The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform
Yu Zhen - One of the best experts on this subject based on the ideXlab platform.
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enumeration of microalgae in different cell growth phases with sandwich hybridization integrated with Nuclease Protection Assay
Indian Journal of Geo-Marine Sciences, 2018Co-Authors: Yu ZhenAbstract:Sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH), which takes advantage of high copy number of rRNA molecules in cells, has been developed to quantify microalgae. Seven microalgae in different cell growth phases were analysed by NPA-SH to find how the amounts of RNA had influence on enumeration of microalgae with NPA-SH. It was found that the calibration curves of NPA-SH from samples in different growth phases would influence the quantification results. Although the cell densities enumerated with the calibration curves from stable samples were higher than those by microscopy in the logarithmic growth phase, when rRNA greatly accumulated, it was very significant to detect samples from natural marine environments with NPA-SH. The results from this method can reflect the growth potentiality of microalgae, which will present the evidence for pre-warning of harmful algal blooms.
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Detection of Prorocentrum minimum (Pavillard) Schiller with the electrochemiluminescence-molecular probe
Huan jing ke xue= Huanjing kexue, 2012Co-Authors: Xia Zhu, Yu Zhen, Zhen-ming ChiAbstract:In this study, the electrochemiluminescence-molecular probe (ECL-MP) was established based on the sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH) improved by electrochemiluminescence (ECL). It can be used for detecting Prorocentrum minimum (Pavillard) Schiller qualitatively and quantitatively. NPA probes of P. minimum were designed based on the NPA-SH. After labeled with Ru (bpy)3(2+) and biotin, they bounded with streptavidin-coupled magnetic beads and generated electrochemiluminescence in an ECL analyzer. The ECL counts per second (CPS) was drawn against the cell number obtained from microscopy to establish a calibration curve of P. minimum. The results showed that the labeled NPA probes had good specificity and practicability, the optimal usage of magnetic beads was 4 microg for detecting 20 microL hybridization mixture, the detection range of P. minimum cell numbers was 6.25 x 10(2) - 4 x 10(4), and there was no significant difference between the data gained from ECL-MP and microscopy with 95% confidence level (t-test) when treated with cultured and mixed samples. ECL-MP was a convenient new tool for rapid assessment of P. minimum in marine environment.
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Quantification methods for Alexandrium catenella, a toxic dinoflagellate: Comparison of competitive enzyme-linked immunosorbent Assay and sandwich hybridization integrated with a Nuclease Protection Assay
Harmful Algae, 2011Co-Authors: Yu Zhen, Tiezhu Mi, Zhigang YuAbstract:Abstract Alexandrium catenella (Whedon et Kofoid) Balech, a toxic dinoflagellate, is a bloom-forming planktonic species in cold water coastal regions. It produces strong paralytic shellfish poisoning (PSP) toxins which are transmitted via tainted shellfish. These toxins can affect humans, other mammals, fish and birds. In this study, polyclonal antiserum against A. catenella was produced, and a competitive enzyme-linked immunosorbent Assay (cELISA) was developed to detect A. catenella. The antiserum against A. catenella showed good specificity, the linear detection range was relatively large, between 38 and 600,000 cells. In addition, specific probes were designed to target the small subunit ribosomal RNA (SSU rRNA) of A. catenella, and quantitative sandwich hybridization integrated with a Nuclease Protection Assay (NPA-SH) was established in order to identify and quantify A. catenella. The NPA-SH Assay did not show good specificity as well as cELISA, by which A. catenella and A. tamarense could not be distinguished. Samples in different cell growth phases were analyzed with cELISA and NPA-SH. The results showed that the cell concentration calculated by cELISA was very similar with microscopy, while that of NPA-SH was sometimes higher than that of microscopy, especially in log phase. Comparing the two methods, both Assays allow rapid identification of A. catenella without time-consuming microscopy when multiple sites need to be considered in routine monitoring. Meanwhile, cELISA was more specific and accurate in detection of A. catenella than NPA-SH.
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Detection of several harmful algal species by sandwich hybridization integrated with a Nuclease Protection Assay
Harmful Algae, 2009Co-Authors: Yu ZhenAbstract:The frequent occurrence of harmful algal blooms (HABs) is a pressing topic in marine research. An integrated sandwich hybridization and Nuclease Protection Assay was established to qualitatively and quantitatively detect 12 harmful algal species. This method demonstrated good reliability, specificity and accuracy for analyzing samples from individual and mixed cultures, as well as field collection, and cell volumes were positively correlated to the slopes of calibration curves. The lowest quantitative detection limits were those concentrations observed during blooms; thus, this technique provides an efficient alternative to microscopy for rapid identification and quantitation of harmful algal species and could be routinely used to monitor phytoplankton in field surveys.
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Detection of Phaeocystis globosa using sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH).
Journal of environmental sciences (China), 2008Co-Authors: Yu ZhenAbstract:Abstract Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China. In this study, sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH) was used to qualitatively and quantitatively detect P. globosa. Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields. The linear regression equation for P. globosa was obtained, and the lowest detection number of cells was 1.8 × 104 cells. Statistics showed that there was no distinct difference between the results of detecting the microalgae by NPA-SH and traditional microscopy. This technique has good reliability, accuracy, and can give a remarkably high sample processing rate. Sandwich hybridization integrated with Nuclease Protection Assay will provide an efficient alternative to microscopic method for monitoring and investigating the bloom of P. globosa.
Michael J. Bannon - One of the best experts on this subject based on the ideXlab platform.
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GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.
Neurochemistry international, 1993Co-Authors: Yue Xia, Gregory Kapatos, Michael S. Poosch, Christopher J. Whitty, Michael J. BannonAbstract:A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by Nuclease Protection Assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by Nuclease Protection Assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.
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Quantitation of rat dopamine transporter mRNA : effects of cocaine treatment and withdrawal
Journal of neurochemistry, 1992Co-Authors: Yue Xia, Gregory Kapatos, Dennis J. Goebel, Michael J. BannonAbstract:Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive Nuclease Protection Assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.
Gregory Kapatos - One of the best experts on this subject based on the ideXlab platform.
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Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA
Neurochemistry international, 1995Co-Authors: Kei Hirayama, Gregory KapatosAbstract:6-Pyruvoyl tetrahydropterin synthase catalyzes the second step in the biosynthesis of tetrahydrobiopterin. In the present study, the reverse transcription-polymerase chain reaction technique was used to clone a portion of 6-pyruvoyl tetrahydropterin synthase cDNA from rat pineal gland RNA. The sequence of this cDNA was found to be essentially identical to that previously reported for the rat liver. 6-Pyruvoyl tetrahydropterin synthase mRNA levels in various rat tissues, including the brain, were then analyzed by Northern blot and Nuclease Protection Assay. A single 1.35 kb transcript of 6-pyruvoyl tetrahydropterin synthase mRNA was detected by Northern blot analysis in the rat adrenal gland, brain-stem, and liver. Quantitation by Nuclease Protection Assay demonstrated that 6-pyruvoyl tetrahydropterin synthase mRNA was most abundant in the adrenal gland, kidney, and pineal gland (19.5-25.5 amol/microgram RNA). Relatively homogeneous levels of 6-pyruvoyl tetrahydropterin synthase mRNA were found in various brain regions including the cerebellum, substantia nigra and locus coeruleus (4.12-12 amol/microgram RNA). In the adrenal gland, 6-pyruvoyl tetrahydropterin synthase and tyrosine hydroxylase mRNAs were elevated between 3 and 4-fold 24 h after a single dose of reserpine (10 mg/kg), a treatment known to increase tetrahydrobiopterin levels in this tissue. This result suggests that although 6-pyruvoyl tetrahydropterin synthase is not believed to be rate-limiting in the tetrahydrobiopterin biosynthetic pathway, control of gene expression for this enzyme may play an essential role in regulating the synthesis of this important cofactor.
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Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.
Journal of neurochemistry, 1993Co-Authors: Kei Hirayama, Stephen I. Lentz, Gregory KapatosAbstract:: GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, Nuclease Protection Assay, and in situ hybridisation. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1,2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by Nuclease Protection Assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2-and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by Nuclease Protection Assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.
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GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.
Neurochemistry international, 1993Co-Authors: Yue Xia, Gregory Kapatos, Michael S. Poosch, Christopher J. Whitty, Michael J. BannonAbstract:A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by Nuclease Protection Assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by Nuclease Protection Assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.
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Quantitation of rat dopamine transporter mRNA : effects of cocaine treatment and withdrawal
Journal of neurochemistry, 1992Co-Authors: Yue Xia, Gregory Kapatos, Dennis J. Goebel, Michael J. BannonAbstract:Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive Nuclease Protection Assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a greater than 40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72-h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down-regulation of dopamine transporter gene expression in dopamine neurons.
Qingsong Cai - One of the best experts on this subject based on the ideXlab platform.
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Detection of two diatoms using sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH)
Hydrobiologia, 2006Co-Authors: Yu Zhen, Qingsong CaiAbstract:Nuclease Protection Assay (NPA) probes were designed to target the rRNA of Chaetoceros curvisetus and Skeletonema costatum, and quantitative sandwich hybridization integrated with Nuclease Protection Assay (NPA-SH) was developed to detect C. curvisetus and S. costatum in culture and field samples in Jiaozhou Bay, China. The specificity and validity of the NPA-SH technique were tested with cultured pure strains, mixed strains and field samples, and by comparison with that of microscopy observation. The linear detection range for C. curvisetus was 4.2 × 104 to 1.2 × 106 cells with a detection limit of 42 cells ml−1. The linear range for S. costatum was 6.0 × 104 to 1.0 × 106 cells with a detection limit of 60 cells ml−1. The NPA-SH in this study provides a convenient tool for rapid assessment of HAB species in marine environments.
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Detection of two Prorocentrum species using sandwich hybridization integrated with Nuclease Protection Assay
Harmful Algae, 2006Co-Authors: Qingsong Cai, Yu ZhenAbstract:Abstract A novel Assay method using Nuclease Protection Assay integrated with sandwich hybridization (NPA-SH) for qualitative and quantitative detection of microalgae has been developed. Two species-specific Nuclease-Protection-Assay (NPA) probes targeted 28S ribosomal RNA of Prorocentrum minimum and Prorocentrum micans , respectively, were designed in this study. The Assay consists of S1 Nuclease Protection, sandwich hybridization and signal detection. The specificity of the probes was verified with cultured algae in the laboratory and field sample from Jiaozhou Bay, and the quantity by NPA-SH analysis showed good agreement with that of cell-counting with a light microscope. The optical absorbance of probe binding on the target showed good linear fit with cell amount. A standard curve for P. minimum was established to correlate the optical absorbance to cell density on a basis in the linear range between 15 and 475 cells ml −1 seawater, and the equation deducted was ‘ y = 0.0053 × x + 0.0658’ ( R 2 = 0.992, n = 4). The Assay was sensitive to detect 15 cells ml −1 seawater. And for P. micans , with linear range between 0.6 and 20 cells ml −1 seawater, the equation deducted was ‘ y = 0.1174 × x + 0.1106’ ( R 2 = 0.996, n = 4); the Assay was sensitive to detect less than 1 cell ml −1 seawater. The inter-Assay coefficients of variation (CVs) were 12.4 and 10.9%, respectively. The good specificity, sensitivity and reproducibility of the NPA-SH implied that this new technique could be extremely useful for qualitative and quantitative Assay of P. minimum and P. micans at low abundance.
Gustavo F. Doncel - One of the best experts on this subject based on the ideXlab platform.
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a quantitative multiplex Nuclease Protection Assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation
Toxicology and Applied Pharmacology, 2015Co-Authors: Raina N. Fichorova, Kevin Mendonca, Hidemi S. Yamamoto, Ryan Murray, Neelima Chandra, Gustavo F. DoncelAbstract:Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative Nuclease Protection Assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.
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A quantitative multiplex Nuclease Protection Assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.
Toxicology and applied pharmacology, 2015Co-Authors: Raina N. Fichorova, Kevin Mendonca, Hidemi S. Yamamoto, Ryan Murray, Neelima Chandra, Gustavo F. DoncelAbstract:Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative Nuclease Protection Assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P
