether PAPS might serve as a photoaffinity ligand for sulfotransferase enzymes. UV irradiation of (35S)PAPS with partially purified human liver thermostable (TS) phenol sulfotransferase (PST) radioactively labeled a protein with a molecular mass of 35 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoaffinity labeling of TS PST with (35S) PAPS did not require the presence of a phenolic substrate but rather was inhibited by p-nitrophenol, a sulfate acceptor substrate for TS PST. Inhibitors of TS PST enzymatic activity, including 3'-phosphoadenosine-5'-phosphate, ATP, ADP, and 2,6-dichloro-4-nitrophenol, also inhibited photoaffinity labeling of the 35-kDa protein with (35S)PAPS, in a concentration-dependent fashion, with IC50 values of 14 microM, 2.1 mM, 7.7 mM, and 91 microM, respectively. The 35-kDa protein that was radioactively labeled by (35S)PAPS in the presence of UV light coeluted with TS PST enzymatic activity during gel filtration high performance liquid chromatography. (35S)PAPS was then used to photoaffinity label another sulfotransferase enzyme, the thermolabile (TL) form of PST partially purified from human liver. Therefore, (35S)PAPS appears to be a photoaffinity ligand that could be used to study a variety of PAPS-dependent sulfotransferases. Photoaffinity labeling of TS and TL PST, as well as other PAPS-dependent sulfotransferases, should enhance our ability to purify this important group of enzymes and to determine amino acid sequences at or near their active sites.

  2. The important roles of steroid sulfatase and sulfotransferases in gynecological diseases

    Directory of Open Access Journals (Sweden)

    Tea eLanisnik Rizner


    Full Text Available Gynecological diseases such as endometriosis, adenomyosis and uterine fibroids, and gynecological cancers including endometrial cancer and ovarian cancer, affect a large proportion of women. These diseases are estrogen dependent, and their progression often depends on local estrogen formation. In peripheral tissues, estrogens can be formed from the inactive precursors dehydroepiandrosterone sulfate and estrone sulfate. Sulfatase and sulfotransferases have pivotal roles in these processes, where sulfatase hydrolyzes estrone sulfate to estrone, and dehydroepiandrosterone sulfate to dehydroepiandrosterone, and sulfotransferases catalyze the reverse reactions. Further activation of estrone to the most potent estrogen, estradiol, is catalyzed by 17-ketosteroid reductases, while estradiol can be formed from dehydroepiandrosterone by the sequential actions of 3-hydroxysteroid dehydrogenase-4-isomerase, aromatase, and 17-ketosteroid reductase. This review introduces the sulfatase and sulfotransferase enzymes, in terms of their structures and reaction mechanisms, and the regulation and different transcripts of their genes, together with the importance of their currently known single nucleotide polymorphisms. Data on expression of sulfatase and sulfotransferases in gynecological diseases are also reviewed. There are often unchanged mRNA and protein levels in diseased tissue, with higher sulfatase activities in cancerous endometrium, ovarian cancer cell lines, and adenomyosis. This can be indicative of a disturbed balance between the sulfatase and sulfotransferases enzymes, defining the potential for sulfatase as a drug target for treatment of gynecological diseases. Finally, clinical trials with sulfatase inhibitors are discussed, where two inhibitors have already concluded phase II trials, although so far with no convincing clinical outcomes for patients with endometrial cancer and endometriosis.

  3. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason


    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  4. Nanoparticle-based assays in automated flow systems: A review

    Energy Technology Data Exchange (ETDEWEB)

    Passos, Marieta L.C. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Pinto, Paula C.A.G., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Santos, João L.M., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Saraiva, M. Lúcia M.F.S., E-mail: [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Araujo, André R.T.S. [LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, n° 228, 4050-313 Porto (Portugal); Unidade de Investigação para o Desenvolvimento do Interior, Instituto Politécnico da Guarda, Av. Dr. Francisco de Sá Carneiro, n° 50, 6300-559 Guarda (Portugal)


    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section.

  5. Comparison of 2 Luminex-based Multiplexed Protein Assays for Quantifying Microglia Activation and Inflammatory Proteins (United States)


    streptavidin-phycoerythrin (PE) similar to sandwich enzyme-linked immunosorbent assays (ELISAs). The 3 fluorescent markers (2 beads plus PE) allow for...least expensive platform. It uses a magnetic plate to create a monolayer of beads that can be imaged with a light-emitting-diode-based imager capable... Magnetic Luminex Screening Assay Rat Premixed Multi-Analyte Kit, a kit was purchased that included all of the 17 analytes included in company’s catalog

  6. A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology. (United States)

    Abderrahim, Mohamed; Arribas, Silvia M; Condezo-Hoyos, Luis


    Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H2O2) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL(-1)), HRP (1UmL(-1)) and H2O2 (250µmolL(-1)) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL(-1)) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H2O2 as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL(-1) (R(2)=0.993) and very sensitive with limits of detection (LOD) of 0.005UmL(-1) and quantitation (LOQ) of 0.01UmL(-1). PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples.

  7. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned. (United States)

    Maddox, Clinton B; Rasmussen, Lynn; White, E Lucile


    In recent years, cell-based phenotypic assays have emerged as an effective and robust addition to the array of assay technologies available for drug discovery in the high throughput screening arena. Previously, biochemical target-based assays have been the technology of choice. With the emergence of stem cells as a basis for a new screening technology, it is important to keep in mind the lessons that have been learned from the adaptation of existing stable cell lines onto the high throughput screening drug discovery platform, with special consideration being given to assay miniaturization, liquid handling complications and instrument-introduced artifacts. We present an overview of the problems encountered with the implementation of multiple cell-based assays at the High Throughput Screening Center at Southern Research Institute as well as empirically defined effective solutions to these problems. These include examples of artifacts induced by temperature differences throughout the screening campaign, cell plating conditions including the effect of room temperature incubation on assay consistency, DMSO carry-over, and incubator induced artifacts.

  8. Time-stretch microscopy on a DVD for high-throughput imaging cell-based assay. (United States)

    Tang, Anson H L; Yeung, P; Chan, Godfrey C F; Chan, Barbara P; Wong, Kenneth K Y; Tsia, Kevin K


    Cell-based assay based on time-stretch imaging is recognized to be well-suited for high-throughput phenotypic screening. However, this ultrafast imaging technique has primarily been limited to suspension-cell assay, leaving a wide range of solid-substrate assay formats uncharted. Moreover, time-stretch imaging is generally restricted to intrinsic biophysical phenotyping, but lacks the biomolecular signatures of the cells. To address these challenges, we develop a spinning time-stretch imaging assay platform based on the functionalized digital versatile disc (DVD). We demonstrate that adherent cell culture and biochemically-specific cell-capture can now be assayed with time-stretch microscopy, thanks to the high-speed DVD spinning motion that naturally enables on-the-fly cellular imaging at an ultrafast line-scan rate of >10MHz. As scanning the whole DVD at such a high speed enables ultra-large field-of-view imaging, it could be favorable for scaling both the assay throughput and content as demanded in many applications, e.g. drug discovery, and rare cancer cell screening.

  9. A versatile microparticle-based immunoaggregation assay for macromolecular biomarker detection and quantification.

    Directory of Open Access Journals (Sweden)

    Haiyan Wu

    Full Text Available The rapid, sensitive and low-cost detection of macromolecular biomarkers is critical in clinical diagnostics, environmental monitoring, research, etc. Conventional assay methods usually require bulky, expensive and designated instruments and relative long assay time. For hospitals and laboratories that lack immediate access to analytical instruments, fast and low-cost assay methods for the detection of macromolecular biomarkers are urgently needed. In this work, we developed a versatile microparticle (MP-based immunoaggregation method for the detection and quantification of macromolecular biomarkers. Antibodies (Abs were firstly conjugated to MP through streptavidin-biotin interaction; the addition of macromolecular biomarkers caused the aggregation of Ab-MPs, which were subsequently detected by an optical microscope or optical particle sizer. The invisible nanometer-scale macromolecular biomarkers caused detectable change of micrometer-scale particle size distributions. Goat anti-rabbit immunoglobulin and human ferritin were used as model biomarkers to demonstrate MP-based immunoaggregation assay in PBS and 10% FBS to mimic real biomarker assay in the complex medium. It was found that both the number ratio and the volume ratio of Ab-MP aggregates caused by biomarker to all particles were directly correlated to the biomarker concentration. In addition, we found that the detection range could be tuned by adjusting the Ab-MP concentration. We envision that this novel MP-based immunoaggregation assay can be combined with multiple detection methods to detect and quantify macromolecular biomarkers at the nanogram per milliliter level.

  10. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate. (United States)

    Bi, Xiaodong; Liu, Zhen


    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  11. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan


    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  12. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne


    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...... the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...

  13. HIV-1 Reverse Transcriptase based assay to determine cellular dNTP concentrations (United States)

    Hollenbaugh, Joseph A.; Kim, Baek


    Summary Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis are tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection. PMID:26714705

  14. Long term response of a Concanavalin-A based fluorescence glucose sensing assay (United States)

    Locke, Andrea K.; Cummins, Brian M.; Abraham, Alexander A.; Coté, Gerard L.


    Competitive binding assays comprised of the protein Concanavalin A (ConA) have shown potential for use in continuous glucose monitoring devices. However, its time-dependent, thermal instability can impact the lifetime of these ConA based assays. In an attempt to design sensors with longer in vivo lifetimes, different groups have immobilized the protein to various surfaces. For example, Ballerstadt et al. have shown that immobilizing ConA onto the interior of a micro-dialysis membrane and allowing dextran to be freely suspended within solution allowed for successful in vivo glucose sensing up to 16 days. This work explores the glucose response of an assay comprised of modified ConA and a single fluorescently labeled competing ligand in free solution to increase the in vivo sensing lifetime without immobilization,. The behavior of this assay in the presence of varying glucose concentrations is monitored via fluorescence anisotropy over a 30 day period.

  15. In Silico Prediction of Human Sulfotransferase 1E1 Activity Guided by Pharmacophores from Molecular Dynamics Simulations. (United States)

    Rakers, Christin; Schumacher, Fabian; Meinl, Walter; Glatt, Hansruedi; Kleuser, Burkhard; Wolber, Gerhard


    Acting during phase II metabolism, sulfotransferases (SULTs) serve detoxification by transforming a broad spectrum of compounds from pharmaceutical, nutritional, or environmental sources into more easily excretable metabolites. However, SULT activity has also been shown to promote formation of reactive metabolites that may have genotoxic effects. SULT subtype 1E1 (SULT1E1) was identified as a key player in estrogen homeostasis, which is involved in many physiological processes and the pathogenesis of breast and endometrial cancer. The development of an in silico prediction model for SULT1E1 ligands would therefore support the development of metabolically inert drugs and help to assess health risks related to hormonal imbalances. Here, we report on a novel approach to develop a model that enables prediction of substrates and inhibitors of SULT1E1. Molecular dynamics simulations were performed to investigate enzyme flexibility and sample protein conformations. Pharmacophores were developed that served as a cornerstone of the model, and machine learning techniques were applied for prediction refinement. The prediction model was used to screen the DrugBank (a database of experimental and approved drugs): 28% of the predicted hits were reported in literature as ligands of SULT1E1. From the remaining hits, a selection of nine molecules was subjected to biochemical assay validation and experimental results were in accordance with the in silico prediction of SULT1E1 inhibitors and substrates, thus affirming our prediction hypotheses.

  16. Pivotal Role of Carbohydrate Sulfotransferase 15 in Fibrosis and Mucosal Healing in Mouse Colitis.

    Directory of Open Access Journals (Sweden)

    Kenji Suzuki

    Full Text Available Induction of mucosal healing (MH is an important treatment goal in inflammatory bowel disease (IBD. Although the molecular mechanisms underlying MH in IBD is not fully explored, local fibrosis would contribute to interfere mucosal repair. Carbohydrate sulfotransferase 15 (CHST15, which catalyzes sulfation of chondroitin sulfate to produce rare E-disaccharide units, is a novel mediator to create local fibrosis. Here we have used siRNA-based approach of silencing CHST15 in dextran sulfate sodium (DSS induced colitis in mice, human colon fibroblasts and cancer cell lines. In a DSS-induced acute colitis model, CHST15 siRNA reduced CHST15 mRNA in the colon, serum IL-6, disease activity index (DAI and accumulation of F4/80+ macrophages and ER-TR7+ fibroblasts, while increased Ki-67+ epithelial cells. In DSS-induced chronic colitis models, CHST15 siRNA reduced CHST15 mRNA in the colon, DAI, alpha-smooth muscle actin+ fibroblasts and collagen deposition, while enhanced MH as evidenced by reduced histological and endoscopic scores. We also found that endoscopic submucosal injection achieved effective pancolonic delivery of CHST15 siRNA in mice. In human CCD-18 Co cells, CHST15 siRNA inhibited the expression of CHST15 mRNA and selectively reduced E-units, a specific product biosynthesized by CHST15, in the culture supernatant. CHST15 siRNA significantly suppressed vimentin in both TGF-ß-stimulated CCD18-Co cells and HCT116 cells while up-regulated BMP7 and E-cadherin in HCT116 cells. The present study demonstrated that blockade CHST15 represses colonic fibrosis and enhances MH partly though reversing EMT pathway, illustrating a novel therapeutic opportunity to refractory and fibrotic lesions in IBD.

  17. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. (Univ. of Pittsburgh School of Medicine, PA (USA))


    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

  18. Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay. (United States)

    Dossou, K S S; Devkota, K P; Kavanagh, P V; Beutler, J A; Egan, J M; Moaddel, R


    Cannabinoid (CB) receptors are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed antiproliferative activity against breast cancer and prostate cancer in animal models. To study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [(3)H]Win55-212-2 for CB1 and [(3)H]CP55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630, and AM6545. The data obtained were consistent with previously reported values, thereby confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors. In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.

  19. Processing of nanolitre liquid plugs for microfluidic cell-based assays

    Directory of Open Access Journals (Sweden)

    Junji Fukuda, Shintaro Takahashi, Tatsuya Osaki, Naoto Mochizuki and Hiroaki Suzuki


    Full Text Available Plugs, i.e. droplets formed in a microchannel, may revolutionize microfluidic cell-based assays. This study describes a microdevice that handles nanolitre-scale liquid plugs for the preparation of various culture setups and subsequent cellular assays. An important feature of this mode of liquid operation is that the recirculation flow generated inside the plug promotes the rapid mixing of different solutions after plugs are merged, and it keeps cell suspensions homogeneous. Thus, serial dilutions of reagents and cell suspensions with different cell densities and cell types were rapidly performed using nanolitres of solution. Cells seeded through the plug processing grew well in the microdevice, and subsequent plug processing was used to detect the glucose consumption of cells and cellular responses to anticancer agents. The plug-based microdevice may provide a useful platform for cell-based assay systems in various fields, including fundamental cell biology and drug screening applications.

  20. Capacitance-based assay for real-time monitoring of endocytosis and cell viability. (United States)

    Lee, Rimi; Kim, Jihun; Kim, Sook Young; Jang, Seon Mi; Lee, Sun-Mi; Choi, In-Hong; Park, Seung Woo; Shin, Jeon-Soo; Yoo, Kyung-Hwa


    Label-free cell-based assays have emerged as a promising means for high-throughput screening. Most label-free sensors are based on impedance measurements that reflect the passive electrical properties of cells. Here we introduce a capacitance-based assay that measures the dielectric constant (capacitance) of biological cells, and demonstrate the feasibility of analyzing endocytosis and screening chemotherapeutic agents with this assay. Endocytosis induces a change in the zeta potential, leading to a change in the dielectric constant which enables real-time endocytosis monitoring using the capacitance sensor. Additionally, since the dielectric constant is proportional to cell radius and cell volume, cell viability can be estimated from the change in capacitance. Therefore, the capacitance sensor array can also be used for cytotoxicity testing for large-scale chemotherapeutic screening.

  1. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay. (United States)

    Sonavane, Manoj; Creusot, Nicolas; Maillot-Maréchal, Emmanuelle; Péry, Alexandre; Brion, François; Aїt-Aïssa, Selim


    Endocrine disrupting chemicals (EDCs) act on the endocrine system through multiple mechanisms of action, among them interaction with estrogen receptors (ERs) is a well-identified key event in the initiation of adverse outcomes. As the most commonly used estrogen screening assays are either yeast- or human-cell based systems, the question of their (eco)toxicological relevance when assessing risks for aquatic species can be raised. The present study addresses the use of zebrafish (zf) derived reporter gene assays, both in vitro (i.e. zf liver cell lines stably expressing zfERα, zfERβ1 and zfERβ2 subtypes) and in vivo (i.e. transgenic cyp19a1b-GFP zf embryos), to assess estrogenic contaminants in river waters. By investigating 20 French river sites using passive sampling, high frequencies of in vitro zfER-mediated activities in water extracts were measured. Among the different in vitro assays, zfERβ2 assay was the most sensitive and responsive one, enabling the detection of active compounds at all investigated sites. In addition, comparison with a conventional human-based in vitro assay highlighted sites that were able to active zfERs but not human ER, suggesting the occurrence of zf-specific ER ligands. Furthermore, a significant in vivo estrogenic activity was detected at the most active sites in vitro, with a good accordance between estradiol equivalent (E2-EQ) concentrations derived from both in vitro and in vivo assays. Overall, this study shows the relevance and usefulness of such novel zebrafish-based assays as screening tools to monitor estrogenic activities in complex mixtures such as water extracts. It also supports their preferred use compared to human-based assays to assess the potential risks caused by endocrine disruptive chemicals for aquatic species such as fish.

  2. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening. (United States)

    Caballero, Iván; Lafuente, María José; Gamo, Francisco-Javier; Cid, Concepción


    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

  3. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. (United States)

    Christopher-Hennings, Jane; Araujo, Karla P C; Souza, Carlos J H; Fang, Ying; Lawson, Steven; Nelson, Eric A; Clement, Travis; Dunn, Michael; Lunney, Joan K


    Bead-based multiplex assays (BBMAs) are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several to 50-500 analytes within a single, small sample volume). Currently, few assays are commercially available for veterinary applications, but they are available to identify and measure various cytokines, growth factors and their receptors, inflammatory proteins, kinases and inhibitors, neurobiology proteins, and pathogens and antibodies in human beings, nonhuman primates, and rodent species. In veterinary medicine, various nucleic acid and protein-coupled beads can be used in, or for the development of, antigen and antibody BBMAs, with the advantage that more data can be collected using approximately the same amount of labor as used for other antigen and antibody assays. Veterinary-related BBMAs could be used for detection of pathogens, genotyping, measurement of hormone levels, and in disease surveillance and vaccine assessment. It will be important to evaluate whether BBMAs are "fit for purpose," how costs and efficiencies compare between assays, which assays are published or commercially available for specific veterinary applications, and what procedures are involved in the development of the assays. It is expected that many veterinary-related BBMAs will be published and/or become commercially available in the next few years. The current review summarizes the BBMA technology and some of the currently available BBMAs developed for veterinary settings. Some of the human diagnostic BBMAs are also described, providing an example of possible templates for future development of new veterinary-related BBMAs.

  4. C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-O-sulfated heparan sulfate domains. (United States)

    Préchoux, Aurélie; Halimi, Célia; Simorre, Jean-Pierre; Lortat-Jacob, Hugues; Laguri, Cédric


    Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments.

  5. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides (United States)

    Rogers, Kim R.; Wang, Yi; Mulchandani, Ashok; Mulchandani, P.; Chen, Wilfred


    We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphate pesticides. This assay is based on a substrate-dependant change in pH near the active site of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC) which is covalently immobilized to the enzyme. This method employs FITC-labeled enzyme adsorbed to polymethylmethacrylate beads. Analytes were measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 (mu) M to 400 (mu) M for paraoxon with a detection limit of 8 (mu) M. This assay compared favorably to an HPLC method for monitoring the concentration of coumaphos in bioremediation filtrate samples.

  6. Cultivar origin and admixture detection in Turkish olive oils by SNP-based CAPS assays. (United States)

    Uncu, Ali Tevfik; Frary, Anne; Doganlar, Sami


    The aim of this study was to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils. To reach this aim, we sequenced short fragments from five olive genes for SNP (single nucleotide polymorphism) identification and developed CAPS (cleaved amplified polymorphic DNA) assays for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. We also tested the efficiency and limit of our approach for detecting olive oil admixtures. As a result of the analysis, we were able to detect admixing down to a limit of 20%. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.

  7. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity. (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I


    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

  8. A robotics-based automated assay for inorganic and organic phosphates. (United States)

    Cogan, E B; Birrell, G B; Griffith, O H


    Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.

  9. Crystal Structures of the Glycopeptide Sulfotransferase Teg12 in a Complex with the Teicoplanin Aglycone

    Energy Technology Data Exchange (ETDEWEB)

    Bick, Matthew J.; Banik, Jacob J.; Darst, Seth A.; Brady, Sean F. (Rockefeller)


    The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, -13, and -14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures: an apo structure of Teg12, Teg12 bound to the desulfated cosubstrate 3{prime}-phosphoadenosine 5{prime}-phosphate, and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding, and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site-directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.

  10. Conference report: the 5th cell-based assay and bioanalytical method development conference. (United States)

    Ma, Mark


    Approximately 80 participants met at the Marriot Hotel, San Francisco, CA, USA, between the 4th and 6th October 2010 to share novel techniques and discuss the emerging approaches in the evolving field of cell-based assay and bioanalytical method development. This report highlights the discussion and summary of the meeting.

  11. An assay for secologanin in plant tissues based on enzymatic conversion into strictosidine

    DEFF Research Database (Denmark)

    Hallard, Didier; van der Heijden, Robert; Contin, Adriana;


    The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding...

  12. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    NARCIS (Netherlands)

    Nicolas, J.A.Y.


    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby rep

  13. Uncertainty budget for final assay of a pharmaceutical product based on RP-HPLC

    DEFF Research Database (Denmark)

    Heydorn, Kaj; Anglov, Thomas; Byrialsen, Kirsten


    Compliance with specified limits for the content of active substance in a pharmaceutical drug requires knowledge of the uncertainty of the final assay. The uncertainty of measurement is based on the ISO recommendation as expressed in the Guide to the Expression of Uncertainty in Measurement (GUM...

  14. Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India.

    Directory of Open Access Journals (Sweden)

    Shanmugam Vanithamani

    Full Text Available Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS was evaluated by enzyme linked immunosorbent assay (ELISA, dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA. Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%, Autumnalis (11.7%, Ballum (25.8%, Grippotyphosa (12.5%, Pomona (10% and were used as antigens in the diagnostics to detect IgM antibodies in patients' sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05.The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.

  15. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo


    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  16. Serological assays based on recombinant viral proteins for the diagnosis of arenavirus hemorrhagic fevers. (United States)

    Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru


    The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  17. Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

    Directory of Open Access Journals (Sweden)

    Phairote Teeranaipong


    Full Text Available Introduction: A dual split reporter protein system (DSP, recombining Renilla luciferase (RL and green fluorescent protein (GFP split into two different constructs (DSP1–7 and DSP8–11, was adapted to create a novel rapid phenotypic tropism assay (PTA for HIV-1 infection (DSP-Pheno. Methods: DSP1–7 was stably expressed in the glioma-derived NP-2 cell lines, which expressed CD4/CXCR4 (N4X4 or CD4/CCR5 (N4R5, respectively. An expression vector with DSP8–11 (pRE11 was constructed. The HIV-1 envelope genes were subcloned in pRE11 (pRE11-env and transfected into 293FT cells. Transfected 293FT cells were incubated with the indicator cell lines independently. In developing the assay, we selected the DSP1–7-positive clones that showed the highest GFP activity after complementation with DSP8–11. These cell lines, designated N4R5-DSP1–7, N4X4-DSP1–7 were used for subsequent assays. Results: The env gene from the reference strains (BaL for R5 virus, NL4-3 for X4 virus, SF2 for dual tropic virus subcloned in pRE11 and tested, was concordant with the expected co-receptor usage. Assay results were available in two ways (RL or GFP. The assay sensitivity by RL activity was comparable with those of the published phenotypic assays using pseudovirus. The shortest turnaround time was 5 days after obtaining the patient's plasma. All clinical samples gave positive RL signals on R5 indicator cells in the fusion assay. Median RLU value of the low CD4 group was significantly higher on X4 indicator cells and suggested the presence of more dual or X4 tropic viruses in this group of patients. Comparison of representative samples with Geno2Pheno [co-receptor] assay was concordant. Conclusions: A new cell-fusion-based, high-throughput PTA for HIV-1, which would be suitable for in-house studies, was developed. Equipped with two-way reporter system, RL and GFP, DSP-Pheno is a sensitive test with short turnaround time. Although maintenance of cell lines and

  18. Identification and localization of soluble sulfotransferases in the human gastrointestinal tract


    Teubner, Wera; Meinl, Walter; Florian, Simone; Kretzschmar, Michael; Glatt, Hansruedi


    Abstract Soluble sulfotransferases (SULTs) are important in the regulation of messenger molecules and the elimination of xenobiotics. However, sulfo-conjugation of various substrates can also lead to the formation of reactive metabolites that may induce cancer and cause other damage. Our aim was to identify the SULT forms expressed in the human gastrointestinal tract, especially colon and rectum (common sites for cancer) and to determine their cellular localization. Normal colonic ...

  19. Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay. (United States)

    Wasserstrom, Adam; Frumkin, Dan; Davidson, Ariane; Shpitzen, Moshe; Herman, Yael; Gafny, Ron


    Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This

  20. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风


    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  1. [Changes in serum vitamin D assay usage and the need for evidence-based recommendations]. (United States)

    Pilon, Antoine; Lim, Soo-Kyung; Guéchot, Jérôme


    Result of renewed interest due to the large amount of literature that reported numerous epidemiological data demonstrating the high prevalence of vitamin D deficiency, the number of prescriptions of serum vitamin D assays has grown exponentially in recent years with a cost for health insurance that increased almost fivefold in four years. The quantitative and qualitative analysis of assays carried out from 2007 to 2011 in a French university adult short-stay hospital shows changes in practices not only quantitatively but also qualitatively resulting in an overtime increase in the frequency of prescriptions in patients younger, less vitamin D deficient and more frequently male. In the absence of French guidelines, this development cannot be qualified as deviant but justifies the urgent need to establish evidence-based recommendations for good prescriptions and adequate assays of blood vitamin D.

  2. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. (United States)

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger


    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  3. Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

    Institute of Scientific and Technical Information of China (English)

    陈宝玉; 郭勤; 郭智; 王希成


    This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid-reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N-phospho-L-arginine (PArg) formed in the forward catalysis reaction.The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized.For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L)-1 · cm-1 for the phosphate.Standard curves for phosphate show a good linearity of 0.999.Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies.This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

  4. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification. (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng


    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  5. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays. (United States)

    Langer, Gernot


    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  6. Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics. (United States)

    Tao, Ye; Rotem, Assaf; Zhang, Huidan; Chang, Connie B; Basu, Anindita; Kolawole, Abimbola O; Koehler, Stephan A; Ren, Yukun; Lin, Jeffrey S; Pipas, James M; Feldman, Andrew B; Wobus, Christiane E; Weitz, David A


    A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.

  7. Measuring immunoglobulin g antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin with single-antigen enzyme-linked immunosorbent assays and a bead-based multiplex assay. (United States)

    Reder, Sabine; Riffelmann, Marion; Becker, Christian; Wirsing von König, Carl Heinz


    Bead-based assay systems offer the possibility of measuring several specific antibodies in one sample simultaneously. This study evaluated a vaccine panel of a multianalyte system that measures antibodies to tetanus toxin, diphtheria toxin, and pertussis toxin (PT) from Bordetella pertussis. The antibody concentrations of human immunoglobulin G (IgG) to PT, tetanus toxin, and diphtheria toxin were measured in 123 serum pairs (total of 246 sera) from a vaccine study. The multianalyte bead assay was compared to a standardized in-house IgG- anti-PT enzyme-linked immunosorbent assay (ELISA) of the German reference laboratory for bordetellae, as well as to various commercially available ELISAs for anti-PT IgG, anti-tetanus IgG, and anti-diphtheria IgG. The results of the multiplex assay regarding the antibodies against diphtheria toxin compared favorably with a regression coefficient of 0.938 for values obtained with an ELISA from the same manufacturer used as a reference. Similarly, antibodies to tetanus toxin showed a correlation of 0.910 between the reference ELISA and the multianalyte assay. A correlation coefficient of 0.905 was found when an "in-house" IgG anti-PT and the multiplex assay were compared. Compared to single ELISA systems from two other manufacturers, the multiplex assay performed similarly well or better. The multianalyte assay system was a robust system with fast and accurate results, analyzing three parameters simultaneously in one sample. The system was well suited to quantitatively determine relevant vaccine induced antibodies compared to in-house and commercially available single-antigen ELISA systems.

  8. Quantification of microglial phagocytosis by a flow cytometer-based assay. (United States)

    Pul, Refik; Chittappen, Kandiyil Prajeeth; Stangel, Martin


    Microglia represent the largest population of phagocytes in the CNS and have a principal role in immune defense and inflammatory responses in the CNS. Their phagocytic activity can be studied by a variety of techniques, including a flow cytometry-based approach utilizing polystyrene latex beads. The flow cytometry-based microglial phagocytosis assay, which is presented here, offers the advantage of rapid and reliable analysis of thousands of cells in a quantitative fashion.

  9. Cloning,Expression and Identification of Sulfotransferase from S. piezotolerans WP3%Shewanella piezotolerans WP3中sulfotransferase基因的克隆表达与活性研究

    Institute of Scientific and Technical Information of China (English)

    韦海澜; 李艳霞; 李升康


      Shewanella piezotolerans WP3分离自西太平洋1914 m的深海沉积物中,是一株革兰氏阴性,耐冷、耐压且兼性厌氧的杆状细菌.初步研究证明海洋细菌中含有磺基转移酶(sulfotransferase,ST)活性.本实验中构建了WP3两个磺基转移酶基因的原核表达载体,在E. coli中表达并纯化了重组ST,通过底物的化学反应,证明两个重组磺基转移酶均具有转磺酶活性.研究结果为后续实验的WP3胞外寡糖的离体硫酸化修饰工作奠定了基础.%S.piezotolerans WP3 is psychrotolerant and piezotolerant deep sea bacterium with a broad pressure optimum,extending from atmospheric pressure to about 20 MPa. The sulfotransferase(ST)of the bacterium is studied in this paper. Two genes,Swp1579 and Swp 4147,encoding the STs were cloned by PCR method. The gene Swp 1579 contains an open reading frame (ORF) of 849 bp,encoding a 526 aa putative ST protein, while the ORF of Swp 4147 consists of 1707 bp, encoding 568aa WP3 arylsulfate sulfotransferase. The 900bp and 1700 bp PCR product is obtained and ligated to the pQE30 vector and pQE70 vector,respectively. Utilizing the IPTG treatment,the cloned ST genes was expressed in Escherichia coli M15. The enzymatic activity assays of recombination proteins showed both had ST activities.

  10. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Directory of Open Access Journals (Sweden)

    Iole Macchia


    Full Text Available The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs and tumor-associated antigens (TAAs and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM- based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

  11. Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions. (United States)

    Tan, Yen Nee; Su, Xiaodi; Liu, Edison T; Thomsen, Jane S


    Gold nanoparticles (AuNPs) are widely used as colorimetric probes for biosensing, relying on their unique particle size-dependent and/or interparticle distance-dependent extinction spectrum and solution color. Herein, we describe an AuNP-based colorimetric assay to detect binding interactions between nuclear hormone receptors and their corresponding DNA-binding elements, particularly the human estrogen receptors (ERalpha and ERbeta) and their cognate estrogen response elements (EREs). We found that the protein-DNA (ER-ERE) complexes can stabilize citrate anion-capped AuNPs against salt-induced aggregation to a larger extent than the protein (ER) or the DNA (ERE) alone, due to their unique molecular size and charge properties that provide a strong electrosteric protection. Moreover, our results show that the extent of stabilization is sequence-dependent and can distinguish a single base variation in the ERE associated with minor changes in protein-DNA binding affinity. With this assay, many important parameters of protein-DNA binding events (e.g., sequence selectivity, distinct DNA binding properties of protein subtypes, binding stoichiometry, and sequence-independent transient binding) can be determined instantly without using labels, tedious sample preparations, and sophisticated instrumentation. These benefits, in particular the high-throughput potential, could enable this assay to become the assay of choice to complement conventional techniques for large scale characterization of protein-DNA interactions, a key aspect in biological research.

  12. Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F. (United States)

    Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole


    Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.

  13. Comparison of microscopy and Alamar blue reduction in a larval based assay for schistosome drug screening.

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    Nuha R Mansour

    Full Text Available BACKGROUND: In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS to identify hits amongst large compound libraries. METHODOLOGY/PRINCIPAL FINDINGS: We have been carrying out low throughput (24-well plate in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB, a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes. CONCLUSION: We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome

  14. Determination of protease subsite preference on SPOT peptide array by fluorescence quenching-based assay. (United States)

    Kim, Do-Hyun; Shin, Dong-Sik; Lee, Yoon-Sik


    A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc-amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl-X-X-P(2)-Arg-X-X-X-Lys(FITC) (X: equimolar mixture of 19 amino acids, P(2): one of 19 amino acids) was synthesized on a chitosan-grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching-based assay. The P(2) subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P(2) subsite showed the highest specificity for thrombin based on both the fluorescence quenching-based assay and the solution phase assay. From these results, we confirmed that our mixture-based peptide SPOT array format on the chitosan-grafted glass chips could be used to determine protease subsite preference.

  15. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

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    Van Neste Leander


    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  16. Phylogenomic approaches to common problems encountered in the analysis of low copy repeats: The sulfotransferase 1A gene family example

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    Benner Steven A


    Full Text Available Abstract Background Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs. Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT 1A genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage. Results Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULT1A paralogs defined a new LCR family. The stem hominoid SULT1A progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULT1A expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript. Conclusion SULT1A genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently one after the divergence of chimpanzees and humans. Thus, LCRs may

  17. Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis. (United States)

    Rascoe, Lisa N; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W; Handali, Sukwan


    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States.

  18. Strategies to develop strain-specific PCR based assays for probiotics. (United States)

    Treven, P


    Since health benefits conferred by probiotics are strain-specific, identification to the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotic in a product or in a gastrointestinal tract. Compared to standard plate counts, the reduced duration of the assays and higher specificity makes PCR-based methods (standard PCR and quantitative PCR) very appropriate for detection or quantification of probiotics. Development of strain-specific assay consists of 4 main stages: (1) strain-specific marker identification; (2) construction of potential strain-specific primers; (3) validation on DNA from pure cultures of target and related strains; and (4) validation on spiked samples. The most important and also the most challenging step is the identification of strain-specific sequences, which can be subsequently targeted by specific primers or probes. Such regions can be identified on sequences derived from 16S-23S internally transcribed spacers, randomly amplified polymorphic DNA, representational difference analysis and suppression subtractive hybridisation. Already known phenotypic or genotypic characteristics of the target strain can also be used to develop the strain-specific assay. However, the initial stage of strain-specific assay development can be replaced by comparative genomics analysis of target genome with related genomes in public databases. Advances in whole genome sequencing (WGS) have resulted in a cost reduction for bacterial genome sequencing and consequently have made this approach available to most laboratories. In the present paper I reviewed the available literature on PCR and qPCR assays developed for detection of a specific probiotic strain and discussed future WGS and comparative genomics-based approaches.

  19. A simple technique for reducing edge effect in cell-based assays. (United States)

    Lundholt, Betina Kerstin; Scudder, Kurt M; Pagliaro, Len


    Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.

  20. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay. (United States)

    Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H; Xia, Menghang


    In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling.

  1. Development of a cell-based, high-throughput screening assay for ATM kinase inhibitors. (United States)

    Guo, Kexiao; Shelat, Anang A; Guy, R Kiplin; Kastan, Michael B


    The ATM (ataxia-telangiectasia, mutated) protein kinase is a major regulator of cellular responses to DNA double-strand breaks (DSBs), DNA lesions that can be caused by ionizing irradiation (IR), oxidative damage, or exposure to certain chemical agents. In response to DSBs, the ATM kinase is activated and subsequently phosphorylates numerous downstream substrates, including p53, Chk2, BRCA1, and KAP1, which affect processes such as cell cycle progression and DNA repair. Numerous studies have demonstrated that loss of ATM function results in enhanced sensitivity to ionizing irradiation in clinically relevant dose ranges, suggesting that ATM kinase is an attractive therapeutic target for enhancing tumor cell kill with radiotherapy. Previously identified small-molecule ATM kinase inhibitors, such as CP466722 and Ku55933, were identified using in vitro kinase assays carried out with recombinant ATM kinase isolated from mammalian cells. Since it has not been feasible to express full-length recombinant ATM in bacterial or baculovirus systems, a robust in vitro screening tool has been lacking. We have developed a cell-based assay that is robust, straightforward, and sensitive. Using this high-throughput assay, we screened more than 7000 compounds and discovered additional small molecules that inhibit the ATM kinase and further validated these hits by secondary assays.

  2. A highly sensitive and selective diagnostic assay based on virus nanoparticles (United States)

    Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon


    Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

  3. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA. (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman


    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

  4. GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

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    Beatriz A Santillan

    Full Text Available Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

  5. Assessing the applicability of FISH-based prematurely condensed dicentric chromosome assay in triage biodosimetry. (United States)

    Suto, Yumiko; Gotoh, Takaya; Noda, Takashi; Akiyama, Miho; Owaki, Makiko; Darroudi, Firouz; Hirai, Momoki


    The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

  6. Fluorescence-based assay as a new screening tool for toxic chemicals (United States)

    Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey


    Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

  7. Electrochemical Genotoxicity Assay Based on a SOS/umu Test Using Hydrodynamic Voltammetry in a Droplet


    Kazuharu Sugawara; Masami Fukushima; Shigeru Taguchi; Noriko Hata; Kazuto Sazawa; Yasuaki Nanayama; Hideki Kuramitz


    The SOS/umu genotoxicity assay evaluates the primary DNA damage caused by chemicals from the β-galactosidase activity of S. typhimurium. One of the weaknesses of the common umu test system based on spectrophotometric detection is that it is unable to measure samples containing a high concentration of colored dissolved organic matters, sediment, and suspended solids. However, umu tests with electrochemical detection techniques prove to be a better strategy because it causes less inter...

  8. Performance of a MALDI-TOF MS-based imipenem hydrolysis assay incorporating zinc sulfate. (United States)

    Knox, James; Palombo, Enzo


    A MALDI-TOF MS(1)-based imipenem hydrolysis assay was modified by adding ZnSO4. This improved detection of metallo-β-lactamase producing strains without compromising detection of other carbapenemase types. Using 129 genetically characterized Gram-negative bacilli, the sensitivity and specificity were 98.5% (95% confidence interval [CI]: 91.9-99.7%) and 100% (95% CI: 94.3-100%), respectively.

  9. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes


    Alvaro Díaz-Badillo; María de Lourdes Muñoz; Gerardo Perez-Ramirez; Victor Altuzar; Juan Burgueño; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Alejandro Cisneros; Joel Navarrete-Espinosa; Feliciano Sanchez-Sinencio


    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybrid...

  10. The use of nanocrystal quantum dot as fluorophore reporters in molecular beacon-based assays (United States)

    Adegoke, Oluwasesan; Park, Enoch Y.


    The utilization of molecular beacon (MB) biosensor probes to detect nucleic acid targets has received enormous interest within the scientific community. This interest has been stimulated by the operational qualities of MB-based probes with respect to their unique sensitivity and specificity. The design of MB biosensors entails not only optimizing the sequence of the loop to hybridize with the nucleic acid target or optimization of the length of the stem to tune the sensitivity but also the selection of the appropriate fluorophore reporter to generate the signal transduction read-out upon hybridization of the probe with the target sequence. Traditional organic fluorescent dyes are mostly used for signal reporting in MB assays but their optical properties in comparison to semiconductor fluorescent quantum dot (Qdot) nanocrystals are at a disadvantage. This review highlights the progress made in exploiting Qdot as fluorophore reporters in MB-based assays with the aim of instigating further development in the field of Qdot-MB technology. The development reported to date indicates that unparalleled fluorescence signal reporting in MB-based assays can be achieved using well-constructed Qdot fluorophores.

  11. A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms. (United States)

    Kim, Young Hwa; Kim, Eung Soo; Ko, Byong Seob; Oh, Seung-Eun; Ryuk, Jin-Ah; Chae, Seong Wook; Lee, Hye Won; Choi, Go Ya; Seo, Doo Won; Lee, Mi Young


    This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.

  12. A novel mass spectrometry-based assay for GSK-3β activity

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    Gan Bing Siang


    Full Text Available Abstract Background As a component of the progression from genomic to proteomic analysis, there is a need for accurate assessment of protein post-translational modifications such as phosphorylation. Traditional kinase assays rely heavily on the incorporation of γ-P32 radiolabeled isotopes, monoclonal anti-phospho-protein antibodies, or gel shift analysis of substrate proteins. In addition to the expensive and time consuming nature of these methods, the use of radio-ligands imposes restrictions based on the half-life of the radionucleotides and pose potential health risks to researchers. With the shortcomings of traditional assays in mind, the aim of this study was to develop a high throughput, non-radioactive kinase assay for screening Glycogen Synthase Kinase-3beta (GSK-3β activity. Results Synthetic peptide substrates designed with a GSK-3β phosphorylation site were assayed with both recombinant enzyme and GSK-3β immunoprecipitated from NIH 3T3 fibroblasts. A molecular weight shift equal to that of a single phosphate group (80 Da. was detected by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS in a GSK-3β target peptide (2B-Sp. Not only was there a dose-dependent response in molecular weight shift to the amount of recombinant GSK-3β used in this assay, this shift was also inhibited by lithium chloride (LiCl, in a dose-dependent manner. Conclusion We present here a novel method to sensitively measure peptide phosphorylation by GSK-3β that, due to the incorporation of substrate controls, is applicable to either purified enzyme or cell extracts. Future studies using this method have the potential to elucidate the activity of GSK-3β in vivo, and to screen enzyme activity in relation to a variety of GSK-3β related disorders.

  13. A new scintillation proximity assay-based approach for the detection of KRAS mutations

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    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.


    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  14. Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo.

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    Xiaoming Yang

    Full Text Available Hydroxysteroid sulfotransferase 2B1b (SULT2B1b is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC.

  15. Comparative cytotoxic and genotoxic potential of 13 drinking water disinfection by-products using a microplate-based cytotoxicity assay and a developed SOS/umu assay. (United States)

    Zhang, Shao-Hui; Miao, Dong-Yue; Tan, Li; Liu, Ai-Lin; Lu, Wen-Qing


    The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively.

  16. Decline in arylsulfatase B and Increase in chondroitin 4-sulfotransferase combine to increase chondroitin 4-sulfate in traumatic brain injury. (United States)

    Bhattacharyya, Sumit; Zhang, Xiaolu; Feferman, Leo; Johnson, David; Tortella, Frank C; Guizzetti, Marina; Tobacman, Joanne K


    In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-β1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-β1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-β1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the

  17. Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

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    Edilaine Mauricia Gelinski Grabicoski


    Full Text Available Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max. Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction primer set and seed soaking (without DNA extraction for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA. The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence and one naturally infected seed in a 300-seed sample (0.33 % incidence. The PCR-based assay was rapid (< 9 h, did not require DNA extraction and was very sensitive.

  18. Three-dimensional paper-based microfluidic device for assays of protein and glucose in urine. (United States)

    Sechi, Deidre; Greer, Brady; Johnson, Jesse; Hashemi, Nastaran


    The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

  19. An enzyme thermistor-based assay for total and free cholesterol. (United States)

    Raghavan, V; Ramanathan, K; Sundaram, P V; Danielsson, B


    A method to evaluate the free (FC) and total cholesterol (TC) in human serum, bile and gallstone extract using an enzyme thermistor (ET)-based flow injection analysis (FIA) is presented. The cholesterol in high-density (HDL-C) and low density lipoprotein (LDL-C) have also been evaluated. A heparin functionalized Sepharose column was employed for the isolation of HDL and LDL fractions from serum. The estimation of cholesterol and its esters was based on their reaction with cholesterol oxidase (CO), cholesterol esterase (CE) and catalase (CAT). Three different enzyme columns, i.e. co-immobilized CO/CAT (column A), only CE (column B) and co-immobilized CO/CE/CAT (column C) were prepared by cross-linking the enzymes on glass beads using glutaraldehyde. Column A was used for estimating FC and column C was used for estimating total cholesterol (cholesterol plus esterified cholesterol). Column B was used as a pre-column which could be switched 'in' or 'out' in conjunction with column A for the estimation of TC or FC, respectively. A calibration between 1.0 and 8.0 mmol/l for FC and 0. 25 and 4.0 mmol/l for TC was obtained. For more than 2000 assays with the ET device a C.V. of less than 4% was obtained. The assay time was approximately 4 min per assay. The cholesterol estimations on the ET correlated well with similar estimations using a commercially available cholesterol diagnostic kit.

  20. Novel PCR Assays Complement Laser Biosensor-Based Method and Facilitate Listeria Species Detection from Food. (United States)

    Kim, Kwang-Pyo; Singh, Atul K; Bai, Xingjian; Leprun, Lena; Bhunia, Arun K


    The goal of this study was to develop the Listeria species-specific PCR assays based on a house-keeping gene (lmo1634) encoding alcohol acetaldehyde dehydrogenase (Aad), previously designated as Listeria adhesion protein (LAP), and compare results with a label-free light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology). PCR primer sets targeting the lap genes from the species of Listeria sensu stricto were designed and tested with 47 Listeria and 8 non-Listeria strains. The resulting PCR primer sets detected either all species of Listeria sensu stricto or individual L. innocua, L. ivanovii and L. seeligeri, L. welshimeri, and L. marthii without producing any amplified products from other bacteria tested. The PCR assays with Listeria sensu stricto-specific primers also successfully detected all species of Listeria sensu stricto and/or Listeria innocua from mixed culture-inoculated food samples, and each bacterium in food was verified by using the light scattering sensor that generated unique scatter signature for each species of Listeria tested. The PCR assays based on the house-keeping gene aad (lap) can be used for detection of either all species of Listeria sensu stricto or certain individual Listeria species in a mixture from food with a detection limit of about 10⁴ CFU/mL.

  1. SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

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    Wai-Hoe Lau


    Full Text Available Abstract Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P

  2. An extended set of yeast-based functional assays accurately identifies human disease mutations (United States)

    Sun, Song; Yang, Fan; Tan, Guihong; Costanzo, Michael; Oughtred, Rose; Hirschman, Jodi; Theesfeld, Chandra L.; Bansal, Pritpal; Sahni, Nidhi; Yi, Song; Yu, Analyn; Tyagi, Tanya; Tie, Cathy; Hill, David E.; Vidal, Marc; Andrews, Brenda J.; Boone, Charles; Dolinski, Kara; Roth, Frederick P.


    We can now routinely identify coding variants within individual human genomes. A pressing challenge is to determine which variants disrupt the function of disease-associated genes. Both experimental and computational methods exist to predict pathogenicity of human genetic variation. However, a systematic performance comparison between them has been lacking. Therefore, we developed and exploited a panel of 26 yeast-based functional complementation assays to measure the impact of 179 variants (101 disease- and 78 non-disease-associated variants) from 22 human disease genes. Using the resulting reference standard, we show that experimental functional assays in a 1-billion-year diverged model organism can identify pathogenic alleles with significantly higher precision and specificity than current computational methods. PMID:26975778

  3. Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis. (United States)

    Yen, Ching-Yu; Wu, Yu-Wei; Hsiung, Chao-Nan; Yeh, Min-I; Lin, Yi-Ming; Lee, Sheng-Yang


    Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

  4. Best practice recommendations for the transfer of cell-based assays for the measurement of neutralizing anti-drug antibodies. (United States)

    Belouski, Shelley S; Born, Danika; Jacques, Susan; Harder, Brandon; Reynhardt, Kai; Kaliyaperumal, Arunan; Gupta, Shalini


    We recommend the application of a strategically designed step-wise approach to transfer cell-based assays that includes assessing analytical performance (through a fit for purpose validation and/or design of experiment robustness characterization), clinical performance (i.e., concordance) and performance or proficiency testing for long-term method monitoring. Here we focus on the application of this strategy to cell-based assays for the measurement of neutralizing anti-drug antibodies. This application is unique in that it requires a custom cell-based assay to be used over a long period of time (potentially phase 1a through the life of a marketed product) with the confidence of consistent method performance and result reporting. But, the process is adaptable to a variety of assay types and applications. We present lessons learned from two cell-based assay transfers that met relevant challenges while implementing alternative permutations of the recommended method transfer process.

  5. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR. (United States)

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R


    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  6. A pseudovirus-based hemagglutination-inhibition assay as a rapid, highly sensitive, and specific assay for detecting avian influenza A (H7N9 antibodies

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    Anli Zhang


    Full Text Available Background Increased surveillance of avian-origin influenza A (H7N9 virus infection is critical to assess the risk of new outbreaks in China. A high-throughput assay with a good safety profile, sensitivity, and specificity is urgently needed. Methods We used a hemagglutination-inhibition (HI assay based on an H7N9-enveloped pseudovirus to assess serum neutralization antibodies level in 40 H7N9 positive sera and 40 H7N9 negative sera and compared the efficacy of the assay with traditional HI test and micro-neutralization (MN test. Results Spearman’s rank correlation coefficient analysis showed pseudovirus HI (PHI titers correlated well with both HI titers and MN titers. Receiver operating characteristic (ROC curves test revealed using a PHI cut-off titer of 10, the sensitivity and specificity reached 1.0. Conclusions PHI can be used in H7N9-related serological studies. This assay is high-throughput, very sensitive and specific, and cost effective.

  7. Novel microwell-based spectrophotometric assay for determination of atorvastatin calcium in its pharmaceutical formulations

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    Abdel-Rahman Hamdy M


    Full Text Available Abstract The formation of a colored charge-transfer (CT complex between atorvastatin calcium (ATR-Ca as a n-electron donor and 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ as a π-electron acceptor was investigated, for the first time. The spectral characteristics of the CT complex have been described, and the reaction mechanism has been proved by computational molecular modeling. The reaction was employed in the development of a novel microwell-based spectrophotometric assay for determination of ATR-Ca in its pharmaceutical formulations. The proposed assay was carried out in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm by microwell-plate absorbance reader. The optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, linear relationship with good correlation coefficient (0.9995 was found between the absorbance and the concentration of ATR-Ca in the range of 10-150 μg/well. The limits of detection and quantitation were 5.3 and 15.8 μg/well, respectively. No interference was observed from the additives that are present in the pharmaceutical formulation or from the drugs that are co-formulated with ATR-Ca in its combined formulations. The assay was successfully applied to the analysis of ATR-Ca in its pharmaceutical dosage forms with good accuracy and precision. The assay described herein has great practical value in the routine analysis of ATR-Ca in quality control laboratories, as it has high throughput property, consumes minimum volume of organic solvent thus it offers the reduction in the exposures of the analysts to the toxic effects of organic solvents, and reduction in the analysis cost by 50-fold. Although the proposed assay was validated for ATR-Ca, however, the same methodology could be used for any electron-donating analyte for which a CT reaction can be performed.

  8. Histochemical analysis of heparan sulfate 3-O-sulfotransferase expression in mouse brain. (United States)

    Yabe, Tomio; Maeda, Nobuaki


    In situ hybridization provides information for understanding the localization of gene expression in various tissues. The relative expression levels of mRNAs in a single cell can be sensitively visualized by this technique. Furthermore, since in situ hybridization is a histological technique, tissue structure is maintained after fixation, and it is possible to accurately identify cell types. We have examined the expression of heparan sulfate sulfotransferases by in situ hybridization to better understand the functions of heparan sulfate in the development of mouse nervous system. This chapter describes methods of in situ hybridization analyses using cRNA probes labeled with nonradioactive nucleotides.

  9. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes. (United States)

    Ding, Xiaokang; Yang, Kun-Lin


    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  10. A novel in vitro image-based assay identifies new drug leads for giardiasis. (United States)

    Hart, Christopher J S; Munro, Taylah; Andrews, Katherine T; Ryan, John H; Riches, Andrew G; Skinner-Adams, Tina S


    Giardia duodenalis is an intestinal parasite that causes giardiasis, a widespread human gastrointestinal disease. Treatment of giardiasis relies on a small arsenal of compounds that can suffer from limitations including side-effects, variable treatment efficacy and parasite drug resistance. Thus new anti-Giardia drug leads are required. The search for new compounds with anti-Giardia activity currently depends on assays that can be labour-intensive, expensive and restricted to measuring activity at a single time-point. Here we describe a new in vitro assay to assess anti-Giardia activity. This image-based assay utilizes the Perkin-Elmer Operetta(®) and permits automated assessment of parasite growth at multiple time points without cell-staining. Using this new approach, we assessed the "Malaria Box" compound set for anti-Giardia activity. Three compounds with sub-μM activity (IC50 0.6-0.9 μM) were identified as potential starting points for giardiasis drug discovery.

  11. A simple, versatile and sensitive cell-based assay for prions from various species.

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    Zaira E Arellano-Anaya

    Full Text Available Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

  12. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants. (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan


    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  13. EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis. (United States)

    Bandeira-Melo, Christianne; Paiva, Ligia Almeida; Amorim, Natália R T; Weller, Peter F; Bozza, Patricia T


    Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.

  14. Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes. (United States)

    Warrilow, David; Northill, Judith A; Pyke, Alyssa; Smith, Greg A


    Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.

  15. An Acetylcholinesterase-Based Chronoamperometric Biosensor for Fast and Reliable Assay of Nerve Agents

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    Rene Kizek


    Full Text Available The enzyme acetylcholinesterase (AChE is an important part of cholinergic nervous system, where it stops neurotransmission by hydrolysis of the neurotransmitter acetylcholine. It is sensitive to inhibition by organophosphate and carbamate insecticides, some Alzheimer disease drugs, secondary metabolites such as aflatoxins and nerve agents used in chemical warfare. When immobilized on a sensor (physico-chemical transducer, it can be used for assay of these inhibitors. In the experiments described herein, an AChE- based electrochemical biosensor using screen printed electrode systems was prepared. The biosensor was used for assay of nerve agents such as sarin, soman, tabun and VX. The limits of detection achieved in a measuring protocol lasting ten minutes were 7.41 × 10−12 mol/L for sarin, 6.31 × 10−12 mol /L for soman, 6.17 × 10−11 mol/L for tabun, and 2.19 × 10−11 mol/L for VX, respectively. The assay was reliable, with minor interferences caused by the organic solvents ethanol, methanol, isopropanol and acetonitrile. Isopropanol was chosen as suitable medium for processing lipophilic samples.

  16. Sensitive measurement of thrombopoietin by a monoclonal antibody based sandwich enzyme-linked immunosorbent assay. (United States)

    Folman, C C; von dem Borne, A E; Rensink, I H; Gerritsen, W; van der Schoot, C E; de Haas, M; Aarden, L


    In this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs). The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 +/- 0.8 A.U./ml, mean +/- sd) was lower than the concentration found in controls (11 +/- 8 A.U./ml, mean +/- sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma. We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels. In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

  17. Applications of monolithic solid-phase extraction in chromatography-based clinical chemistry assays. (United States)

    Bunch, Dustin R; Wang, Sihe


    Complex matrices, for example urine, serum, plasma, and whole blood, which are common in clinical chemistry testing, contain many non-analyte compounds that can interfere with either detection or in-source ionization in chromatography-based assays. To overcome this problem, analytes are extracted by protein precipitation, solid-phase extraction (SPE), and liquid-liquid extraction. With correct chemistry and well controlled material SPE may furnish clean specimens with consistent performance. Traditionally, SPE has been performed with particle-based adsorbents, but monolithic SPE is attracting increasing interest of clinical laboratories. Monoliths, solid pieces of stationary phase, have bimodal structures consisting of macropores, which enable passage of solvent, and mesopores, in which analytes are separated. This structure results in low back-pressure with separation capabilities similar to those of particle-based adsorbents. Monoliths also enable increased sample throughput, reduced solvent use, varied support formats, and/or automation. However, many of these monoliths are not commercially available. In this review, application of monoliths to purification of samples from humans before chromatography-based assays will be critically reviewed.

  18. Naked-eye quantitative aptamer-based assay on paper device. (United States)

    Zhang, Yun; Gao, Dong; Fan, Jinlong; Nie, Jinfang; Le, Shangwang; Zhu, Wenyuan; Yang, Jiani; Li, Jianping


    This work initially describes the design of low-cost, naked-eye quantitative aptamer-based assays by using microfluidic paper-based analytical device (μPAD). Two new detection motifs are proposed for quantitative μPAD measurement without using external electronic readers, which depend on the length of colored region in a strip-like μPAD and the number of colorless detection microzones in a multi-zone μPAD. The length measuring method is based on selective color change of paper from colorless to blue-black via formation of iodine-starch complex. The counting method is conducted on the basis of oxidation-reduction reaction between hydrogen peroxide and potassium permanganate. Their utility is well demonstrated with sensitive, specific detection of adenosine as a model analyte with the naked eye in buffer samples and undiluted human serum. These equipment-free quantitative methods proposed thus hold great potential for the development of more aptamer-based assays that are simple, cost-efficient, portable, and user-friendly for various point-of-care applications particularly in resource-constrained environments.

  19. Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

    Institute of Scientific and Technical Information of China (English)

    Pei-Yuan Li; Xiao-Jun Zhou; Lan Yao; Xin-Hua Fang; Jiang-Nan Ren; Jia-Wu Song


    AIM:To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rsS099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 103-104 white cells/lμL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray

  20. Diagnosis of Morquio Syndrome in Dried Blood Spots Based on a New MRM-MS Assay.

    Directory of Open Access Journals (Sweden)

    Claudia Cozma

    Full Text Available Mucopolysaccharidosis IVA (MPS IVA; Morquio A disease is an autosomal recessive disease caused and characterized by a decreased activity of N-acetylgalactosamine-6-sulfate sulfatase (GALNS, resulting in accumulation of keratan sulfate and chondroitin-6-sulfate in tissues and secondary organ damage. Recently approved enzyme replacement therapy renders the easy and early identification of MPS IVA of out-most importance.We propose a completely new assay for the stable and reproducible detection of GALNS deficiency in dry blood spots (DBS. For the validation blood samples were taken from 59 healthy individuals and 24 randomly selected genetically confirmed MPS IVA patients. The material extracted from DBS was incubated with a 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate as a specific substrate. Final enzymatic product, 4-methylumbelliferone, obtained after adding exogenous beta-galactosidase, was quantified by LC/MRM-MS (liquid-chromatography/multiple-reaction-monitoring mass-spectrometry. 4-propyl-5-hydroxy-7-methyl-2h-chromen-2-one was used as internal standard, a compound with a similar molecular structure and fragmentation pattern in negative ion mode as 4-methylumbelliferone.The enzymatic assay yielded a positive and negative predictive value of 1.0 for genetically confirmed MPS IVA patients (GALNS activity of 0.35 ± 0.21 μmol/L/h and for controls with normal GALNS activity (23.1 ± 5.3 μmol/L /h. With present enzymatic conditions, the reaction yield in dried blood spots is at least 20 fold higher than any previously reported data with other assays.The present LC/MRM-MS based assay for MPS IVA diagnosis provides an easy, highly-standardized, accurate and innovative quantification of the enzymatic product in vitro and distinguishes perfectly between MPS IVA affected patients and normal controls. This technique will significantly simplify the early detection of MPS IVA patients.

  1. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    NARCIS (Netherlands)

    Agren, J.; Hamidjaja, R.A.; Hansen, T.; Ruuls, R.C.; Thierry, S.; Vigre, H.; Janse, I.; Sundström, A.; Segerman, B.; Koene, M.G.J.; Löfström, Ch.; Rotterdam, van B.; Derzelle, S.


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely o

  2. Evaluation of a novel PCR-based assay for detection and identification of Chlamydia trachomatis serovars in cervical specimens.

    NARCIS (Netherlands)

    Quint, K.D.; Porras, C.; Safaeian, M.; Gonzalez, P.; Hildesheim, A.; Quint, W.G.V.; Doorn, L.J. van; Silva, S.; Melchers, W.J.G.; Schiffman, M.; Rodriguez, A.C.; Wacholder, S.; Freer, E.; Cortes, B.; Herrero, R.


    The aims of this study were to compare a novel PCR-based Chlamydia trachomatis detection and genotyping (Ct-DT) assay with the FDA-approved, commercially available C. trachomatis detection Hybrid Capture 2 (HC2) assay and to investigate the C. trachomatis serovar distribution among young women in a

  3. A facile low-cost enzymatic paper-based assay for the determination of urine creatinine. (United States)

    Talalak, Kwanrutai; Noiphung, Julaluk; Songjaroen, Temsiri; Chailapakul, Orawon; Laiwattanapaisal, Wanida


    Creatinine is one of many markers used to investigate kidney function. This paper describes a low-cost enzymatic paper-based analytical device (enz-PAD) for determining urine creatinine. The disposable dead volumes of creatinine enzyme reagents from an automatic analyser cassette were utilised. Whatman No. 3 paper was cut into long rectangular shapes (4×40 mm(2)) on which the enzyme reagents, R1 and R2, were adsorbed in two consecutive regions. The assay was performed by immersing test strips into urine samples contained in microwells to allow creatinine in the sample to react with immobilised active ingredients and, then, traverse via capillary action to the detection area where chromogen products accumulated. The method is based on hydrogen peroxide (H2O2) formation via creatinine conversion using creatininase, creatinase, and sarcosine oxidase. The liberated H2O2 reacts with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid to form quinoneimine with a pink-red colour at the detection zone. The linear range of the creatinine assay was 2.5-25 mg dL(-1) (r(2)=0.983), and the detection limit was 2.0 mg dL(-1). The colorimetric enz-PAD for the creatinine assay was highly correlated with a conventional alkaline picrate method when real urine samples were evaluated (r(2)=0.977; n=40). This simple and nearly zero-cost paper-based device provides a novel alternative method for screening urinary creatinine and will be highly beneficial for developing countries.

  4. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins. (United States)

    Li, Hua; Fang, Xueen; Cao, Hongmei; Kong, Jilie


    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis.

  5. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay (United States)

    Zhou, Huijuan; Wu, Baoyan


    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  6. Detection of ESKAPE Bacterial Pathogens at the Point of Care Using Isothermal DNA-Based Assays in a Portable Degas-Actuated Microfluidic Diagnostic Assay Platform. (United States)

    Renner, Lars D; Zan, Jindong; Hu, Linda I; Martinez, Manuel; Resto, Pedro J; Siegel, Adam C; Torres, Clint; Hall, Sara B; Slezak, Tom R; Nguyen, Tuan H; Weibel, Douglas B


    An estimated 1.5 billion microbial infections occur globally each year and result in ∼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ∼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.

  7. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis. (United States)

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman


    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  8. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements. (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David


    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

  9. Comparison of histopathology and PCR based assay for detection of experimentally induced toxoplasmosis in murine model

    Institute of Scientific and Technical Information of China (English)

    Vikrant Sudan; A K Tewari; R Singh; Harkirat Singh


    Objective:To compare histopathology and PCR based detection in diagnosis of experimentally induced toxoplasmosis of RH human strain of the parasite in murine models. Methods:A comparison of histopathology and PCR based detection was done to diagnose experimentally induced toxoplasmosis in ten inbred swiss albino mice after intraperitoneal inoculation of 100 tachyzoites of laboratory mantained human RH strain of the parasite. Tissue samples from lung, liver, spleen, brain, heart and kidney were taken and processed for histopathological examination while all the samples also were subjected to PCR, using primers directed to the multicopy of SAG 3 gene, in dublicates. Results: Histopathology revealed presence of tachyzoites only in liver while along with lung, liver, spleen and brain tissue yielded desired positive PCR amplicons. Conclusions:The SAG 3 based PCR is able to diagnose toxoplasmosis in those tissues which are declared negative by histopathological assay.

  10. A practical method for extending the biuret assay to protein determination of corn-based products. (United States)

    Liu, Zelong; Pan, Junhui


    A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.

  11. Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay. (United States)

    Khan, Sadia Afrin; DeGrasse, Jeffrey A; Yakes, Betsy Jean; Croley, Timothy R


    Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV-Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.

  12. Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design (United States)

    Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R.; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S.; Williams, Steven A.


    Background The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Methodology/Principal Findings Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. Conclusions/Significance The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other

  13. Heteropolymeric triplex-based genomic assay to detect pathogens or single-nucleotide polymorphisms in human genomic samples.

    Directory of Open Access Journals (Sweden)

    Jasmine I Daksis

    Full Text Available Human genomic samples are complex and are considered difficult to assay directly without denaturation or PCR amplification. We report the use of a base-specific heteropolymeric triplex, formed by native duplex genomic target and an oligonucleotide third strand probe, to assay for low copy pathogen genomes present in a sample also containing human genomic duplex DNA, or to assay human genomic duplex DNA for Single Nucleotide Polymorphisms (SNP, without PCR amplification. Wild-type and mutant probes are used to identify triplexes containing FVL G1691A, MTHFR C677T and CFTR mutations. The specific triplex structure forms rapidly at room temperature in solution and may be detected without a separation step. YOYO-1, a fluorescent bis-intercalator, promotes and signals the formation of the specific triplex. Genomic duplexes may be assayed homogeneously with single base pair resolution. The specific triple-stranded structures of the assay may approximate homologous recombination intermediates, which various models suggest may form in either the major or minor groove of the duplex. The bases of the stable duplex target are rendered specifically reactive to the bases of the probe because of the activity of intercalated YOYO-1, which is known to decondense duplex locally 1.3 fold. This may approximate the local decondensation effected by recombination proteins such as RecA in vivo. Our assay, while involving triplex formation, is sui generis, as it is not homopurine sequence-dependent, as are "canonical triplexes". Rather, the base pair-specific heteropolymeric triplex of the assay is conformation-dependent. The highly sensitive diagnostic assay we present allows for the direct detection of base sequence in genomic duplex samples, including those containing human genomic duplex DNA, thereby bypassing the inherent problems and cost associated with conventional PCR based diagnostic assays.

  14. Mapping of the phenol sulfotransferase gene (STP) to human chromosome 16p12. 1-p11. 2 and to mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Dooley, T.P.; Obermoeller, R.D. (Southwest Foundation for Biomedical Research, San Antonio, TX (United States)); Leiter, E.H.; Chapman, H.D. (Jackson Lab., Bar Harbor, ME (United States)); Falany, C.N. (Univ. of Alabama, Birmingham, AL (United States)); Deng, Z.; Siciliano, M.J. (Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States))


    The authors have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotransferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5[prime] end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, [beta]1 polypeptide (PRKCB1), in the region from the distal portion of 16p11.2 to p12.1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, the authors have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers. 18 refs., 2 figs., 1 tab.

  15. Subcellular location and molecular mobility of human cytosolic sulfotransferase 1C1 in living human embryonic kidney 293 cells. (United States)

    Sheng, Jonathan J; Acquaah-Mensah, George K


    Cytosolic sulfotransferases were first isolated from the hepatic cytosol, and they have been localized in the cytoplasm of formaldehyde-fixed human cell samples. The current work was carried out to determine the subcellular localization and molecular mobility of cytosolic sulfotransferases in living human embryonic kidney (HEK) 293 cells. In this work, the subcellular location of human cytosolic sulfotransferase 1C1 (SULT1C1) was studied in cultured HEK293 cells using confocal laser-scanning microscopy. A green fluorescent protein (GFP)-tagged SULT1C1 protein was localized in the cytoplasm of living HEK293 cells. This is consistent with results from previous studies on several other cytosolic sulfotransferase isoforms. Fluorescence recovery after photobleaching microscopy was performed to assess the molecular mobility of the expressed GFP-SULT1C1 molecules. The results suggested that the expressed recombinant GFP-SULT1C1 molecules in living HEK293 cells may include both mobile and immobile populations. To obtain additional insights into the subcellular location of SULT1C1, two machine learning algorithms, Sequential Minimal Optimization and Multilayer Perceptron, were used to compute the probability distribution for the localization of SULT1C1 in nine selected cellular compartments. The resulting probability distribution suggested that the most likely subcellular location of SULT1C1 is the cytosol.

  16. Human cytosolic sulfotransferase SULT1C4 mediates the sulfation of doxorubicin and epirubicin. (United States)

    Luo, Lijun; Zhou, Chunyang; Hui, Ying; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh


    Doxorubicin, an anthracycline, has been reported to be excreted in sulfate conjugated form. The current study aimed to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of sulfating doxorubicin and its analog epirubicin, and to verify whether sulfation of doxorubicin and epirubicin may occur under metabolic conditions. A systematic analysis of thirteen known human SULTs, previously cloned, expressed, and purified, revealed SULT1C4 as the only human SULT capable of sulfating doxorubicin and epirubicin. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [(35)S]sulfate in the presence of different concentrations of doxorubicin or epirubicin. Analysis of spent labeling media showed the generation and release of [(35)S]sulfated doxorubicin and epirubicin by HepG2 cells and Caco-2 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of SULT1C4 in both HepG2 cells and Caco-2 cells. These results provided a molecular basis underlying the previous finding that sulfate-conjugated doxorubicin was excreted in the urine of patients treated with doxorubicin.

  17. Sulfation of 6-Gingerol by the Human Cytosolic Sulfotransferases: A Systematic Analysis. (United States)

    Luo, Lijun; Mei, Xue; Xi, Yuecheng; Zhou, Chunyang; Hui, Ying; Kurogi, Katsuhisa; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh


    Previous studies have demonstrated the presence of the sulfated form of 6-gingerol, a major pharmacologically active component of ginger, in plasma samples of normal human subjects who were administered 6-gingerol. The current study was designed to systematically identify the major human cytosolic sulfotransferase enzyme(s) capable of mediating the sulfation of 6-gingerol. Of the 13 known human cytosolic sulfotransferases examined, six (SULT1A1, SULT1A2, SULT1A3, SULT1B1, SULT1C4, SULT1E1) displayed significant sulfating activity toward 6-gingerol. Kinetic parameters of SULT1A1, SULT1A3, SULT1C4, and SULT1E1 that showed stronger 6-gingerol-sulfating activity were determined. Of the four human organ samples tested, small intestine and liver cytosols displayed considerably higher 6-gingerol-sulfating activity than those of the lung and kidney. Moreover, sulfation of 6-gingerol was shown to occur in HepG2 human hepatoma cells and Caco-2 human colon adenocarcinoma cells under the metabolic setting. Collectively, these results provided useful information relevant to the metabolism of 6-gingerol through sulfation both in vitro and in vivo.

  18. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)


    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  19. A parallel and quantitative cell migration assay using a novel multi-well-based device. (United States)

    Quan, Qianghua; Zhang, Shuwen; Wang, Xudong; Ouyang, Qi; Wang, Yugang; Yang, Gen; Luo, Chunxiong


    Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-β(TGF-β) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-β + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

  20. A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape. (United States)

    Quereda, Juan J; Pizarro-Cerdá, Javier; Balestrino, Damien; Bobard, Alexandre; Danckaert, Anne; Aulner, Nathalie; Shorte, Spencer; Enninga, Jost; Cossart, Pascale


    Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.

  1. Gold nanoparticle aggregation-based colorimetric assay for β-casein detection in bovine milk samples. (United States)

    Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Song, F; Lu, S Y; Ren, H L; Hu, P; Liu, Z S; Zhang, J H


    Traditional Kjeldahl method, used for quality evaluation of bovine milk, has intrinsic defects of time-consuming sample preparation and two analyses to determine the difference between non-protein nitrogen content and total protein nitrogen content. Herein, based upon antibody functionalized gold nanoparticles (AuNPs), we described a colorimetric method for β-casein (β-CN) detection in bovine milk samples. The linear dynamic range and the LOD were 0.08-250 μg mL(-1), and 0.03 μg mL(-1) respectively. In addition, the real content of β-CN in bovine milk was measured by using the developed assay. The results are closely correlated with those from Kjeldahl method. The advantages of β-CN triggered AuNP aggregation-based colorimetric assay are simple signal generation, the high sensitivity and specificity as well as no need of complicated sample preparation, which make it for on-site detection of β-CN in bovine milk samples.

  2. Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses. (United States)

    Sudarshana, M R; Reddy, D V


    A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5-1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4 degrees C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.

  3. Epigenetic inactivation of heparan sulfate (glucosamine 3-O-sulfotransferase 2 in lung cancer and its role in tumorigenesis.

    Directory of Open Access Journals (Sweden)

    Jung-Ah Hwang

    Full Text Available BACKGROUND: This study was aimed at investigating the functional significance of heparan sulfate (glucosamine 3-O-sulfotransferase 2 (HS3ST2 hypermethylation in non-small cell lung cancer (NSCLC. METHODOLOGY/ PRINCIPAL FINDINGS: HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting and EpiTYPER(TM assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2'-deoxycytidine (5-Aza-dC. A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman's rank correlation. HS3ST2 hypermethylation was found in 95 (32% of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25-3.58, P = 0.005 compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation. CONCLUSIONS/ SIGNIFICANCE: The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.

  4. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells. (United States)

    Wang, Xiao; Peden, Keith; Murata, Haruhiko


    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  5. Upconversion nanoparticle-based fluorescence resonance energy transfer assay for organophosphorus pesticides. (United States)

    Long, Qian; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo


    This paper reports a novel nanosensor for organophosphorus pesticides based on the fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs). The detection mechanism is based on the facts that AuNPs quench the fluorescence of UCNPs and organophosphorus pesticides (OPs) inhibit the activity of acetylcholinesterase (AChE) which catalyzes the hydrolysis of acetylthiocholine (ATC) into thiocholine. Under the optimized conditions, the logarithm of the pesticides concentration was proportional to the inhibition efficiency. The detection limits of parathion-methyl, monocrotophos and dimethoate reached 0.67, 23, and 67 ng/L, respectively. Meanwhile, the biosensor shows good sensitivity, stability, and could be successfully applied to detection of OPs in real food samples, suggesting the biosensor has potentially extensive application clinic diagnoses assays.

  6. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye. (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta


    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  7. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF


    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

  8. A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

    NARCIS (Netherlands)

    van Ameijde, Jeroen; Overvoorde, John; Knapp, Stefan; den Hertog, Jeroen; Ruijtenbeek, Rob; Liskamp, Rob M J


    A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production

  9. Microplate assay for screening the antibacterial activity of Schiff bases derived from substituted benzopyran-4-one. (United States)

    Amin, Rehab M; Abdel-Kader, Nora S; El-Ansary, Aida L


    Schiff bases (SB(1)-SB(3)) were synthesized from the condensation of 6-formyl-7-hydroxy-5-methoxy-2-methylbenzopyran-4-one with 2-aminopyridine (SB(1)), p-phenylenediamine (SB(2)) and o-phenylenediamine (SB(3)), while Schiff bases (SB(4)-SB(6)) were synthesized by condensation of 5,7-dihydroxy-6-formyl-2-methylbenzopyran-4-one with 2-aminopyridine (SB(4)), p-phenylenediamine (SB(5)) and o-phenylenediamine (SB(6)). Schiff bases were characterized using elemental analysis, IR, UV-Vis, (1)H NMR, (13)C NMR and mass spectroscopy. These compounds were screened for antibacterial activities by micro-plate assay technique. Escherichia coli and Staphylococcus capitis were exposed to different concentrations of the Schiff bases. Results showed that the antibacterial effect of these Schiff bases on Gram-negative bacteria were higher than that on Gram-positive bacteria moreover, the Schiff bases containing substituent OCH(3) on position five have higher antibacterial activity than that containing hydroxy group on the same position.

  10. A fourier transform infrared spectroscopy (FTIR) based assay for Candida parapsilosis ATCC 7330 mediated oxidation of aryl alcohols. (United States)

    Sudhakara, Sneha; Chadha, Anju


    We present an FTIR based assay to monitor the whole cell mediated oxidation of aryl alcohols by measuring the characteristic IR absorption of the hydroxyl group [OH] of the substrate and the carbonyl group [CO] of the corresponding oxidized product. This method expedites the analysis of whole cell mediated catalysis which is usually done by GC and/or HPLC. The FTIR assay had linearity with R(2)≥0.980 and sensitivity up to 10μM. The accuracy and precision of FTIR assay was found ≥81% and ≥94%, respectively. This assay was validated by GC which exhibited ≥82% accuracy and ≥79% precision. The time of analysis taken by this assay was 2-3min per sample in comparison with 20-40min by GC.

  11. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen


    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  12. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna


    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  13. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies. (United States)

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A


    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

  14. A High-Throughput MALDI-TOF Mass Spectrometry-Based Assay of Chitinase Activity (United States)

    A high-throughput MALDI-TOF mass spectrometric assay is described for assay of chitolytic enzyme activity. The assay uses unmodified chitin oligosaccharide substrates, and is readily achievable on a microliter scale (2 µL total volume, containing 2 µg of substrate and 1 ng of protein). The speed a...

  15. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

    Directory of Open Access Journals (Sweden)

    Chan Koon H


    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  16. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay.

    Directory of Open Access Journals (Sweden)

    Yiyi Sun

    Full Text Available Adiponectin, the adipose-derived hormone, plays an important role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. It has been shown that up-regulation of adiponectin or adiponectin receptor has a number of therapeutic benefits. Given that it is hard to convert the full size adiponectin protein into a viable drug, adiponectin receptor agonists could be designed or identified using high-throughput screening. Here, we report on the development of a two-step screening process to identify adiponectin agonists. First step, we developed a high throughput screening assay based on fluorescence polarization to identify adiponectin ligands. The fluorescence polarization assay reported here could be adapted to screening against larger small molecular compound libraries. A natural product library containing 10,000 compounds was screened and 9 hits were selected for validation. These compounds have been taken for the second-step in vitro tests to confirm their agonistic activity. The most active adiponectin receptor 1 agonists are matairesinol, arctiin, (--arctigenin and gramine. The most active adiponectin receptor 2 agonists are parthenolide, taxifoliol, deoxyschizandrin, and syringin. These compounds may be useful drug candidates for hypoadiponectin related diseases.

  17. SDS-PAGE-Based Quantitative Assay for Screening of Kidney Stone Disease

    Directory of Open Access Journals (Sweden)

    Wing-Seng Leong


    Full Text Available Abstract Kidney stone disease is a common health problem in industrialised nations. We developed a SDS-PAGE-based method to quantify Tamm Horsfall glycoprotein (THP for screening of kidney stone disease. Urinary proteins were extracted by using ammonium sulphate precipitation at 0.27 g salt/mL urine. The resulted pellet was dissolved in TSE buffer. Ten microliters of the urinary proteins extract was loaded and separated on 10% SDS-PAGE under reducing condition. THP migrated as single band in SDS-PAGE. The assay reproducibility and repeatability were 4.8% CV and 2.6% CV, respectively. A total of 117 healthy subjects and 58 stone patients were tested using this assay, and a distinct cut-off (P < 0.05 at 5.6 μg/mL THP concentration was used to distinguish stone patients from healthy subjects. The sensitivity and specificity of the method were 92.3% and 83.3%, respectively.

  18. Recommendations for the development and validation of flow cytometry-based receptor occupancy assays. (United States)

    Green, Cherie L; Stewart, Jennifer J; Högerkorp, Carl-Magnus; Lackey, Alan; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Ferbas, John; Moulard, Maxime; Czechowska, Kamila; Mc Closkey, Thomas W; van der Strate, Barry W A; Wilkins, Danice E C; Lanham, David; Wyant, Timothy; Litwin, Virginia


    Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

  19. Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers

    Institute of Scientific and Technical Information of China (English)

    Kai SUN; Qian WANG; Xiao-hui HUANG; Mao-chuan ZHEN; Wen LI; Long-juan ZHANG


    Aim: The multiplexed, microsphere-based flow cytometric assay (MFCA) for mul- tiple human tumor markers was established for the early screening and detection of suspected cancer patients. Methods: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were iden- tified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. Results: The identifications revealed that the coupling proce- dures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality con- trol of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. Conclusion: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

  20. Miniaturized analytical instrumentation for electrochemiluminescence assays: a spectrometer and a photodiode-based device. (United States)

    Neves, Marta M P S; Bobes-Limenes, Pablo; Pérez-Junquera, Alejandro; González-García, María Begoña; Hernández-Santos, David; Fanjul-Bolado, Pablo


    Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (μSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the μSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol-H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.

  1. Novel arginine deiminase-based method to assay L-arginine in beverages. (United States)

    Stasyuk, N Ye; Gayda, G Z; Fayura, L R; Boretskyy, Y R; Gonchar, M V; Sibirny, A A


    A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 μM to 24 μM with the detection limit of 0.25 μM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 μM to 50 μM with the detection limit of 0.55 μM. The new method was tested on real samples of wines and juices. A high correlation (R=0.978) was shown for the results obtained with the proposed and the reference enzymatic method.

  2. A High-resolution Typing Assay for Uropathogenic Escherichia coli Based on Fimbrial Diversity. (United States)

    Ren, Yi; Palusiak, Agata; Wang, Wei; Wang, Yi; Li, Xiao; Wei, Huiting; Kong, Qingke; Rozalski, Antoni; Yao, Zhi; Wang, Quan


    Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, causing cystitis, pyelonephritis, and renal failure. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTIs. Accurate and rapid discrimination of UPEC lineages is useful for epidemiological surveillance. Fimbriae are necessary for the adherence of UPEC strains to host uroepithelia, and seem to be abundant and diverse in UPEC strains. By analyzing all the possible fimbrial operons in UPEC strains, we found that closely related strains had similar types of chaperone-usher fimbriae, and the diversity of fimbrial genes was higher than that of multilocus sequence typing (MLST) genes. A typing assay based on the polymorphism of four gene sequences (three fimbrial genes and one housekeeping gene) and the diversity of fimbriae present was developed. By comparison with the MLST, whole-genome sequence (WGS) and fumC/fimH typing methods, this was shown to be accurate and have high resolution, and it was also relatively inexpensive and easy to perform. The assay can supply more discriminatory information for UPEC lineages, and have the potential to be applied in epidemiological surveillance of UPEC isolates.

  3. Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors (United States)

    Andresen, Liis; Varik, Vallo; Tozawa, Yuzuru; Jimmy, Steffi; Lindberg, Stina; Tenson, Tanel; Hauryliuk, Vasili


    The stringent response is a central adaptation mechanism that allows bacteria to adjust their growth and metabolism according to environmental conditions. The functionality of the stringent response is crucial for bacterial virulence, survival during host invasion as well as antibiotic resistance and tolerance. Therefore, specific inhibitors of the stringent response hold great promise as molecular tools for disarming and pacifying bacterial pathogens. By taking advantage of the valine amino acid auxotrophy of the Bacillus subtilis stringent response-deficient strain, we have set up a High Throughput Screening assay for the identification of stringent response inhibitors. By screening 17,500 compounds, we have identified a novel class of antibacterials based on the 4-(6-(phenoxy)alkyl)-3,5-dimethyl-1H-pyrazole core. Detailed characterization of the hit compounds as well as two previously identified promising stringent response inhibitors – a ppGpp-mimic nucleotide Relacin and cationic peptide 1018 – showed that neither of the compounds is sufficiently specific, thus motivating future application of our screening assay to larger and more diverse molecular libraries. PMID:27775002

  4. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase. (United States)

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga


    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  5. A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol. (United States)

    Hassan, Rabeay Y A; Bilitewski, Ursula


    Candida albicans is an opportunistic fungal pathogen with comparably high respiratory activity. Thus, we established a viability test based on 2,6-dichlorophenolindophenol (DCIP), a membrane-permeable electron transfer agent. NADH dehydrogenases catalyze the reduction of DCIP by NADH, and the enzymatic activity can be determined either electrochemically via oxidation reactions of DCIP or photometrically. Among the specific respiratory chain inhibitors, only the complex I inhibitor rotenone decreased the DCIP signal from C. albicans, leaving residual activity of approximately 30%. Thus, the DCIP-reducing activity of C. albicans was largely dependent on complex I activity. C. albicans is closely related to the complex I-negative yeast Saccharomyces cerevisiae, which had previously been used in DCIP viability assays. Via comparative studies, in which we included the pathogenic complex I-negative yeast Candida glabrata, we could define assay conditions that allow a distinction of complex I-negative and -positive organisms. Basal levels of DCIP turnover by S.cerevisiae and C. glabrata were only 30% of those obtained from C. albicans but could be increased to the C. albicans level by adding glucose. No significant increases were observed with galactose. DCIP reduction rates from C. albicans were not further increased by any carbon source.

  6. Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system. (United States)

    StGelais, Corine; Tuthill, Tobias J; Clarke, Dean S; Rowlands, David J; Harris, Mark; Griffin, Stephen


    Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.

  7. Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay. (United States)

    Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan


    This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

  8. Detection of Cronobacter sakazakii in powdered infant formula using an immunoliposome-based immunomagnetic concentration and separation assay (United States)

    Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Jung Hyun; Cho, Hyunjeong; Lee, Eun Ju; Kim, Myunghee


    This study aimed to optimize the applicability of an immunoliposome-based immunomagnetic concentration and separation assay to facilitate rapid detection of Cronobacter sakazakii in powdered infant formula (PIF). To determine the detection limit, specificity, and pre-enrichment incubation time (0, 4, 6, and 8 h), assay tests were performed with different cell numbers of C. sakazakii (2 × 100 and 2 × 101 CFU/ml) inoculated in 10 g of PIF. The assay was able to detect as few as 2 cells of C. sakazakii/10 g of PIF sample after 6 h of pre-enrichment incubation with an assay time of 2 h 30 min. The assay was assessed for cross-reactivity with other bacterial strains and exhibited strong specificity to C. sakazakii. Moreover, the assay method was applied to the detection of C. sakazakii in PIF without pre-enrichment steps, and the results were compared with INC-ELISA and RT-PCR. The developed method was able to detect C. sakazakii in spiked PIF without pre-enrichment, whereas INC-ELISA failed to detect C. sakazakii. In addition, when compared with the results obtained with RT-PCR, our developed assay required lesser detection time. The developed assay was also not susceptible to any effect of the food matrix or background contaminant microflora. PMID:27721500

  9. Investigation of parameters that affect the success rate of microarray-based allele-specific hybridization assays.

    Directory of Open Access Journals (Sweden)

    Lena Poulsen

    Full Text Available BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%. Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios.

  10. Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay. (United States)

    Rücker, Hannelore; Amslinger, Sabine


    The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

  11. Flow cytometry-based assay to evaluate human serum MUC1-Tn antibodies

    DEFF Research Database (Denmark)

    Van Elssen, Catharina H M J; Clausen, Henrik; Germeraad, Wilfred T V;


    to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary......-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique...... in vaccination studies as well as for functional assays....

  12. The regulation of circulating ghrelin - with recent updates from cell-based assays. (United States)

    Iwakura, Hiroshi; Kangawa, Kenji; Nakao, Kazuwa


    Ghrelin is a stomach-derived orexigenic hormone with a wide range of physiological functions. Elucidation of the regulation of the circulating ghrelin level would lead to a better understanding of appetite control in body energy homeostasis. Earlier studies revealed that circulating ghrelin levels are under the control of both acute and chronic energy status: at the acute scale, ghrelin levels are increased by fasting and decreased by feeding, whereas at the chronic scale, they are high in obese subjects and low in lean subjects. Subsequent studies revealed that nutrients, hormones, or neural activities can influence circulating ghrelin levels in vivo. Recently developed in vitro assay systems for ghrelin secretion can assess whether and how individual factors affect ghrelin secretion from cells. In this review, on the basis of numerous human, animal, and cell-based studies, we summarize current knowledge on the regulation of circulating ghrelin levels and enumerate the factors that influence ghrelin levels.

  13. A carbon nanotubes based ATP apta-sensing platform and its application in cellular assay. (United States)

    Zhang, Libing; Wei, Hui; Li, Jing; Li, Tao; Li, Dan; Li, Yunhui; Wang, Erkang


    In this paper, a sensitive and selective fluorescent aptasensor for adenosine triphosphate (ATP) detection is constructed, based on the noncovalent assembly of dye-labeled ATP aptamer and single-walled carbon nanotubes (SWNTs). In the absence of ATP, the dye tethered to the ATP aptamer is close to SWNTs, which can effectively quench fluorescence of the dye. Upon adding ATP, the fluorophore keeps away from the quencher, since ATP specifically binds to the aptamer and competes with carbon nanotubes, resulting in an increase in the fluorescence intensity. This enables ATP to be detected down to 4.5nM. To the best of our knowledge, this is the most sensitive fluorescent ATP aptasensor. In addition, prominent fluorescence signals were obtained in cellular ATP assays, thus the aptasensor could be used to detect ATP in real samples.

  14. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo


    Full Text Available Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  15. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes (United States)

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano


    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  16. Development of a Luminex-Based DIVA Assay for Serological Detection of African Horse Sickness Virus in Horses. (United States)

    Sánchez-Matamoros, A; Nieto-Pelegrín, E; Beck, C; Rivera-Arroyo, B; Lecollinet, S; Sailleau, C; Zientara, S; Sánchez-Vizcaíno, J M


    African horse sickness (AHS) is considered a fatal re-emergent vector-borne disease of horses. In the absence of any effective treatment for AHS, vaccination remains the most effective form of disease control. The new generation of vaccines, such as one based on purified, inactivated AHS virus (AHSV, serotype 4), which does not induce antibodies against non-structural protein 3 (NS3), enables the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA assays). As detecting AHS in AHSV-free countries may lead to restrictions on international animal movements and thereby cause significant economic damage, these DIVA assays are crucial for reducing movement restrictions. In this article, we describe a Luminex-based multiplex assay for DIVA diagnosis of AHS, and we validate it in a duplex format to detect antibodies against structural protein 7 (VP7) and NS3 in serum samples from horses vaccinated with inactivated AHSV4 vaccine or infected with a live virus of the same serotype. Results of the Luminex-based assay for detecting anti-NS3 antibodies showed good positive correlation with results from an in-house enzyme-linked immunosorbent assay (ELISA). Thus, the Luminex-based technique described here may allow multiplex DIVA antibody detection in a single sample in less than 2 h, and it may prove adaptable for the development of robust, multiplex serological assays.

  17. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences


    Ågren, Joakim; Raditijo A Hamidjaja; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; van Rotterdam, Bart; Derzelle, Sylviane


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. ...

  18. Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples. (United States)

    Maher, M; Glennon, M; Martinazzo, G; Turchetti, E; Marcolini, S; Smith, T; Dawson, M T


    We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay. PMID:8862607

  19. Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Robert G Rutledge

    Full Text Available BACKGROUND: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of +/-25% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4-8 determinations from each amplification reaction. FINDINGS: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustrated by varying reaction volume and amplification efficiency, which also demonstrates how LRE can be used to model PCR. Examining the dynamic range of LRE further demonstrates that quantitative accuracy can be maintained down to a single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution. Essential to the universality of optical calibration, the fluorescence intensity generated by SYBR Green I (FU/bp is shown to be independent of GC content and amplicon size, further verifying that absolute scale can be established using a single quantitative standard. Two high-performance lambda amplicons are also introduced that in addition to producing highly precise optical calibrations, can be used as benchmarks for performance testing. The utility of limiting dilution assay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performance in terms of absolute accuracy. CONCLUSIONS: Founded on the ability to exploit lambda gDNA as a universal quantitative standard, LRE provides the ability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilities of LRE, this kinetic-based approach has the

  20. Increased knowledge of Francisella genus diversity highlights the benefits of optimised DNA-based assays

    Directory of Open Access Journals (Sweden)

    Ahlinder Jon


    Full Text Available Abstract Background Recent advances in sequencing technologies offer promising tools for generating large numbers of genomes, larger typing databases and improved mapping of environmental bacterial diversity. However, DNA-based methods for the detection of Francisella were developed with limited knowledge about genetic diversity. This, together with the high sequence identity between several Francisella species, means there is a high risk of false identification and detection of the highly virulent pathogen Francisella tularensis. Moreover, phylogenetic reconstructions using single or limited numbers of marker sequences often result in incorrect tree topologies and inferred evolutionary distances. The recent growth in publicly accessible whole-genome sequences now allows evaluation of published genetic markers to determine optimal combinations of markers that minimise both time and laboratory costs. Results In the present study, we evaluated 38 previously published DNA markers and the corresponding PCR primers against 42 genomes representing the currently known diversity of the genus Francisella. The results highlight that PCR assays for Francisella tularensis are often complicated by low specificity, resulting in a high probability of false positives. A method to select a set of one to seven markers for obtaining optimal phylogenetic resolution or diagnostic accuracy is presented. Conclusions Current multiple-locus sequence-typing systems and detection assays of Francisella, could be improved by redesigning some of the primers and reselecting typing markers. The use of only a few optimally selected sequence-typing markers allows construction of phylogenetic topologies with almost the same accuracy as topologies based on whole-genome sequences.

  1. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU


    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  2. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Institute of Scientific and Technical Information of China (English)

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU


    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  3. A digital microfluidic method for multiplexed cell-based apoptosis assays. (United States)

    Bogojevic, Dario; Chamberlain, M Dean; Barbulovic-Nad, Irena; Wheeler, Aaron R


    Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.

  4. Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation. (United States)

    Mota, Caroline M; Ferreira, Marcela D; Costa, Lourenço F; Barros, Patrício S C; Silva, Murilo V; Santiago, Fernanda M; Mineo, José R; Mineo, Tiago W P


    Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.

  5. Estrogen-related receptor ERRα regulation of human hydroxysteroid sulfotransferase (SULT2A1) gene expression in human Caco-2 cells. (United States)

    Huang, Chaoqun; Zhou, Tianyan; Chen, Yue; Sun, Teng; Zhang, Shunfen; Chen, Guangping


    Human hydroxysteroid sulfotransferase, SULT2A1, is important for xenobiotic detoxification and the maintenance of hydroxysteroid homeostasis. Our published report suggested that estrogen-related receptor ERRα downregulates SULT2A1 in Hep G2 cells. The results shown in this study suggest that ERRα upregulates SULT2A1 transcription in Caco-2 cells. The deletion analysis suggested that SULT2A1 promoter region between -65 and -44 is important for this upregulation. Our further investigation suggested that ERRα binding element, ERRE51, mediates ERRα activation of SULT2A1 promoter transcription in Caco-2 cells. The interaction of ERRE51 with ERRα was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Results also suggest that the difference of constitutive androstane receptor transcription levels in Hep G2 and Caco-2 cells at least partially contribute to the cell type dependent ERRα modulation of SULT2A1 promoter transcription. ERRα regulates human SULT2A1 transcription by competing with other nuclear receptors binding to the DNA-promoter region.

  6. MicroRNA-191 targets N-deacetylase/N-sulfotransferase 1 and promotes cell growth in human gastric carcinoma cell line MGC803

    Institute of Scientific and Technical Information of China (English)

    Xuejun Shi; Shicang Su; Jian Long; Bing Mei; Yajun Chen


    As a family of post-transcriptional regulator of gene expression,the microRNAs (miRNAs) control a wide array of biological processes including cell differentiation,proliferation and apoptosis,and the dysregulation of miRNAs is a hallmark of cancer.Here,we found that the microRNA-191 (miR-191) was at a high-expression level in human gastric adenocarcinoma cell line MGC803 and human gastric cancer tissues.The results of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays showed that miR-191 could promote cell growth and suppress apoptosis of MGC803 cells.The N-deacetylase/N-sulfotransferase 1 (NDST1) was confirmed to be a direct target gene of miR-191 by enhanced green fluorescent protein reporter experiment.The mRNA and protein levels of NDST1 were inversely correlated with miR-191 in MGC803 cells,suggesting the negative regulation of NDST1 by miR-191.Furthermore,NDST1 played an inhibitory role and could suppress MGC803 cell proliferation.Our findings suggested that miR-191 could act as an oncogene in MGC803 cells,and the cellular function was partially due to its negative regulation of NDST1.

  7. Transcriptional Regulation of Cytosolic Sulfotransferase 1C2 by Intermediates of the Cholesterol Biosynthetic Pathway in Primary Cultured Rat Hepatocytes. (United States)

    Rondini, Elizabeth A; Pant, Asmita; Kocarek, Thomas A


    Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 μM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 μM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1β. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.

  8. Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

    Directory of Open Access Journals (Sweden)

    Haifeng Geng

    Full Text Available Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs, inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA activity found in patients with metachromatic leukodystrophy (MLD, a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS, detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an

  9. Cell-based galactosemia diagnosis system based on a galactose assay using a bioluminescent Escherichia coli array. (United States)

    Woo, Min-Ah; Kim, Moon Il; Cho, Daeyeon; Park, Hyun Gyu


    A new cell-based galactose assay system, which is comprised of two bioluminescent Escherichia coli strains immobilized within an agarose gel arrayed on a well plate, has been developed. For this purpose, a galT knockout strain [galT(-) cell] of E. coli was genetically constructed so that cell growth is not promoted by galactose but rather by glucose present in a sample. Another E. coli W strain (normal cell), which grows normally in the presence of either glucose or galactose, was employed. A luminescent reporter gene, which produces luminescence as cells grow, was inserted into both of the E. coli strains, so that cell growth could be monitored in a facile manner. The two strains were separately grown for 4 h on gel arrays to which test samples were individually supplied. The relative luminescence unit (RLU) values caused by cell growth were determined for each array, one of which is resulted by glucose only and the other of which is resulted by both glucose and galactose present in the sample. By employing this protocol, galactose concentrations present in the test sample are reflected in the differences between the RLU values for each array. The practical utility of the new assay system was demonstrated by its use in determining galactose levels in clinical blood spot specimens coming from newborn babies. Because it can be employed to diagnosis of galactosemia in newborn babies in a more rapid, convenient, and cost-effective manner, this cell-based solid-phase galactose assay system should become a powerful alternative to conventional methods, which require labor-intensive and time-consuming procedures and/or complicated and expensive equipment.

  10. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay. (United States)

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung


    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm(2). A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times.

  11. Liquid crystal based sensors monitoring lipase activity: a new rapid and sensitive method for cytotoxicity assays. (United States)

    Hussain, Zakir; Zafiu, Christian; Küpcü, Seta; Pivetta, Lucineia; Hollfelder, Nadine; Masutani, Akira; Kilickiran, Pinar; Sinner, Eva-Kathrin


    In this work we present liquid crystal (LC) based sensor devices to monitor cell viability. The sensing layer is composed by the LC and a planar monolayer of phospholipids. In the presence of minute traces of phospholipases, which hydrolyze enzymatically phospholipids, the LC-lipid interface is disintegrated. This event causes a change in orientation of the LC, which was followed in a polarized microscope. The lipase activity can be used to measure the cell viability, since members of this enzyme family are released by cells, as they undergo necrosis. The described sensor was used to monitor the presence of the lipases released from three different cell lines, which were either exposed to highly cytotoxic model compounds (sodium azide and paracetamol) or subjected to freeze-thaw cycles to induce cell death by a non-chemical based inducer for apoptosis, such as temperature. Finally, the comparison of lipase activity detected by a state-of-the-art fluorescence assay to the LC based system resulted in the superiority of the LC system concerning incubation time and sensitivity.

  12. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.


    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  13. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources. (United States)

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar


    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

  14. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

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    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  15. CCL18 in a multiplex urine-based assay for the detection of bladder cancer.

    Directory of Open Access Journals (Sweden)

    Virginia Urquidi

    Full Text Available The early detection of bladder cancer (BCa is pivotal for successful patient treatment and management. Through genomic and proteomic studies, we have identified a number of bladder cancer-associated biomarkers that have potential clinical utility. In a case-control study, we examined voided urines from 127 subjects: 64 tumor-bearing subjects and 63 controls. The urine concentrations of the following proteins were assessed by enzyme-linked immunosorbent assay (ELISA; C-C motif chemokine 18 (CCL18, Plasminogen Activator Inhibitor 1 (PAI-1 and CD44. Data were compared to a commercial ELISA-based BCa detection assay (BTA-Trak© and voided urinary cytology. We used analysis of the area under the curve of receiver operating characteristic curves to compare the ability of CCL18, PAI-1, CD44, and BTA to detect BCa in voided urine samples. Urinary concentrations of CCL18, PAI-1, and BTA were significantly elevated in subjects with BCa. CCL18 was the most accurate biomarker (AUC; 0.919; 95% confidence interval [CI], 0.8704-0.9674. Multivariate regression analysis highlighted CCL18 (OR; 18.31; 95% CI, 4.95-67.70, p<0.0001 and BTA (OR; 6.43; 95% CI, 1.86-22.21, p = 0.0033 as independent predictors of BCa in voided urine samples. The combination of CCL18, PAI-1 and CD44 improved the area under the curve to 0.938. Preliminary results indicate that CCL18 was a highly accurate biomarker for BCa detection in this cohort. Monitoring CCL18 in voided urine samples has the potential to improve non-invasive tests for BCa diagnosis. Furthermore using the combination of CCL18, PAI-1 and CD44 may make the model more robust to errors to detect BCa over the individual biomarkers or BTA.

  16. Prediction of Non-Genotoxic Carcinogenicity Based on Genetic Profiles of Short Term Exposure Assays (United States)

    Pérez, Luis Orlando; González-José, Rolando; García, Pilar Peral


    Non-genotoxic carcinogens are substances that induce tumorigenesis by non-mutagenic mechanisms and long term rodent bioassays are required to identify them. Recent studies have shown that transcription profiling can be applied to develop early identifiers for long term phenotypes. In this study, we used rat liver expression profiles from the NTP (National Toxicology Program, Research Triangle Park, USA) DrugMatrix Database to construct a gene classifier that can distinguish between non-genotoxic carcinogens and other chemicals. The model was based on short term exposure assays (3 days) and the training was limited to oxidative stressors, peroxisome proliferators and hormone modulators. Validation of the predictor was performed on independent toxicogenomic data (TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System, Osaka, Japan). To build our model we performed Random Forests together with a recursive elimination algorithm (VarSelRF). Gene set enrichment analysis was employed for functional interpretation. A total of 770 microarrays comprising 96 different compounds were analyzed and a predictor of 54 genes was built. Prediction accuracy was 0.85 in the training set, 0.87 in the test set and increased with increasing concentration in the validation set: 0.6 at low dose, 0.7 at medium doses and 0.81 at high doses. Pathway analysis revealed gene prominence of cellular respiration, energy production and lipoprotein metabolism. The biggest target of toxicogenomics is accurately predict the toxicity of unknown drugs. In this analysis, we presented a classifier that can predict non-genotoxic carcinogenicity by using short term exposure assays. In this approach, dose level is critical when evaluating chemicals at early time points. PMID:27818731

  17. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers. (United States)

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick


    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  18. Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay. (United States)

    Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro


    Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP ). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

  19. Regulation of Sulfotransferase and UDP-Glucuronosyltransferase Gene Expression by the PPARs

    Directory of Open Access Journals (Sweden)

    Melissa Runge-Morris


    Full Text Available During phase II metabolism, a substrate is rendered more hydrophilic through the covalent attachment of an endogenous molecule. The cytosolic sulfotransferase (SULT and UDP-glucuronosyltransferase (UGT families of enzymes account for the majority of phase II metabolism in humans and animals. In general, phase II metabolism is considered to be a detoxication process, as sulfate and glucuronide conjugates are more amenable to excretion and elimination than are the parent substrates. However, certain products of phase II metabolism (e.g., unstable sulfate conjugates are genotoxic. Members of the nuclear receptor superfamily are particularly important regulators of SULT and UGT gene transcription. In metabolically active tissues, increasing evidence supports a major role for lipid-sensing transcription factors, such as peroxisome proliferator-activated receptors (PPARs, in the regulation of rodent and human SULT and UGT gene expression. This review summarizes current information regarding the regulation of these two major classes of phase II metabolizing enzyme by PPARs.

  20. Sulfation of afimoxifene, endoxifen, raloxifene, and fulvestrant by the human cytosolic sulfotransferases (SULTs): A systematic analysis. (United States)

    Hui, Ying; Luo, Lijun; Zhang, Lingtian; Kurogi, Katsuhisa; Zhou, Chunyang; Sakakibara, Yoichi; Suiko, Masahito; Liu, Ming-Cheh


    Previous studies demonstrated that sulfate conjugation is involved in the metabolism of three commonly used breast cancer drugs, tamoxifen, raloxifene and fulvestrant. The current study was designed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating raloxifene, fulvestrant, and two active metabolites of tamoxifen, afimoxifene and endoxifen. A systematic analysis using 13 known human SULTs revealed SULT1A1 and SULT1C4 as the major SULTs responsible for the sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant. Kinetic parameters of these two human SULTs in catalyzing the sulfation of these drug compounds were determined. Sulfation of afimoxifene, endoxifen, raloxifene and fulvestrant under metabolic conditions was examined using HepG2 human hepatoma cells and MCF-7 breast cancer cells. Moreover, human intestine, kidney, liver, and lung cytosols were examined to verify the presence of afimoxifene/endoxifen/raloxifene/fulvestrant-sulfating activity.

  1. Thermolabile phenol sulfotransferase gene (STM): Localization to human chromosome 16p11.2

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    Aksoy, I.A.; Her, C.; Weinshilboum, M. [Mayo Medical School, Rochester, MN (United States)] [and others


    Thermolabile (TL) phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic monoamine neurotransmitters such as dopamine and serotonin. We recently cloned a cDNA for human liver TL PST and expressed it in COS-1 cells. We now report the chromosomal localization of the human TL PST gene (STM) as well as its partial sequence. DNA from NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panels 1 and 2 was screened by use of the PCR, and the STM gene was mapped to chromosome 16. Regional localization to 16p11.2 was performed by PCR analysis of a high-resolution mouse/human somatic cell hybrid panel that contained defined portions of human chromosome 16. 15 refs., 2 figs.

  2. Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua. (United States)

    Chikeka, Ijeuru; Matute, Armando J; Dumler, J Stephen; Woods, Christopher W; Mayorga, Orlando; Reller, Megan E


    Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.

  3. Identification and characterization of a novel kaempferol sulfotransferase from Arabidopsis thaliana. (United States)

    Hashiguchi, Takuyu; Sakakibara, Yoichi; Hara, Yosuke; Shimohira, Takehiko; Kurogi, Katsuhisa; Akashi, Ryo; Liu, Ming-Cheh; Suiko, Masahito


    In plants, flavonoids have been shown to be subjected to conjugation modifications such as glycosylation, methylation, and sulfation. Among these modifications, sulfation is known as an important pathway in the regulation of the levels of endogenous compounds such as steroids. Although a large variety of flavonoid sulfates also exist in plants, the detailed biochemical characterization of Arabidopsis thaliana sulfotransferases (AtSULTs) remains to be fully clarified. We report here that uncharacterized AtSULT202E1 (AGI code: At2g03770), a SULT202E subfamily member, shows the sulfating activity toward flavonoids. The general characteristics of the enzyme were studied on the optimum temperature and pH, the effect of divalent cations, and the thermal stability with kaempferol as substrate. A comparative analysis of the sulfation of flavonoids by AtSULT202E1, AtSULT202B1 and AtSULT202A1 revealed that three AtSULTs have differential substrate specificities. Surprisingly, 3-hydroxyflavone was sulfated only by AtSULT202A1 while 7-hydroxyflavone was highly sulfated by AtSULT202E1 and AtSULT202B1. These results indicate that flavonols might be sulfated in a position specific manner. In conclusion, our studies indicate that a novel AtSULT202E1 has the sulfating activity toward flavonoids together with AtSULT202B1 and AtSULT202A1. The existence of three flavonoid sulfotransferases in A. thaliana suggests that sulfation of flavonoids have an important role in regulation of their functions.

  4. Small interfering RNA therapy against carbohydrate sulfotransferase 15 inhibits cardiac remodeling in rats with dilated cardiomyopathy. (United States)

    Watanabe, Kenichi; Arumugam, Somasundaram; Sreedhar, Remya; Thandavarayan, Rajarajan A; Nakamura, Takashi; Nakamura, Masahiko; Harima, Meilei; Yoneyama, Hiroyuki; Suzuki, Kenji


    Carbohydrate sulfotransferase 15 (CHST15) is a sulfotransferase responsible for biosynthesis of chondroitin sulfate E (CS-E), which plays important roles in numerous biological events such as biosynthesis of proinflammatory cytokines. However, the effects of CHST15 siRNA in rats with chronic heart failure (CHF) after experimental autoimmune myocarditis (EAM) have not yet been investigated. CHF was elicited in Lewis rats by immunization with cardiac myosin, and after immunization, the rats were divided into two groups and treated with either CHST15 siRNA (2μg/week) or vehicle. Age matched normal rats without immunizations were also included in this study. After 7weeks of treatment, we investigated the effects of CHST15 siRNA on cardiac function, proinflammatory cytokines, and cardiac remodeling in EAM rats. Myocardial functional parameters measured by hemodynamic and echocardiographic studies were significantly improved by CHST15 siRNA treatment in rats with CHF compared with that of vehicle-treated CHF rats. CHST15 siRNA significantly reduced cardiac fibrosis, and hypertrophy and its marker molecules (left ventricular (LV) mRNA expressions of transforming growth factor beta1, collagens I and III, and atrial natriuretic peptide) compared with vehicle-treated CHF rats. CHF-induced increased myocardial mRNA expressions of proinflammatory cytokines [interleukin (IL)-6, IL-1β], monocyte chemoattractant protein-1, and matrix metalloproteinases (MMP-2 and -9), and CHST15 were also suppressed by the treatment with CHST15 siRNA. Western blotting study has confirmed the results obtained from mRNA analysis as CHST15 siRNA treated rats expressed reduced levels of inflammatory and cardiac remodeling marker proteins. Our results demonstrate for the first time, that CHST15 siRNA treatment significantly improved LV function and ameliorated the progression of cardiac remodeling in rats with CHF after EAM.

  5. Protective role of hydroxysteroid sulfotransferase in lithocholic acid—in—duced liver toxicity

    Institute of Scientific and Technical Information of China (English)

    YamaY; KitaH


    Supplement of 1% lithocholic acid (LCA) in the diet for 5-9d resulted in elevated levels of the marker for liver damage AST and ALP activities in both FXR-null and wild-type female mice.The levels were clearly higher in wild-type mice than in FXR-null mice,in spite of the diminished expression of a bile salt export pump(Bsep) in the latter.Consistent with liver toxicity marker activities,serum and liver levels of bile acide,particularly LCA and tauro LCA,were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement.Marked increases in hepatic sulfating activity for LCA(5.5-fold) and hydroxysteroid sulfotransferase St2a(5.8-fold) were detected in liver of FXR-null mice.Liver St2a content was inversely correlated with levels of ALP.In contrast,microsomal LCA 6-hydroxylation was not increased and in fact lower in FXR-null mice compared in wild-type mice.Clear decreases in mRNA encoding Ntcp,Oatpl and Lst-1 transporters function in bild acid import were detected in LCA fed mice.These transporter levels are higher in FXR-null mice than in wild-type mice after 1% LCA supplement.No obvious changes were detected in the Mrp2,Mrp3 and Mrp4 mRNAs.These results indicate that hydroxysteroid sulfotransferase is reuired for protection against LCA-induced liver damage.

  6. Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation. (United States)

    Choulier, Laurence; Nominé, Yves; Zeder-Lutz, Gabrielle; Charbonnier, Sebastian; Didier, Bruno; Jung, Marie-Louise; Altschuh, Danièle


    We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 μM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 μM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 μM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 μM range. We propose that a medium (μM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

  7. A simple dot-blot-Sirius red-based assay for collagen quantification. (United States)

    Rodríguez-Rodríguez, Pilar; Arribas, Silvia M; de Pablo, Angel Luis López; González, M Carmen; Abderrahim, Fatima; Condezo-Hoyos, Luis


    The assessment of collagen content in tissues is important in biomedical research, since this protein is altered in numerous diseases. Hydroxyproline and Sirius red based assays are the most common methods for collagen quantification. However, these procedures have some pitfalls, such as the requirement of oxygen-free medium or expensive equipment and large sample size or being unsuitable for hydrolyzed collagen, respectively. Our objective was to develop a specific, versatile, and user-friendly quantitative method applicable to small tissue samples and extracts obtained from elastin purification, therefore, suitable for simultaneous quantification of elastin. This method is based on the binding of Sirius red to collagen present in a sample immobilized on a PVDF membrane, as in the dot-blot technique, and quantified by a scanner and image analysis software. Sample loading, Sirius red concentration, temperature and incubation time, type of standard substance, albumin interference, and quantification time are optimized. The method enabled the quantification of (1) intact collagen in several rat tissue homogenates, including small resistance-sized arteries, (2) partially hydrolyzed collagen obtained from NaOH extracts, compatible with elastin purification, and (3) for the detection of differences in collagen content between hypertensive and normotensive rats. We conclude that the developed technique can be widely used since it is versatile (quantifies intact and hydrolyzed collagen), requires small sample volumes, is user-friendly (low-cost, easy to use, minimum toxic materials, and reduced time of test), and is specific (minimal interference with serum albumin).

  8. Analytical assays based on chromogenic and fluorogenic chemosensors for the detection of cyanide

    Directory of Open Access Journals (Sweden)

    Vanderléia Gava Marini


    Full Text Available Cyanide (CN– is an anion well–known for its toxicity, being a chemical agent often related to cases of homicide and suicide. Despite being responsible for the toxicity of many animals and plants, it is used in several industrial activities, with innumerous implications in terms of the environment. Due to its high toxicity, the maximum level of CN– concentration allowed by the World Health Organization in potable water is 1.7 µmol/L. This low concentration limit requires methods of visual detection and quantitative determination which are ever more sensitive, simple, reliable, and economical. Advancements in the field of chromogenic and fluorogenic chemosensors for anionic analytes have led to the development of several methodologies for the detection of CN–. Therefore, this review aims to present the main strategies that have been used in the study of quantitative and naked–eye detection of CN– by means of chromogenic and fluorogenic chemosensors. Aspects related to CN–, such as its reactivity, toxicity, applications, and implications in different domains of knowledge, are presented. Recent work involving the development of chemosensors for CN– based on acid–base reactions, chemodosimeters, chromoreactands, and competition assays is also described. In addition, recent studies that make use of nanotechnology to develop strategies for the detection of CN– are also discussed, as well as the prospects envisioned in this field.

  9. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening. (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun


    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions.

  10. A high-throughput pipeline for designing microarray-based pathogen diagnostic assays

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    Reifman Jaques


    Full Text Available Abstract Background We present a methodology for high-throughput design of oligonucleotide fingerprints for microarray-based pathogen diagnostic assays. The oligonucleotide fingerprints, or DNA microarray probes, are designed for identifying target organisms in environmental or clinical samples. The design process is implemented in a high-performance computing software pipeline that incorporates major algorithmic improvements over a previous version to both reduce computation time and improve specificity assessment. Results The algorithmic improvements result in significant reduction in runtimes, with the updated pipeline being nearly up to five-times faster than the previous version. The improvements in specificity assessment, based on multiple specificity criteria, result in robust and consistent evaluation of cross-hybridization with nontarget sequences. In addition, the multiple criteria provide finer control on the number of resulting fingerprints, which helps in obtaining a larger number of fingerprints with high specificity. Simulation tests for Francisella tularensis and Yersinia pestis, using a well-established hybridization model to estimate cross-hybridization with nontarget sequences, show that the improved specificity criteria yield a larger number of fingerprints as compared to using a single specificity criterion. Conclusion The faster runtimes, achieved as the result of algorithmic improvements, are critical for extending the pipeline to process multiple target genomes. The larger numbers of identified fingerprints, obtained by considering broader specificity criteria, are essential for designing probes for hard-to-distinguish target sequences.

  11. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays. (United States)

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark


    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

  12. Microtiter plate based colorimetric assay for characterization of dehalogenation activity of GAC/Fe0 composite

    DEFF Research Database (Denmark)

    Hwang, Yuhoon; Salatas, Apostolos; Mines, Paul D.


    of nZVI and its composite with granular activated carbon (GAC). The assay focused on analysis of reaction products rather than its mother compounds, which gives more accurate quantification of reductive activity. The colorimetric assays were developed to quantify three reaction products, ammonia...

  13. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat


    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  14. Membrane-based assay for iodide ions based on anti-leaching of gold nanoparticles. (United States)

    Shen, Yu-Wei; Hsu, Pang-Hung; Unnikrishnan, Binesh; Li, Yu-Jia; Huang, Chih-Ching


    We report a label-free colorimetric strategy for the highly selective and sensitive detection of iodide (I(-)) ions in human urine sample, seawater and edible salt. A poly(N-vinyl-2-pyrrolidone)-stabilized Au nanoparticle (34.2-nm) was prepared to detect I(-) ions using silver (Ag(+)) and cyanide (CN(-)) ions as leaching agents in a glycine-NaOH (pH 9.0) solution. For the visual detection of the I(-) ions by naked eye, and for long time stability of the probe, Au nanoparticles (NPs) decorated mixed cellulose ester membrane (MCEM) was prepared (Au NPs/MCEM). The Au NPs-based probe (CN(-)/Ag(+)-Au NPs/MCEM) operates on the principle that Ag(+) ions form a monolyar silver atoms/ions by aurophilic/argentophilic interactions on the Au NPs and it accelerates the leaching rate of Au atoms in presence of CN(-) ions. However, when I(-) is introduced into this system, it inhibits the leaching of Au atoms because of the strong interactions between Ag/Au ions and I(-) ions. Inductively coupled plasma mass spectrometry, surface-assisted laser desorption/ionization time-of-flight mass spectrometry were used to characterize the surface properties of the Au NPs in the presence of Ag(+) and I(-). Under optimal solution conditions, the CN(-)/Ag(+)-Au NPs/MCEM probe enabled the detection of I(-) by the naked eye at nanomolar concentrations with high selectivity (at least 1000-fold over other anions). In addition, this cost-effective probe allowed the determination of I(-) ions in complex samples, such as urine, seawater, and edible salt samples.

  15. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

    DEFF Research Database (Denmark)

    Ørsted, Michael; Roslev, Peter


    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl o...... that the fluorescence based hydrolytic enzyme activity assay (FLEA assay) can be used as an index of D. magna stress. Combining enzyme activity with fluorescence measurements may be applied as a simple and quantitative supplement for toxicity testing with D. magna....

  16. A dendritic cell-based assay for measuring memory T cells specific to dengue envelope proteins in human peripheral blood. (United States)

    Sun, Peifang; Beckett, Charmagne; Danko, Janine; Burgess, Timothy; Liang, Zhaodong; Kochel, Tadeusz; Porter, Kevin


    Dengue envelope (E) protein is a dominant immune inducer and E protein-based vaccines elicited partial to complete protection in non-human primates. To study the immunogenicity of these vaccines in humans, an enzyme linked immunospot (ELISPOT) assay for measuring interferon gamma (IFN-γ) production was developed. Cells from two subject groups, based on dengue-exposure, were selected for assay development. The unique feature of the IFN-γ ELISPOT assay is the utilization of dendritic cells pulsed with E proteins as antigen presenting cells. IFN-γ production, ranging from 53-513 spot forming units per million peripheral blood mononuclear cells (PBMCs), was observed in dengue-exposed subjects as compared to 0-45 IFN-γ spot forming units in dengue-unexposed subjects. Further, both CD4(+) and CD8(+) T cells, and cells bearing CD45RO memory marker, were the major sources of IFN-γ production. The assay allowed quantification of E-specific IFN-γ-secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Results suggested that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.

  17. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum. (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai


    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

  18. Genetic linkage mapping of the dehydroepiandrosterone sulfotransferase (STD) gene on the chromosome 19q13.3 region

    Energy Technology Data Exchange (ETDEWEB)

    Durocher, F.; Morissette, J.; Dufort, I.; Simard, J.; Luu-The, V. [Laval Univ. Quebec (Canada)


    In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3{beta}-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Genethon and NIH/CEPH. The closest linkage was observed with D19S412 (Z{sub max} = 9.23; {theta}{sub max} 0.038) and HRC (Z{sub max} =5.95; {theta}{sub max}0.036), located on the 19q13.3 region. A framework map including six Genethon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter-D19S414-D19S224-D19S420-D19S217-(APOC2-D19S412)-(STD-HRC)-KLK-D19S22-D19S180-PRKCG-D19S418-tel. 24 refs., 2 figs.

  19. Altered UDP-Glucuronosyltransferase and Sulfotransferase Expression and Function during Progressive Stages of Human Nonalcoholic Fatty Liver Disease


    Hardwick, Rhiannon N.; Ferreira, Daniel W.; More, Vijay R.; Lake, April D.; Lu, Zhenqiang; Manautou, Jose E.; Slitt, Angela L.; Cherrington, Nathan J


    The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic...

  20. Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics (United States)

    Joubert, Marisa K.; Deshpande, Meghana; Yang, Jane; Reynolds, Helen; Bryson, Christine; Fogg, Mark; Baker, Matthew P.; Herskovitz, Jonathan; Goletz, Theresa J.; Zhou, Lei; Moxness, Michael; Flynn, Gregory C.; Narhi, Linda O.; Jawa, Vibha


    An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes. However, the response in the assay is a measure of T-cell functional activity and the alignment with clinical immunogenicity depends on several other factors. The assay was sensitive to sequence variants and could differentiate single point mutations of the same biotherapeutic. Nine mAbs that were highly aggregated by stirring induced a higher response in the assay than the original mAbs before stirring stress, in a manner that did not match the relative T-cell response of the original mAbs. In contrast, mAbs that were glycated by different sugars (galactose, glucose, and mannose) showed little to no increase in response in the assay above the response to the original mAbs before glycation treatment. The assay was also used successfully to assess similarity between multiple lots of the same mAb, both from the same manufacturer and from different manufacturers (biosimilars). A strategy for using the IVCIA assay for immunogenicity risk assessment during the entire lifespan development of biopharmaceuticals is proposed. PMID:27494246

  1. Exploring the dark side of MTT viability assay of cells cultured onto electrospun PLGA-based composite nanofibrous scaffolding materials. (United States)

    Qi, Ruiling; Shen, Mingwu; Cao, Xueyan; Guo, Rui; Tian, Xuejiao; Yu, Jianyong; Shi, Xiangyang


    One major method used to evaluate the biocompatibility of porous tissue engineering scaffolding materials is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT cell viability assay is based on the absorbance of the dissolved MTT formazan crystals formed in living cells, which is proportional to the number of viable cells. Due to the strong dye sorption capability of porous scaffolding materials, we propose that the cell viability determined from the MTT assay is likely to give a false negative result. In this study, we aim to explore the effect of the adsorption of MTT formazan on the accuracy of the viability assay of cells cultured onto porous electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers, HNTs (halloysite nanotubes)/PLGA, and CNTs (multiwalled carbon nanotubes)/PLGA composite nanofibrous mats. The morphology of electrospun nanofibers and L929 mouse fibroblasts cultured onto the nanofibrous scaffolds were observed using scanning electron microscopy. The viability of cells proliferated for 3 days was evaluated through the MTT assay. In the meantime, the adsorption of MTT formazan onto the same electrospun nanofibers was evaluated and the standard concentration-absorbance curve was obtained in order to quantify the contribution of the adsorbed MTT formazan during the MTT cell viability assay. We show that the PLGA, and the HNTs- or CNTs-doped PLGA nanofibers display appreciable MTT formazan dye sorption, corresponding to 35.6-50.2% deviation from the real cell viability assay data. The better dye sorption capability of the nanofibers leads to further deviation from the real cell viability. Our study gives a general insight into accurate MTT cytotoxicity assessment of various porous tissue engineering scaffolding materials, and may be applicable to other colorimetric assays for analyzing the biological properties of porous scaffolding materials.

  2. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences. (United States)

    Ågren, Joakim; Hamidjaja, Raditijo A; Hansen, Trine; Ruuls, Robin; Thierry, Simon; Vigre, Håkan; Janse, Ingmar; Sundström, Anders; Segerman, Bo; Koene, Miriam; Löfström, Charlotta; Van Rotterdam, Bart; Derzelle, Sylviane


    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

  3. A superstructure-based electrochemical assay for signal-amplified detection of DNA methyltransferase activity. (United States)

    Zhang, Hui; Yang, Yin; Dong, Huilei; Cai, Chenxin


    DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a superstructure-based electrochemical assay for signal-amplified detection of DNA MTase activity using M.SssI as an example. First, low-density coverage of DNA duplexes on the surface of the gold electrode was achieved by immobilized mercaptohexanol, followed by immobilization of DNA duplexes. The duplex can be cleaved by BstUI endonuclease in the absence of DNA superstructures. However, the cleavage is blocked after the DNA is methylated by M.SssI. The DNA superstructures are formed with the addition of helper DNA. By using an electroactive complex, RuHex, which can bind to DNA double strands, the activity of M.SssI can be quantitatively detected by differential pulse voltammetry. Due to the high site-specific cleavage by BstUI and signal amplification by the DNA superstructure, the biosensor can achieve ultrasensitive detection of DNA MTase activity down to 0.025U/mL. The method can be used for evaluation and screening of the inhibitors of MTase, and thus has potential in the discovery of methylation-related anticancer drugs.

  4. "Molecular beacon"-based fluorescent assay for selective detection of glutathione and cysteine. (United States)

    Xu, Hui; Hepel, Maria


    We report on the development of a fluorescence turn-on "molecular beacon" probe for the detection of glutathione (GSH) and cysteine (Cys). The method is based on a competitive ligation of Hg(2+) ions by GSH/Cys and thymine-thymine (T-T) mismatches in a DNA strand of the self-hybridizing beacon strand. The assay relies on the distance-dependent optical properties of the fluorophore/quencher pair attached to the ends of the molecular beacon DNA strand. In a very selective coordination of Hg(2+) to GSH/Cys, the fluorophore/quencher distance increases concomitantly with the dehybridization and dissociation of the beacon stem T-Hg(2+)-T due to the extraction of Hg(2+) ions. This process results in switching the molecular beacon to the "on" state. The concentration range of the probe is 4-200 nM with the limit of detection (LOD) of 4.1 nM for GSH and 4.2 nM Cys. The probe tested satisfactorily against interference for a range of amino acids including sulfur-containing methionine.

  5. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening. (United States)

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua


    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  6. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay. (United States)

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues


    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  7. Functional screening with a live cell imaging-based random cell migration assay. (United States)

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob


    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  8. A novel flow-based procedure for automation of respirometric assays in soils. (United States)

    Silva, Claudineia R; Oliveira, Eliezer; Zagatto, Elias A G; Henriquez, Camelia


    A flow-based strategy involving a gas-diffusion sampling probe was proposed for evaluating the respiration rate in soils. The amount of CO2 collected after a pre-defined time interval was proportional to the free CO2 released by the soil ecosystem. The 500-mL incubation flasks typically used for soil respirometric assays were adapted and a special cover was designed for connecting a tubular gas diffusion membrane, a fan, and a septum for adding the CO2(g) standards required for calibration. The method relied on the pH-dependent absorbance variations resulting from the CO2 collection. A 1.3mmolL(-1) bromothymol blue solution (pH 7.0) acted as both acceptor and carrier streams. In order to widen the dynamical working range to 0.003-0.2mmol CO2, two analytical curves were obtained, each related to a different time interval for the CO2 collection. Kinetic curves related to CO2 release by the soil samples were straightforwardly attained. Repeatability and detection limit were estimated as 2.0% and 0.001mmol CO2 (n=10), and accuracy was assessed in relation to a recommended titrimetric procedure.

  9. LC-MS and LC-MS/MS studies of incorporation of 34SO3 into glycosaminoglycan chains by sulfotransferases. (United States)

    Shi, Xiaofeng; Shao, Chun; Mao, Yang; Huang, Yu; Wu, Zhengliang L; Zaia, Joseph


    The specificities of glycosaminoglycan (GAG) modification enzymes, particularly sulfotransferases, and the locations and concentrations of these enzymes in the Golgi apparatus give rise to the mature GAG polysaccharides that bind protein ligands. We studied the substrate specificities of sulfotransferases with a stable isotopically labeled donor substrate, 3'-phosphoadenosine-5'-phosphosulfate. The sulfate incorporated by in vitro sulfation using recombinant sulfotransferases was easily distinguished from those previously present on the GAG chains using mass spectrometry. The enrichment of the [M + 2] isotopic peak caused by (34)S incorporation, and the [M + 2]/[M + 1] ratio, provided reliable and sensitive measures of the degree of in vitro sulfation. It was found that both CHST3 and CHST15 have higher activities at the non-reducing end (NRE) units of chondroitin sulfate, particularly those terminating with a GalNAc monosaccharide. In contrast, both NDST1 and HS6ST1 showed lower activities at the NRE of heparan sulfate (HS) chains than at the interior of the chain. Contrary to the traditional view of HS biosynthesis processes, NDST1 also showed activity on O-sulfated GlcNAc residues.

  10. Chondroitin 4-O-sulfotransferases are required for cell adhesion and morphogenesis in the Ciona intestinalis embryo. (United States)

    Nakamura, Jun; Tetsukawa, Akira; Fujiwara, Shigeki


    Chondroitin sulfate (CS) is a carbohydrate component of proteoglycans. Several types of sulfotransferases determine the pattern of CS sulfation, and thus regulate the biological functions of proteoglycans. The protochordate ascidians are the closest relatives of vertebrates, but the functions of their sulfotransferases have not been investigated. Here, we show that two chondroitin 4-O-sulfotransferases (C4STs) play important roles in the embryonic morphogenesis of the ascidian Ciona intestinalis. Ci-C4ST-like1 is predominantly expressed in the epidermis and muscle. Epidermal and muscle cells became spherical upon the injection of a Ci-C4ST-like1-specific morpholino oligo (MO), thus suggesting weakened cell adhesion. Co-injection of a Ci-C4ST-like1-expressing transgene rescued the phenotype, suggesting that the effects of the MO were specific. Ci-C4ST-like3 was expressed in the central nervous system, muscle, and mesenchyme. A specific MO appeared to affect cell adhesion in the epidermis and muscle. Convergent extension movement of notochordal cells was also impaired. Forced expression of Ci-C4ST-like3 restored normal morphogenesis, suggesting that the effects of the MO were specific. The present study suggests that Ci-C4ST-like1 and Ci-C4ST-like3 are required for cell adhesion mainly in the epidermis and muscle.

  11. Graphene and graphene-like two-denominational materials based fluorescence resonance energy transfer (FRET) assays for biological applications. (United States)

    Tian, Feng; Lyu, Jing; Shi, Jingyu; Yang, Mo


    In the past decades, Förster resonance energy transfer (FRET) has been applied in many biological applications to reveal the biological information at the nanoscale. Recently, graphene and graphene-like two-dimensional (2D) nanomaterials started to be used in FRET assays as donors or acceptors including graphene oxide (GO), graphene quantum dot (GQD), graphitic-carbon nitride nanosheets (g-C3N4) and transition metal dichalcogenides (e.g. MoS2, MnO2, and WS2). Due to the remarkable properties such as large surface to volume ratio, tunable energy band, photoluminescence and excellent biocompatibility, these 2D nanomaterials based FRET assays have shown great potential in various biological applications. This review summarizes the recent development of graphene and graphene-like 2D nanomaterials based FRET assays in applications of biosensing, bioimaging, and drug delivery monitoring.

  12. An aptamer based competition assay for protein detection using CNT activated gold-interdigitated capacitor arrays. (United States)

    Qureshi, Anjum; Roci, Irena; Gurbuz, Yasar; Niazi, Javed H


    An aptamer can specifically bind to its target molecule, or hybridize with its complementary strand. A target bound aptamer complex has difficulty to hybridize with its complementary strand. It is possible to determine the concentration of target based on affinity separation system for the protein detection. Here, we exploited this property using C-reactive protein (CRP) specific RNA aptamers as probes that were immobilized by physical adsorption on carbon nanotubes (CNTs) activated gold interdigitated electrodes of capacitors. The selective binding ability of RNA aptamer with its target molecule was determined by change in capacitance after allowing competitive binding with CRP and complementary RNA (cRNA) strands in pure form and co-mixtures (CRP:cRNA=0:1, 1:0, 1:1, 1:2 and 2:1). The sensor showed significant capacitance change with pure forms of CRP/cRNA while responses reduced considerably in presence of CRP:cRNA in co-mixtures (1:1 and 1:2) because of the binding competition. At a critical CRP:cRNA ratio of 2:1, the capacitance response was dramatically lost because of the dissociation of adsorbed aptamers from the sensor surface to bind when excess CRP. Binding assays showed that the immobilized aptamers had strong affinity for cRNA (K(d)=1.98 μM) and CRP molecules (K(d)=2.4 μM) in pure forms, but low affinity for CRP:cRNA ratio of 2:1 (K(d)=8.58 μM). The dynamic detection range for CRP was determined to be 1-8 μM (0.58-4.6 μg/capacitor). The approach described in this study is a sensitive label-free method to detect proteins based on affinity separation of target molecules that can potentially be used for probing molecular interactions.

  13. Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

    Directory of Open Access Journals (Sweden)

    Milla Luis A


    Full Text Available Abstract Background The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1 and novel Hh-regulated genes in zebrafish embryos. Results The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. Conclusion A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in

  14. Flavocytochrome b2-Based Enzymatic Method of L-Lactate Assay in Food Products

    Directory of Open Access Journals (Sweden)

    Oleh Smutok


    Full Text Available L-lactate, a key metabolite of the anaerobic glycolytic pathway, plays an important role as a biomarker in medicine, in the nutritional sector and food quality control. For these reasons, there is a need for very specific, sensitive, and simple analytical methods for the accurate L-lactate measuring. A new highly selective enzymatic method for L-lactate determination based on the use of flavocytochrome b2 (EC; FC b2 isolated from the recombinant strain of the yeast Hansenula polymorpha has been developed. A proposed enzymatic method exploits an enzymatic oxidation of L-lactate to pyruvate coupled with nitrotetrazolium blue (NTZB reduction to a colored product, formazan. The maximal absorption peak of the colored product is near λ=525 nm and the linear range is observed in the interval 0.005–0.14 mM of L-lactate. The main advantages of the proposed method when compared to the LDH-based routine approaches are a higher sensitivity (2.0 μM of L-lactate, simple procedure of analysis, usage of inexpensive, nontoxic reagents, and small amount of the enzyme. Enzymatic oxidation of L-lactate catalyzed by flavocytochrome b2 and coupled with formazan production from nitrotetrazolium blue was shown to be used for L-lactate assay in food samples. A high correlation between results of the proposed method and reference ones proves the possibility to use flavocytochrome b2-catalysed reaction for enzymatic measurement of L-lactate in biotechnology and food chemistry.

  15. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases

    NARCIS (Netherlands)

    van der Linden, Lonneke; Ulferts, Rachel; Nabuurs, Sander B; Kusov, Yuri; Liu, Hong; George, Shyla; Lacroix, Céline; Goris, Nesya; Lefebvre, David; Lanke, Kjerstin H W; De Clercq, Kris; Hilgenfeld, Rolf; Neyts, Johan; van Kuppeveld, Frank J M


    Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases

  16. Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system. (United States)

    Karadeniz, Hakan; Erdem, Arzum; Kuralay, Filiz; Jelen, Frantisek


    An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N=3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.

  17. Template reporter bacteriophage platform and multiple bacterial detection assays based thereon (United States)

    Goodridge, Lawrence (Inventor)


    The invention is a method for the development of assays for the simultaneous detection of multiple bacteria. A bacteria of interest is selected. A host bacteria containing plasmid DNA from a T even bacteriophage that infects the bacteria of interest is infected with T4 reporter bacteriophage. After infection, the progeny bacteriophage are plating onto the bacteria of interest. The invention also includes single-tube, fast and sensitive assays which utilize the novel method.

  18. Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays


    Traver Hart; Alice Zhao; Ankit Garg; Swetha Bolusani; Marcotte, Edward M.


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential ap...

  19. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays


    Iole Macchia; Francesca Urbani; Enrico Proietti


    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometr...

  20. Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator. (United States)

    Chen, Chia-Hung; Sarkar, Aniruddh; Song, Yong-Ak; Miller, Miles A; Kim, Sung Jae; Griffith, Linda G; Lauffenburger, Douglas A; Han, Jongyoon


    We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.

  1. Tuning a 96-Well Microtiter Plate Fluorescence-Based Assay to Identify AGE Inhibitors in Crude Plant Extracts

    Directory of Open Access Journals (Sweden)

    Luc Séro


    Full Text Available Advanced glycation end-products (AGEs are involved in the pathogenesis of numerous diseases. Among them, cellular accumulation of AGEs contributes to vascular complications in diabetes. Besides using drugs to lower blood sugar, a balanced diet and the intake of herbal products potentially limiting AGE formation could be considered beneficial for patients’ health. The current paper presents a simple and cheap high-throughput screening (HTS assay based on AGE fluorescence and suitable for plant extract screening. We have already implemented an HTS assay based on vesperlysines-like fluorescing AGEs quickly (24 h formed from BSA and ribose under physiological conditions. However, interference was noted when fluorescent compounds and/or complex mixtures were tested. To overcome these problems and apply this HTS assay to plant extracts, we developed a technique for systematic quantification of both vesperlysines (λexc 370 nm; λem 440 nm and pentosidine-like (λexc 335 nm; λem 385 nm AGEs. In a batch of medicinal and food plant extracts, hits were selected as soon as fluorescence decreased under a fixed threshold for at least one wavelength. Hits revealed during this study appeared to contain well-known and powerful anti-AGE substances, thus demonstrating the suitability of this assay for screening crude extracts (0.1 mg/mL. Finally, quercetin was found to be a more powerful reference compound than aminoguanidine in such assay.

  2. Antitumour efficacy of Piper tuberculatum and piplartine based on the hollow fiber assay. (United States)

    Bezerra, Daniel P; Ferreira, Paulo Michel P; Machado, Camila Maria L; de Aquino, Nayara C; Silveira, Edilberto R; Chammas, Roger; Pessoa, Claudia


    Piper tuberculatum, popularly known in Brazil as "jaborandi falso" and "pimenta darta", is widely used in folk medicine for the treatment of several diseases. In this study, the in vivo hollow fiber assay was used to investigate the antitumour efficacy of the crude extract and piplartine obtained from P. tuberculatum roots. Human glioblastoma (SF-295) and colon carcinoma (HCT-8) cell lines were used. In vitro cytotoxicity was assayed by the MTT assay. In the hollow fiber assay, nude mice implanted with tumour cells in hollow fibers were treated for four consecutive days via the intraperitoneal route, and tumour cell populations were assessed by the MTT assay. Both the crude extract and piplartine displayed cytotoxicity. In the hollow fiber assay, tumour growth inhibition rates were 24.6-54.8 % for the crude extract and 33.7-62.2 % for piplartine. No signal of toxicity was noticed. In conclusion, the crude extract and piplartine obtained from P. tuberculatum roots displayed in vitro and in vivo anticancer efficacy.

  3. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays (United States)

    Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen


    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

  4. Medical Devices; Immunology and Microbiology Devices; Classification of Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay. Final order. (United States)


    The Food and Drug Administration (FDA) is classifying a gastrointestinal microorganism multiplex nucleic acid-based assay into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

  5. Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016). (United States)

    Berns, Eric J; Cabezas, Maria D; Mrksich, Milan


    On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

  6. Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. (United States)

    Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M


    Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31).

  7. Evaluation of a gp63-PCR based assay as a molecular diagnosis tool in canine leishmaniasis in Tunisia.

    Directory of Open Access Journals (Sweden)

    Souheila Guerbouj

    Full Text Available A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania (L. parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture or serological (IFAT techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18 and control dogs (N = 45 originating from non-endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8-95.4% or serology IFAT technique (47.4%, 95% CI: 23.5-71.3%. However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia.

  8. Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia (United States)

    Guerbouj, Souheila; Djilani, Fattouma; Bettaieb, Jihene; Lambson, Bronwen; Diouani, Mohamed Fethi; Ben Salah, Afif; Ben Ismail, Riadh; Guizani, Ikram


    A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia. PMID:25153833

  9. Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

    Energy Technology Data Exchange (ETDEWEB)

    Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.; Kuhn, Eric; Liu, Tao; Massoni, Sam A.; Thomas, Stefani N.; Townsend, Reid; Zimmerman, Lisa J.; Boja, Emily; Chen, Jing; Crimmins, Daniel L.; Davies, Sherri; Gao, Yuqian; Hiltke, Tara R.; Ketchum, Karen; Kinsinger, Christopher; Mesri, Mehdi; Meyer, Matthew R.; Qian, Weijun; Schoenherr, Regine M.; Scott, Mitchell; Shi, Tujin; Whiteley, Gordon; Wrobel, John; Wu, Chaochao; Ackermann, Bradley L.; Aebersold, Ruedi; Barnidge, David R.; Bunk, David M.; Clarke, Nigel; Fishman, Jordan B.; Grant, Russ P.; Kusebauch, Ulrike; Kushnir, Mark M.; Lowenthal, Mark S.; Moritz, Robert; Neubert, Hendrik; Patterson, Scott D.; Rockwood, Alan L.; Rogers, John; Singh, Ravinder J.; Van Eyk, Jennifer; Wong, Steven H.; Zhang, Shucha; Chan, Daniel W.; Chen, Xian; Ellis, Matthew J.; Liebler, Daniel; Rodland, Karin D.; Rodriguez, Henry; Smith, Richard D.; Zhang, Zhen; Zhang, Hui; Paulovich, Amanda G.


    The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

  10. Making the cut: Innovative methods for optimizing perfusion-based migration assays. (United States)

    Holt, Andrew W; Howard, William E; Ables, Elizabeth T; George, Stephanie M; Kukoly, Cindy A; Rabidou, Jake E; Francisco, Jake T; Chukwu, Angel N; Tulis, David A


    Application of fluid shear stress to adherent cells dramatically influences their cytoskeletal makeup and differentially regulates their migratory phenotype. Because cytoskeletal rearrangements are necessary for cell motility and migration, preserving these adaptations under in vitro conditions and in the presence of fluid flow are physiologically essential. With this in mind, parallel plate flow chambers and microchannels are often used to conduct in vitro perfusion experiments. However, both of these systems currently lack capacity to accurately study cell migration in the same location where cells were perfused. The most common perfusion/migration assays involve cell perfusion followed by trypsinization which can compromise adaptive cytoskeletal geometry and lead to misleading phenotypic conclusions. The purpose of this study was to quantitatively highlight some limitations commonly found with currently used cell migration approaches and to introduce two new advances which use additive manufacturing (3D printing) or laser capture microdissection (LCM) technology. The residue-free 3D printed insert allows accurate cell seeding within defined areas, increases cell yield for downstream analyses, and more closely resembles the reported levels of fluid shear stress calculated with computational fluid dynamics as compared to other residue-free cell seeding techniques. The LCM approach uses an ultraviolet laser for "touchless technology" to rapidly and accurately introduce a custom-sized wound area in otherwise inaccessible perfusion microchannels. The wound area introduced by LCM elicits comparable migration characteristics compared to traditional pipette tip-induced injuries. When used in perfusion experiments, both of these newly characterized tools were effective in yielding similar results yet without the limitations of the traditional modalities. These innovative methods provide valuable tools for exploring mechanisms of clinically important aspects of cell

  11. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  12. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine


    on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S......Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely......-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal...

  13. A fluorescence turn on assay for alkaline phosphatase based on the Cu(2+) catalyzed Fenton-like reaction. (United States)

    Zhang, Qingfeng; Zhang, Cuiyun; Shahzad, Sohail Anjum; Yu, Cong


    A fluorescence turn-on assay was established for ALP (alkaline phosphatase) based on Cu(2+) catalyzed Fenton-like reaction and Graphene Oxide (GO). GO was utilized to quench the fluorescence of fluorescein (FAM) labeled single strand DNA (F-DNA). ALP can remove the phosphate group in sodium ascorbyl phosphate (SAP), and convert it into reducing ascorbate. Highly reactive hydroxyl radicals (·OH) were generated in the presence of ascorbate and Cu(2+) through the Fenton-like reaction. The reactive radicals generated in situ caused the cleavage of F-DNA into small fragments. When GO was added, the fluorescence emission of the sample without ALP was quenched and fluorescence emission recovered in the presence of ALP. The intensity of the recovered fluorescence was directly related to the concentration of ALP in the assay solution, and a sensitive and selective facile ALP assay is therefore established.

  14. Toward the authentication of wines of Nemea denomination of origin through cleaved amplified polymorphic sequence (CAPS)-based assay. (United States)

    Spaniolas, Stelios; Tsachaki, Maroussa; Bennett, Malcolm J; Tucker, Gregory A


    In the present study, we developed a cleaved amplified polymorphic sequence (CAPS)-based assay as a first attempt to detect fraud in grapevine musts with a long-term objective to establish an analytical methodology to authenticate wines of Nemea denomination of origin (Agiorgitiko). The analytical assay makes use of a single nucleotide polymorphism that discriminates Agiorgitiko and Cabernet Sauvignon varieties. The latter grape variety is one of the major adulterants for Nemea wines. Agiorgitiko grapevine must was spiked with Cabernet Sauvignon in several ratios (v/v) from 50 down to 10%, and the subsequent mixes were subjected to alcoholic microfermentation. DNA was extracted from all mixture samples up to the end of the fermentation process and was subjected to the CAPS assay. Both standard agarose gel and lab-on-a-chip capillary electrophoresis illustrated the ability of the method to detect the presence of Cabernet Sauvignon down to 10% throughout the whole fermentation process.

  15. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus? (United States)

    Morton, C Oliver; White, P Lewis; Barnes, Rosemary A; Klingspor, Lena; Cuenca-Estrella, Manuel; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem; Mengoli, Carlo; Caliendo, Angela M; Cogliati, Massimo; Debets-Ossenkopp, Yvette; Gorton, Rebecca; Hagen, Ferry; Halliday, Catriona; Hamal, Petr; Harvey-Wood, Kathleen; Jaton, Katia; Johnson, Gemma; Kidd, Sarah; Lengerova, Martina; Lass-Florl, Cornelia; Linton, Chris; Millon, Laurence; Morrissey, C Orla; Paholcsek, Melinda; Talento, Alida Fe; Ruhnke, Markus; Willinger, Birgit; Donnelly, J Peter; Loeffler, Juergen


    A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

  16. Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates. (United States)

    Mirazizi, Fatemeh; Bahrami, Azita; Haghbeen, Kamahldin; Shahbani Zahiri, Hossein; Bakavoli, Mehdi; Legge, Raymond L


    Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.

  17. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity. (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John


    Dipeptidyl peptidase 1 (DPP1) (EC; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  18. Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays

    Directory of Open Access Journals (Sweden)

    Yu Jingjing


    Full Text Available Abstract Background The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP and methylated DNA (MeDIP represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. Results To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. Conclusion The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation

  19. Dose-response curve of a microfluidic magnetic bead-based surface coverage sandwich assay. (United States)

    Cornaglia, Matteo; Trouillon, Raphaël; Tekin, H Cumhur; Lehnert, Thomas; Gijs, Martin A M


    Magnetic micro- and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fgmL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8μm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0μm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves.

  20. Validation of a quantitative flow cytometer assay for monitoring HER-2/neu expression level in cell-based cancer immunotherapy products. (United States)

    Randlev, Britta; Huang, Li-chun; Watatsu, Mitsuko; Marcus, Matthew; Lin, Andy; Shih, Shian-Jiun


    GVAX immunotherapy for prostate cancer is comprised of two genetically modified prostate cancer cell lines, CG1940 and CG8711, engineered to secrete granulocyte macrophage-colony-stimulating factor. As part of the matrix of potency assays, CG1940 and CG8711 are tested for the expression level of cell surface HER-2/neu using a quantitative flow cytometer assay. This assay reports the antibody binding capacity value of the cells as a measure of HER-2/neu expression using cells immediately after thawing from cryogenic storage. With optimized cell handling and staining procedure and appropriate system suitability controls, the assay was validated as a quantitative assay. The validation results showed that assay accuracy, specificity, precision, linearity, and range were suitable for the intended use of ensuring lot-to-lot consistency of HER-2/neu expression. Assay robustness was demonstrated using design of experiments that evaluated critical assay parameters. Finally, the assay was successfully transferred to a current good manufacturing practice Quality Control laboratory in a separate facility. Since the overall precision of this assay is better than that of ELISA methods and it can be performed with ease and high throughput, quantitative flow cytometer-based assays may be an appropriate immunological assay platform for Quality Control laboratories for characterization and release of cell-based therapies.

  1. Fast and automated DNA assays on a compact disc (CD)-based microfluidic platform (United States)

    Jia, Guangyao

    Nucleic acid-based molecular diagnostics offers enormous potential for the rapid and accurate diagnosis of infectious diseases. However, most of the existing commercial tests are time-consuming and technically complicated, and are thus incompatible with the need for rapid identification of infectious agents. We have successfully developed a CD-based microfluidic platform for fast and automated DNA array hybridization and a low cost, disposable plastic microfluidic platform for polymerase chain reaction (PCR). These platforms have proved to be a promising approach to meet the requirements in terms of detection speed and operational convenience in diagnosis of infectious diseases. In the CD-based microfluidic platform for DNA hybridization, convection is introduced to the system to enhance mass transport so as to accelerate the hybridization rate since DNA hybridization is a diffusion limited reaction. Centrifugal force is utilized for sample propulsion and surface force is used for liquid gating. Standard microscope glass slides are used as the substrates for capture probes owing to their compatibility with commercially available instrumentation (e.g. laser scanners) for detection. Microfabricated polydimethylsiloxane (PDMS) structures are used to accomplish the fluidic functions required by the protocols for DNA hybridization. The assembly of the PDMS structure and the glass slide forms a flow-through hybridization unit that can be accommodated onto the CD platform for reagent manipulation. The above scheme has been validated with oligonucleotides as the targets using commercially available enzyme-labeled fluorescence (ELF 97) for detection of the hybridization events, and tested with amplicons of genomic staphylococcus DNA labeled with Cy dye. In both experiments, significantly higher fluorescence intensities were observed in the flow-through hybridization unit compared to the passive assays. The CD fluidic scheme was also adapted to the immobilization of

  2. Development of a Cryptosporidium oocyst assay using an automated fiber optic-based biosensor

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    Kramer Marianne F


    Full Text Available Abstract An intestinal protozoan parasite, Cryptosporidium parvum, is a major cause of waterborne gastrointestinal disease worldwide. Detection of Cryptosporidium oocysts in potable water is a high priority for the water treatment industry to reduce potential outbreaks among the consumer populace. Anti-Cryptosporidium oocyst polyclonal and monoclonal antibodies were tested as capture and detection reagents for use in a fiber optic biosensor assay for the detection of Cryptosporidium oocysts. Antibodies were validated using enzyme-linked immunosorbent assays, flow cytometry, Western blotting and fluorescent microscopy. Oocysts could be detected at a concentration of 105 oocysts/ml when the polyclonal antibodies were used as the capture and detection reagents. When oocysts were boiled prior to detection, a ten-fold increase in sensitivity was achieved using the polyclonal antibody. Western blotting and immunofluorescence revealed that both the monoclonal and polyclonal antibodies recognize a large (>300 kDa molecular weight mucin-like antigen present on the surface of the oocyst wall. The polyclonal antibody also reacted with a small (105 kDa molecular weight antigen that was present in boiled samples of oocysts. Preliminary steps to design an in-line biosensor assay system have shown that oocysts would have to be concentrated from water samples and heat treated to allow detection by a biosensor assay.

  3. Sensitivity and Selectivity on Aptamer-Based Assay: The Determination of Tetracycline Residue in Bovine Milk

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    Sohee Jeong


    Full Text Available A competitive enzyme-linked aptamer assay (ELAA to detect tetracycline in milk was performed by using two different aptamers individually; one is 76 mer-DNA aptamer and the other is 57 mer-RNA aptamer. The best optimum condition was obtained without monovalent ion, Na+ and also by adding no Mg2+ ion in the assay buffer, along with RT incubation. The optimized ELAA showed a good sensitivity (LOD of 2.10 × 10−8 M with a wide dynamic range (3.16 × 10−8 M ~ 3.16 × 10−4 M. In addition, the average R.S.D. across all data points of the curve was less than 2.5% with good recoveries (~101.8% from the milk media. Thus, this method provides a good tool to monitor tetracycline in milk from MRLs’ point of view. However, this ELAA method was not superior to the ELISA method in terms of specificity. This paper describes that it does not always give better sensitivity and specificity in assays even though aptamers have several advantages over antibodies and have been known to be good binders for binding assays.

  4. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae (United States)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  5. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.


    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two...

  6. Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-e; WANG Cheng-ke; LIU Dian-jun; WANG Zhen-xin


    The interactions of antibiotic with living cells were studied by iectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.

  7. Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate. (United States)

    Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred


    Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry.

  8. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites. (United States)

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; Terkawi, Mohamed Alaa; Youssef, Mohamed Ahmed; El Said, El Said El Shirbini; Elsayed, Gehad; El-Khodery, Sabry; El-Ashker, Maged; Elsify, Ahmed; Omar, Mosaab; Salama, Akram; Yokoyama, Naoaki; Igarashi, Ikuo


    A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  9. Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. (United States)

    Ai, L; Dong, S J; Zhang, W Y; Elsheikha, H M; Mahmmod, Y S; Lin, R Q; Yuan, Z G; Shi, Y L; Huang, W Y; Zhu, X Q


    Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.

  10. Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites.

    Directory of Open Access Journals (Sweden)

    Mohamed Abdo Rizk

    Full Text Available A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10% were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equine Babesia and Theileria parasites. The IC50 values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC50 values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for in vitro large-scale drug screening against Babesia and Theileria parasites that infect cattle and horses.

  11. Rapid on-chip apoptosis assay on human carcinoma cells based on annexin-V/quantum dot probes. (United States)

    Montón, Helena; Medina-Sánchez, Mariana; Soler, Joan Antoni; Chałupniak, Andrzej; Nogués, Carme; Merkoçi, Arben


    Despite all the efforts made over years to study the cancer expression and the metastasis event, there is not a clear understanding of its origins and effective treatment. Therefore, more specialized and rapid techniques are required for studying cell behaviour under different drug-based treatments. Here we present a quantum dot signalling-based cell assay carried out in a segmental microfluidic device that allows studying the effect of anti-cancer drugs in cultured cell lines by monitoring phosphatidylserine translocation that occurs in early apoptosis. The developed platform combines the automatic generation of a drug gradient concentration, allowing exposure of cancer cells to different doses, and the immunolabeling of the apoptotic cells using quantum dot reporters. Thereby a complete cell-based assay for efficient drug screening is performed showing a clear correlation between drug dose and amount of cells undergoing apoptosis.

  12. Evaluation of a new serological test for syphilis based on chemiluminescence assay in a tertiary care hospital

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    Aseem K Tiwari


    Full Text Available Context: Syphilis is a transfusion transmissible infections and it is mandatory to do serological test for syphilis (STS on all donor blood samples. STS is usually based on detection of antibodies against the cardiolipin-lecithin antigen or against the Treponema-specific antigen. STS with good sensitivity and specificity helps enhance blood safety and consolidation of STS along with other transfusion transmittable infections such as human immunodeficiency virus, hepatitis-C virus, and hepatitis-B virus helps in reducing the errors and enhances efficiency. Aims: This study was designed to evaluate the performance of newly introduced VITROS ® syphilis Treponema pallidum agglutination (TPA assay based on enhanced chemiluminescence principle for its analytical performance for use as a STS on donor blood samples at a tertiary care health center in National Capital Region, India. Materials and Methods: A total of 108 random blood units collected from the donors (both voluntary and replacement donors and 28 known syphilis sero-reactive samples stored at −20°C, were used to evaluate the performance of VITROS ® syphilis TPA assay based on enhanced chemiluminescence assay on VITROS ® ECiQ immunodiagnostics system along with its analytical performance in terms of its sensitivity, precision, cross-reactivity and interference studies. Results: VITROS ® syphilis TPA showed 100% sensitivity and specificity with precision (20 days study of <10% co-efficient of variation. There was no cross-reactivity with other viral and auto-immune antibodies. No interference was observed from endogenous interfering substances like free hemoglobin or fats. Conclusions: Performance of the VITROS ® syphilis TPA assay meets the requirements for its use as STS in blood bank, thus allowing consolidation with other transfusion transmittable infections screening assay on chemiluminescence platform, which is highly valuable for optimizing workflow and efficiency.

  13. Genetic diversity in Silene sennenii Pau (Caryophyllaceae assayed through DNA-based techniques

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    Blanché, C.


    Full Text Available Silene sennenii is a narrow endemic species from the NE of the Iberian Peninsula. It is considered as EN (“Endangered” according to the IUCN criteria and is under legal protection in Catalonia. In the present work we report an assay using three different approaches for surveying the genetic diversity in this rare, endangered campion: analysis of chloroplast haplotypes, AFLPs and transferability of microsatellites previously developed for Silene latifolia. None of the nine chloroplast regions sequenced showed any variability. Five out of the 21 microsatellite loci tested were polymorphic although more loci are required in order to perform a robust population genetics study. Regarding the AFLP analysis, five out of the 26 pairs of primers tested exhibited moderate levels of variability and therefore they could be useful for further investigating the genetic structure of S. sennenii. Although preliminary, our results based on three different genetic markers are in agreement with the low values of genetic variation already reported for this species employing allozymes.Silene sennenii es una especie endémica, circunscrita a un área extremadamente reducida al NE de la Península Ibérica. Está catalogada como EN («En Peligro» según criterios UICN y se encuentra legalmente protegida en Cataluña. En el presente trabajo se expone el ensayo de tres aproximaciones diferentes al estudio de su diversidad genética: análisis de haplotipos cloroplásticos, AFLPs y transferibilidad de microsatélites diseñados previamente para Silene latifolia. Ninguna de las nueve regiones cloroplásticas secuenciadas ha presentado variabilidad. Se han obtenido cinco loci microsatélites polimórficos de los 21 ensayados, cantidad insuficiente para llevar a cabo un estudio robusto sobre genética poblacional. En cuanto a AFLPs, cinco combinaciones de cebadores de las 26 probadas han mostrado niveles moderados de variabilidad siendo así útiles para posteriores

  14. High throughput microwell spectrophotometric assay for olmesartan medoxomil in tablets based on its charge-transfer reaction with DDQ

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A.


    Full Text Available The study describes the development and validation of a new microwell-based spectrophotometric assay for determination of olmesartan medoxomil (OLM in tablets. The formation of a colored charge-transfer (CT complex between OLM as an n-electron donor and 2,3-dichloro- -5,6-dicyano-1,4-benzoquinone (DDQ as a p-electron acceptor was investigated, and employed as the basis for the development of the new assay. The proposed assay was conducted in 96-microwell plates. The absorbance of the colored-CT complex was measured at 460 nm with a microplate reader. Optimum conditions of the reaction and the analytical procedures of the assay were established. Under the optimum conditions, a linear relationship with a good correlation coefficient was found between the absorbance and the concentration of OLM in the range of 2-200 μg per well. The limits of detection and quantitation were 0.53 and 1.61 μg per well, respectively. No interference was observed from the excipients present in OLM tablets or from hydrochlorothiazide and amlodipine besylate that were co-formulated with OLM in some of its formulations. The assay was successfully applied to the analysis of OLM in tablets with good accuracy and precision. The assay described herein has a great practical value in the routine analysis of OLM in quality control laboratories, since it has a high throughput property and consumes low volumes of organic solvent. It thus offers a reduction in the exposure of analysts to the toxic effects of organic solvents, as well as a reduction in the cost of analysis.

  15. Zebrafish 3-O-sulfotransferase-4 generated heparan sulfate mediates HSV-1 entry and spread.

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    Thessicar E Antoine

    Full Text Available Rare modification of heparan sulfate (HS by glucosaminyl 3-O sulfotransferase (3-OST isoforms generates an entry receptor for herpes simplex virus type-1 (HSV-1. In the zebrafish (ZF model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1 cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.

  16. The impact of ligands on the structure and flexibility of sulfotransferases: a molecular dynamics simulation study. (United States)

    Zhao, Li; Zhang, Pupu; Long, Shiyang; Wang, Linlin; Tian, Pu


    Sulfotransferases catalyze transfer of the sulfuryl-group (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a large number of substrates. They play an important role in phase II metabolic process. The impact of the cofactor (PAPS) on the structure and flexibility of the enzyme has been studied extensively, and the response of the active-cap region to cofactor binding was proposed as the molecular basis for substrate selectivity. In this study, individual and cooperative effects of the cofactor and substrate on the structure and flexibility of the enzyme were investigated. Molecular dynamics simulations were performed for four systems, including free enzyme, binary complexes (cofactor or substrate bound enzyme) and ternary complex (both cofactor and substrate bound enzyme). The influence of ligands (the cofactor and the substrate) on the structure and flexibility of the enzyme, especially that of the active-site cap region, was analyzed. Moreover, mutual structural impact of the ligands was examined as well. The results show that the impact of both the cofactor and the substrate was significant. Our study indicated that the substrate, such as lithocholic acid (LCA), participated in regulating the structure and flexibility of the enzyme actively rather than merely being selected passively. Additionally, the observed synergistic effects of the cofactor and the substrate demonstrated the importance of examining both ligands in understanding enzymes.

  17. Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase. (United States)

    Liu, Chunhui; Sheng, Juzheng; Krahn, Juno M; Perera, Lalith; Xu, Yongmei; Hsieh, Po-Hung; Dou, Wenfang; Liu, Jian; Pedersen, Lars C


    Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.

  18. Genetic variation, expression and ontogeny of sulfotransferase SULT2A1 in humans. (United States)

    Ekström, L; Rane, A


    Sulfotransferases (SULTs) are enzymes involved in the metabolism of several endogenous molecules. The activity and expression exhibit inter- and intra-individual variations due to age and genetic variation. The aims of this study were to compare the gene expression of SULT2A1 in fetal and adult livers, to study the intra-individual tissue distribution, and investigate if expression is associated with a SULT2A1 copy number variation polymorphism. In contrast to other drug metabolizing enzyme systems the expression of SULT2A1 did not differ between fetal and adult liver samples and it was not affected by maternal smoking or gestational age. Gene expression in relation to sex could not be assessed as the sex of the fetuses was unknown. SULT2A1 was consistently expressed in livers and adrenals, being seven times more abundant in adrenals, but was absent in the lungs. The SULT2A1 copy number variation was proportional to gene expression in liver and adrenals. Our results show that SULT2A1 is important in the first trimester; particularly in the adrenals.

  19. Paradigms of sulfotransferase catalysis: the mechanism of SULT2A1. (United States)

    Wang, Ting; Cook, Ian; Falany, Charles N; Leyh, Thomas S


    Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of signaling small molecules via transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. Sulfonation controls the affinities of ligands for their targets, and thereby regulates numerous receptors, which, in turn, regulate complex cellular responses. Despite their biological and medical relevance, basic SULT mechanism issues remain unresolved. To settle these issues, and to create an in-depth model of SULT catalysis, the complete kinetic mechanism of a representative member of the human SULT family, SULT2A1, was determined. The mechanism is composed of eight enzyme forms that interconvert via 22 rate constants, each of which was determined independently. The result is a complete quantitative description of the mechanism that accurately predicts complex enzymatic behavior. This is the first description of a SULT mechanism at this resolution, and it reveals numerous principles of SULT catalysis and resolves previously ambiguous issues. The structures and catalytic behaviors SULTs are highly conserved; hence, the mechanism presented here should prove paradigmatic for the family.

  20. Triclosan causes spontaneous abortion accompanied by decline of estrogen sulfotransferase activity in humans and mice. (United States)

    Wang, Xiaoli; Chen, Xiaojiao; Feng, Xuejiao; Chang, Fei; Chen, Minjian; Xia, Yankai; Chen, Ling


    Triclosan (TCS), an antibacterial agent, is identified in serum and urine of humans. Here, we show that the level of urinary TCS in 28.3% patients who had spontaneous abortion in mid-gestation were increased by 11.3-fold (high-TCS) compared with normal pregnancies. Oral administration of TCS (10 mg/kg/day) in mice (TCS mice) caused an equivalent urinary TCS level as those in the high-TCS abortion patients. The TCS-exposure from gestation day (GD) 5.5 caused dose-dependently fetal death during GD12.5-16.5 with decline of live fetal weight. GD15.5 TCS mice appeared placental thrombus and tissue necrosis with enhancement of platelet aggregation. The levels of placenta and plasma estrogen sulfotransferase (EST) mRNA and protein in TCS mice or high-TCS abortion patients were not altered, but their EST activities were significantly reduced compared to controls. Although the levels of serum estrogen (E2) in TCS mice and high-TCS abortion patients had no difference from controls, their ratio of sulfo-conjugated E2 and unconjugated E2 was reduced. The estrogen receptor antagonist ICI-182,780 prevented the enhanced platelet aggregation and placental thrombosis and attenuated the fetal death in TCS mice. The findings indicate that TCS-exposure might cause spontaneous abortion probably through inhibition of EST activity to produce placental thrombosis.

  1. Structural plasticity in the human cytosolic sulfotransferase dimer and its role in substrate selectivity and catalysis. (United States)

    Tibbs, Zachary E; Rohn-Glowacki, Katie Jo; Crittenden, Frank; Guidry, Amber L; Falany, Charles N


    The cytosolic sulfotransferases (SULTs) are dimeric enzymes that help maintain homeostasis through the modulation of hormone and drug activity by catalyzing their transformation into hydrophilic sulfate esters and increasing their excretion. Each of the thirteen active human SULT isoforms displays a unique substrate specificity pattern that underlies its individual role in our bodies. These specificities have proven to be complex, in some cases masking the biological role of specific isoforms. The first part of this review offers a short summary of historical underpinnings of human SULTs, primarily centered on the characterization of each isoform's kinetic and structural properties. Recent structural investigations have revealed each SULT has an active site "lid" that undergoes restructuring once the cofactor/sulfonate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), binds to the enzyme. This structural rearrangement can alter substrate-binding profiles, therefore complicating enzyme/substrate interactions and making substrate/cosubstrate concentrations and binding order important considerations in enzyme functionality. Molecular dynamic simulations have recently been employed to describe this restructuring in an attempt to offer insight to its effects on substrate selectivity. In addition to reviewing new data on SULT molecular dynamics, we will discuss the contribution of PAPS concentrations and SULT dimerization in the regulation of SULT activity within the human body.

  2. The multi-protein family of sulfotransferases in plants: Composition, occurrence, substrate specificity and functions

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    Felix eHirschmann


    Full Text Available All members of the sulfotransferase (SOT, EC 2.8.2.- protein family transfer a sulfuryl group from the donor 3´-phosphoadenosine 5´-phosphosulfate (PAPS to an appropriate hydroxyl group of several classes of substrates. The primary structure of these enzymes is characterized by a histidine residue in the active site, defined PAPS binding sites and a longer SOT domain. Proteins with this SOT domain occur in all organisms from all three domains, usually as a multi-protein family. Arabidopsis thaliana SOTs, the best characterized SOT multi-protein family, contains 21 members. The substrates for several plant enzymes have already been identified, such as glucosinolates, brassinosteroids, jasmonates, flavonoids, and salicylic acid. Much information has been gathered on desulfo-glucosinolate (dsGl SOTs in A. thaliana. The three cytosolic dsGl SOTs show slightly different expression patterns. The recombinant proteins reveal differences in their affinity to indolic and aliphatic dsGls. Also the respective recombinant dsGl SOTs from different A. thaliana ecotypes differ in their kinetic properties. However, determinants of substrate specificity and the exact reaction mechanism still need to be clarified. Probably, the three-dimensional structures of more plant proteins need to be solved to analyze the mode of action and the responsible amino acids for substrate binding. In addition to A. thaliana, more plant species from several families need to be investigated to fully elucidate the diversity of sulfated molecules and the way of biosynthesis catalyzed by SOT enzymes.

  3. The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

    Directory of Open Access Journals (Sweden)

    Ilana Berger

    Full Text Available Cytosolic sulfotransferases (SULTs are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.

  4. Sulphation of acetaminophen by the human cytosolic sulfotransferases: a systematic analysis. (United States)

    Yamamoto, Akihiro; Liu, Ming-Yih; Kurogi, Katsuhisa; Sakakibara, Yoichi; Saeki, Yuichi; Suiko, Masahito; Liu, Ming-Cheh


    Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo. This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.

  5. Induction of Epoxide Hydrolase, Glucuronosyl Transferase, and Sulfotransferase by Phenethyl Isothiocyanate in Male Wistar Albino Rats

    Directory of Open Access Journals (Sweden)

    Ahmad Faizal Abdull Razis


    Full Text Available Phenethyl isothiocyanate (PEITC is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin. The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT, N-acetyltransferase (NAT, glucuronosyl transferase (UDP, and epoxide hydrolase (EH following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake, 0.6 µmol/g (medium dose, and 6.0 µmol/g (high dose, and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention.

  6. Characterization of kidney sulfotransferases during lead-induced nephrotoxicity in rats

    Energy Technology Data Exchange (ETDEWEB)

    Templer, L.A.; Kong, J.; Ronis, M.J.J.; Ringer, D.P. [Univ. Arkansas Medical School, Little Rock, AR (United States)


    Kidney sulfotransferases (ST) have been shown to be involved in the biotransformation of steroid and thyroid hormones as well as xenobiotics varying from carcinogenic heterocyclic amines to drugs such as acetaminophen. In order to examine the impact of lead-induced nephrotoxicity on kidney aryl, estrogen and DHEA STs during growth and development, time-impregnated female Sprague-Dawley rats were exposed ad libitum to lead acetate (0.6%) in drinking water from gestational day 5 and continuing in male and female pups until they were sacrificed at day 85. Cytosols from male rat kidneys showed levels of estrogen ST activity (59% of females) that were significantly lowered (P{le}0.05) after lead exposure (6-20% of male). Aryl ST activity was relatively unchanged in male rats after rat kidney cytosol. Immunochemical analysis of cytosols from normal males and females with the antiserums to the three STs substantiated the presence of only the aryl and estrogen STs. Immunohistochemical techniques localized the aryl and estrogen STs primarily to the S3 section of the proximal tubules. These findings indicate that kidney STs may be differently modulated during lead exposure.

  7. Reduced sulfotransferase SULT2A1 activity in patients with Alzheimer's disease. (United States)

    Vaňková, M; Hill, M; Velíková, M; Včelák, J; Vacínová, G; Lukášová, P; Vejražková, D; Dvořáková, K; Rusina, R; Holmerová, I; Jarolímová, E; Vaňková, H; Bendlová, B


    Steroids are important components in the pathophysiology of Alzheimer's disease (AD). Although their role has been studied, the corresponding metabolomic data is limited. In the present study we evaluate the role of steroid sulfotransferase SULT2A1 in the pathophysiology of AD on the basis of circulating steroids (measured by GC-MS), in which the sulfation catalyzed by SULT2A1 dominates over glucuronidation (pregnenolone/sulfate, DHEA/sulfate, androstenediol/sulfate and 5alpha-reduced pregnane and androstane catabolites). To estimate a general trend of SUL2A1 activity in AD patients we compared the ratios of steroid conjugates to their unconjugated counterparts (C/U) in controls (11 men and 22 women) and AD patients (18 men and 16 women) for individual circulating steroids after adjustment for age and BMI using ANCOVA model including the factors AD status and gender. Decreased C/U ratio for the C19 steroids demonstrate an association between attenuated sulfation of C19 steroids in adrenal zona reticularis and the pathophysiology of AD.

  8. Phylogeny of algal sequences encoding carbohydrate sulfotransferases, formylglycine-dependent sulfatases and putative sulfatase modifying factors

    Directory of Open Access Journals (Sweden)

    Chai-Ling eHo


    Full Text Available Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs. Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute towards future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering.

  9. Regulation of chondroitin-4-sulfotransferase (CHST11) expression by opposing effects of arylsulfatase B on BMP4 and Wnt9A. (United States)

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K


    In this report, the gene regulatory mechanism by which decline in arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) reduces CHST11 (chondroitin-4-sulfotransferase; C4ST) mRNA expression in human colonic epithelial cells and in colonic epithelium of ARSB-deficient mice is presented. ARSB controls the degradation of chondroitin 4-sulfate (C4S) by removing the 4-sulfate group at the non-reducing end of the C4S chain, but has not previously been shown to affect C4S biosynthesis. The decline in CHST11 expression following ARSB reduction is attributable to effects of ARSB on bone morphogenetic protein (BMP)4, since BMP4 expression and secretion declined when ARSB was silenced. Inhibition of BMP4 by neutralizing antibody also reduced CHST11 expression. When C4S was more sulfated due to decline in ARSB, more BMP4 was sequestered by C4S in the cell membrane, and CHST11 expression declined. Exogenous recombinant BMP4, acting through a phospho-Smad3 binding site in the CHST11 promoter, increased the mRNA expression of CHST11. In contrast to the decline in BMP4 that followed decline in ARSB, Wnt9A mRNA expression was previously shown to increase when ARSB was silenced and C4S was more highly sulfated. Galectin-3 bound less to the more highly sulfated C4S, leading to increased nuclear translocation and enhanced galectin-3 interaction with Sp1 in the Wnt9A promoter. Silencing Wnt9A increased the expression of CHST11 in the colonic epithelial cells, and chromatin immunoprecipitation assay demonstrated enhancing effects of Wnt9A siRNA and exogenous BMP4 on the CHST11 promoter through the pSmad3 binding site. These findings suggest that cellular processes mediated by differential effects of Wnt9A and BMP4 can result from opposing effects on CHST11 expression.

  10. Conversion of Suspected Food Carcinogen 5-Hydroxymethylfurfural by Sulfotransferases and Aldehyde Dehydrogenases in Postmitochondrial Tissue Preparations of Humans, Mice, and Rats. (United States)

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H


    The food contaminant 5-hydroxymethylfurfural (HMF) is formed by heat- and acid-catalyzed reactions from carbohydrates. More than 80% of HMF is metabolized by oxidation of the aldehyde group in mice and rats. Sulfo conjugation yields mutagenic 5-sulfoxymethylfurfural, the probable cause for the neoplastic effects observed in HMF-treated rodents. Considerable metabolic differences between species hinder assessing the tumorigenic risk associated with human dietary HMF uptake. Here, we assayed HMF turnover catalyzed by sulfotransferases or by aldehyde dehydrogenases (ALDHs) in postmitochondrial preparations from liver, kidney, colon, and lung of humans, mice, and rats. The tissues-specific clearance capacities of HMF sulfo conjugation (CL(SC)) and ALDH-catalyzed oxidation (CL(OX)) were concentrated to the liver. The hepatic clearance CL(SC) in mice (males: 487 µl/min/kg bw, females: 2520 µl/min/kg bw) and rats (males: 430 µl/min/kg bw, females: 198 µl/min/kg bw) were considerably higher than those in humans (males: 21.2 µl/min/kg bw, females: 32.2 µl/min/kg bw). The ALDH-related clearance rates CLOX in mice (males: 3400 ml/min/kg bw, females: 1410 ml/min/kg bw) were higher than those of humans (males: 436 ml/min/kg bw, females: 646 ml/min/kg bw) and rats (males: 627 ml/min/kg bw, females: 679 ml/min/kg bw). The ratio of CL(OX) to CL(SC) was lowest in female mice. This finding indicated that HMF sulfo conjugation was most substantial in the liver of female mice, a target tissue for HMF-induced neoplastic effects, and that humans may be less sensitive regarding HMF sulfo conjugation compared with the rodent models.

  11. Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells. (United States)

    Sun, Hua; Wang, Xiao; Zhou, Xiaotong; Lu, Danyi; Ma, Zhiguo; Wu, Baojian


    Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

  12. Preclinical detection of porcine circovirus type 2 infection using an ultrasensitive nanoparticle DNA probe-based PCR assay.

    Directory of Open Access Journals (Sweden)

    Yong Huang

    Full Text Available Porcine circovirus type 2 (PCV2 has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.

  13. High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction

    Institute of Scientific and Technical Information of China (English)

    Hong-qiu HE; Xiao-hui MA; Bin LIU; Xiao-yi ZHANG; Wei-zu CHEN; Cun-xin WANG; Shao-hui CHENG


    Aim: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening.Methods: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluores-cence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3'end.IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal param-eters were obtained. Moreover, 2 IN inhibitors were employed to test the perfor-mance of this assay in antiviral compound screening.Results: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recom-binant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn2+. The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay.Conclusion: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

  14. A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry. (United States)

    de Boer, Arjen R; Bruyneel, Ben; Krabbe, Johannes G; Lingeman, Henk; Niessen, Wilfried M A; Irth, Hubertus


    The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.

  15. Development of fully automated determination of marker-specific immunoglobulin G (IgG) avidity based on the avidity competition assay format: application for Abbott Architect cytomegalovirus and Toxo IgG Avidity assays. (United States)

    Curdt, Ingo; Praast, Gerald; Sickinger, Eva; Schultess, Jan; Herold, Iris; Braun, Hans Bertram; Bernhardt, Stephanie; Maine, Gregory T; Smith, Darwin D; Hsu, Stephen; Christ, Heike M; Pucci, Dominick; Hausmann, Michael; Herzogenrath, Jörg


    Determination of the avidity of immunoglobulin G (IgG) directed against a specific marker has become an established diagnostic tool for identifying or excluding acute infections with pathogens. A novel assay format termed AVIcomp (avidity competition based on mass action) circumventing the conventional chaotropic format has been developed for determination of the avidity of marker-specific IgG in patient specimens. Its applications for cytomegalovirus (CMV) and Toxoplasma gondii are presented. Specific high-avidity IgG from the patient specimen is selectively blocked using a soluble antigen in a sample pretreatment reagent, and the amount of remaining specific low-avidity IgG is determined relative to that in an untreated control. The comparison of the conventional chaotropic format, represented by the Radim CMV IgG Avidity assay, and the newly developed AVIcomp method, as exemplified by the Architect CMV IgG Avidity assay, on blood drawn within 4 months after seroconversion revealed a sensitivity of 100% (97.3% by an alternative calculation) for the AVIcomp format versus 87.5% (75.7% by an alternative calculation) for the chaotropic avidity assay. The specificity on 312 CMV IgG reactive and CMV IgM nonreactive specimens from pregnant women was 100% for the AVIcomp assay and 99.7% for the conventional avidity assay. The Architect Toxo IgG Avidity assay showed an agreement of 97.2% with the bioMérieux Vidas Toxo IgG Avidity Assay employing chaotropic reagents. These performance data suggest that the AVIcomp format shows superior sensitivity and equivalent specificity for the determination of IgG avidity to assays based on the chaotropic method and that the AVIcomp format may also be applicable to other disease states.

  16. Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors. (United States)

    Doughan, Samer; Noor, M Omair; Han, Yi; Krull, Ulrich J


    Quantum dots (QDs) and upconverting nanoparticles (UCNPs) are luminescent nanoparticles (NPs) commonly used in bioassays and biosensors as resonance energy transfer (RET) donors. The narrow and tunable emissions of both QDs and UCNPs make them versatile RET donors that can be paired with a wide range of acceptors. Ratiometric signal processing that compares donor and acceptor emission in RET-based transduction offers improved precision, as it accounts for fluctuations in the absolute photoluminescence (PL) intensities of the donor and acceptor that can result from experimental and instrumental variations. Immobilizing NPs on a solid support avoids problems such as those that can arise with their aggregation in solution, and allows for facile layer-by-layer assembly of the interfacial chemistry. Paper is an attractive solid support for the development of point-of-care diagnostic assays given its ubiquity, low-cost, and intrinsic fluid transport by capillary action. Integration of nanomaterials with paper-based analytical devices (PADs) provides avenues to augment the analytical performance of PADs, given the unique optoelectronic properties of nanomaterials. Herein, we describe methodology for the development of PADs using QDs and UCNPs as RET donors for optical transduction of nucleic acid hybridization. Immobilization of green-emitting QDs (gQDs) on imidazole functionalized cellulose paper is described for use as RET donors with Cy3 molecular dye as acceptors for the detection of SMN1 gene fragment. We also describe the covalent immobilization of blue-emitting UCNPs on aldehyde modified cellulose paper for use as RET donors with orange-emitting QDs (oQDs) as acceptors for the detection of HPRT1 gene fragment. The data described herein is acquired using an epifluorescence microscope, and can also be collected using technology such as a typical electronic camera.

  17. Methylation-regulated miR-149 modulates chemoresistance by targeting GlcNAc N-deacetylase/N-sulfotransferase-1 in human breast cancer. (United States)

    He, Dong-Xu; Gu, Xiao-Ting; Li, You-Ran; Jiang, Li; Jin, Jian; Ma, Xin


    Dysregulation of microRNA is strongly implicated in the chemoresistance of cancer. In this study, we found that miR-149 was downregulated and involved in chemoresistance in adriamycin (ADM)-resistant human breast cancer cells (MCF-7/ADM). Downregulation of miR-149 was related to hypermethylation of its 5'-UTR; this methylation also affected the expression of the glypican 1 gene, which is both the host and the target gene of miR-149. Furthermore, we found that miR-149 modulated chemoresistance through targeting the expression of GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST1). With downregulated miR-149, NDST1 expression was increased in chemoresistant MCF-7/ADM cells versus control MCF-7 wild-type cells. The increased NDST1 then activated a heparan sulfate-related pathway involving activation of heparanase. Finally, expression of miR-149 and NDST1 was confirmed in clinical chemoresistant samples of breast cancers receiving anthracycline/taxane-based chemotherapies. The high expression of NDST1 was also an unfavorable predictor for distant relapse-free survival in Her2 and basal breast cancers. Taken together, our findings demonstrate that miR-149 is regulated by methylation, and is a modulator of cancer chemoresistance by targeting NDST1.

  18. Multidimensional GPCR profiling and screening using impedance-based label-free and real-time assay. (United States)

    Ke, Ning; Nguyen, Khanh; Irelan, Jeffery; Abassi, Yama A


    GPCRs constitute one of the most sought-after targets in drug discovery because they are associated with conditions ranging from cardiovascular diseases, autoimmune diseases, inflammation, cancer, and diseases of the nervous system. Moreover, they are one of the most amenable targets for drug discovery because they can be modulated by small molecules, peptides, proteins, and antibodies. Therefore it may not come as a surprise that close to 40 % of the drugs that are currently on the market are targeting GPCRs. It has become evident that GPCR signaling is highly complex and may involve multiple or a subset of pathways depending on the interaction of a GPCR with an agonist or antagonist. It is imperative that any functional screening for GPCR activity integrates this complexity. In this assay protocol, we describe how the xCELLigence RTCA HT impedance-based platform which can be used for functional cell-based GPCR assays can be utilized for GPCR screening.

  19. Paper-Based Digital Microfluidic Chip for Multiple Electrochemical Assay Operated by a Wireless Portable Control System

    DEFF Research Database (Denmark)

    Ruecha, Nipapan; Lee, Jumi; Chae, Heedo


    The printing and modular fabrication of a paper-based active microfluidic lab on a chip implemented with electrochemical sensors (ECSs) is developed and integrated on a portable electrical control system. The electrodes of a chip plate for active electrowetting actuation of digital drops and an ECS...... for multiple analysis assays are fabricated by affordable printing techniques. For enhanced sensitivity of the sensor, the working electrode is modified through the electrochemical method, namely by reducing graphene with voltammetry and coating gold nanoparticles by amperometry. Detachable sensor and absorber...... designed portable power supply and wireless control system, the active paper-based chip platform can be utilized as an advanced point-of-care device for multiple assays in digital microfluidics....

  20. A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain

    DEFF Research Database (Denmark)

    Thorsen, Thor S; Madsen, Kenneth L; Dyhring, Tino


    domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters...... (Z'˜0.7 and Z˜0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 µM to ~193 µM. Binding to the PICK1 PDZ domain...... was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 µM) in the FP assay, and was according...

  1. A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens. (United States)

    Xue, Wenfei; Peng, Jingfu; Yu, Xiaoli; Zhang, Shulin; Zhou, Boping; Jiang, Danqing; Chen, Jianbo; Ding, Bingbing; Zhu, Bin; Li, Yao


    The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.

  2. Colloidal Gold Probe-Based Immunochromatographic Strip Assay for the Rapid Detection of Microbial Transglutaminase in Frozen Surimi

    Directory of Open Access Journals (Sweden)

    Daming Fan


    Full Text Available Adding microbial transglutaminase (MTGase to frozen surimi to enable the surimi to be sold as a higher-grade product at a higher price defrauds surimi product manufacturers and undercuts legitimate industry prices. Therefore, it is important to develop an accurate method of detecting the presence of MTGase in surimi. In this study, an immunochromatographic strip assay with a colloidal gold antibody probe was successfully developed and used to rapidly and qualitatively detect MTGase in surimi samples. The results were obtained in less than 10 min. The limit for the qualitative detection of MTGase using the immunochromatographic strip assay was identified as 1.0 μg/mL. The results of the immunochromatographic strip analysis of frozen surimi samples were verified by comparison with the results of a sandwich enzyme-linked immunosorbent assay. The colloidal gold probe-based immunochromatographic strip assay was thus found to be a rapid, economical, and user friendly method of detecting MTGase in surimi.

  3. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization. (United States)

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J


    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  4. Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry. (United States)

    Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Antelo, Alvaro; Vieytes, Mercedes R; Botana, Luis M


    The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.

  5. A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

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    Xian Zhang


    Full Text Available A novel enzyme-linked immunosorbent assay based on magnetic nanoparticles and biotin/streptavidin-HRP (MNP-bsELISA was developed for rapid and sensitive detection of zearalenone (ZEN. The detection signal was enhanced and the sensitivity of the assay was improved by combined use of antibody-conjugated magnetic nanoparticles and biotin-streptavidin system. Under the optimized conditions, the regression equation for quantification of ZEN was y = −0.4287x + 0.3132 (R2 = 0.9904. The working range was 0.07–2.41 ng/mL. The detection limit was 0.04 ng/mL and IC50 was 0.37 ng/mL. The recovery rates of intra-assay and inter-assay ranged from 92.8%–111.9% and 91.7%–114.5%, respectively, in spiked corn samples. Coefficients of variation were less than 10% in both cases. Parallel analysis of cereal and feed samples showed good correlation between MNP-bsELISA and liquid chromatograph-tandem mass spectrometry (R2 = 0.9283. We conclude that this method is suitable for rapid detection of zearalenone in cereal and feed samples in relevant laboratories.

  6. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays. (United States)

    Hart, Traver; Zhao, Alice; Garg, Ankit; Bolusani, Swetha; Marcotte, Edward M


    Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFkappaB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon), and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  7. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Directory of Open Access Journals (Sweden)

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  8. Development of a Filtration-Based Bioluminescence Assay for Detection of Microorganisms in Tea Beverages. (United States)

    Shinozaki, Yohei; Igarashi, Toshinori; Harada, Yasuhiro


    The market for tea drinks as healthy beverages has been steadily expanding, and ready-to-drink beverages in polyethylene terephthalate bottles have been popular. To more rapidly and accurately test tea beverages bottled in polyethylene terephthalate for microbial contamination, a newly developed filtration device and a washing method with a commercial bioluminescence assay were combined to detect low numbers of bacterial spores, fungal conidia, and ascospores. Washing buffers were formulated with nonionic detergents from the Tween series. Commercially available tea beverages were used to evaluate the filtration capacity of the filtration device, the effect of washing buffers, and the performance of the assay. The assay was tested with serially diluted suspensions of colonies of two bacterial strains, spores of three Bacillus strains, conidia of five fungal strains, and ascospores of four fungal strains. The filtration device enabled filtration of a large sample volume (100 to 500 ml), and the washing buffer significantly decreased the background bioluminescence intensity of tea samples when compared with the no-washing method. Low numbers (1 to 10 CFU/100 ml) of the tested strains of bacteria were detected within 8 to 18 h of cultivation, and fungi were detected within 24 to 48 h. Furthermore, a whole bottle (500 ml) of mixed tea was filtered through the filtration device and microbes were detected. This method could be used for quality control of bottled beverages without preincubation.

  9. Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots. (United States)

    Ullal, Anirudh J; Millington, David S; Bali, Deeksha S


    Mucopolysaccharidosis type IVA or Morquio type-A disease is a hereditary lysosomal storage disorder caused by deficient activity of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The disease is caused by lysosomal accumulation of unprocessed glycosaminoglycans (GAGs) that manifests with severe to mild skeletal and cardiopulmonary abnormalities. We have developed a modified microtiter plate-based enzyme activity assay using dried blood spots and a fluorescent substrate for measuring specific GALNS activity to identify patients with MPS IVA.

  10. Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics (United States)

    The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

  11. Assessment of Chemical Skin-Sensitizing Potency by an In Vitro Assay Based on Human Dendritic Cells


    Lambrechts, Nathalie; Vanheel, Hanne; Nelissen, Inge; Witters, Hilda; VAN DEN HEUVEL Rosette; Van Tendeloo, Viggo; Schoeters, Greet; HOOYBERGHS, Jef


    The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemica...

  12. A nanostructure-initiator mass spectrometry-based enzyme activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Siuzdak, Gary; Northen, Trent R.; Lee, Jinq-Chyi; Hoang, Linh; Raymond, Jason; Hwang, Der-Ren; Yannone, Steven M.; Wong, Chi-Huey; Siuzdak, Gary


    We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This 'soft' immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing {beta}-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65 C and 5.5, respectively, and the activity was inhibited by both phenylethyl-{beta}-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced {gamma}-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis. The interest in leveraging mass spectrometry for studying enzyme activities in complex biological samples derives from its high sensitivity and specificity; however, signal suppression and significant sample preparation requirements limit its overall utility (1). Here we describe a Nanostructure-Initiator Mass Spectrometry (NIMS

  13. Development of an Assay for the Detection of PrPres in Blood and Urine Based on PMCA Assay and ELISA Methods (United States)


    inoculated together in 3 deer13. The results from our studies were presented at the Prion2006 meeting and elicited strong interest especially on the part...the ORIGEN technology (the instrument is produced by BioVeris, Gaithersburg, MD) to measure PrP in brain and plasma of scrapie infected animals. We...the ORIGEN assay. To improve the PrPc assay sensitivity an aliquot of the PK-treated plasma was mixed with the 3F4 immuno affinity resin. The resin

  14. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana


    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors.

  15. DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors (United States)

    Deka, Jashmini; Mojumdar, Aditya; Parisse, Pietro; Onesti, Silvia; Casalis, Loredana


    Helicase are essential enzymes which are widespread in all life-forms. Due to their central role in nucleic acid metabolism, they are emerging as important targets for anti-viral, antibacterial and anti-cancer drugs. The development of easy, cheap, fast and robust biochemical assays to measure helicase activity, overcoming the limitations of the current methods, is a pre-requisite for the discovery of helicase inhibitors through high-throughput screenings. We have developed a method which exploits the optical properties of DNA-conjugated gold nanoparticles (AuNP) and meets the required criteria. The method was tested with the catalytic domain of the human RecQ4 helicase and compared with a conventional FRET-based assay. The AuNP-based assay produced similar results but is simpler, more robust and cheaper than FRET. Therefore, our nanotechnology-based platform shows the potential to provide a useful alternative to the existing conventional methods for following helicase activity and to screen small-molecule libraries as potential helicase inhibitors. PMID:28287182

  16. Automated reporter quantification in vivo: high-throughput screening method for reporter-based assays in zebrafish.

    Directory of Open Access Journals (Sweden)

    Steven L Walker

    Full Text Available Reporter-based assays underlie many high-throughput screening (HTS platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv. ARQiv differs from current "high-content" (e.g., confocal imaging-based whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1 Rapid; achieving true HTS capacities (i.e., >50,000 units per day, 2 Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5, and 3 Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1 Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors, 2 Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3 Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.

  17. Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study (United States)

    Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; ,; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.


    A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

  18. Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure

    Directory of Open Access Journals (Sweden)

    Ranjeva Raoul


    Full Text Available Abstract Background The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS, a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds. The aim of this work was to optimize the radiolabelling of Nod factors with 35S. Results The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step, the sulfate donor for the NodH sulfotransferase activity (second step. By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti. Conclusions By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO Nod factors to a specific radioactivity of about 800 Ci.mmol-1 with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules.

  19. Expression of N-acetyl-glucosamine-6-O-sulfotransferase in the endometrium of implantation window stage from infertile patients

    Institute of Scientific and Technical Information of China (English)

    Dai Hui-hua; Zhang Hong-mei; Liu Jia-yin


    Objective: To observe the expression of N-acetyl-glucosamine-6-O-sulfotransferase (GN-6-ST)in the endometrium during the window stage of implantation from infertile women before IVF-ET treatment, we compared the GN-6-ST gene expression level between the women with succeeded and failed implantation, and investigated the roles of selectin and its ligands in the embryo implantation.Methods: The hysteroscopy and endometrial biopsies were performed in patients prior to undergoing IVF-ET treatment in the IVF Center of the First Affiliated Hospital of Nanjing Medical University from July 2004 to March 2005.Fourteen patients who succeeded in implantation were taken as study group, while the 28 infertile patients with failed implantation served as control group.The RT-PCR method was used to detect the mRNA levels of N-ac-etyl-glucosamine-6-O-sulfotransferase in the endometrium during the window stage of imp-lantation of the women from both groups.Results: For these infertile patients with succeeded implantation, the average mRNA expression level of acetylglucosamine-6-O-sulfotransferase in the endometrium during the window stage of implantation was (0.65±0.33),while for those with failed implantation cycle, the average mRNA expression level was (0.41±0.36), which was significantly lower than that of study group, P<0.05.Conclusions: The combination of the selectin and ligands may play a role in the embryo implantation capacibility.

  20. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.

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    Piwen Wang

    Full Text Available BACKGROUND: The chemopreventive effect of green tea polyphenols, such as (--epigallocatechin-3-gallate (EGCG, has been well demonstrated in cell culture studies. However, a wide range of IC(50 concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature. METHODOLOGY/PRINCIPAL FINDINGS: We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP and deoxynucleic acid (DNA showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95. However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC(50 concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.

  1. Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays (United States)

    Breveglieri, Giulia; Travan, Anna; D’Aversa, Elisabetta; Cosenza, Lucia Carmela; Pellegatti, Patrizia; Guerra, Giovanni; Gambari, Roberto


    The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week. PMID:28235086

  2. An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

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    Marua Prevato

    Full Text Available Antibodies (Ab to neuraminidase (NA play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA which relies on hemagglutinin (HA mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs containing only the NA antigen (NA-PPs with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI assay. Both swine A/California/07/2009 (H1N1 and avian A/turkey/Turkey/01/2005 (H5N1 N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.

  3. A membrane vesicle-based assay to enable prediction of human biliary excretion. (United States)

    Colombo, Federico; Poirier, Hugo; Rioux, Nathalie; Montecillo, Maria Arias; Duan, Jianmin; Ribadeneira, Maria D


    1. Prediction of biliary excretion is a challenge due to the lack of in vitro assays. Our laboratory previously demonstrated a highly significant correlation between in vitro IC50 values against mrp2 using rat canalicular liver plasma membrane vesicles and in vivo biliary excretion (Colombo et al., 2012). This study explores the possibility of predicting in vivo biliary excretion in human using membrane vesicles prepared from MDCKII cells transfected with human ABCC2. 2. In vitro MRP2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in inside-out membrane vesicles isolated from MDCK-ABCC2 cells. CDCF uptake was time- and concentration-dependent (Km of 4.0 ± 1.2 µM and a Vmax of 7.8 ± 0.9 pmol/mg/min) and inhibited by benzbromarone and MK-571 with IC50 values of 1.2 and 7.6 µM, respectively. 3. A significant linear correlation (r(2 )= 0.790) between the in vitro IC50 values from the described MRP2 assay and in vivo biliary excretion in humans was observed using 11 well-documented drugs covering low to high biliary excretions. 4. This study demonstrates, for the first time, that inhibition of CDCF uptake in MDCKII-ABCC2 vesicles not only provides a screening assay to assess MRP2 drug-drug interaction potential, but is also predictive of human MRP2-mediated biliary excretion.

  4. Aptamer-based Sandwich Assay and its Clinical Outlooks for Detecting Lipocalin-2 in Hepatocellular Carcinoma (HCC) (United States)

    Lee, Kyeong-Ah; Ahn, Ji-Young; Lee, Sang-Hee; Singh Sekhon, Simranjeet; Kim, Dae-Ghon; Min, Jiho; Kim, Yang-Hoon


    We validated a single-stranded, DNA aptamer-based, diagnostic method capable of detecting Lipocalin-2 (LCN2), a biomarker from clinically relevant hepatocellular carcinoma (HCC) patient serum, in the sandwich assay format. Nine aptamers (LCN2_apta1 to LCN2_apta9) for LCN2 were screened with SELEX processes, and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen using surface plasmon resonance (SPR) and dot blotting analysis. The result of the proposed aptamer sandwich construction shows that LCN2 was sensitively detected in the concentration range of 2.5–500 ng mL−1 with a limit of detection of 0.6 ng mL−1. Quantitative measurement tests in HCC patients were run on straight serum and were compared with the performance of the conventional antibody-based ELISA kit. The aptamer sandwich assay demonstrated an excellent dynamic range for LCN2 at clinically relevant serum levels, covering sub-nanogram per mL concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. It consists of functionalization, hybridization and signal read-out, and no dilution is required. The results of the study demonstrate the capability of the aptamer sandwich assay platform for diagnosing HCC and its potential applicability to the point-of-care testing (POCT) system. PMID:26039737

  5. Multiple detection of single nucleotide polymorphism by microarray-based resonance light scattering assay with enlarged gold nanoparticle probes. (United States)

    Gao, Jiaxue; Ma, Lan; Lei, Zhen; Wang, Zhenxin


    The mapping of specific single nucleotide polymorphisms (SNPs) in patients' genome is a critical process for the development of personalized therapy. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of breast cancer related SNPs with high sensitivity and selectivity. After hybridization of the desired target single-stranded DNAs (ssDNAs) with the ssDNA probes on a microarray, the polyvalent ssDNA modified 13 nm gold nanoparticles (GNPs) are employed to label the hybridization reaction through the formation of a three-stranded DNA system. The H2O2-mediated enlargement of GNPs is then used to enhance the RLS signal. The microarray-based RLS assay provides a detection limit of 10 pM (S/N = 3) for the target ssDNA and determines an allele frequency as low as 1.0% in the target ssDNA cocktail. Combined with an asymmetric PCR technique, the proposed assay shows good accuracy and sensitivity in profiling 4 SNPs related to breast cancer of three selected cell lines.

  6. Assessment of combined antiandrogenic effects of binary parabens mixtures in a yeast-based reporter assay. (United States)

    Ma, Dehua; Chen, Lujun; Zhu, Xiaobiao; Li, Feifei; Liu, Cong; Liu, Rui


    To date, toxicological studies of endocrine disrupting chemicals (EDCs) have typically focused on single chemical exposures and associated effects. However, exposure to EDCs mixtures in the environment is common. Antiandrogens represent a group of EDCs, which draw increasing attention due to their resultant demasculinization and sexual disruption of aquatic organisms. Although there are a number of in vivo and in vitro studies investigating the combined effects of antiandrogen mixtures, these studies are mainly on selected model compounds such as flutamide, procymidone, and vinclozolin. The aim of the present study is to investigate the combined antiandrogenic effects of parabens, which are widely used antiandrogens in industrial and domestic commodities. A yeast-based human androgen receptor (hAR) assay (YAS) was applied to assess the antiandrogenic activities of n-propylparaben (nPrP), iso-propylparaben (iPrP), methylparaben (MeP), and 4-n-pentylphenol (PeP), as well as the binary mixtures of nPrP with each of the other three antiandrogens. All of the four compounds could exhibit antiandrogenic activity via the hAR. A linear interaction model was applied to quantitatively analyze the interaction between nPrP and each of the other three antiandrogens. The isoboles method was modified to show the variation of combined effects as the concentrations of mixed antiandrogens were changed. Graphs were constructed to show isoeffective curves of three binary mixtures based on the fitted linear interaction model and to evaluate the interaction of the mixed antiandrogens (synergism or antagonism). The combined effect of equimolar combinations of the three mixtures was also considered with the nonlinear isoboles method. The main effect parameters and interaction effect parameters in the linear interaction models of the three mixtures were different from zero. The results showed that any two antiandrogens in their binary mixtures tended to exert equal antiandrogenic activity

  7. World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays. (United States)

    Oh, Kwang W; Park, Chinsung; Namkoong, Kak; Kim, Jintae; Ock, Kyeong-Sik; Kim, Suhyeon; Kim, Young-A; Cho, Yoon-Kyoung; Ko, Christopher


    We report a practical world-to-chip microfluidic interfacing method with built-in valves suitable for microscale multichamber chip-based assays. One of the primary challenges associated with the successful commercialization of fully integrated microfluidic systems has been the lack of reliable world-to-chip microfluidic interconnections. After sample loading and sealing, leakage tests were conducted at 100 degrees C for 30 min and no detectable leakage flows were found during the test for 100 microchambers. To demonstrate the utility of our world-to-chip microfluidic interface, we designed a microscale PCR chip with four chambers and performed PCR assays. The PCR results yielded a 100% success rate with no contamination or leakage failures. In conclusion, we have introduced a simple and inexpensive microfluidic interfacing system for both sample loading and sealing with no dead volume, no leakage flow and biochemical compatibility.

  8. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay. (United States)

    Zhang, Huafei; An, Dong; Liu, Wei; Mao, Qunying; Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai


    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial ( Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

  9. Insights on antioxidant assays for biological samples based on the reduction of copper complexes-the importance of analytical conditions. (United States)

    Marques, Sara S; Magalhães, Luís M; Tóth, Ildikó V; Segundo, Marcela A


    Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples.

  10. Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions

    Directory of Open Access Journals (Sweden)

    Sara S. Marques


    Full Text Available Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E, uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples.

  11. p53 Promoter-based Reporter Gene in vitro Assays for Quick Assessment of Agents with Genotoxic Potential

    Institute of Scientific and Technical Information of China (English)

    Huaixing LI; Ke SHI; Ruiwen CHEN; Yan HE; Dan WU; Shuhan SUN


    The p53 promoter-based green fluorescent protein (GFP) and luciferase reporter gene assays have been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or 5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cells exposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of the cells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reporter gene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.

  12. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase. (United States)

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu


    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  13. Detection of pathogens in food using a SERS-based assay in just a few hours (United States)

    Shende, Chetan; Sengupta, Atanu; Huang, Hermes; Farquharson, Stuart


    In 2011 Escherichia, Listeria, and Salmonella species infected over 1.2 million people in the United States, resulting in over 23,000 hospitalizations and 650 deaths. In January 2013 President Obama signed into law the Food and Drug Administration (FDA) Food Safety Modernization Act (FSMA), which requires constant microbial testing of food processing equipment and food to minimize contamination and distribution of food tainted with pathogens. The challenge to preventing distribution and consumption of contaminated foods lies in the fact that just a few bacterial cells can rapidly multiply to millions, reaching infectious doses within a few days. Unfortunately, current methods used to detect these few cells rely on similar growth steps to multiply the cells to the point of detection, which also takes a few days. Consequently, there is a critical need for an analyzer that can rapidly extract and detect foodborne pathogens at 1000 colony forming units per gram of food in 1-2 hours (not days), and with a specificity that differentiates from indigenous microflora, so that false alarms are eliminated. In an effort to meet this need, we have been developing an assay that extracts such pathogens from food, selectively binds these pathogens, and produces surface-enhanced Raman spectra (SERS) when read by a Raman analyzer. Here we present SERS measurements of these pathogens in actual food samples using this assay.

  14. Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    SHENG Jianwu; HE Miao; YU Shaoqing; SHI Hanchang; QIAN Yi


    Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established and used to detect microcystin-LR (MC-LR) in drinking and surface waters.The concentration of coating antigen was 5 μg/mL,the dilution of monoclonal antibody MC10E7 was 1:3 000,the dilution of enzyme tracer (goat anti-mouse IgG-peroxidase) was 1:3 000,the standard concentration of MC-LR ranged from 0.001 μg/L to 30 μg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC) with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 μg/L and up to 5.1 μg/L.The quantitative detection range was from 0.03 μg/L to 3 μg/L,and the antibody had high specificity for [4-arginine]microcystins.It performed well in spite of the influence of the real samples.

  15. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Directory of Open Access Journals (Sweden)

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  16. Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and surface-enhanced Raman scattering. (United States)

    Kong, Xianming; Yu, Qian; Lv, Zhongpeng; Du, Xuezhong


    Tandem assays of protein and glucose in combination with mannose-functionalized Fe3 O4 @SiO2 and Ag@SiO2 tag particles have promising potential in effective magnetic separation and highly sensitive and selective SERS assays of biomaterials. It is for the first time that tandem assay of glucose is developed using SERS based on the Con A-sandwiched microstructures between the functionalized magnetic and tag particles.

  17. Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

    Directory of Open Access Journals (Sweden)

    Lina Ma


    Full Text Available This work reports on a simple and feasible fluorescence resonance energy transfer (FRET assay for detecting reactive oxygen species (ROS both in solution and living cell using polydopamine nanoparticle (PDA NP as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.

  18. Rapid detection of abrin in foods with an up-converting phosphor technology-based lateral flow assay (United States)

    Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Zhang, Pingping; Qiu, Jingfu; Yang, Ruifu; Zhou, Lei


    Abrin is a natural plant toxin found in the seeds of Abrus precatorius. It may be used for food poisoning or bioterrorism, seriously endangering public health. In this study, a reliable method for the rapid detection of abrin in foods was developed, based on an up-converting phosphor technology-based lateral flow assay (abrin-UPT-LFA). Nine high-affinity monoclonal antibodies (mAbs) against abrin were prepared, and the optimum mAbs (mAb-6F4 and mAb-10E11) were selected for use in the assay in double-antibody-sandwich mode. The assay was confirmed to be specific for abrin, with a detection sensitivity of 0.1 ng mL−1 for standard abrin solutions. Good linearity was observed for abrin quantitation from 0.1 to 1000 ng mL−1 (r = 0.9983). During the analysis of various abrin-spiked food samples, the assay showed strong sample tolerance and a satisfactory limit of detection for abrin (0.5–10 ng g−1 for solid and powdered samples; 0.30–0.43 ng mL−1 for liquid samples). The analysis of suspected food samples, from sample treatment to result feed-back, could be completed by non-professionals within 20 min. Therefore, the abrin-UPT-LFA is a rapid, sensitive, and reliable method for the on-site detection of abrin in foods. PMID:27703269

  19. Prognostic impact of chondroitin-4-sulfotransferase CHST11 in ovarian cancer. (United States)

    Oliveira-Ferrer, L; Heßling, A; Trillsch, F; Mahner, S; Milde-Langosch, K


    Ovarian cancer (OvCa) accounts for the highest tumor-related mortality among gynecological malignancies, but the underlying mechanisms are poorly understood. Glycosaminoglycans are abundantly present in ovarian tumors, and there is rising evidence that chondroitin sulfate (CS) as well as diverse carbohydrate sulfotransferases (CHSTs), the enzymes involved in the sulfation process of these structures, plays an important role in metastatic spread of tumor cells. mRNA expression levels of CHST3/7/11/12/13/15 were compared between malignant (86 OvCas) and non-malignant tumors (6 borderline tumors and 3 cystadenomas). CHST11 and CHST15 were further chosen for Western blot analysis in a cohort of 216 OvCas. Protein expression levels were correlated with clinicopathologic prognostic parameters and survival data. A significantly higher mRNA expression of CHST11, CHST12, and CHST15 was measured in ovarian cancer samples in comparison to non-malignant ones, and the same trend was observed for CHST13. For CHST3 and CHST7, no significant differences were found between the two groups. At protein level, high CHST11 expression was independently associated with unfavorable progression-free survival (PFS; p = 0.027). A similar trend was observed for CHST15, showing a nearly significant correlation between high expression levels and shorter recurrence-free survival in patients without macroscopic residual tumor after surgery (p = 0.053). We conclude that CHSTs involved in the synthesis of CS-A and CS-E might influence ovarian cancer progression, and we suggest CHST11 as independent unfavorable prognostic factor in this entity.

  20. 硫酸基转移酶与乳腺癌%Sulfotransferase and breast cancer

    Institute of Scientific and Technical Information of China (English)

    蒋一维; 周力恒


    Sulfotransferase (SULT) enzymes play important roles in the biosynthesis of estrogen.These enzymes catalyze the sulfation of estrogens to form biologically inactive molecules. Several studies have shown that Arg213His polymorphism in SULT1A1 gene may be a risk factor for breast cancer. In addition, a low mRNA expression of SULT has been significantly associated with reduced recurrence and improved overall survival in patients with estrogen receptor (ER)-positive tumors. Furthermore, SULT may be a crucial factor in the response to tamoxifen treatment. As an important part of estrogen synthetic pathways, SULTs are closely re lated to breast cancer.%硫酸基转移酶(SULT)在雌激素的合成通路中发挥着重要的作用,它将雌激素硫酸化形成无活性的硫酸化雌激素.众多研究表明SULT1A1在Arg213His上的突变可能与乳腺癌的发病存在一定的关联,而SULT mRNA的低表达与乳腺癌复发风险的降低及总生存率改善在雌激素受体阳性的患者中也显著相关.此外,SULT有可能将对三苯氧胺的治疗有预测作用.SULT作为雌激素形成中的重要通路与乳腺癌存在着密不可分的联系.

  1. Expression of Estrogen Sulfotransferase 1E1 and Steroid Sulfatase in Breast Cancer: A Immunohistochemical Study

    Directory of Open Access Journals (Sweden)

    D. Poisson Paré


    Full Text Available It is known that the steroid sulfatase (STS and the estrogen sulfotransferase (EST1E1 are commonly expressed in human breast carcinomas. STS and EST1E1 combined action could maintain the equilibrium between sulfated (inactive and unconjugated (active estrogens, which might have effects on development of hormone dependent breast cancer. We studied the expression of the STS and EST1E1 in 88 breast carcinomas and 57 adjacent non-malignant tissues by immunohistochemistry. The results were correlated with the tumor expression of estrogen receptor α (ER-α and β (ER-β, progesterone receptor A (PR-A and B (PR-B and the proliferation marker CDC47, the tumoral type and stage and the age at surgery. STS expression was higher in carcinoma specimens than in adjacent normal tissues, although not to a significant level (p = 0.064 and it was positively associated with CDC47 expression (p 0.05. These observations support the hypothesis that STS is overexpressed in breast cancer and associated with a worse prognosis. EST1E1 was observed for the first time in the nuclei of epithelial and tumoral cells. Tumor expression of EST1E1 was positively correlated with ER-β (p 0.01 and PR-B (p 0.05, two steroid receptors already associated with an improve prognosis for breast cancer. Controlling the STS overexpression in carcinomas could be a way to inhibit cancer growth. The significance of the association between EST1E1 and ER-β or PR-B should be further studied since these two receptors are transcription activators and may regulate the expression of protective enzymes like EST1E1.

  2. Salivary gland hypofunction in tyrosylprotein sulfotransferase-2 knockout mice is due to primary hypothyroidism.

    Directory of Open Access Journals (Sweden)

    Andrew D Westmuckett

    Full Text Available BACKGROUND: Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2. We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were (≈ 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine-induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes were restored to normal or near normal by thyroid hormone supplementation. CONCLUSIONS/SIGNIFICANCE: Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism.

  3. Melanoma Cell Adhesion and Migration Is Modulated by the Uronyl 2-O Sulfotransferase (United States)

    Nikolovska, Katerina; Spillmann, Dorothe; Haier, Jörg; Ladányi, Andrea; Stock, Christian; Seidler, Daniela G.


    Although the vast majority of melanomas are characterized by a high metastatic potential, if detected early, melanoma can have a good prognostic outcome. However, once metastasised, the prognosis is bleak. We showed previously that uronyl-2-O sulfotransferase (Ust) and 2-O sulfation of chondroitin/dermatan sulfate (CS/DS) are involved in cell migration. To demonstrate an impact of 2-O sulfation in metastasis we knocked-down Ust in mouse melanoma cells. This significantly reduced the amount of Ust protein and enzyme activity. Furthermore, in vitro cell motility and adhesion were significantly reduced correlating with the decrease of cellular Ust protein. Single cell migration of B16VshUst(16) cells showed a decreased cell movement phenotype. The adhesion of B16V cells to fibronectin depended on α5β1 but not αvβ3 integrin. Inhibition of glycosaminoglycan sulfation or blocking fibroblast growth factor receptor (FgfR) reduced α5 integrin in B16V cell lines. Interestingly, FgfR1 expression and activation was reduced in Ust knock-down cells. In vivo, pulmonary metastasis of B16VshUst cells was prevented due to a reduction of α5 integrin. As a proof of concept UST knock-down in human melanoma cells also showed a reduction in ITGa5 and adhesion. This is the first study showing that Ust, and consequently 2-O sulfation of the low affinity receptor for FgfR CS/DS, reduces Itga5 and leads to an impaired adhesion and migration of melanoma cells. PMID:28107390

  4. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)


    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  5. An improved sensitive assay for the detection of PSP toxins with neuroblastoma cell-based impedance biosensor. (United States)

    Zou, Ling; Wu, Chunsheng; Wang, Qin; Zhou, Jie; Su, Kaiqi; Li, Hongbo; Hu, Ning; Wang, Ping


    Paralytic shellfish poisoning (PSP) toxins are well-known sodium channel-blocking marine toxins, which block the conduction of nerve impulses and lead to a series of neurological disorders symptoms. However, PSP toxins can inhibit the cytotoxicity effect of compounds (e.g., ouabain and veratridine). Under the treatment of ouabain and veratridine, neuroblastoma cell will swell and die gradually, since veratridine causes the persistent inflow of Na(+) and ouabain inhibits the activity of Na(+)/K(+)-ATPases. Therefore, PSP toxins with antagonism effect can raise the chance of cell survival by blocking inflow of Na(+). Based on the antagonism effect of PSP toxins, we designed an improved cell-based assay to detect PSP toxins using a neuroblastoma cell-based impedance biosensor. The results demonstrated that this biosensor showed high sensitivity and good specificity for saxitoxins detection. The detection limit of this biosensor was as low as 0.03 ng/ml, which was lower than previous reported cell-based assays and mouse bioassays. With the improvement of biosensor performance, the neuroblastoma cell-based impedance biosensor has great potential to be a universal PSP screening method.

  6. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    Directory of Open Access Journals (Sweden)

    Belete Teferedegne

    Full Text Available A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses. The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours. In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50. Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

  7. Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay. (United States)

    Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui


    Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

  8. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    Milk contains many different proteins of which the larger constituents like the caseins and major whey constituents are well characterized. We have for some time been studying the structure and function of proteins associated with the milk fat globule membrane like lactadherin, MUC1/15, xanthine...... oxidoreductase along with minor whey constituents like osteopontin, EPV20 etc. The enterocyte migration rate is a key parameter in maintaining intestinal homeostasis and intestinal repair when recovering from infection or intestinal diseases like Crohns and ulcerative colitis. We developed a novel in vitro wound...... healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  9. Polymerase chain reaction-based assays for the diagnosis of human brucellosis. (United States)

    Wang, Ying; Wang, Zhanli; Zhang, Yaxian; Bai, Liyun; Zhao, Yue; Liu, Chunfang; Ma, An; Yu, Hui


    Polymerase chain reaction (PCR) is an in vitro technique for the nucleic acid amplification, which is commonly used to diagnose infectious diseases. The use of PCR for pathogens detection, genotyping and quantification has some advantages, such as high sensitivity, high specificity, reproducibility and technical ease. Brucellosis is a common zoonosis caused by Brucella spp., which still remains as a major health problem in many developing countries around the world. The direct culture and immunohistochemistry can be used for detecting infection with Brucella spp. However, PCR has the potential to address limitations of these methods. PCR are now one of the most useful assays for the diagnosis in human brucellosis. The aim of this review was to summarize the main PCR techniques and their applications for diagnosis and follow-up of patients with brucellosis. Moreover, advantages or limitation of the different PCR methods as well as the evaluation of PCR results for treatment and follow-up of human brucellosis were also discussed.

  10. Tetrazolium-based assays for cellular viability: a critical examination of selected parameters affecting formazan production. (United States)

    Vistica, D T; Skehan, P; Scudiero, D; Monks, A; Pittman, A; Boyd, M R


    The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.

  11. High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

    Directory of Open Access Journals (Sweden)

    Du Yuefen


    Full Text Available Abstract Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA. Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

  12. Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay for the Analysis of Jasmonates in Plants

    Institute of Scientific and Technical Information of China (English)

    Aixing Deng; Weiming Tan; Suping He; Wei Liu; Tiegui Nan; Zhaohu Li; Baomin Wang; Qing X.Li


    Methyl jasmonate (MeJA) and its free-acid form,jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants.In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples,a monoclonal antibody (MAb) (designated as MAb 3E5D7C4B6) against MeJA was derived from a JAbovine serum albumin (BSA) conjugate as an immunogen.The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 x 10-9 M.MAb3E5D7C4B6 is very specific to MeJA.It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA),conventional and simplified indirect competitive ELISAs (icELISA).JA was derivatized into MeJA for the ELISA analysis.The IC50 value and detection range for MeJA were,respectively,34 and 4-257 ng/mL by the conventional icELISA,21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA.The dcELISA was more sensitive than either the conventional or simplified icELISA.The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves.The increased jasmonates content indicated its role in response to the drought stress and pathogens.

  13. Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

    Directory of Open Access Journals (Sweden)

    Manuel A F V Gonçalves

    Full Text Available Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and

  14. Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples. (United States)

    Chow, Maggie L; Winn, Mary E; Li, Hai-Ri; April, Craig; Wynshaw-Boris, Anthony; Fan, Jian-Bing; Fu, Xiang-Dong; Courchesne, Eric; Schork, Nicholas J


    Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples.

  15. Evaluation of the IrisPlex DNA-based eye color prediction assay in a United States population. (United States)

    Dembinski, Gina M; Picard, Christine J


    DNA phenotyping is a rapidly developing area of research in forensic biology. Externally visible characteristics (EVCs) can be determined based on genotype data, specifically based on single nucleotide polymorphisms (SNPs). These SNPs are chosen based on their association with genes related to the phenotypic expression of interest, with known examples in eye, hair, and skin color traits. DNA phenotyping has forensic importance when unknown biological samples at a crime scene do not result in a criminal database hit; a phenotypic profile of the sample can therefore be used to develop investigational leads. IrisPlex, an eye color prediction assay, has previously shown high prediction rates for blue and brown eye color in a Dutch European population. The objective of this work was to evaluate its utility in a North American population. We evaluated six SNPs included in the IrisPlex assay in population sample collected from a USA college campus. We used a quantitative method of eye color classification based on (RGB) color components of digital photographs of the eye taken from each study volunteer so that each eye was placed in one of three eye color categories: brown, intermediate, or blue. Objective color classification was shown to correlate with basic human visual determination making it a feasible option for use in future prediction assay development. Using these samples and various models, the maximum prediction accuracies of the IrisPlex system after allele frequency adjustment was 58% and 95% brown and blue eye color predictions, respectively, and 11% for intermediate eye colors. Future developments should include incorporation of additional informative SNPs, specifically related to the intermediate eye color, and we recommend the use of a Bayesian approach as a prediction model as likelihood ratios can be determined for reporting purposes.

  16. A short-term sublethal toxicity assay with zebra fish based on preying rate and its integration with mortality. (United States)

    Abdel-moneim, Ahmed; Moreira-Santos, Matilde; Ribeiro, Rui


    Contaminant-induced feeding inhibition has direct and immediate consequences at higher levels of biological organization, by depressing the population consumption and thus hampering ecosystem functioning (e.g. grazing, organic matter decomposition). Thus, similarly to lethality and avoidance, feeding is mechanistically linked to ecosystem processes and is therefore an unequivocal ecologically meaningful response. The objective of the present study was to develop a short-term assay with the small freshwater fish Danio rerio, based on feeding. For this, a methodology to easily and precisely quantify feeding was first optimized: each fish was allowed to prey on ten live Daphnia magna juveniles, for 1h, just before the end of a 48-h exposure test period. Secondly, copper sensitivity of feeding relatively to survival and growth was evaluated. At the growth EC20 (40 μg L(-1)), feeding was inhibited by 53%, and at the feeding EC50 (36 μg L(-1)), mortality was negligible (1.3%). Integrating feeding and survival revealed a 97% depression in the population consumption at the LC50 (61 μg L(-1)). Thirdly, the influence of pH, conductivity and hardness on the feeding background variability was assessed by assaying waters collected at eight reference sites and was found to be negligible, within tested ranges. Fourthly, feeding assays with natural waters contaminated with acid mine drainage confirmed the integration of lethality and feeding to be pertinent at estimating contaminant effects at higher levels of biological organization.

  17. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification. (United States)

    Duan, Ya-Bing; Ge, Chang-Yan; Zhang, Xiao-Ke; Wang, Jian-Xin; Zhou, Ming-Guo


    Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP) with hydroxynaphthol blue dye (HNB). The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3) ng µL(-1) of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2) ng µL(-1)). Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2%) were confirmed as positive by LAMP, 172 (90.1%) positive by the tissue separation, while 147 (77.0%) positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  18. A 1,536-well-based kinetic HTS assay for inhibitors of Schistosoma mansoni thioredoxin glutathione reductase. (United States)

    Lea, Wendy A; Jadhav, Ajit; Rai, Ganesha; Sayed, Ahmed A; Cass, Cynthia L; Inglese, James; Williams, David L; Austin, Christopher P; Simeonov, Anton


    Abstract: Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-mul final volume assay is based on the Ellman reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.

  19. Determining the concentration of procalcitonin using a magnetic particles-based chemiluminescence assay for the clinical diagnosis of sepsis. (United States)

    Qi, Suwen; Li, Qiaoliang; Rao, Wei; Liu, Xinyu; Yin, Li; Zhang, Huisheng


    Our objective is to develop an assay based on magnetic particles (MPs) to determine the concentration of procalcitonin (PCT) using a chemiluminescence immunoassay (CLIA). Fluorescein isothiocyanate (FITC) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) were used to label two different anti-procalcitonin (PCT) monoclonal antibodies. The labeled antibodies, the PCT antigen, and the anti-FITC antibody-coated MPs formed a double-sandwiched immunocomplex. The measured relative light units (RLUs) of ABEI in the substrate solution were directly proportional to the amount of PCT present in the samples. The proposed method was linear to 600 ng/mL with a detection limit of 0.03 ng/mL. The coefficient of variation (CV) was <5% and <6% for the intra- and inter-assay precision, respectively. The average recoveries were between 95 and 107%. The linearity-dilution effect gave a linear correlation coefficient of 0.9912. This proposed assay provided an alternative method to quantitatively measure PCT in serum for the diagnosis of sepsis.

  20. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites. (United States)

    Fu, Ying; Lu, Danqin; Lin, Bin; Sun, Qianqian; Liu, Kai; Xu, Lili; Zhang, Shengping; Hu, Chen; Wang, Chuangui; Xu, Zhiai; Zhang, Wen


    Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

  1. Ultrasensitive and quantitative gold nanoparticle-based immunochromatographic assay for detection of ochratoxin A in agro-products. (United States)

    Majdinasab, Marjan; Sheikh-Zeinoddin, Mahmoud; Soleimanian-Zad, Sabihe; Li, Peiwu; Zhang, Qi; Li, Xin; Tang, Xiaoqian


    In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensitivity of this technique is questioned. In order to overcome these limitations, an ultrasensitive and quantitative immunochromatographic assay (ICA) for rapid and sensitive quantitation of ochratoxin A (OTA) was developed. The assay was based on a competitive format and its sensitivity was improved by using a sensitive and selective OTA monoclonal antibody (OTA-mAb). The visible ICA results were obtained within 15 min, and in addition to visual examination, they were read by the rapid color intensity portable strip reader. The visual and computational detection limits (vLOD and cLOD, respectively) for ochratoxin A were 0.2 and 0.25 ng mL(-1), respectively. These values were lower than those reported by previous studies in a range 5-2500 folds. For validation, contaminated samples including wheat, maize, rice and soybean were assayed by ICA and a standard high performance liquid chromatography (HPLC). The results were in good agreement for both ICA and HPLC methods. The average recoveries of the HPLC were in the range 72-120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method.

  2. Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium. (United States)

    Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin


    Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

  3. Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores. (United States)

    Nikiforov, Theo T; Beechem, Joseph M


    We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.

  4. A label-free G-quadruplex-based luminescent switch-on assay for the selective detection of histidine. (United States)

    He, Hong-Zhang; Wang, Modi; Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Qiu, Jian-Wen; Ma, Dik-Lung


    A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5'-TG4AG3TG4AG3TG4A2G2-3')/cupric ion (Cu(2+)) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu(2+) ions due to the Cu(2+)-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu(2+) ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 μM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated.

  5. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid. (United States)

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher


    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity.

  6. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Ya-Bing Duan

    Full Text Available Botrytis cinerea is a devastating plant pathogen that causes grey mould disease. In this study, we developed a visual detection method of B. cinerea based on the Bcos5 sequence using loop-mediated isothermal amplification (LAMP with hydroxynaphthol blue dye (HNB. The LAMP reaction was optimal at 63 °C for 45 min. When HNB was added prior to amplification, samples with B. cinerea DNA developed a characteristic sky blue color after the reaction but those without DNA or with DNA of other plant pathogenic fungi did not. Results of HNB staining method were reconfirmed when LAMP products were subjected to gel electrophoresis. The detection limit of this LAMP assay for B. cinerea was 10(-3 ng µL(-1 of genomic DNA per reaction, which was 10-fold more sensitive than conventional PCR (10(-2 ng µL(-1. Detection of the LAMP assay for inoculum of B. cinerea was possible in the inoculated tomato and strawberry petals. In the 191 diseased samples, 180 (94.2% were confirmed as positive by LAMP, 172 (90.1% positive by the tissue separation, while 147 (77.0% positive by PCR. Because the LAMP assay performed well in aspects of sensitivity, specificity, repeatability, reliability, and visibility, it is suitable for rapid detection of B. cinerea in infected plant materials prior to storage and during transportation, such as cut flowers, fruits and vegetables.

  7. Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

    Directory of Open Access Journals (Sweden)

    Pace Umberto


    Full Text Available Abstract Background We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i products of the proteolytic (e.g. tryptic digestion of the protein to be identified and (ii unique to the target protein, as far as one can know from the published sequences. Results In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. Conclusion The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plate


    AbstractThree related forms of phenol sulfotransferase (PSULT), thermostable ST1A2 (SULT1A2hum) and ST1A3 (SULT1A1hum) and a thermolabile TL-PST (SULT1A3hum), are known to exist in human livers. Thermostable forms, whose activities are polymorphically distributed, hav...

  9. Sulfotransferase activity in plucked hair follicles predicts response to topical minoxidil in the treatment of female androgenetic alopecia. (United States)

    Roberts, Janet; Desai, Nisha; McCoy, John; Goren, Andy


    Two percent topical minoxidil is the only US Food and Drug Administration-approved drug for the treatment of female androgenetic alopecia (AGA). Its success has been limited by the low percentage of responders. Meta-analysis of several studies reporting the number of responders to 2% minoxidil monotherapy indicates moderate hair regrowth in only 13-20% of female patients. Five percent minoxidil solution, when used off-label, may increase the percentage of responders to as much as 40%. As such, a biomarker for predicting treatment response would have significant clinical utility. In a previous study, Goren et al. reported an association between sulfotransferase activity in plucked hair follicles and minoxidil response in a mixed cohort of male and female patients. The aim of this study was to replicate these findings in a well-defined cohort of female patients with AGA treated with 5% minoxidil daily for a period of 6 months. Consistent with the prior study, we found that sulfotransferase activity in plucked hair follicles predicts treatment response with 93% sensitivity and 83% specificity. Our study further supports the importance of minoxidil sulfation in eliciting a therapeutic response and provides further insight into novel targets for increasing minoxidil efficacy.

  10. Role of Heparan Sulfate 2-O-Sulfotransferase in Prostate Cancer Cell Proliferation, Invasion, and Growth Factor Signaling

    Directory of Open Access Journals (Sweden)

    Brent W. Ferguson


    Full Text Available Heparan-sulfate proteoglycans (HSPGs are required for maximal growth factor signaling in prostate cancer progression. The degree of sulfate modification on the covalently attached heparan sulfate (HS chains is one of the determining factors of growth factor-HSPG interactions. Sulfate groups are transferred to HS chains via a series of O-sulfotransferases. In the present study, we demonstrate that Heparan sulfate 2-O-sulfotransferase (2OST is essential for maximal proliferation and invasion of prostate cancer cells in the LNCaP-C4-2B model. We also show that a decrease in invasion due to 2OST siRNA is associated with an increase in actin and E-cadherin accumulation at the cell surface. 2OST expression correlates with increasing metastatic potential in this model. We demonstrate that 2OST expression is upregulated by the stress-inducible transcription factors HIF1α, ATF2, and NFκB. Chromatin immunoprecipitation analysis suggests that HIF1α and ATF2 act directly on the 2OST promoter, while NFκB acts indirectly.

  11. Characterization of bovine phenol sulfotransferases: evidence of a major role for SULT1B1 in the liver. (United States)

    Choughule, Kanika V; Locuson, Charles W; Coughtrie, Michael W H


    1. Cattle are an important component of the human food chain. Drugs used either legally or illegally in cattle may therefore enter the food chain and it is thus important to understand pathways of drug metabolism in this species, including sulfation catalyzed by the sulfotransferases (SULTs). 2. In this study, we have analyzed the sulfation of 4-nitrophenol and other compounds in male and female bovine liver and characterized recombinant bovine SULT isoforms 1A1 and 1B1 expressed in Escherichia coli. 3. We found that, in contrast to most other mammalian species, the major phenol sulfotransferase SULT1A1 is not expressed in bovine liver. Rather SULT1B1 seems to be a major form in both male and female bovine liver. 4. We also identified kinetic differences between bovine and human SULT1A1 and, using the human SULT1A1 crystal structure, identified two amino acid positions in the active site of bovine SULT1A1 (Ile89Val and Phe247Val) that may be responsible for these differences.

  12. Ablation of GalNAc-4-sulfotransferase-1 enhances reproduction by altering the carbohydrate structures of luteinizing hormone in mice. (United States)<