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Sample records for assay reveals munc18-1

  1. Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites*

    PubMed Central

    Smyth, Annya M.; Yang, Lei; Martin, Kirsty J.; Hamilton, Charlotte; Lu, Weiping; Cousin, Michael A.; Rickman, Colin; Duncan, Rory R.

    2013-01-01

    Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites. PMID:23223447

  2. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation

    PubMed Central

    Giles, Nichole; Morrow, Isabel C.; Collins, Brett M.

    2016-01-01

    Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body–like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-SynWT and that of the Parkinson’s disease–causing α-SynA30P mutant, an effect rescued by Munc18-1WT expression, indicative of chaperone activity. Coexpression of the α-SynA30P mutant with Munc18-1 reduced the number of α-SynA30P aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. PMID:27597756

  3. Munc18-1 is a molecular chaperone for α-synuclein, controlling its self-replicating aggregation.

    PubMed

    Chai, Ye Jin; Sierecki, Emma; Tomatis, Vanesa M; Gormal, Rachel S; Giles, Nichole; Morrow, Isabel C; Xia, Di; Götz, Jürgen; Parton, Robert G; Collins, Brett M; Gambin, Yann; Meunier, Frédéric A

    2016-09-12

    Munc18-1 is a key component of the exocytic machinery that controls neurotransmitter release. Munc18-1 heterozygous mutations cause developmental defects and epileptic phenotypes, including infantile epileptic encephalopathy (EIEE), suggestive of a gain of pathological function. Here, we used single-molecule analysis, gene-edited cells, and neurons to demonstrate that Munc18-1 EIEE-causing mutants form large polymers that coaggregate wild-type Munc18-1 in vitro and in cells. Surprisingly, Munc18-1 EIEE mutants also form Lewy body-like structures that contain α-synuclein (α-Syn). We reveal that Munc18-1 binds α-Syn, and its EIEE mutants coaggregate α-Syn. Likewise, removal of endogenous Munc18-1 increases the aggregative propensity of α-Syn(WT) and that of the Parkinson's disease-causing α-Syn(A30P) mutant, an effect rescued by Munc18-1(WT) expression, indicative of chaperone activity. Coexpression of the α-Syn(A30P) mutant with Munc18-1 reduced the number of α-Syn(A30P) aggregates. Munc18-1 mutations and haploinsufficiency may therefore trigger a pathogenic gain of function through both the corruption of native Munc18-1 and a perturbed chaperone activity for α-Syn leading to aggregation-induced neurodegeneration. © 2016 Chai et al.

  4. The Munc18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

    PubMed Central

    Kasula, Ravikiran; Bademosi, Adekunle T.; Harper, Callista B.; Morrow, Isabel C.; Hosy, Eric; Collins, Brett M.; Choquet, Daniel

    2016-01-01

    Munc18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc18-1Δ317-333) in PC12 cells engineered to knockdown Munc18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Munc18-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc18-1WT mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc18-1Δ317-333, suggesting that a conformational change in the Munc18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNARE complex formation during priming. PMID:27646276

  5. Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner.

    PubMed

    Cijsouw, Tony; Weber, Jens P; Broeke, Jurjen H; Broek, Jantine A C; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid; Verhage, Matthijs; Toonen, Ruud F

    2014-03-03

    Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon-synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.

  6. Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

    PubMed Central

    Cijsouw, Tony; Weber, Jens P.; Broeke, Jurjen H.; Broek, Jantine A.C.; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid

    2014-01-01

    Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity. PMID:24590174

  7. Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1.

    PubMed

    Schmitz, Sabine K; King, Cillian; Kortleven, Christian; Huson, Vincent; Kroon, Tim; Kevenaar, Josta T; Schut, Desiree; Saarloos, Ingrid; Hoetjes, Joost P; de Wit, Heidi; Stiedl, Oliver; Spijker, Sabine; Li, Ka Wan; Mansvelder, Huibert D; Smit, August B; Cornelisse, Lennart Niels; Verhage, Matthijs; Toonen, Ruud F

    2016-06-01

    Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.

  8. Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons.

    PubMed

    Meijer, Marieke; Cijsouw, Tony; Toonen, Ruud F; Verhage, Matthijs

    2015-01-01

    The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions. Furthermore, Munc18-1a is the major splice variant in the Calyx of Held. Synaptic transmission was analyzed in autaptic hippocampal munc18-1 KO neurons re-expressing either Munc18-1a or Munc18-1b. The two splice variants supported synaptic transmission to a similar extent, but Munc18-1b was slightly more potent in sustaining synchronous release during high frequency stimulation. Our data suggest that alternative splicing of Munc18-1 support synaptic transmission to a similar extent, but could modulate presynaptic short-term plasticity.

  9. Synaptic Effects of Munc18-1 Alternative Splicing in Excitatory Hippocampal Neurons

    PubMed Central

    Toonen, Ruud F.; Verhage, Matthijs

    2015-01-01

    The munc18-1 gene encodes two splice-variants that vary at the C-terminus of the protein and are expressed at different levels in different regions of the adult mammalian brain. Here, we investigated the expression pattern of these splice variants within the brainstem and tested whether they are functionally different. Munc18-1a is expressed in specific nuclei of the brainstem including the LRN, VII and SOC, while Munc18-1b expression is relatively low/absent in these regions. Furthermore, Munc18-1a is the major splice variant in the Calyx of Held. Synaptic transmission was analyzed in autaptic hippocampal munc18-1 KO neurons re-expressing either Munc18-1a or Munc18-1b. The two splice variants supported synaptic transmission to a similar extent, but Munc18-1b was slightly more potent in sustaining synchronous release during high frequency stimulation. Our data suggest that alternative splicing of Munc18-1 support synaptic transmission to a similar extent, but could modulate presynaptic short-term plasticity. PMID:26407320

  10. Normal Molecular Specification and Neurodegenerative Disease-Like Death of Spinal Neurons Lacking the SNARE-Associated Synaptic Protein Munc18-1.

    PubMed

    Law, Chris; Schaan Profes, Marcos; Levesque, Martin; Kaltschmidt, Julia A; Verhage, Matthijs; Kania, Artur

    2016-01-13

    The role of synaptic activity during early formation of neural circuits is a topic of some debate; genetic ablation of neurotransmitter release by deletion of the Munc18-1 gene provides an excellent model to answer the question of whether such activity is required for early circuit formation. Previous analysis of Munc18-1(-/-) mouse mutants documented their grossly normal nervous system, but its molecular differentiation has not been assessed. Munc18-1 deletion in mice also results in widespread neurodegeneration that remains poorly characterized. In this study, we demonstrate that the early stages of spinal motor circuit formation, including motor neuron specification, axon growth and pathfinding, and mRNA expression, are unaffected in Munc18-1(-/-) mice, demonstrating that synaptic activity is dispensable for early nervous system development. Furthermore, we show that the neurodegeneration caused by Munc18-1 loss is cell autonomous, consistent with apparently normal expression of several neurotrophic factors and normal GDNF signaling. Consistent with cell-autonomous degeneration, we demonstrate defects in the trafficking of the synaptic proteins Syntaxin1a and PSD-95 and the TrkB and DCC receptors in Munc18-1(-/-) neurons; these defects do not appear to cause ER stress, suggesting other mechanisms for degeneration. Finally, we demonstrate pathological similarities to Alzheimer's disease, such as altered Tau phosphorylation, neurofibrillary tangles, and accumulation of insoluble protein plaques. Together, our results shed new light upon the neurodegeneration observed in Munc18-1(-/-) mice and argue that this phenomenon shares parallels with neurodegenerative diseases. In this work, we demonstrate the absence of a requirement for regulated neurotransmitter release in the assembly of early neuronal circuits by assaying transcriptional identity, axon growth and guidance, and mRNA expression in Munc18-1-null mice. Furthermore, we characterize the neurodegeneration

  11. Normal Molecular Specification and Neurodegenerative Disease-Like Death of Spinal Neurons Lacking the SNARE-Associated Synaptic Protein Munc18-1

    PubMed Central

    Law, Chris; Schaan Profes, Marcos; Levesque, Martin; Kaltschmidt, Julia A.; Verhage, Matthijs

    2016-01-01

    The role of synaptic activity during early formation of neural circuits is a topic of some debate; genetic ablation of neurotransmitter release by deletion of the Munc18-1 gene provides an excellent model to answer the question of whether such activity is required for early circuit formation. Previous analysis of Munc18-1−/− mouse mutants documented their grossly normal nervous system, but its molecular differentiation has not been assessed. Munc18-1 deletion in mice also results in widespread neurodegeneration that remains poorly characterized. In this study, we demonstrate that the early stages of spinal motor circuit formation, including motor neuron specification, axon growth and pathfinding, and mRNA expression, are unaffected in Munc18-1−/− mice, demonstrating that synaptic activity is dispensable for early nervous system development. Furthermore, we show that the neurodegeneration caused by Munc18-1 loss is cell autonomous, consistent with apparently normal expression of several neurotrophic factors and normal GDNF signaling. Consistent with cell-autonomous degeneration, we demonstrate defects in the trafficking of the synaptic proteins Syntaxin1a and PSD-95 and the TrkB and DCC receptors in Munc18-1−/− neurons; these defects do not appear to cause ER stress, suggesting other mechanisms for degeneration. Finally, we demonstrate pathological similarities to Alzheimer's disease, such as altered Tau phosphorylation, neurofibrillary tangles, and accumulation of insoluble protein plaques. Together, our results shed new light upon the neurodegeneration observed in Munc18-1−/− mice and argue that this phenomenon shares parallels with neurodegenerative diseases. SIGNIFICANCE STATEMENT In this work, we demonstrate the absence of a requirement for regulated neurotransmitter release in the assembly of early neuronal circuits by assaying transcriptional identity, axon growth and guidance, and mRNA expression in Munc18-1-null mice. Furthermore, we

  12. Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission.

    PubMed

    Meijer, Marieke; Burkhardt, Pawel; de Wit, Heidi; Toonen, Ruud F; Fasshauer, Dirk; Verhage, Matthijs

    2012-05-02

    Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.

  13. Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission

    PubMed Central

    Meijer, Marieke; Burkhardt, Pawel; de Wit, Heidi; Toonen, Ruud F; Fasshauer, Dirk; Verhage, Matthijs

    2012-01-01

    Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission. PMID:22446389

  14. Munc18-1 haploinsufficiency results in enhanced anxiety-like behavior as determined by heart rate responses in mice.

    PubMed

    Hager, Torben; Maroteaux, Grégoire; Pont, Paula du; Julsing, Joris; van Vliet, Rick; Stiedl, Oliver

    2014-03-01

    Heterozygous (HZ) missense mutations in the gene encoding syntaxin binding protein 1 (Stxbp1 or Munc18-1), a presynaptic protein essential for neurotransmitter release, causes early infantile epileptic encephalopathy, abnormal brain structure and mental retardation in humans. Here we investigated whether the mouse model mimics symptoms of the human phenotype. The effects of the deletion of munc18-1 were studied in HZ and wild-type (WT) mice based on heart rate (HR) and its variability (HRV) as independent measures to expand previous behavioral results of enhanced anxiety and impaired emotional learning suggesting mild cognitive impairments. HR responses were assessed during novelty exposure, during the expression and extinction of conditioned tone-dependent fear and during the diurnal phase. Novelty exposure yielded no differences in activity patterns between the two genotypes, while maximum HR differed significantly (WT: 770 bpm; HZ: 790 bpm). Retention tests after both auditory delay and trace fear conditioning showed a delayed extinction of the conditioned HR response in HZ mice compared to WT mice. Since the HR versus HRV correlation and HR dynamics assessed by nonlinear methods revealed similar function in HZ and WT mice, the higher HR responses of munc18-1 HZ mice to different emotional challenges cannot be attributed to differences in autonomic nervous system function. Thus, in contrast to the adverse consequences of deletion of a single allele of munc18-1 in humans, C57BL/6J mice show enhanced anxiety responses based on HR adjustments that extend previous results on the behavioral level without support of cognitive impairment, epileptic seizures and autonomic dysregulation. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly.

    PubMed

    Parisotto, Daniel; Pfau, Maximilian; Scheutzow, Andrea; Wild, Klemens; Mayer, Matthias P; Malsam, Jörg; Sinning, Irmgard; Söllner, Thomas H

    2014-04-04

    Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended α-helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.

  16. Munc18-1 phosphorylation by protein kinase C potentiates vesicle pool replenishment in bovine chromaffin cells.

    PubMed

    Nili, U; de Wit, H; Gulyas-Kovacs, A; Toonen, R F; Sørensen, J B; Verhage, M; Ashery, U

    2006-12-01

    Activation of protein kinase C (PKC) after robust stimulation is necessary for vesicle pool replenishment in secretory cells. Here we studied the contribution of a prominent downstream PKC target, Munc18-1, to this process in bovine chromaffin cells. In these cells, both activation of endogenous PKC and overexpressing of Munc18-1 promote vesicle pool replenishment after an extensive stimulation. In order to study the physiological relevance of PKC-dependent Munc18-1 phosphorylation, we generated two Munc18-1 phospho-mutants; one that mimics a constitutively PKC-phosphorylated Munc18-1 (i.e. a phosphomimetic mutant; Munc18-1(S313D)) and a second that cannot be PKC-phosphorylated (Munc18-1(3A)). Overexpression of Munc18-1(3A) caused a significant decrease in vesicle pool replenishment following a depleting stimulation, while Munc18-1(S313D) caused a significant increase in vesicle pool replenishment. These findings suggested that the phosphorylation of Munc18-1 by PKC potentiates vesicle pool replenishment. This hypothesis was further strengthened by the finding that overexpression of wild type Munc18-1 in the presence of a PKC inhibitor caused a significant reduction in vesicle pool replenishment, similar to that observed with Munc18-1(3A). Moreover, overexpression of Munc18-1(S313D) in the presence of the PKC inhibitor partly alleviated this attenuation, elucidating Munc18-1's unique contribution to vesicle pool replenishment. Finally, we demonstrate that Munc18-1 promotes vesicle docking in a phosphorylation-independent manner. This is deduced from the findings that both the wild type and the two Munc18-1 phospho-mutants enhanced docking to the same extent in bovine chromaffin cells. We conclude that Munc18-1 facilitates docking in a PKC phosphorylation-independent manner, and that its phosphorylation by PKC potentiates vesicle pool replenishment following a depleting stimulation, at a post-docking stage.

  17. Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size

    PubMed Central

    Toonen, Ruud F. G.; Wierda, Keimpe; Sons, Michèle S.; de Wit, Heidi; Cornelisse, L. Niels; Brussaard, Arjen; Plomp, Jaap J.; Verhage, Matthijs

    2006-01-01

    Prompt recovery after intense activity is an essential feature of most mammalian synapses. Here we show that synapses with reduced expression of the presynaptic gene munc18-1 suffer from increased depression during intense stimulation at glutamatergic, GABAergic, and neuromuscular synapses. Conversely, munc18-1 overexpression makes these synapses recover faster. Concomitant changes in the readily releasable vesicle pool and its refill kinetics were found. The number of vesicles docked at the active zone and the total number of vesicles per terminal correlated with both munc18-1 expression levels and the size of the releasable vesicle pool. These data show that varying expression of a single gene controls synaptic recovery by modulating the number of docked, release-ready vesicles and thereby replenishment of the secretion capacity. PMID:17110441

  18. Early Golgi abnormalities and neurodegeneration upon loss of presynaptic proteins Munc18-1, syntaxin-1 or SNAP-25.

    PubMed

    Santos, Tatiana C; Wierda, Keimpe; Broeke, Jurjen H; Toonen, Ruud F; Verhage, Matthijs

    2017-03-27

    The loss of presynaptic proteins Munc18-1, syntaxin-1 or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and dorsal root ganglion neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 days in vitro (DIV) upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using Tetanus Neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed cis-Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normal cis-Golgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that dorsal root ganglion neurons are the only Munc18-1 KO neurons that do not degenerate in vivo or in vitro In these neurons, cis-Golgi abnormalities were less severe, with no changes in Golgi shape. Together these data demonstrate that cell death upon Munc18-1, syntaxin-1 or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving early cis-Golgi abnormalities, distinct from apoptosis.SIGNIFICANCE STATEMENTThis study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), while other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive

  19. Munc18-1 Is Critical for Plasma Membrane Localization of Syntaxin1 but Not of SNAP-25 in PC12 Cells

    PubMed Central

    Arunachalam, Lakshmanan; Han, Liping; Tassew, Nardos G.; He, Yu; Wang, Li; Xie, Li; Fujita, Yoshihito; Kwan, Edwin; Davletov, Bazbek; Monnier, Philippe P.; Gaisano, Herbert Y.

    2008-01-01

    Although Munc18-1 was originally identified as a syntaxin1–interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1. PMID:18077557

  20. Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

    PubMed Central

    Urigüen, L; Gil-Pisa, I; Munarriz-Cuezva, E; Berrocoso, E; Pascau, J; Soto-Montenegro, M L; Gutiérrez-Adán, A; Pintado, B; Madrigal, J L M; Castro, E; Sánchez-Blázquez, P; Ortega, J E; Guerrero, M J; Ferrer-Alcon, M; García-Sevilla, J A; Micó, J A; Desco, M; Leza, J C; Pazos, Á; Garzón, J; Meana, J J

    2013-01-01

    Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D1 receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder. PMID:23340504

  1. Structure-Function Study of Mammalian Munc18-1 and C. elegans UNC-18 Implicates Domain 3b in the Regulation of Exocytosis

    PubMed Central

    Graham, Margaret E.; Prescott, Gerald R.; Johnson, James R.; Jones, Mathew; Walmesley, Alice; Haynes, Lee P.; Morgan, Alan; Burgoyne, Robert D.; Barclay, Jeff W.

    2011-01-01

    Munc18-1 is an essential synaptic protein functioning during multiple stages of the exocytotic process including vesicle recruitment, docking and fusion. These functions require a number of distinct syntaxin-dependent interactions; however, Munc18-1 also regulates vesicle fusion via syntaxin-independent interactions with other exocytotic proteins. Although the structural regions of the Munc18-1 protein involved in closed-conformation syntaxin binding have been thoroughly examined, regions of the protein involved in other interactions are poorly characterised. To investigate this we performed a random transposon mutagenesis, identifying domain 3b of Munc18-1 as a functionally important region of the protein. Transposon insertion in an exposed loop within this domain specifically disrupted Mint1 binding despite leaving affinity for closed conformation syntaxin and binding to the SNARE complex unaffected. The insertion mutation significantly reduced total amounts of exocytosis as measured by carbon fiber amperometry in chromaffin cells. Introduction of the equivalent mutation in UNC-18 in Caenorhabditis elegans also reduced neurotransmitter release as assessed by aldicarb sensitivity. Correlation between the two experimental methods for recording changes in the number of exocytotic events was verified using a previously identified gain of function Munc18-1 mutation E466K (increased exocytosis in chromaffin cells and aldicarb hypersensitivity of C. elegans). These data implicate a novel role for an exposed loop in domain 3b of Munc18-1 in transducing regulation of vesicle fusion independent of closed-conformation syntaxin binding. PMID:21445306

  2. Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

    PubMed Central

    Genç, Özgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf

    2014-01-01

    Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001 PMID:24520164

  3. Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release.

    PubMed

    Genc, Ozgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf

    2014-02-11

    Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001.

  4. The Role of Rab3a in Secretory Vesicle Docking Requires Association/Dissociation of Guanidine Phosphates and Munc18-1

    PubMed Central

    van Weering, Jan R.T.; Toonen, Ruud F.; Verhage, Matthijs

    2007-01-01

    Rab3a is a small GTPase that binds selectively to secretory vesicles and switches between active, GTP-bound and inactive, GDP-bound conformations. In yeast, Rab and SM-genes interact genetically to promote vesicle targeting/fusion. We tested different Rab3a conformations and genetic interactions with the SM-gene munc18-1 on the docking function of Rab3a in mammalian chromaffin cells. We expressed Rab3a mutants locked in the GTP- or GDP-bound form in wild-type and munc18-1 null mutant cells and analyzed secretory vesicle distribution. We confirmed that wild-type Rab3a promotes vesicle docking in wild-type cells. Unexpectedly, both GTP- and GDP-locked Rab3a mutants did not promote docking. Furthermore, wild-type Rab3a did not promote docking in munc18-1 null cells and GTP- and GDP-Rab3a both decreased the amount of docked vesicles. The results show that GTP- and GDP-locked conformations do not support a Munc18-1 dependent role of Rab3a in docking. This suggests that nucleotide cycling is required to support docking and that this action of Rab3a is upstream of Munc18-1. PMID:17637832

  5. Munc18-1 and the Syntaxin-1 N-terminus Regulate Open-Closed States in a t-SNARE complex

    PubMed Central

    Dawidowski, Damian; Cafiso, David S.

    2016-01-01

    Summary Neuronal exocytosis is mediated by SNARE proteins, which assemble into a highly stable four helical bundle in a process that is not well-understood. In the present study, EPR spectroscopy was used to examine how the t-SNAREs syntaxin and SNAP25 assemble in the presence and absence of the regulatory protein Munc18-1. Syntaxin and SNAP25 form a 2:1 complex, which is structurally heterogeneous and persists in the presence of excess SNAP25. Munc18-1 dissociates this 2:1 complex, but a 1:1 complex is retained where syntaxin is in a closed state. In the absence of an N-terminal fragment of syntaxin, Munc18-1 also stabilizes a 1:1 complex of sytaxin:SNAP25; however, syntaxin now samples an open state. These data demonstrate that the open-closed syntaxin equilibrium is shifted towards the open state when syntaxin and Munc18-1 are associated with SNAP-25, and the results indicate that a syntaxin:SNAP25:Munc18-1 complex is a likely starting point for SNARE assembly. PMID:26876096

  6. Deletion of Munc18-1 in 5-HT Neurons Results in Rapid Degeneration of the 5-HT System and Early Postnatal Lethality

    PubMed Central

    Dudok, Jacobus J.; Groffen, Alexander J. A.; Toonen, Ruud F. T.; Verhage, Matthijs

    2011-01-01

    The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival. PMID:22140524

  7. A Pivotal Role for Pro-335 in Balancing the Dual Functions of Munc18-1 Domain-3a in Regulated Exocytosis*

    PubMed Central

    Han, Gayoung Anna; Park, Seungmee; Bin, Na-Ryum; Jung, Chang Hun; Kim, Byungjin; Chandrasegaram, Prashanth; Matsuda, Maiko; Riadi, Indira; Han, Liping; Sugita, Shuzo

    2014-01-01

    Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation. PMID:25326390

  8. Cytokine pathway disruption in a mouse model of schizophrenia induced by Munc18-1a overexpression in the brain

    PubMed Central

    2014-01-01

    Background An accumulating body of evidence points to the significance of neuroinflammation and immunogenetics in schizophrenia, and an imbalance of cytokines in the central nervous system (CNS) has been suggested to be associated with the disorder. Munc18-overexpressing mice (Munc18-OE) have provided a model for the study of the alterations that may underlie the symptoms of subjects with schizophrenia. The aim of the present study was to elucidate the involvement of neuroinflammation and cytokine imbalance in this model. Methods Cytokines were evaluated in the cortex and the striatum of Munc18-OE and wild-type (WT) mice by enzyme-linked immunosorbent assay (ELISA). Protein levels of specific microglia and macrophage, astrocytic and neuroinflammation markers were quantified by western blot in the cortex and the striatum of Munc18-OE and WT mice. Results Each cytokine evaluated (Interferon-gamma (IFN-γ), Tumor Necrosis Factor-alpha (TNF-α), Interleukin-2 (IL-2) and CCL2 chemokine) was present at higher levels in the striatum of Munc18-OE mice than WT. Cortical TNF-α and IL-2 levels were significantly lower in Munc18-OE mice than WT mice. The microglia and macrophage marker CD11b was lower in the cortexes of Munc18-OE mice than WT, but no differences were observed in the striatum. Glial Fibrillary Acidic Protein (GFAP) and Nuclear Factor-kappaB (NF-κB)p65 levels were not different between the groups. Interleukin-1beta (IL-1β) and IL-6 levels were beneath detection limits. Conclusions The disrupted levels of cytokines detected in the brain of Munc18-OE mice was found to be similar to clinical reports and endorses study of this type for analysis of this aspect of the disorder. The lower CD11b expression in the cortex but not in the striatum of the Munc18-OE mice may reflect differences in physiological activity. The cytokine expression pattern observed in Munc18-OE mice is similar to a previously published model of schizophrenia caused by maternal immune

  9. TNFα induces co-trafficking of TRPV1/TRPA1 in VAMP1-containing vesicles to the plasmalemma via Munc18-1/syntaxin1/SNAP-25 mediated fusion.

    PubMed

    Meng, Jianghui; Wang, Jiafu; Steinhoff, Martin; Dolly, James Oliver

    2016-02-18

    Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis, and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content, and both resided in close proximity, indicating co-trafficking. Syntaxin 1-interacting protein, Munc18-1, proved necessary for the response to TNFα, and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca(2+) influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins' known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.

  10. Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ

    PubMed Central

    Pertsinidis, Alexandros; Mukherjee, Konark; Sharma, Manu; Pang, Zhiping P.; Park, Sang Ryul; Zhang, Yunxiang; Brunger, Axel T.; Südhof, Thomas C.; Chu, Steven

    2013-01-01

    Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ. PMID:23821748

  11. Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion.

    PubMed

    Liu, Xiaoxia; Seven, Alpay Burak; Xu, Junjie; Esser, Victoria; Su, Lijing; Ma, Cong; Rizo, Josep

    2017-09-01

    This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca(2+)-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca(2+) sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

  12. Preliminary assay reveals quality of Mars blend

    SciTech Connect

    Rhodes, A.K.

    1996-06-10

    Shell Oil Co. has assayed a preproduction well sample of Mars blend, from the Louisiana Gulf of Mexico. The assay shows the crude to have an API gravity of 31{degree} and a sulfur content of 2.0 wt%. Distillation data for the crude are shown in this paper. When partners Shell (71.5%) and BP (28.5%) begin commercial production in July, several producing zones will be tapped through a number of wells. Shell says the final sales quality of Mars Blend may vary from the qualities of this representative sample. The crude will be produced from a tension-leg platform (TLP) at a water depth of 2,940 ft (OGJ, Apr. 8, p. 23). The TLP is anchored in Mississippi Canyon block 807, about 130 miles southwest of New Orleans (map). Mars field will be the principal source of the crude initially. Shell says production will escalate to about 100,000 b/d crude and 110 MMcfd natural gas in 1997. Ultimate recovery is estimated to be 700 million BOE.

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

    PubMed Central

    Paskin, Taylor R.; Jellies, John; Bacher, Jessica; Beane, Wendy S.

    2014-01-01

    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. PMID:25493551

  14. What is Comet assay not telling us: AFLP reveals wider aspects of genotoxicity.

    PubMed

    Šrut, Maja; Štambuk, Anamaria; Klobučar, Göran I V

    2013-06-01

    DNA damage detected by genotoxicity biomarkers such as the Comet assay is not always a reliable indicator of the consequences that genotoxic agents can have on the genome integrity of the exposed organisms. Therefore, to reveal the existence of more permanent alterations of DNA structure after genotoxic stress, the RTG-2 rainbow trout cell line was exposed for 3 days to benzo[a]pyrene (B[a]P, 0.1-10 μM) and ethyl methanesulfonate (EMS, 0.1-1mM) followed by 3 days of recovery period. Primary DNA damage was evaluated by the Comet assay and DNA alterations were assessed using AFLP (amplified fragment length polymorphism). Qualitative and quantitative modifications in AFLP profiles were analyzed in order to detect genetic alterations arising from mutation events and/or DNA damage. Significant induction in DNA damage measured by the Comet assay was noticed after B[a]P treatment at all concentrations but values returned to the control level after recovery. Exposure to EMS induced significant DNA damage only at the highest concentration and damage persisted after the recovery period. AFLP profiles detected DNA alterations even when Comet assay indicated complete DNA repair, revealing more persistent damage. Since such DNA damage can impair its structure and function, Comet assay results should preferably be supplemented with other methods in order to predict the consequences of genotoxic insult more accurately.

  15. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

    PubMed Central

    Suauam, Pitipreya; Yingyongnarongkul, Boon-ek; Palaga, Tanapat; Miyakawa, Tokichi; Yompakdee, Chulee

    2015-01-01

    Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant. PMID:26313553

  16. Fluorescence-based retention assays reveals sustained release of vascular endothelial growth factor from bone grafts.

    PubMed

    Kang, Wonmo; Yun, Ye-Rang; Lee, Dong-Sung; Kim, Tae-Hyun; Kim, Joong-Hyun; Kim, Hae-Won; Jang, Jun-Hyeog

    2016-01-01

    The sustained release of growth factors following their implantation in vivo is essential for successful outcomes in bone tissue engineering. In this study, we evaluated the release kinetics and delivery efficacies of vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, incorporated into calcium phosphate bone grafts (BGs). We evaluated the release profile of VEGF from BGs using a novel fluorescence-based retention assay, which revealed that VEGF loaded on BGs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. In contrast, an ELISA-based release assay showed VEGF to have an early burst-release profile for the first week. However, the biological activity of VEGF released from the BGs was preserved over the 7-week release period, which is consistent with the sustained-release profile observed in the fluorescence-based retention assay. Furthermore, the in vivo bone-forming action of the VEGF-loaded BGs was well demonstrated in a rat subcutaneous model. Taken together, the sustained release of VEGF loaded onto BGs was effective in stimulating proliferation, angiogenesis and osteogenesis, suggesting the ultimate value of VEGF-engineered BGs for bone tissue engineering.

  17. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

    PubMed Central

    Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

    2011-01-01

    Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

  18. Cytokine Production Assays Reveal Discriminatory Immune Defects in Adults with Recurrent Infections and Noninfectious Inflammation

    PubMed Central

    van de Veerdonk, Frank L.; Joosten, Leo A. B.; Simon, Anna; van Crevel, Reinout; Kullberg, Bart-Jan; Gyssens, Inge C.; van der Meer, Jos W. M.; van Deuren, Marcel; Netea, Mihai G.

    2014-01-01

    Cytokine production assays have been primarily used in research settings studying novel immunodeficiencies. We sought to determine the diagnostic value of cytokine production assays in patients with recurrent and/or severe infectious diseases (IDs) without known immunodeficiencies and unclassified noninfectious inflammatory disorders (NIIDs). We retrospectively examined cytokine production in whole-blood and peripheral blood mononuclear cell samples from 157 adult patients. A cytokine production rate of <5% of that of healthy controls was considered defective. While monocyte-derived cytokine (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], and IL-6) production was rarely affected, 30% of all included patients had deficient production of interferon gamma (IFN-γ), IL-17A, or IL-22. Twenty-five percent of the NIID patients displayed defective IFN-γ production, whereas IL-17A production was generally unaffected. In the group of ID patients, defective IFN-γ production was found in 19% and 14% of the patients with viral and bacterial infections, respectively, and in 38%, 24%, and 50% of patients with mycobacterial, mucocutaneous, and invasive fungal infections, respectively. Defective IL-17A and IL-22 production was mainly confined to ID patients with mucocutaneous fungal infections. In conclusion, cytokine production assays frequently detect defective Th1 responses in patients with mycobacterial or fungal infections, in contrast to patients with respiratory tract infections or isolated bacterial infections. Defective IL-17A and IL-22 production was primarily found in patients with fungal infections, while monocyte-derived cytokine production was unaffected. Thus, lymphocyte-derived cytokine production assays are helpful in the diagnostic workup of patients with recurrent infections and suspected immunodeficiencies and have the potential to reveal immune defects that might guide adjunctive immunomodulatory therapy. PMID:24872512

  19. A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis.

    PubMed

    Miwako, Ishido; Schmid, Sandra L

    2006-05-01

    The low density lipoprotein receptor-related protein (LRP) binds multiple, distinct ligands and participates in constitutive endocytosis and signal transduction. Using an in vitro reconstitution system and a new biochemical complementation assay, we have explored the limiting cytosolic requirements for endocytosis of LRP from isolated plasma membranes. We find that clathrin, AP2 and dynamin do not support efficient LRP uptake and that additional factors present in a 30% ammonium sulfate supernatant fraction of bovine brain cytosol (AS supt) are required. Fractionation of the AS supt revealed that multiple and redundant factors are required to support LRP endocytosis. Among these, we identified Hsc70, synaptojanin1 and CRMP-2 by mass spectrometry. Our data suggest that LRP, which bears several distinct endocytic motifs in its cytoplasmic domain, may use multiple pathways for endocytosis in vitro.

  20. A kinetic fluorescence assay reveals unusual features of Ca++ uptake in Plasmodium falciparum-infected erythrocytes

    PubMed Central

    2014-01-01

    Background To facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes. Methods Based on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum. Results Both uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++. Conclusions Nondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development. PMID:24885754

  1. Single-molecule peptide-lipid affinity assay reveals interplay between solution structure and partitioning.

    PubMed

    Matin, Tina R; Sigdel, Krishna P; Utjesanovic, Milica; Marsh, Brendan P; Gallazzi, Fabio; Smith, Virginia F; Kosztin, Ioan; King, Gavin M

    2017-03-27

    Interactions between short protein segments and phospholipid bilayers dictate fundamental aspects of cellular activity and have important applications in biotechnology. Yet, a lack of suitable methodology for directly probing these interactions has hindered mechanistic understanding. We developed a precision atomic force microscope (AFM)-based single-molecule force spectroscopy assay and probed partitioning into lipid bilayers by measuring the mechanical force experienced by a peptide. Protein segments were constructed from the peripheral membrane protein SecA, a key ATPase in bacterial secretion. We focused on the first 10 amino-terminal residues of SecA (SecA2-11) which are known to be lipophilic. In addition to the core SecA2-11 sequence, constructs with nearly identical chemical composition but with differing geometry were used: two copies of SecA2-11 linked in series, and two copies in parallel. Lipid bilayer partitioning interactions of peptides with differing structures were distinguished. To model the energetic landscape, a theory of diffusive barrier crossing was extended to incorporate a superposition of potential barriers with variable weights. Analysis revealed two dissociation pathways for the core SecA2-11 sequence with well-separated intrinsic dissociation rates. Molecular dynamics simulations showed that the three peptides had significant conformational differences in solution that correlated well with measured variations in the propensity to partition into the bilayer. The methodology is generalizable and can be applied to other peptide and lipid species.

  2. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution.

    PubMed

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-07-15

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.

  3. Clonogenic assay of type a influenza viruses reveals noninfectious cell-killing (apoptosis-inducing) particles.

    PubMed

    Ngunjiri, John M; Sekellick, Margaret J; Marcus, Philip I

    2008-03-01

    Clonogenic (single-cell plating) assays were used to define and quantify subpopulations of two genetically closely related variants of influenza virus A/TK/OR/71 that differed primarily in the size of the NS1 gene product; they expressed a full-size (amino acids [aa] 1 to 230) or truncated (aa 1 to 124) NS1 protein. Monolayers of Vero cells were infected with different amounts of virus, monodispersed, and plated. Cell survival curves were generated from the fraction of cells that produced visible colonies as a function of virus multiplicity. The exponential loss of colony-forming capacity at low multiplicities demonstrated that a single virus particle sufficed to kill a cell. The ratios of cell-killing particles (CKP) to plaque-forming particles (PFP) were 1:1 and 7:1 in populations of variants NS1(1-124) and NS1(1-230), respectively. This study revealed a new class of particles in influenza virus populations-noninfectious CKP. Both infectious and noninfectious CKP were 6.3 times more resistant to UV radiation than PFP activity. Based on UV target theory, a functional polymerase subunit was implicated in a rate-limiting step in cell killing. Since influenza viruses kill cells by apoptosis (programmed cell death), CKP are functionally apoptosis-inducing particles. Noninfectious CKP are present in excess of PFP in virus populations with full-size NS1 and induce apoptosis that is temporally delayed and morphologically different than that initiated by infectious CKP present in the virus population expressing truncated NS1. The identification and quantification of both infectious and noninfectious CKP defines new phenotypes in influenza virus populations and presents a challenge to determine their role in regulating infectivity, pathogenesis, and vaccine efficacy.

  4. Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay.

    PubMed

    Caviglia, C; Zór, K; Canepa, S; Carminati, M; Larsen, L B; Raiteri, R; Andresen, T L; Heiskanen, A; Emnéus, J

    2015-05-21

    We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

  5. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    PubMed Central

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane

    2015-01-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  6. Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination.

    PubMed

    Barkal, Layla J; Walsh, Naomi M; Botts, Michael R; Beebe, David J; Hull, Christina M

    2016-05-16

    Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination.

  7. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.

    PubMed

    Maass, Katie F; Kulkarni, Chethana; Quadir, Mohiuddin A; Hammond, Paula T; Betts, Alison M; Wittrup, Karl Dane

    2015-12-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.

  8. Leveraging a high resolution microfluidic assay reveals insights into pathogenic fungal spore germination

    PubMed Central

    Barkal, Layla J.; Walsh, Naomi M.; Botts, Michael R.; Beebe, David J.; Hull, Christina M.

    2016-01-01

    Germination of spores into actively growing cells is a process essential for survival and pathogenesis of many microbes. Molecular mechanisms governing germination, however, are poorly understood in part because few tools exist for evaluating and interrogating the process. Here, we introduce an assay that leverages developments in microfluidic technology and image processing to quantitatively measure germination with unprecedented resolution, assessing both individual cells and the population as a whole. Using spores from Cryptococcus neoformans, a leading cause of fatal fungal disease in humans, we developed a platform to evaluate spores as they undergo morphological changes during differentiation into vegetatively growing yeast. The assay uses pipet-accessible microdevices that can be arrayed for efficient testing of diverse microenvironmental variables, including temperature and nutrients. We discovered that temperature influences germination rate, a carbon source alone is sufficient to induce germination, and the addition of a nitrogen source sustains it. Using this information, we optimized the assay for use with fungal growth inhibitors to pinpoint stages of germination inhibition. Unexpectedly, the clinical antifungal drugs amphotericin B and fluconazole did not significantly alter the process or timing of the transition from spore to yeast, indicating that vegetative growth and germination are distinct processes in C. neoformans. Finally, we used the high temporal resolution of the assay to determine the precise defect in a slow-germination mutant. Combining advances in microfluidics with a robust fungal molecular genetic system allowed us to identify and alter key temporal, morphological, and molecular events that occur during fungal germination. PMID:27026574

  9. Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay.

    PubMed

    Nguyen, Kiet T; Hu, Xubo; Pei, Dehua

    2004-06-01

    A new spectrophotometric/fluorimetric assay for peptide deformylase (PDF) has been developed by coupling the PDF reaction with that of dipeptidyl peptidase I (DPPI) and using N-formyl-Met-Lys-AMC as substrate. Removal of the N-terminal formyl group by PDF renders the dipeptide an efficient substrate of DPPI, which subsequently removes the dipeptidyl units to release 7-amino-4-methylcoumarin as the chromophore/fluorophore. The PDF reaction is conveniently monitored on a UV-Vis spectrophotometer or a fluorimeter in a continuous fashion. The utility of the assay was demonstrated by determining the catalytic activity of PDF and the inhibition constants of PDF inhibitors. These studies revealed the slow-binding behavior of a previously reported macrocyclic PDF inhibitor. This method offers several advantages over the existing PDF assays and should be particularly useful for screening PDF inhibitors in the continuous fashion.

  10. A Genetic Assay for Transcription Errors Reveals Multilayer Control of RNA Polymerase II Fidelity

    PubMed Central

    Irvin, Jordan D.; Kireeva, Maria L.; Gotte, Deanna R.; Shafer, Brenda K.; Huang, Ingold; Kashlev, Mikhail; Strathern, Jeffrey N.

    2014-01-01

    We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing. PMID:25232834

  11. A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.

    PubMed

    Irvin, Jordan D; Kireeva, Maria L; Gotte, Deanna R; Shafer, Brenda K; Huang, Ingold; Kashlev, Mikhail; Strathern, Jeffrey N

    2014-09-01

    We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing.

  12. Competition assays revealed Paenibacillus pasadenensis strain R16 as a novel antifungal agent.

    PubMed

    Passera, Alessandro; Venturini, Giovanni; Battelli, Giovanna; Casati, Paola; Penaca, Francesca; Quaglino, Fabio; Bianco, Piero Attilio

    2017-05-01

    The development of new sustainable containment strategies of plant diseases is very important to guarantee food security while reducing the environmental impact of agriculture. Research of new biocontrol agents is a long and difficult process that involves several steps that start from the identification of possible candidates which, for example, show antibiotic activities, and ends with in field, large scale trials. In this study, the plant growth promoting potential and antifungal effect exerted by a novel, putative candidate biocontrol agent, strain R16, identified as Paenibacillus pasadenensis by sequence analysis of 16S rRNA and rpoB genes, against three important plant pathogenic fungi (Botrytis cinerea, Fusarium verticillioides, and Phomopsis viticola), were assessed. Biochemical assays to determine plant growth promoting potential gave negative results for siderophore production and phosphate solubilization, and positive results for ACC-deamination and IAA production. Further biochemical assays for endophytic lifestyle and antifungal activity gave positive results for catalase and chitinase activity, respectively. In vitro antagonism assays showed that strain R16 is effective against B. cinerea, reducing mycelial growth both in dual-culture and through volatile substances, characterized to be mostly composed by farnesol, and inhibiting conidia germination. Good antagonistic potential was also observed in vitro towards P. viticola, but not towards F. verticillioides. Moreover, in vivo assays confirmed the strain R16 activity reduced the infection rate on B. cinerea-inoculated berries. The obtained results firstly proved that P. pasadenesis strain R16 is a putative plant growth promoter and effective against phytopathogenic fungi. Further studies will be needed to investigate the possible application of this strain as a biocontrol agent. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Factors Essential for Prostate Cancer Metastasis Revealed Through a Novel 3D Microtissue Assay

    DTIC Science & Technology

    2016-06-01

    and destruction. Growing evidence suggest that the micro-dissemination of bone metastatic prostate cancer (BMPCa) to bone marrow (BM) may be...A Novel 3D osteoblastic niche Assay designed to assess the ability of bone -metastatic Cancer homing to Bone Marrow Niche (Background) Many patients...microenvironment. It is known that cancer-stromal cell interaction in bone marrow is a pivotal step in determining bone metastasis and tumor behavior

  14. Minor antigen graft-versus-host reactions revealed in irradiated spleen and popliteal lymph node assays

    SciTech Connect

    Claman, H.N.; Jaffee, B.D.

    1984-10-01

    The graft-versus-hot (GVH) reaction across minor (non-H-2) histocompatibility barriers was studied in mice, in vivo. To increase GVH potential and to mimic clinical bone marrow transplantation protocols, we modified the popliteal lymph node (PLN) and the splenomegaly assays by irradiating the recipients before they received allogeneic lymphoid cell suspensions. In several combinations across major (H-2), minor (non-H-2) and multiple minor (non-H-2 plus minor lymphocyte stimulation) barriers, increased recipient organ weight (a measure of GVH activity) was seen with irradiated F1 recipients of parental cells. The irradiated splenomegaly (x-splenomegaly) assay was more sensitive than the (x-PLN) assay, but both correlated with in vivo GVH experiments of the P----F1 variety. The x-splenomegaly test indicated histoincompatibility in a system (B10.D2----BALB/c) in which the primary in vitro mixed leukocyte reactions is nonreactive, but in which systemic GVH can be induced. The x-splenomegaly test should be useful in analyzing complex reactions involving minor histocompatibility antigens in vivo.

  15. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors

    PubMed Central

    Levis, Mark; Brown, Patrick; Smith, B. Douglas; Stine, Adam; Pham, Rosalyn; Stone, Richard; DeAngelo, Daniel; Galinsky, Ilene; Giles, Frank; Estey, Elihu; Kantarjian, Hagop; Cohen, Pamela; Wang, Yanfeng; Roesel, Johannes; Karp, Judith E.; Small, Donald

    2006-01-01

    We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. PMID:16857987

  16. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

    PubMed

    Wikstrom, Jakob D; Sereda, Samuel B; Stiles, Linsey; Elorza, Alvaro; Allister, Emma M; Neilson, Andy; Ferrick, David A; Wheeler, Michael B; Shirihai, Orian S

    2012-01-01

    The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  17. Bacterial regrowth in water reclamation and distribution systems revealed by viable bacterial detection assays.

    PubMed

    Lin, Yi-wen; Li, Dan; Gu, April Z; Zeng, Si-yu; He, Miao

    2016-02-01

    Microbial regrowth needs to be managed during water reclamation and distribution. The aim of present study was to investigate the removal and regrowth of Escherichia coli (E. coli) and Salmonella in water reclamation and distribution system by using membrane integrity assay (PMA-qPCR), reverse transcriptional activity assay (Q-RT-PCR) and culture-based assay, and also to evaluate the relationships among bacterial regrowth, and environmental factors in the distribution system. The results showed that most of the water reclamation processes potentially induced bacteria into VBNC state. The culturable E. coli and Salmonella regrew 1.8 and 0.7 log10 in distribution system, which included reactivation of bacteria in the viable but non-culturable (VBNC) state and reproduction of culturable bacteria. The regrowth of culturable E. coli and Salmonella in the distribution system mainly depended on the residual chlorine levels, with correlations (R(2)) of -0.598 and -0.660. The abundances of membrane integrity and reverse transcriptional activity bacteria in reclamation effluents had significant correlations with the culturable bacteria at the end point of the distribution system, demonstrating that PMA-qPCR and Q-RT-PCR are sensitive and accurate tools to determine and predict bacterial regrowth in water distribution systems. This study has improved our understanding of microbial removal and regrowth in reclaimed water treatment and distribution systems. And the results also recommended that more processes should be equipped to remove viable bacteria in water reclamation plants for the sake of inhibition microbial regrowth during water distribution and usages. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. A rapid dehydration leaf assay reveals stomatal response differences in grapevine genotypes

    PubMed Central

    Hopper, Daniel W; Ghan, Ryan; Cramer, Grant R

    2014-01-01

    A simple and reliable way of phenotyping plant responses to dehydration was developed. Fully-developed leaves were detached and placed in a closed plastic box containing a salt solution to control the atmospheric water potential in the container. Three hours of dehydration (weight loss of the leaf) was optimal for measuring changes in stomatal response to dehydration. Application of the plant hormone abscisic acid (ABA) prior to leaf detachment decreased the amount of water loss, indicating that the assay was able to detect differences based on a stomatal response to dehydration. Five different Vitis genotypes (V. riparia, V. champinii, V. vinifera cv. Shiraz, V. vinifera cv. Grenache and V. vinifera cv. Cabernet Sauvignon) with known differences in drought tolerance were screened for their dehydration response and the results obtained corresponded to previous reports of stomatal responses in the vineyard. Significant differences in stomatal density along with differences in the amount and rate of water lost indicate differences in dehydration sensitivity among the genotypes screened. Differences in stomatal response to ABA were also detected. Shiraz had the lowest stomatal density and the highest ABA sensitivity among the genotypes screened, yet Shiraz lost the most amount of water, indicating that it was the least sensitive to dehydration. Despite having the highest stomatal density and intermediate stomatal sensitivity to ABA, V. riparia lost the smallest amount of water, indicating that it was the most sensitive to dehydration. The assay presented here represents a simple and reliable phenotyping method for plant responses to leaf dehydration. PMID:26504528

  19. Genetic diversity and relationships in mulberry (genus Morus) as revealed by RAPD and ISSR marker assays

    PubMed Central

    Awasthi, Arvind K; Nagaraja, GM; Naik, GV; Kanginakudru, Sriramana; Thangavelu, K; Nagaraju, Javaregowda

    2004-01-01

    Background The genus Morus, known as mulberry, is a dioecious and cross-pollinating plant that is the sole food for the domesticated silkworm, Bombyx mori. Traditional methods using morphological traits for classification are largely unsuccessful in establishing the diversity and relationships among different mulberry species because of environmental influence on traits of interest. As a more robust alternative, PCR based marker assays including RAPD and ISSR were employed to study the genetic diversity and interrelationships among twelve domesticated and three wild mulberry species. Results RAPD analysis using 19 random primers generated 128 discrete markers ranging from 500–3000 bp in size. One-hundred-nineteen of these were polymorphic (92%), with an average of 6.26 markers per primer. Among these were a few putative species-specific amplification products which could be useful for germplasm classification and introgression studies. The ISSR analysis employed six anchored primers, 4 of which generated 93 polymorphic markers with an average of 23.25 markers per primer. Cluster analysis of RAPD and ISSR data using the WINBOOT package to calculate the Dice coefficient resulted into two clusters, one comprising polyploid wild species and the other with domesticated (mostly diploid) species. Conclusion These results suggest that RAPD and ISSR markers are useful for mulberry genetic diversity analysis and germplasm characterization, and that putative species-specific markers may be obtained which can be converted to SCARs after further studies. PMID:14715088

  20. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers.

    PubMed

    Parker, Hugo J; Sauka-Spengler, Tatjana; Bronner, Marianne; Elgar, Greg

    2014-01-01

    The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

  1. Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay: A Biophysical Chemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Shafirovich, Vladimir; Singh, Carolyn; Geacintov, Nicholas E.

    2003-11-01

    Oxidative damage of DNA molecules associated with electron-transfer reactions is an important phenomenon in living cells, which can lead to mutations and contribute to carcinogenesis and the aging processes. This article describes the design of several simple experiments to explore DNA damage initiated by photoinduced electron-transfer reactions sensitized by the acridine derivative, proflavine (PF). A supercoiled DNA agarose gel nicking assay is employed as a sensitive probe of DNA strand cleavage. A low-cost experimental and computer-interfaced imaging apparatus is described allowing for the digital recording and analysis of the gel electrophoresis results. The first experiment describes the formation of direct strand breaks in double-stranded DNA induced by photoexcitation of the intercalated PF molecules. The second experiment demonstrates that the addition of the well-known electron acceptor, methylviologen, gives rise to a significant enhancement of the photochemical DNA strand cleavage effect. This occurs by an electron transfer step to methylviologen that renders the inital photoinduced charge separation between photoexcited PF and DNA irreversible. The third experiment demonstrates that the action spectrum of the DNA photocleavage matches the absorption spectrum of DNA-bound, intercalated PF molecules, which differs from that of free PF molecules. This result demonstrates that the photoinduced DNA strand cleavage is initiated by intercalated rather than free PF molecules.

  2. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

  3. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  4. Heritable Differences in Schooling Behavior among Threespine Stickleback Populations Revealed by a Novel Assay

    PubMed Central

    Taylor, Elspeth M.; Yoshida, Kohta; Peichel, Catherine L.

    2011-01-01

    Identifying the proximate and ultimate mechanisms of social behavior remains a major goal of behavioral biology. In particular, the complex social interactions mediating schooling behavior have long fascinated biologists, leading to theoretical and empirical investigations that have focused on schooling as a group-level phenomenon. However, methods to examine the behavior of individual fish within a school are needed in order to investigate the mechanisms that underlie both the performance and the evolution of schooling behavior. We have developed a technique to quantify the schooling behavior of an individual in standardized but easily manipulated social circumstances. Using our model school assay, we show that threespine sticklebacks (Gasterosteus aculeatus) from alternative habitats differ in behavior when tested in identical social circumstances. Not only do marine sticklebacks show increased association with the model school relative to freshwater benthic sticklebacks, they also display a greater degree of parallel swimming with the models. Taken together, these data indicate that marine sticklebacks exhibit a stronger tendency to school than benthic sticklebacks. We demonstrate that these population-level differences in schooling tendency are heritable and are shared by individuals within a population even when they have experienced mixed-population housing conditions. Finally, we begin to explore the stimuli that elicit schooling behavior in these populations. Our data suggest that the difference in schooling tendency between marine and benthic sticklebacks is accompanied by differential preferences for social vs. non-social and moving vs. stationary shelter options. Our study thus provides novel insights into the evolution of schooling behavior, as well as a new experimental approach to investigate the genetic and neural mechanisms that underlie this complex social behavior. PMID:21464914

  5. The peptide equivalent of the 16SrRNA assay: Revealing Phylogeny and Function

    NASA Astrophysics Data System (ADS)

    May, D. H.; Nunn, B. L.; Timmins-Schiffman, E.; Mikan, M.; Harvey, H. R.; Noble, W. S.

    2016-02-01

    Metagenomics has taught us that ocean microbes employ a diverse suite of metabolic strategies to break down complex heterogeneous particles and catalyze chemical transformations. However, to date, there has been limited success in establishing connections between microbial diversity and functions associated with subgroups of these microbes. Linking function and phylogeny is essential to developing more accurate models of the `microbial engines' of ocean biogeochemical cycles, and to understanding the relationship between diversity, ecosystem stability and functional redundancy. Ecosystem modelers are now demanding higher-resolution empirical data on microbial phylogeny and physiological responses to disturbances, to help them elucidate functional redundancy on a taxonomic level (i.e., phylum, class, order, etc.). Proteins are the only representative analyte that can resolve functional redundancy within a community of organisms by relating phylogeny and function. Unlike genes and transcripts, these dynamic macromolecules answer "who is doing what" at the time of collection, making them essential candidate biomarkers of changing chemical environments. Mass spectrometry (MS) based metaproteomics provides direct, quantitative evidence of the expression of thousands of proteins synthesized by a microbial community, the information necessary to characterize microbial responses that mitigate temporal disturbances. Our multi-disciplinary team has demonstrated that, within a metaproteome database, we can identify unique peptide sequences that resolve protein functions at different taxonomic levels. Here, we present our progress on the protein equivalent of a 16SrRNA assay, providing insight into both phylogeny and function. We integrated existing proteomics software and novel algorithms into a metaproteomics pipeline for resolving taxonomy-specific protein functions, allowing us to parameterize ecosystem processes as a function of chemistry, environment, space and time.

  6. Life table assay of field-caught Mediterranean fruit flies, Ceratitis capitata, reveals age bias

    PubMed Central

    Kouloussis, Nikos A.; Papadopoulos, Nikos T.; Müller, Hans-Georg; Wang, Jane-Ling; Mao, Meng; Katsoyannos, Byron I.; Duyck, Pierre-François; Carey, James R.

    2012-01-01

    Though traps are used widely to sample phytophagous insects for research or management purposes, and recently in aging research, possible bias stemming from differential response of individuals of various ages to traps has never been examined. In this paper, we tested the response of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) males and females of four ages (spanning from 1 to 40 days) to McPhail-type traps baited with a synthetic food attractant in field cages and found that the probability of trapping was significantly influenced by age. The type of food on which flies were maintained before testing (sugar or protein) also had a strong effect and interacted with age. In another experiment, we collected wild C. capitata adults of unknown age using 1–3 methods and then reared them in the laboratory until death. The survival schedules of these flies were subsequently used in a life table assay to infer their age at the time of capture. Results showed that on a single sampling date, males captured in traps baited with a food attractant were younger compared with males aspirated from fruiting host trees, or males captured in traps baited with a sex attractant. Likewise, females captured in food-baited traps were younger compared with aspirated females. In addition to providing the first evidence of age-dependent sampling bias for a phytophagous insect species, this paper also provides a novel approach to estimate the differences in the age composition of samples collected with different techniques. These findings are of utmost importance for several categories of insects, medically important groups notwithstanding. PMID:22844133

  7. Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains.

    PubMed

    Crauwels, Sam; Van Opstaele, Filip; Jaskula-Goiris, Barbara; Steensels, Jan; Verreth, Christel; Bosmans, Lien; Paulussen, Caroline; Herrera-Malaver, Beatriz; de Jonge, Ronnie; De Clippeleer, Jessika; Marchal, Kathleen; De Samblanx, Gorik; Willems, Kris A; Verstrepen, Kevin J; Aerts, Guido; Lievens, Bart

    2017-01-01

    Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man-made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off-)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces-related phenolic off-flavors 4-ethylguaiacol and 4-ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off-flavor production. Sequence analysis of genes related to phenolic off-flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.

  8. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

    PubMed

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-03-03

    NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

  9. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

    PubMed Central

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-01-01

    NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

  10. New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration.

    PubMed

    Yokoyama, Sho; Matsui, Tsubasa S; Deguchi, Shinji

    2017-01-22

    Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Cadmium-transformed cells in the in vitro cell transformation assay reveal different proliferative behaviours and activated pathways.

    PubMed

    Forcella, M; Callegaro, G; Melchioretto, P; Gribaldo, L; Frattini, M; Stefanini, F M; Fusi, P; Urani, C

    2016-10-01

    The in vitro Cell Transformation Assay (CTA) is a powerful tool for mechanistic studies of carcinogenesis. The endpoint is the classification of transformed colonies (foci) by means of standard morphological features. To increase throughput and reliability of CTAs, one of the suggested follow-up activities is to exploit the comprehension of the mechanisms underlying cell transformation. To this end, we have performed CTAs testing CdCl2, a widespread environmental contaminant classified as a human carcinogen with the underlying mechanisms of action not completely understood. We have isolated and re-seeded the cells at the end (6weeks) of in vitro CTAs to further identify the biochemical pathways underlying the transformed phenotype of foci. Morphological evaluations and proliferative assays confirmed the loss of contact-inhibition and the higher proliferative rate of transformed clones. The biochemical analysis of EGFR pathway revealed that, despite the same initial carcinogenic stimulus (1μM CdCl2 for 24h), transformed clones are characterized by the activation of two different molecular pathways: proliferation (Erk activation) or survival (Akt activation). Our preliminary results on molecular characterization of cell clones from different foci could be exploited for CTAs improvement, supporting the comprehension of the in vivo process and complementing the morphological evaluation of foci.

  12. Functional assays and metagenomic analyses reveals differences between the microbial communities inhabiting the soil horizons of a Norway spruce plantation.

    PubMed

    Uroz, Stéphane; Ioannidis, Panos; Lengelle, Juliette; Cébron, Aurélie; Morin, Emmanuelle; Buée, Marc; Martin, Francis

    2013-01-01

    In temperate ecosystems, acidic forest soils are among the most nutrient-poor terrestrial environments. In this context, the long-term differentiation of the forest soils into horizons may impact the assembly and the functions of the soil microbial communities. To gain a more comprehensive understanding of the ecology and functional potentials of these microbial communities, a suite of analyses including comparative metagenomics was applied on independent soil samples from a spruce plantation (Breuil-Chenue, France). The objectives were to assess whether the decreasing nutrient bioavailability and pH variations that naturally occurs between the organic and mineral horizons affects the soil microbial functional biodiversity. The 14 Gbp of pyrosequencing and Illumina sequences generated in this study revealed complex microbial communities dominated by bacteria. Detailed analyses showed that the organic soil horizon was significantly enriched in sequences related to Bacteria, Chordata, Arthropoda and Ascomycota. On the contrary the mineral horizon was significantly enriched in sequences related to Archaea. Our analyses also highlighted that the microbial communities inhabiting the two soil horizons differed significantly in their functional potentials according to functional assays and MG-RAST analyses, suggesting a functional specialisation of these microbial communities. Consistent with this specialisation, our shotgun metagenomic approach revealed a significant increase in the relative abundance of sequences related glycoside hydrolases in the organic horizon compared to the mineral horizon that was significantly enriched in glycoside transferases. This functional stratification according to the soil horizon was also confirmed by a significant correlation between the functional assays performed in this study and the functional metagenomic analyses. Together, our results suggest that the soil stratification and particularly the soil resource availability impact the

  13. A kinetic fluorescence assay reveals unusual features of Ca⁺⁺ uptake in Plasmodium falciparum-infected erythrocytes.

    PubMed

    Zipprer, Elizabeth M; Neggers, McKinzie; Kushwaha, Ambuj; Rayavara, Kempaiah; Desai, Sanjay A

    2014-05-18

    To facilitate development within erythrocytes, malaria parasites increase their host cell uptake of diverse solutes including Ca++. The mechanism and molecular basis of increased Ca++ permeability remains less well studied than that of other solutes. Based on an appropriate Ca++ affinity and its greater brightness than related fluorophores, Fluo-8 was selected and used to develop a robust fluorescence-based assay for Ca++ uptake by human erythrocytes infected with Plasmodium falciparum. Both uninfected and infected cells exhibited a large Ca++-dependent fluorescence signal after loading with the Fluo-8 dye. Probenecid, an inhibitor of erythrocyte organic anion transporters, abolished the fluorescence signal in uninfected cells; in infected cells, this agent increased fluorescence via mechanisms that depend on parasite genotype. Kinetic fluorescence measurements in 384-well microplates revealed that the infected cell Ca++ uptake is not mediated by the plasmodial surface anion channel (PSAC), a parasite nutrient channel at the host membrane; it also appears to be distinct from mammalian Ca++ channels. Imaging studies confirmed a low intracellular Ca++ in uninfected cells and higher levels in both the host and parasite compartments of infected cells. Parasite growth inhibition studies revealed a conserved requirement for extracellular Ca++. Nondestructive loading of Fluo-8 into human erythrocytes permits measurement of Ca++ uptake kinetics. The greater Ca++ permeability of cells infected with malaria parasites is apparent when probenecid is used to inhibit Fluo-8 efflux at the host membrane. This permeability is mediated by a distinct pathway and may be essential for intracellular parasite development. The miniaturized assay presented here should help clarify the precise transport mechanism and may identify inhibitors suitable for antimalarial drug development.

  14. Zebrafish-based reporter gene assays reveal different estrogenic activities in river waters compared to a conventional human-derived assay.

    PubMed

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

    2016-04-15

    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.

  15. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells.

    PubMed

    Hulme, Amy E; Kelley, Z; Foley, Deirdre; Hope, Thomas J

    2015-05-01

    During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2. The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine the effect of two

  16. Complementary Assays Reveal a Low Level of CA Associated with Viral Complexes in the Nuclei of HIV-1-Infected Cells

    PubMed Central

    Hulme, Amy E.; Kelley, Z; Foley, Deirdre

    2015-01-01

    ABSTRACT During uncoating, the conical capsid of HIV disassembles by dissociation of the p24 capsid protein (CA). Uncoating is known to be required for HIV replication, but the mechanism is poorly defined. Here, we examined the timing and effect of two capsid binding drugs (PF74 and BI2) on infectivity and capsid integrity in HIV-1-infected cells. The virus remained susceptible to the action of PF74 and BI2 for hours after uncoating as defined in parallel drug addition and cyclosporine (CsA) washout assays to detect the kinetics of drug susceptibility and uncoating, respectively. Resistance mutations in CA decreased the potency of these compounds, demonstrating that CA is the target of drug action. However, neither drug altered capsid integrity in a fluorescence microscopy-based assay. These data suggest that PF74 and BI2 do not alter HIV-1 uncoating but rather affect a later step in viral replication. Because both drugs bind CA, we hypothesized that a residual amount of CA associates with the viral complex after the loss of the conical capsid to serve as a target for these drugs. Superresolution structured illumination microscopy (SIM) revealed that CA localized to viral complexes in the nuclei of infected cells. Using image quantification, we determined that viral complexes localized in the nucleus displayed a smaller amount of CA than complexes at the nuclear membrane, in the cytoplasm, or in controls. Collectively, these data suggest that a subset of CA remains associated with the viral complex after uncoating and that this residual CA is the target of PF74 and BI2. IMPORTANCE The HIV-1 capsid is a target of interest for new antiviral therapies. This conical capsid is composed of monomers of the viral CA protein. During HIV-1 replication, the capsid must disassemble by a poorly defined process called uncoating. CA has also been implicated in later steps of replication, including nuclear import and integration. In this study, we used cell-based assays to examine

  17. Quantitative Proteomic Analysis Reveals Molecular Adaptations in the Hippocampal Synaptic Active Zone of Chronic Mild Stress-Unsusceptible Rats

    PubMed Central

    Zhou, Jian; Liu, Zhao; Yu, Jia; Han, Xin; Fan, Songhua; Shao, Weihua; Chen, Jianjun; Qiao, Rui

    2016-01-01

    Background: While stressful events are recognized as an important cause of major depressive disorder, some individuals exposed to life stressors maintain normal psychological functioning. The molecular mechanism(s) underlying this phenomenon remain unclear. Abnormal transmission and plasticity of hippocampal synapses have been implied to play a key role in the pathoetiology of major depressive disorder. Methods: A chronic mild stress protocol was applied to separate susceptible and unsusceptible rat subpopulations. Proteomic analysis using an isobaric tag for relative and absolute quantitation coupled with tandem mass spectrometry was performed to identify differential proteins in enriched hippocampal synaptic junction preparations. Results: A total of 4318 proteins were quantified, and 89 membrane proteins were present in differential amounts. Of these, SynaptomeDB identified 81 (91%) having a synapse-specific localization. The unbiased profiles identified several candidate proteins within the synaptic junction that may be associated with stress vulnerability or insusceptibility. Subsequent functional categorization revealed that protein systems particularly involved in membrane trafficking at the synaptic active zone exhibited a positive strain as potential molecular adaptations in the unsusceptible rats. Moreover, through STRING and immunoblotting analysis, membrane-associated GTP-bound Rab3a and Munc18-1 appear to coregulate syntaxin-1/SNAP25/VAMP2 assembly at the hippocampal presynaptic active zone of unsusceptible rats, facilitating SNARE-mediated membrane fusion and neurotransmitter release, and may be part of a stress-protection mechanism in actively maintaining an emotional homeostasis. Conclusions: The present results support the concept that there is a range of potential protein adaptations in the hippocampal synaptic active zone of unsusceptible rats, revealing new investigative targets that may contribute to a better understanding of stress

  18. Infection assays in Arabidopsis reveal candidate effectors from the poplar rust fungus that promote susceptibility to bacteria and oomycete pathogens.

    PubMed

    Germain, Hugo; Joly, David L; Mireault, Caroline; Plourde, Mélodie B; Letanneur, Claire; Stewart, Donald; Morency, Marie-Josée; Petre, Benjamin; Duplessis, Sébastien; Séguin, Armand

    2016-11-21

    Fungi of the Pucciniales order cause rust diseases which, altogether, affect thousands of plant species worldwide and pose a major threat to several crops. How rust effectors-virulence proteins delivered into infected tissues to modulate host functions-contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplar, and its genome encodes 1184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria, we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of green fluorescent protein-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.

  19. Revealing the ability of a novel polysaccharide bioflocculant in bioremediation of heavy metals sensed in a Vibrio bioluminescence reporter assay.

    PubMed

    Sajayan, Arya; Seghal Kiran, G; Priyadharshini, S; Poulose, Navya; Selvin, Joseph

    2017-09-01

    A bioflocculant-producing bacterial strain, designated MSI021, was isolated from the marine sponge Dendrilla nigra and demonstrated 94% flocculation activity in a kaolin clay suspension. MSI021 was identified as Bacillus cereus based on phylogenetic affiliation and biochemical characteristics. The purified extra-cellular bioflocculant was chemically elucidated as a polysaccharide molecule. The polysaccharide bioflocculant was stable under both acidic and alkaline conditions (pH 2.0-10.0) and temperatures up to 100 °C. The purified bioflocculant efficiently nucleated the formation of silver nanoparticles which showed broad spectrum antibacterial activity. The ability of the bioflocculant to remediate heavy metal toxicity was evaluated by measuring the inhibition of bioluminescence expression in Vibrio harveyi. Enrichment of heavy metals such as zinc, mercury and copper at concentrations of 1, 2 and 3 mM in culture media showed significant reduction of bioluminescence in Vibrio, whereas media enriched with heavy metals and bioflocculant showed dose dependent improvement in the expression of bioluminescence. The assay results demonstrated that the polysaccharide bioflocculant effectively mitigates heavy metal toxicity, thereby improving the expression of bioluminescence in Vibrio. This bioluminescence reporter assay can be developed into a high-throughput format to monitor and evaluate of heavy metal toxicity. The findings of this study revealed that a novel polysaccharide bioflocculant produced by a marine B. cereus demonstrated strong flocculating performance and was effective in nucleating the formation antibacterial silver nanoparticles and removing heavy metals. These results suggest that the MSI021 polysaccharide bioflocculant can be used to develop greener waste water treatment systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A Quantitative Toxicogenomics Assay Reveals the Evolution and Nature of Toxicity during the Transformation of Environmental Pollutants

    PubMed Central

    2015-01-01

    The incomplete mineralization of contaminants of emerging concern (CECs) during the advanced oxidation processes can generate transformation products that exhibit toxicity comparable to or greater than that of the original contaminant. In this study, we demonstrated the application of a novel, fast, and cost-effective quantitative toxicogenomics-based approach for the evaluation of the evolution and nature of toxicity along the electro-Fenton oxidative degradation of three representative CECs whose oxidative degradation pathways have been relatively well studied, bisphenol A, triclosan, and ibuprofen. The evolution of toxicity as a result of the transformation of parent chemicals and production of intermediates during the course of degradation are monitored, and the quantitative toxicogenomics assay results revealed the dynamic toxicity changes and mechanisms, as well as their association with identified intermediates during the electro-Fenton oxidation process of the selected CECs. Although for the three CECs, a majority (>75%) of the parent compounds disappeared at the 15 min reaction time, the nearly complete elimination of toxicity required a minimal 30 min reaction time, and they seem to correspond to the disappearance of identified aromatic intermediates. Bisphenol A led to a wide range of stress responses, and some identified transformation products containing phenolic or quinone group, such as 1,4-benzoquinone and hydroquinone, likely contributed to the transit toxicity exhibited as DNA stress (genotoxicity) and membrane stress during the degradation. Triclosan is known to cause severe oxidative stress, and although the oxidative damage potential decreased concomitantly with the disappearance of triclosan after a 15 min reaction, the sustained toxicity associated with both membrane and protein stress was likely attributed at least partially to the production of 2,4-dichlorophenol that is known to cause the production of abnormal proteins and affect the cell

  1. Velocity-Dependent Actomyosin ATPase Cycle Revealed by In Vitro Motility Assay with Kinetic Analysis

    PubMed Central

    Sato, Masaaki K.; Ishihara, Takashi; Tanaka, Hiroto; Ishijima, Akihiko; Inoue, Yuichi

    2012-01-01

    The actomyosin interaction plays a key role in a number of cellular functions. Single-molecule measurement techniques have been developed to study the mechanism of the actomyosin contractile system. However, the behavior of isolated single molecules does not always reflect that of molecules in a complex system such as a muscle fiber. Here, we developed a simple method for studying the kinetic parameters of the actomyosin interaction using small numbers of molecules. This approach does not require the specialized equipment needed for single-molecule measurements, and permits us to observe behavior that is more similar to that of a complex system. Using an in vitro motility assay, we examined the duration of continuous sliding of actin filaments on a sparsely distributed heavy meromyosin-coated surface. To estimate the association rate constant of the actomyosin motile system, we compared the distribution of experimentally obtained duration times with a computationally simulated distribution. We found that the association rate constant depends on the sliding velocity of the actin filaments. This technique may be used to reveal new aspects of the kinetics of various motor proteins in complex systems. PMID:22947932

  2. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  3. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

    PubMed

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-03-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

  4. Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

    PubMed Central

    Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

    2014-01-01

    β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

  5. Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules

    PubMed Central

    Alper, Joshua D.; Tovar, Miguel; Howard, Jonathon

    2013-01-01

    In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules. PMID:23663842

  6. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    PubMed

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. Copyright © 2011 John Wiley & Sons, Ltd.

  7. Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico.

    PubMed

    Pérez-López, Edel; Rodríguez-Martínez, Douglas; Olivier, Chrystel Y; Luna-Rodríguez, Mauricio; Dumonceaux, Tim J

    2017-04-19

    Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

  8. Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay

    SciTech Connect

    Kuo, Chun-Chen; Chu, Chi-Yuan; Lin, Jing-Jer; Lo, Lee-Chiang

    2010-01-01

    Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.

  9. Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

    PubMed Central

    Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

    2016-01-01

    Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

  10. Subtle conformational changes between CX3CR1 genetic variants as revealed by resonance energy transfer assays.

    PubMed

    Darbandi-Tehrani, Kévin; Hermand, Patricia; Carvalho, Stéphanie; Dorgham, Karim; Couvineau, Alain; Lacapère, Jean-Jacques; Combadière, Christophe; Deterre, Philippe

    2010-11-01

    The chemokine CX3CL1 is expressed as a membrane protein that forms a potent adhesive pair with its unique receptor CX3CR1. This receptor has 3 natural variants, V249-T280 (VT), I249-T280 (IT), and I249-M280 (IM), whose relative frequencies are significantly associated with the incidence of various inflammatory diseases. To assess the adhesive potency of CX3CR1 and the molecular diversity of its variants, we assayed their clustering status and their possible structural differences by fluorescence/bioluminescence resonance energy transfer (FRET or BRET) techniques. FRET assays by flow cytometry showed that the CX3CR1 variants cluster, in comparison with appropriate controls. BRET assays showed low nonspecific signals for VT and IT variants and high specific signals for IM, and thus pointed out a structural difference in this variant. We used molecular modeling to show how natural point mutations of CX3CR1 affect the packing of the 6th and 7th helices of this G-protein coupled receptor. Moreover, we found that the BRET technique is sensitive enough to detect these tiny changes. Consistently with our previous finding that CX3CL1 aggregates, our data here indicate that CX3CR1 clustering may contribute to the adhesiveness of the CX3CL1-CX3CR1 pair and may thus represent a new target for anti-inflammatory therapies.

  11. Genotoxicity of Thermopsis turcica on Allium cepa L. roots revealed by alkaline comet and random amplified polymorphic DNA assays.

    PubMed

    Ciğerci, İbrahim Hakkı; Cenkci, Süleyman; Kargıoğlu, Mustafa; Konuk, Muhsin

    2016-08-01

    This study was undertaken to evaluate genotoxic potential of Thermopsis turcica aqueous extracts on the roots of onion bulb (Allium cepa L.) by comet assay and random amplified polymorphic DNA technique. The Allium root growth inhibition test indicated that the EC50 and 2×EC50 values were 8 and 16 mg/ml concentrations of T. turcica aqueous extracts, respectively. The negative control (distilled water), positive control (methyl methane sulfonate, 10 mg/l) and 8 and 16 mg/ml concentrations of T. turcica extracts were introduced to the roots of onion bulbs for 24 and 96 h. The root growth, DNA damage in root cells and randomly amplified polymorphic DNA (RAPD) profiles of root tissue were used as endpoints of the genotoxicity. The comet assay clearly indicated that dose-dependent single strand DNA breaks in the root nuclei of onions were determined for the treatment concentrations of T. turcica extracts. In comparison to RAPD profile of negative control group, RAPD polymorphisms became evident as disappearance and/or appearance of RAPD bands in treated roots. The diagnostic and phenetic numerical analyses of RAPD profiles obviously indicated dose-dependent genotoxicity induced by Thermopsis extracts. In conclusion, the results clearly indicated that water extract of T. turcica has genotoxic potential on the roots of onion bulbs as shown by comet assay and RAPD technique.

  12. A Novel Entry/Uncoating Assay Reveals the Presence of at Least Two Species of Viral Capsids During Synchronized HIV-1 Infection

    PubMed Central

    Cimarelli, Andrea

    2016-01-01

    To better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial core destabilization. Although reverse transcription drives a faster loss of susceptibility of viral cores to high doses of PF74, it does not lead to higher exposure of the viral genome, implying that viral cores protect the genome irrespectively of reverse transcription. Lastly, IFNα drifts cores from functional to non-functional species, revealing a novel core-destabilizing activity. This assay sheds new light on the behavior of viral cores inside target cells. PMID:27690375

  13. A Novel Entry/Uncoating Assay Reveals the Presence of at Least Two Species of Viral Capsids During Synchronized HIV-1 Infection.

    PubMed

    Da Silva Santos, Claire; Tartour, Kevin; Cimarelli, Andrea

    2016-09-01

    To better characterize the behavior of HIV-1 capsids we developed EURT, for Entry/Uncoating assay based on core-packaged RNA availability and Translation. EURT is an alternative to Blam-Vpr, but as reporter RNA translation relies on core opening, it can be used to study viral capsids behavior. Our study reveals the existence of two major capsid species, a dead end one in which the viral genome is readily exposed to the cytoplasm and a functional one in which such exposure requires artificial core destabilization. Although reverse transcription drives a faster loss of susceptibility of viral cores to high doses of PF74, it does not lead to higher exposure of the viral genome, implying that viral cores protect the genome irrespectively of reverse transcription. Lastly, IFNα drifts cores from functional to non-functional species, revealing a novel core-destabilizing activity. This assay sheds new light on the behavior of viral cores inside target cells.

  14. The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

    PubMed Central

    Rui, Yan-Ning; Xu, Zhen; Chen, Zhihua; Zhang, Sheng

    2015-01-01

    By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study

  15. The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells.

    PubMed

    Rui, Yan-Ning; Xu, Zhen; Chen, Zhihua; Zhang, Sheng

    2015-01-01

    By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study

  16. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk

    PubMed Central

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S.; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Paz-Elizur, Tamar

    2014-01-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case–control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3–16.3), significantly higher than in control subjects—15.1 (14.6–15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1–2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6–4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT. PMID:25355292

  17. Enzymatic MPG DNA repair assays for two different oxidative DNA lesions reveal associations with increased lung cancer risk.

    PubMed

    Leitner-Dagan, Yael; Sevilya, Ziv; Pinchev, Mila; Kremer, Ran; Elinger, Dalia; Rennert, Hedy S; Schechtman, Edna; Freedman, Laurence; Rennert, Gad; Livneh, Zvi; Paz-Elizur, Tamar

    2014-12-01

    DNA repair is a major mechanism for minimizing mutations and reducing cancer risk. Here, we present the development of reproducible and specific enzymatic assays for methylpurine DNA glycosylase (MPG) repairing the oxidative lesions 1,N6-ethenoadenine (εA) and hypoxanthine (Hx) in peripheral blood mononuclear cells protein extracts. Association of these DNA repair activities with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean MPG-εA in case patients was 15.8 units/μg protein (95% CI 15.3-16.3), significantly higher than in control subjects-15.1 (14.6-15.5), *P = 0.011. The adjusted odds ratio for lung cancer associated with a one SD increase in MPG-εA activity (2.48 units) was significantly bigger than 1 (OR = 1.6, 95% CI = 1.1-2.4; *P = 0.013). When activity of OGG1, a different DNA repair enzyme for oxidative damage, was included in the model, the estimated odds ratio/SD for a combined MPG-εA-OGG1 score was 2.6 (95% CI 1.6-4.2) *P = 0.0001, higher than the odds ratio for each single assay. The MPG enzyme activity assays described provide robust functional risk biomarkers, with increased MPG-εA activity being associated with increased lung cancer risk, similar to the behavior of MPG-Hx. This underscores the notion that imbalances in DNA repair, including high DNA repair, usually perceived as beneficial, can cause cancer risk. Such DNA repair risk biomarkers may be useful for risk assessment of lung cancer and perhaps other cancer types, and for early detection techniques such as low-dose CT. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay.

    PubMed

    Byrd, Thomas F; Hoang, Loi T; Kim, Eric G; Pfister, Matthew E; Werner, Erik M; Arndt, Stephen E; Chamberlain, Jeffrey W; Hughey, Jacob J; Nguyen, Bao A; Schneibel, Erik J; Wertz, Laura L; Whitfield, Jonathan S; Wikswo, John P; Seale, Kevin T

    2014-05-30

    Cytometric studies utilizing flow cytometry or multi-well culture plate fluorometry are often limited by a deficit in temporal resolution and a lack of single cell consideration. Unfortunately, many cellular processes, including signaling, motility, and molecular transport, occur transiently over relatively short periods of time and at different magnitudes between cells. Here we demonstrate the multitrap nanophysiometer (MTNP), a low-volume microfluidic platform housing an array of cell traps, as an effective tool that can be used to study individual unattached cells over time with precise control over the intercellular microenvironment. We show how the MTNP platform can be used for hematologic cancer cell characterization by measuring single T cell levels of CRAC channel modulation, non-translational motility, and ABC-transporter inhibition via a calcein-AM efflux assay. The transporter data indicate that Jurkat T cells exposed to indomethacin continue to accumulate fluorescent calcein for over 60 minutes after calcein-AM is removed from the extracellular space.

  19. Why in vivo may not equal in vitro - new effectors revealed by measurement of enzymatic activities under the same in vivo-like assay conditions.

    PubMed

    García-Contreras, Rodolfo; Vos, Paul; Westerhoff, Hans V; Boogerd, Fred C

    2012-11-01

    Does the understanding of the dynamics of biochemical networks in vivo, in terms of the properties of their components determined in vitro, require the latter to be determined all under the same conditions? An in vivo-like assay medium for enzyme activity determination was designed based on the concentrations of the major ionic constituents of the Escherichia coli cytosol: K(+), Na(+), Mg(2+), phosphate, glutamate, sulfate and Cl(-). The maximum capacities (V(max)) of the extracted enzymes of two pathways were determined using both this in vivo-like assay medium and the assay medium specific for each enzyme. The enzyme activities differed between the two assay conditions. Most of the differences could be attributed to unsuspected, pleiotropic effects of K(+) and phosphate. K(+) activated some enzymes (aldolase, enolase and glutamate dehydrogenase) and inhibited others (phosphoglucose isomerase, phosphofructokinase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase), whereas phosphate inhibited all glycolytic enzymes and glutamine synthetase but only activated glutamine 2-oxoglutarate amidotransferase. Neither a high glutamate concentration, nor macromolecular crowding affected the glycolytic or nitrogen assimilation enzymes, other than through the product inhibition of glutamate dehydrogenase by glutamate. This strategy of assessing all pathway enzymes kinetically under the same conditions may be necessary to avoid inadvertent differences between in vivo and in vitro biochemistry. It may also serve to reveal otherwise unnoticed pleiotropic regulation, such as that demonstrated in the present study by K(+) and phosphate. © 2012 The Authors Journal compilation © 2012 FEBS.

  20. Establishment of an In Vitro Transport Assay That Reveals Mechanistic Differences in Cytosolic Events Controlling Cholera Toxin and T-Cell Receptor α Retro-Translocation

    PubMed Central

    Moore, Paul; He, Kaiyu; Tsai, Billy

    2013-01-01

    Following retrograde trafficking to the endoplasmic reticulum (ER), cholera toxin A1 (CTA1) subunit hijacks ER-associated degradation (ERAD) machinery and retro-translocates into the cytosol to induce toxicity. We previously established a cell-based in vivo assay to identify ER components that regulate this process. However, elucidating cytosolic events that govern CTA1 retro-translocation using this assay is difficult as manipulating cytosolic factors often perturbs toxin retrograde transport to the ER. To circumvent this problem, we developed an in vitro assay in semi-permeabilized cells that directly monitors CTA1 release from the ER into the cytosol. We demonstrate CTA1 is released into the cytosol as a folded molecule in a p97- and proteasome-independent manner. Release nonetheless involves a GTP-dependent reaction. Upon extending this assay to the canonical ERAD substrate T-cell receptor α (TCRα), we found the receptor is unfolded when released into the cytosol and degraded by membrane-associated proteasome. In this reaction, p97 initially extracts TCRα from the ER membrane, followed by TCRα discharge into the cytosol that requires additional energy-dependent cytosolic activities. Our results reveal mechanistic insights into cytosolic events controlling CTA1 and TCRα retro-translocation, and provide a reliable tool to further probe this process. PMID:24146777

  1. A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

    PubMed

    Soroush, Fariborz; Zhang, Ting; King, Devon J; Tang, Yuan; Deosarkar, Sudhir; Prabhakarpandian, Balabhaskar; Kilpatrick, Laurie E; Kiani, Mohammad F

    2016-11-01

    A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

  2. Reduction of doxorubicin-induced genotoxicity by Handroanthus impetiginosus in mouse bone marrow revealed by micronucleus assay.

    PubMed

    Boriollo, M F G; Silva, T A; Rodrigues-Netto, M F; Silva, J J; Marques, M B; Dias, C T S; Höfling, J F; Resck, M C C; Oliveira, N M S

    2017-07-10

    Handroanthus impetiginosus has long been used in traditional medicine and various studies have determined the presence of bioactive chemical compounds and potential phytotherapeutics. In this study, the genotoxicity of the lyophilized tincture of H. impetiginosus bark (THI) was evaluated in mouse bone marrow using micronucleus assays. The interaction between THI and genotoxic effects induced by the chemotherapeutic agent, doxorubicin (DXR), was also analyzed. Experimental groups were evaluated 24 to 48 h after treatment with N-nitroso-N-ethylurea (NEU; 50 mg/kg), DXR (5 mg/kg), sodium chloride (NaCl; 150 mM), and THI (0.5-2 g/kg). Antigenotoxic assays were carried out using THI (0.5 g/kg) in combination with NEU or DXR. Analysis of the micronucleated polychromatic erythrocytes (MNPCEs) indicated no significant differences between treatment doses of THI (0.5-2 g/kg) and NaCl. Polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) ratios did not indicate any statistical differences between DXR and THI or NaCl, but there were differences between THI and NaCl. A significant reduction in MNPCEs and PCE/NCE ratios was observed when THI was administered in combination with DXR. This study suggested the absence of THI genotoxicity that was dose-, time-, and gender-independent and the presence of moderate systemic toxicity that was dose-independent, but time- and gender-dependent. The combination of THI and DXR also suggested antigenotoxic effects, indicating that THI reduced genotoxic effects induced by chemotherapeutic agents.

  3. Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gamma-proteobacterial ammonium-oxidizing bacteria.

    PubMed

    Nishigaya, Yuki; Fujimoto, Zui; Yamazaki, Toshimasa

    2016-07-29

    Ammonia-oxidizing bacteria (AOB), ubiquitous chemoautotrophic bacteria, convert ammonia (NH3) to nitrite (NO2(-)) via hydroxylamine as energy source. Excessive growth of AOB, enhanced by applying large amounts of ammonium-fertilizer to the farmland, leads to nitrogen leaching and nitrous oxide gas emission. To suppress these unfavorable phenomena, nitrification inhibitors, AOB specific bactericides, are widely used in fertilized farmland. However, new nitrification inhibitors are desired because of toxicity and weak-effects of currently used inhibitors. Toward development of novel nitrification inhibitors that target hydroxylamine oxidoreductase (HAO), a key enzyme of nitrification in AOB, we established inhibitor evaluation systems that include simplified HAO purification procedure and high-throughput HAO activity assays for the purified enzymes and for the live AOB cells. The new assay systems allowed us to observe distinct inhibitory responses of HAOs from beta-proteobacterial AOB (βAOB) Nitrosomonas europaea (NeHAO) and gamma-proteobacterial AOB (γAOB) Nitrosococcus oceani (NoHAO) against phenylhydrazine, a well-known suicide inhibitor for NeHAO. Consistently, the live cells of N. europaea, Nitrosomonas sp. JPCCT2 and Nitrosospira multiformis of βAOB displayed higher responses to phenylhydrazine than those of γAOB N. oceani. Our homology modeling studies suggest that different inhibitory responses of βAOB and γAOB are originated from different local environments around the substrate-binding sites of HAOs in these two classes of bacteria due to substitutions of two residues. The results reported herein strongly recommend inhibitor screenings against both NeHAO of βAOB and NoHAO of γAOB to develop HAO-targeting nitrification inhibitors with wide anti-AOB spectra. Copyright © 2016. Published by Elsevier Inc.

  4. Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter

    PubMed Central

    Rogge, George A; Shen, Li-Ling; Kuhar, Michael J.

    2010-01-01

    Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action. PMID:20451507

  5. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    SciTech Connect

    Fichorova, Raina N.; Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F.

    2015-06-15

    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. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  6. An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium.

    PubMed Central

    Falk, P; Roth, K A; Borén, T; Westblom, T U; Gordon, J I; Normark, S

    1993-01-01

    Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria

  7. Radioligand-binding assay reveals distinct autoantibody preferences for type I interferons in APS I and myasthenia gravis subgroups.

    PubMed

    Hapnes, Liv; Willcox, Nick; Oftedal, Bergithe E V; Owe, Jone F; Gilhus, Nils Erik; Meager, Anthony; Husebye, Eystein S; Wolff, Anette S Bøe

    2012-04-01

    Patients with autoimmune polyendocrine syndrome type I (APS I) or acquired thymoma-associated myasthenia gravis (MG) surprisingly share several common features, including defective expression of the transcription factor AIRE and autoantibodies against type I interferons. Here, we have adapted and validated the radioligand-binding assay we recently developed against (35)S-Met-interferon-ω, for rapid and specific screening for autoantibodies against interferons-α2 and -α8. We then investigated their potential for diagnosis and for predicting clinical manifestations in patients with APS I and different subgroups of MG. Autoantibodies against interferons-ω, -α2, and -α8 occurred more often in patients with APS I (100%) and MG with thymoma (73%) than in late-onset MG (39%) and early-onset MG (5%). These autoantibodies showed preferences for interferon-ω in APS I and for the interferon-αs in MG, hinting at thymic aberrations in both groups. The exact profile of type I interferon antibodies may indicate MG subtype and may hint at thymoma recurrence.

  8. The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay

    PubMed Central

    Byrd IV, Thomas F.; Hoang, Loi T.; Kim, Eric G.; Pfister, Matthew E.; Werner, Erik M.; Arndt, Stephen E.; Chamberlain, Jeffrey W.; Hughey, Jacob J.; Nguyen, Bao A.; Schneibel, Erik J.; Wertz, Laura L.; Whitfield, Jonathan S.; Wikswo, John P.; Seale, Kevin T.

    2014-01-01

    Cytometric studies utilizing flow cytometry or multi-well culture plate fluorometry are often limited by a deficit in temporal resolution and a lack of single cell consideration. Unfortunately, many cellular processes, including signaling, motility, and molecular transport, occur transiently over relatively short periods of time and at different magnitudes between cells. Here we demonstrate the multitrap nanophysiometer (MTNP), a low-volume microfluidic platform housing an array of cell traps, as an effective tool that can be used to study individual unattached cells over time with precise control over the intercellular microenvironment. We show how the MTNP platform can be used for hematologic cancer cell characterization by measuring single T cell levels of CRAC channel modulation, non-translational motility, and ABC-transporter inhibition via a calcein-AM efflux assay. The transporter data indicate that Jurkat T cells exposed to indomethacin continue to accumulate fluorescent calcein for over 60 minutes after calcein-AM is removed from the extracellular space. PMID:24873950

  9. A yeast-based assay reveals a functional defect of the Q488H polymorphism in human Hsp90{alpha}

    SciTech Connect

    MacLean, Morag J.; Martinez Llordella, Marc; Bot, Nathalie; Picard, Didier . E-mail: picard@cellbio.unige.ch

    2005-11-11

    It has been argued that the molecular chaperone Hsp90 guards the organism against genetic variations by stabilizing variant Hsp90 substrate proteins. However, little is known about polymorphisms affecting its own functions. We have followed up on a recent study describing two polymorphisms that alter the amino acid sequences of the two Hsp90 isoforms Hsp90{alpha} and Hsp90{beta}. Hsp90 is essential for cell proliferation in the budding yeast Saccharomyces cerevisiae, but the human proteins can replace the endogenous ones. In this growth assay, the variant V656M of Hsp90{beta} was indistinguishable from wild-type. In contrast, the Hsp90{alpha} variant Q488H, which carries an alteration of a very highly conserved residue, was severely defective for growth compared to wild-type Hsp90{alpha}. Hence, the characteristics of this yeast-based system-simplicity, rapidity, low cost-make it ideal for phenotype screening of polymorphisms in HSP90 and possibly many other human genes.

  10. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

    PubMed

    Fichorova, Raina N; Mendonca, Kevin; Yamamoto, Hidemi S; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F

    2015-06-15

    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.

  11. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    SciTech Connect

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-05-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group).

  12. A Dual-Color Luciferase Assay System Reveals Circadian Resetting of Cultured Fibroblasts by Co-Cultured Adrenal Glands

    PubMed Central

    Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2012-01-01

    In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co

  13. A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.

    PubMed

    Noguchi, Takako; Ikeda, Masaaki; Ohmiya, Yoshihiro; Nakajima, Yoshihiro

    2012-01-01

    In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co

  14. Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion.

    PubMed

    Ishihara, Naotada; Maeda, Maki; Ban, Tadato; Mihara, Katsuyoshi

    2017-04-27

    Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signaling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential, and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive, and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. 3-Dimensional Patient-Derived Lung Cancer Assays Reveal Resistance to Standards-of-Care Promoted by Stromal Cells but Sensitivity to Histone Deacetylase Inhibitors.

    PubMed

    Onion, David; Argent, Richard H; Reece-Smith, Alexander M; Craze, Madeleine L; Pineda, Robert G; Clarke, Philip A; Ratan, Hari L; Parsons, Simon L; Lobo, Dileep N; Duffy, John P; Atherton, John C; McKenzie, Andrew J; Kumari, Rajendra; King, Peter; Hall, Brett M; Grabowska, Anna M

    2016-04-01

    There is a growing recognition that current preclinical models do not reflect the tumor microenvironment in cellular, biological, and biophysical content and this may have a profound effect on drug efficacy testing, especially in the era of molecular-targeted agents. Here, we describe a method to directly embed low-passage patient tumor-derived tissue into basement membrane extract, ensuring a low proportion of cell death to anoikis and growth complementation by coculture with patient-derived cancer-associated fibroblasts (CAF). A range of solid tumors proved amenable to growth and pharmacologic testing in this 3D assay. A study of 30 early-stage non-small cell lung cancer (NSCLC) specimens revealed high levels of de novo resistance to a large range of standard-of-care agents, while histone deacetylase (HDAC) inhibitors and their combination with antineoplastic drugs displayed high levels of efficacy. Increased resistance was seen in the presence of patient-derived CAFs for many agents, highlighting the utility of the assay for tumor microenvironment-educated drug testing. Standard-of-care agents showed similar responses in the 3D ex vivo and patient-matched in vivo models validating the 3D-Tumor Growth Assay (3D-TGA) as a high-throughput screen for close-to-patient tumors using significantly reduced animal numbers. Mol Cancer Ther; 15(4); 753-63. ©2016 AACR.

  16. Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity.

    PubMed

    Christensen, Sune M; Mortensen, Michael W; Stamou, Dimitrios G

    2011-02-16

    Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca(2+) dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca(2+) accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca(2+) accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) ∼ 1 min and t(mix) ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Large scale integration of drug-target information reveals poly-pharmacological drug action mechanisms in tumor cell line growth inhibition assays

    PubMed Central

    Knight, Richard A.; Gostev, Mikhail; Ilisavskii, Sergei; Willis, Anne E.; Melino, Gerry; Antonov, Alexey V.

    2014-01-01

    Understanding therapeutic mechanisms of drug anticancer cytotoxicity represents a key challenge in preclinical testing. Here we have performed a meta-analysis of publicly available tumor cell line growth inhibition assays (~ 70 assays from 6 independent experimental groups covering ~ 500 000 molecules) with the primary goal of understanding molecular therapeutic mechanisms of cancer cytotoxicity. To implement this we have collected currently available information on protein targets for molecules that were tested in the assays. We used a statistical methodology to identify protein targets overrepresented among molecules exhibiting cancer cytotoxicity with the particular focus of identifying overrepresented patterns consisting of several proteins (i.e. proteins “A” and “B” and “C”). Our analysis demonstrates that targeting individual proteins can result in a significant increase (up to 50-fold) of the observed odds for a molecule to be an efficient inhibitor of tumour cell line growth. However, further insight into potential molecular mechanisms reveals a multi-target mode of action: targeting a pattern of several proteins drastically increases the observed odds (up to 500-fold) for a molecule to be tumour cytotoxic. In contrast, molecules targeting only one protein but not targeting an additional set of proteins tend to be nontoxic. Our findings support a poly-pharmacology drug discovery paradigm, demonstrating that anticancer cytotoxicity is a product, in most cases, of multi-target mode of drug action PMID:24553133

  18. Anteroposterior patterning in Xenopus embryos: egg fragment assay system reveals a synergy of dorsalizing and posteriorizing embryonic domains.

    PubMed

    Fujii, Hidefumi; Nagai, Takeharu; Shirasawa, Hiroki; Doi, Jun-ya; Yasui, Kinya; Nishimatsu, Shin-ichirou; Takeda, Hiroyuki; Sakai, Masao

    2002-12-01

    Two distinct types of axis lacking embryos resulted from partial deletion of the vegetal part of early one-cell-stage embryos. When the deleted volume was 20-40% (relative surface area), the embryos underwent ventral-type gastrulation and formed ventral mesodermal tissues. When the deleted volume was more than 60%, the embryo did not gastrulate nor make mesodermal structures (M. Sakai, 1996, Development 122, 2207-2214). We have designated these two types of embryos as "gastrulating nonaxial embryos (GNEs)" and "permanent blastula-type embryos (PBEs)," respectively. Using these embryos as recipients, a series of Einsteck transplantation experiments were carried out to investigate mechanisms controlling anteroposterior patterning during early Xenopus development. GNEs receiving dorsal marginal zone (DMZ) transplants (GNE/DMZs) elongated and formed posteriorized phenotypes, which had muscle cells, melanocytes, and tail fins. In contrast, PBE/DMZs did not elongate but formed cement glands and brain-like structures showing strong anteriorization. Simultaneous transplantation of the cells from various regions of normal embryos with the DMZ into PBEs revealed that the entire vegetal half of normal embryos, except for the DMZ, showed posteriorizing activity. These results strongly suggest that anteroposterior patterning in Xenopus is not achieved solely by the dorsal marginal zone (the Spemann organizer), but instead by a synergistic mechanism of the dorsalizing domain (DMZ) and the posteriorizing domain (the entire vegetal half except for the DMZ).

  19. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    PubMed

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  20. Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method.

    PubMed

    Volpe, G; Delibato, E; Fabiani, L; Pucci, E; Piermarini, S; D'Angelo, A; Capuano, F; De Medici, D; Palleschi, G

    2016-01-01

    A reliable, low-cost and easy-to-use ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay for detection of Salmonella enterica in irrigation water is presented. Magnetic beads (MBs), coupled to a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8-well/SPE strip), effectively supported a sandwich immunological chain. Enzymatic by-product is quickly measured by chronoamperometry, using a portable instrument. With the goal of developing a method able to detect a wide range of Salmonella serotypes, including S. Napoli and S. Thompson strains responsible for various community alerts, different kinds of MBs, antibodies and blocking agents were tested. The final system employs MBs coated with a broad reactivity monoclonal antibody anti-salmonella and blocked with dry milk. For a simple and rapid assay these two steps were performed in a preliminary phase, while the two sequential incubations for the immuno-recognition events were merged in a single step of 1h. In parallel a Real-Time PCR (RTi-PCR) method, based on a specific locked nucleic acid (LNA) fluorescent probe and an internal amplification control (IAC), was carried out. The selectivity of the ELIME and RTi-PCR assays was proved by inclusivity and exclusivity tests performed analyzing different Salmonella serotypes and non-target microorganisms, most commonly isolated from environmental sources. Furthermore, both methods were applied to experimentally and not experimentally contaminated irrigation water samples. Results confirmed by the ISO culture method, demonstrated the effectiveness of ELIME and RTi-PCR assays to detect a low number of salmonella cells (1-10 CFU/L) reducing drastically the long analysis time usually required to reveal this pathogen.

  1. Stem Cell-Like Differentiation Potentials of Endometrial Side Population Cells as Revealed by a Newly Developed In Vivo Endometrial Stem Cell Assay

    PubMed Central

    Miyazaki, Kaoru; Maruyama, Tetsuo; Masuda, Hirotaka; Yamasaki, Akiko; Uchida, Sayaka; Oda, Hideyuki; Uchida, Hiroshi; Yoshimura, Yasunori

    2012-01-01

    Background Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. Methodology/Principal Findings ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. Conclusions/Significance We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue

  2. Succession patterns of fungi associated to wound-induced agarwood in wild Aquilaria malaccensis revealed from quantitative PCR assay.

    PubMed

    Mohamed, Rozi; Jong, Phai Lee; Nurul Irdayu, Ismail

    2014-09-01

    Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood.

  3. Combined amplicon pyrosequencing assays reveal presence of the apicomplexan "type-N" (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia.

    PubMed

    Slapeta, Jan; Linares, Marjorie C

    2013-01-01

    The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. "type-N" symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa-Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is important not only to understand the evolution of these

  4. A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects.

    PubMed

    Galgoczy, Roland; Pastor, Isabel; Colom, Adai; Giménez, Alicia; Mas, Francesc; Alcaraz, Jordi

    2014-08-01

    The design of 3D culture studies remains challenging due to the limited understanding of extracellular matrix (ECM)-dependent hindered diffusion and the lack of simple diffusivity assays. To address these limitations, we set up a cost-effective diffusivity assay based on a Transwell plate and the spectrophotometer of a Microplate Reader, which are readily accessible to cell biology groups. The spectrophotometer-based assay was used to assess the apparent diffusivity D of FITC-dextrans with molecular weight (4-70kDa) spanning the physiological range of signaling factors in a panel of acellular ECM gels including Matrigel, fibrin and type I collagen. Despite their technical differences, D data exhibited ∼15% relative difference with respect to FRAP measurements. Our results revealed that diffusion hindrance of small particles is controlled by the enhanced viscosity of the ECM gel in conformance with the Stokes-Einstein equation rather than by geometrical factors. Moreover, we provided a strong rationale that the enhanced ECM viscosity is largely contributed to by unassembled ECM macromolecules. We also reported that gels with the lowest D exhibited diffusion hindrance closest to the large physiologic hindrance of brain tissue, which has a typical pore size much smaller than ECM gels. Conversely, sparse gels (≤1mg/ml), which are extensively used in 3D cultures, failed to reproduce the hindered diffusion of tissues, thereby supporting that dense (but not sparse) ECM gels are suitable tissue surrogates in terms of macromolecular transport. Finally, the consequences of reduced diffusivity in terms of optimizing the design of 3D culture experiments were addressed in detail. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Combined Amplicon Pyrosequencing Assays Reveal Presence of the Apicomplexan “type-N” (cf. Gemmocystis cylindrus) and Chromera velia on the Great Barrier Reef, Australia

    PubMed Central

    Šlapeta, Jan; Linares, Marjorie C.

    2013-01-01

    Background The coral is predominantly composed of the metabolically dependent coral host and the photosynthetic dinoflagellate Symbiodinium sp. The system as a whole interacts with symbiotic eukaryotes, bacteria and viruses. Gemmocystiscylindrus (cf. “type-N” symbiont) belonging to the obligatory parasitic phylum Apicomplexa (Alveolata) is ubiquitous in the Caribbean coral, but its presence in the Great Barrier Reef coral has yet to be documented. Approaches allowing identification of the healthy community from the pathogenic or saprobic organisms are needed for sustainable coral reef monitoring. Methods & Principal Findings We investigated the diversity of eukaryotes associated with a common reef-building corals from the southern Great Barrier Reef. We used three tag encoded 454 amplicon pyrosequencing assays targeting eukaryote small-subunit rRNA gene to demonstrate the presence of the apicomplexan type-N and a photosynthetic sister species to Apicomplexa - Chromeravelia. Amplicon pyrosequencing revealed presence of the small-subunit rRNA genes of known eukaryotic pathogens (Cryptosporidium and Cryptococcus). We therefore conducted bacterial tag encoded amplicon pyrosequencing assay for small-subunit rRNA gene to support effluent exposure of the coral. Bacteria of faecal origin (Enterobacteriales) formed 41% of total sequences in contrast to 0-2% of the coral-associated bacterial communities with and without C. velia, respectively. Significance This is the first time apicomplexan type-N has been detected in the Great Barrier Reef. Eukaryote tag encoded amplicon pyrosequencing assays demonstrate presence of apicomplexan type-N and C. Velia in total coral DNA. The data highlight the need for combined approaches for eukaryotic diversity studies coupled with bacterial community assessment to achieve a more realistic goals of defining the holobiont community and assessing coral disease. With increasing evidence of Apicomplexa in coral reef environments, it is

  6. Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

    PubMed

    Perrin, J; Lussato, D; De Méo, M; Durand, P; Grillo, J-M; Guichaoua, M-R; Botta, A; Bergé-Lefranc, J-L

    2007-02-01

    In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

  7. New ex vivo reporter assay system reveals that σ factors of an unculturable pathogen control gene regulation involved in the host switching between insects and plants.

    PubMed

    Ishii, Yoshiko; Kakizawa, Shigeyuki; Oshima, Kenro

    2013-08-01

    Analysis of the environmental regulation of bacterial gene expression is important for understanding the nature, pathogenicity, and infection route of many pathogens. "Candidatus Phytoplasma asteris", onion yellows strain M (OY-M), is a phytopathogenic bacterium that is able to adapt to quite different host environments, including plants and insects, with a relatively small ~850 kb genome. The OY-M genome encodes two sigma (σ) factors, RpoD and FliA, that are homologous to Escherichia coli σ(70) and σ(28) , respectively. Previous studies show that gene expression of OY-M dramatically changes upon the response to insect and plant hosts. However, very little is known about the relationship between the two σ factors and gene regulatory systems in OY-M, because phytoplasma cannot currently be cultured in vitro. Here, we developed an Escherichia coli-based ex vivo reporter assay (EcERA) system to evaluate the transcriptional induction of phytoplasmal genes by the OY-M-derived σ factors. EcERA revealed that highly expressed genes in insect and plant hosts were regulated by RpoD and FliA, respectively. We also demonstrated that rpoD expression was significantly higher in insect than in plant hosts and fliA expression was similar between the hosts. These data indicate that phytoplasma-derived RpoD and FliA play key roles in the transcriptional switching mechanism during host switching between insects and plants. Our study will be invaluable to understand phytoplasmal transmission, virulence expression in plants, and the effect of infection on insect fitness. In addition, the novel EcERA system could be broadly applied to reveal transcriptional regulation mechanisms in other unculturable bacteria.

  8. Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3*

    PubMed Central

    Bart, Geneviève; Vico, Nuria Ortega; Hassinen, Antti; Pujol, Francois M.; Deen, Ashik Jawahar; Ruusala, Aino; Tammi, Raija H.; Squire, Anthony; Heldin, Paraskevi; Kellokumpu, Sakari; Tammi, Markku I.

    2015-01-01

    In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1–3 (HAS1–3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647–23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis. PMID:25795779

  9. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    PubMed

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed

    PubMed Central

    Dale, Renee; Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi; Kato, Naohiro

    2016-01-01

    The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively. PMID:26886551

  11. Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed.

    PubMed

    Dale, Renee; Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi; Kato, Naohiro

    2016-01-01

    The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.

  12. A novel multiregion hybridization assay reveals high frequency of dual inter-subtype infections among HIV-positive individuals in Cameroon, West Central Africa.

    PubMed

    Vidal, Nicole; Diop, Halimatou; Montavon, Céline; Butel, Christelle; Bosch, Stéphanie; Ngole, Eitel Mpoudi; Touré-Kane, Coumba; Mboup, Souleymane; Delaporte, Eric; Peeters, Martine

    2013-03-01

    In West and West Central Africa, multiple subtypes, circulating recombinant forms (CRF), and high proportions of unique recombinant forms (URF) are documented. The predominance of recombinants strongly suggests that dual infections occur frequently. In the present study, we adapted the multi-region hybridization assay (MHA), previously developed to identify dual infections in geographic regions where few HIV-1 variants circulate, to identify HIV-1 variants and dual infections. We designed clade-specific probes in three genomic regions (gag p17, vpu, nef) to detect eight different variants that are common in this part of Africa (A, B/D, C, F, G, CRF02_AG, CRF06_cpx, CRF22_01A1). The assay was validated with 163 samples representing the corresponding HIV-1 variants. Depending on the genomic regions, the global sensitivity of the assay ranged from 86% to 94%, and the global specificity was between 85% and 96%. The assay was then applied on 156 antiretroviral treatment-naive patients from Cameroon. The MHA assay identified 79%, 85% and 90% of the strains in nef, gag and vpu regions, respectively. The subtype/CRF distribution and the proportion of inter-region recombinants obtained by the new MHA assay were in accordance with known subtype/CRF distribution in Cameroon. Moreover, the MHA assay identified 35 (22.4%) patients as dually infected, from which 20 were reactive in more than one region and/or with concordant multigenomic recombination pattern. Despite the high genetic diversity, we successfully developed an hybridization assay allowing identification of eight common HIV-1 variants circulating in West and West Central Africa. We documented high rates of dual infection in a low-risk population group, illustrating that the global evolution of HIV diversity is driven by dual infections. This assay could become a useful screening tool for the global surveillance and monitoring of inter-subtype/CRF dual infections in West and West Central Africa. Copyright © 2012

  13. High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

    EPA Science Inventory

    Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

  14. High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

    EPA Science Inventory

    Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

  15. A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration.

    PubMed

    Fukano, Yasufumi; Okino, Nozomu; Furuya, Shigeki; Ito, Makoto

    2016-09-16

    We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (PTX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the Gαi pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of Gαi. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Safety Evaluation of Chinese Medicine Injections with a Cell Imaging-Based Multiparametric Assay Revealed a Critical Involvement of Mitochondrial Function in Hepatotoxicity

    PubMed Central

    Wang, Meng; Liu, Chen-Xiang; Dong, Ran-Ran; He, Shuang; Liu, Ting-Ting; Zhao, Tie-Chan; Wang, Zhi-Long; Shen, Xi-Ya; Zhang, Bo-Li; Gao, Xiu-Mei; Zhu, Yan

    2015-01-01

    The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods. We have adapted a sensitive and reproducible multiparametric cell-based high-content analysis assay to evaluate the hepatic-safety of four Chinese medicine injections and validated it with classical animal-based toxicity assays. Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents. This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations. PMID:25792997

  17. AKT1, LKB1, and YAP1 revealed as MYC interactors with NanoLuc-based protein-fragment complementation assay. | Office of Cancer Genomics

    Cancer.gov

    The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. Here we report the development of a NanoLuc®-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells.

  18. Antioxidant assays – consistent findings from FRAP and ORAC reveal a negative impact of organic cultivation on antioxidant potential in spinach but not watercress or rocket leaves

    PubMed Central

    Payne, Adrienne C; Mazzer, Alice; Clarkson, Graham J J; Taylor, Gail

    2013-01-01

    Watercress (Rorippa nasturtium-aquaticum), wild rocket (Diplotaxis tenuifolia), and spinach (Spinacia oleracea) are commercial crops reported to have high concentrations of antioxidants, possibly contributing to disease prevention following human consumption. Following analysis of supermarket-purchased salad leaves, we report the antioxidant content potential of these species using two comparable techniques assessing the consistency between the assays – by the ferric reducing antioxidant power (FRAP) assay and the oxygen radical absorbance capacity (ORAC) assay. The leaves were harvested from both conventionally and organically managed crops, to investigate whether organic agriculture results in improved crop quality. Watercress had the highest FRAP and ability to scavenge free radicals, followed by spinach and rocket. For watercress and rocket, there was no significant effect of organic agriculture on FRAP and ORAC, but for spinach, the antioxidant potential was reduced and this was significant at the 5% level of probability for FRAP but not ORAC, although the trend was clear in both tests. We conclude that there is variation in salad crop antioxidant potential and that FRAP and ORAC are useful techniques for measuring antioxidants in these salad crops with similar ranking for each salad crop studied. PMID:24804054

  19. Antioxidant assays - consistent findings from FRAP and ORAC reveal a negative impact of organic cultivation on antioxidant potential in spinach but not watercress or rocket leaves.

    PubMed

    Payne, Adrienne C; Mazzer, Alice; Clarkson, Graham J J; Taylor, Gail

    2013-11-01

    Watercress (Rorippa nasturtium-aquaticum), wild rocket (Diplotaxis tenuifolia), and spinach (Spinacia oleracea) are commercial crops reported to have high concentrations of antioxidants, possibly contributing to disease prevention following human consumption. Following analysis of supermarket-purchased salad leaves, we report the antioxidant content potential of these species using two comparable techniques assessing the consistency between the assays - by the ferric reducing antioxidant power (FRAP) assay and the oxygen radical absorbance capacity (ORAC) assay. The leaves were harvested from both conventionally and organically managed crops, to investigate whether organic agriculture results in improved crop quality. Watercress had the highest FRAP and ability to scavenge free radicals, followed by spinach and rocket. For watercress and rocket, there was no significant effect of organic agriculture on FRAP and ORAC, but for spinach, the antioxidant potential was reduced and this was significant at the 5% level of probability for FRAP but not ORAC, although the trend was clear in both tests. We conclude that there is variation in salad crop antioxidant potential and that FRAP and ORAC are useful techniques for measuring antioxidants in these salad crops with similar ranking for each salad crop studied.

  20. A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues.

    PubMed

    Joanne Wang, C; Li, Xiong; Lin, Benjamin; Shim, Sangwoo; Ming, Guo-Li; Levchenko, Andre

    2008-02-01

    Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.

  1. A highly sensitive prenylation assay reveals in vivo effects of bisphosphonate drug on the Rab prenylome of macrophages outside the skeleton

    PubMed Central

    Ali, Naveid; Jurczyluk, Julie; Shay, Gemma; Tnimov, Zakir; Alexandrov, Kirill; Munoz, Marcia A; Skinner, Oliver P; Pavlos, Nathan J; Rogers, Michael J

    2015-01-01

    Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotropic effects outside the skeleton, most likely via cells of the monocyte/macrophage lineage exposed to nanomolar circulating drug concentrations. However, no effects of such low concentrations of ZOL have been reported using existing approaches. We have optimized a highly sensitive in vitro prenylation assay utilizing recombinant geranylgeranyltransferases to enable the detection of subtle effects of ZOL on the prenylation of Rab- and Rho-family GTPases. Using this assay, we found for the first time that concentrations of ZOL as low as 10nM caused inhibition of Rab prenylation in J774 macrophages following prolonged cell culture. By combining the assay with quantitative mass spectrometry we identified an accumulation of 18 different unprenylated Rab proteins in J774 cells after nanomolar ZOL treatment, with a >7-fold increase in the unprenylated form of Rab proteins associated with the endophagosome pathway (Rab1, Rab5, Rab6, Rab7, Rab11, Rab14 and Rab21). Finally, we also detected a clear effect of subcutaneous ZOL administration in vivo on the prenylation of Rab1A, Rab5B, Rab7A and Rab14 in mouse peritoneal macrophages, confirming that systemic treatment with bisphosphonate drug can inhibit prenylation in myeloid cells in vivo outside the skeleton. These observations begin a new era in defining the precise pharmacological actions of bisphosphonate drugs on the prenylation of small GTPases in vivo. PMID:26399387

  2. In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment.

    PubMed

    Russell, Katie C; Phinney, Donald G; Lacey, Michelle R; Barrilleaux, Bonnie L; Meyertholen, Kristin E; O'Connor, Kim C

    2010-04-01

    In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55-62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with approximately 2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.

  3. Mutagenicity and antimutagenicity testing of six chemicals associated with the pungent properties of specific spices as revealed by the Ames Salmonella/microsomal assay.

    PubMed

    Azizan, A; Blevins, R D

    1995-02-01

    Three compounds, capsaicin, thymol and borneol, were initially screened for mutagenic activity using Salmonella typhimurium strains TA97, TA98 and TA100, with and without S9 metabolic activation, and 20 min standard preincubation time. Three other compounds, allyl isothiocyanate, eugenol and cinnamaldehyde, were screened for mutagenic activity as above, but with a prolonged, nonstandard preincubation time of up to 120 minutes. All six test compounds used in the assays are associated with the pungent properties of some specific spices in which the test compounds can be found to exist naturally. The first objective of this study was to observe if mutagenic activity can be correlated to the pungent properties of these six test compounds. However, due to toxicity and the observation that only capsaicin was mutagenic, using strain TA100 in the presence of S9 metabolic activation, it was not possible to deduce any relationship between mutagenicity and the test chemicals' pungent properties. Naturally occurring capsaicin, found in the spice Capsicum annum, was detected and quantified using thin layer and gas chromatographic techniques. The final objective was to detect the presence of antimutagenic factor(s) in C. annum that would suppress the mutagenicity of capsaicin. When the mutagenic capsaicin and 2-aminoanthracene were assayed in the presence of C. annum acetone extract, using strain TA100 with S9 metabolic activation, the mutagenic response of both the mutagens were reduced by approximately 50%. Assaying capsaicin and 2-aminoanthracene in the presence of chlorophyll, the mutagenic response of the two mutagens was reduced by less than 40%. From this observation it was inferred that chlorophyll can successfully suppress the mutagenicity activities of capsaicin and 2-aminoanthracene, together with other antimutagenic factors that were present in the acetone extract of C. annum.

  4. RAS - Screens & Assays - Drug Discovery

    Cancer.gov

    The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

  5. Hydrophobicity of diverse bacterial populations in activated sludge and biofilm revealed by microbial adhesion to hydrocarbons assay and high-throughput sequencing.

    PubMed

    Chao, Yuanqing; Guo, Feng; Fang, Herbert H P; Zhang, Tong

    2014-02-01

    Cell hydrophobicity is one of the key physicochemical properties of bacteria in activated sludge (AS) and biofilms can influence the efficient operation of wastewater treatment plants (WWTPs). In the present study the cell hydrophobicity of diverse bacterial populations in AS and biofilms from the Shatin and Stanley WWTPs of Hong Kong was characterized by combining the microbial adhesion to hydrocarbons (MATH) assay with the Illumina high-throughput sequencing. The results indicated that, at the phylum level, a majority of bacteria in AS and biofilms showed medium hydrophobicity. Most of the top 20 bacterial genera in the AS samples were hydrophilic. However, the top 20 genera in biofilms showed higher hydrophobicity than in the top 20 genera in AS samples, suggesting more hydrophobic bacteria existed in biofilms than in AS. Meanwhile, the hydrophobicity of two specific bacterial groups, including foaming and biosurfactant-producing bacteria, were also evaluated. The results demonstrated that, by combining the MATH assay with the Illumina sequencing approach, bacterial hydrophobicity could be evaluated with high efficiency and coverage in complex systems with high microbial diversity, e.g. AS and biofilms in WWTPs.

  6. Nongenotoxic effects and a reduction of the DXR-induced genotoxic effects of Helianthus annuus Linné (sunflower) seeds revealed by micronucleus assays in mouse bone marrow

    PubMed Central

    2014-01-01

    Background This research evaluated the genotoxicity of oil and tincture of H. annuus L. seeds using the micronucleus assay in bone marrow of mice. The interaction between these preparations and the genotoxic effects of doxorubicin (DXR) was also analysed (antigenotoxicity test). Methods Experimental groups were evaluated at 24-48 h post treatment with N-Nitroso-N-ethylurea (positive control – NEU), DXR (chemotherapeutic), NaCl (negative control), a sunflower tincture (THALS) and two sources of sunflower oils (POHALS and FOHALS). Antigenotoxic assays were carried out using the sunflower tincture and oils separately and in combination with NUE or DXR. Results For THALS, analysis of the MNPCEs showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. A significant reduction in MNPCE was observed when THALS (2,000 mg.Kg-1) was administered in combination with DXR (5 mg.Kg-1). For POHALS or FOHALS, analysis of the MNPCEs also showed no significant differences between treatment doses (250–2,000 mg.Kg-1) and NaCl. However, the combination DXR + POHALS (2,000 mg.Kg-1) or DXR + FOHALS (2,000 mg.Kg-1) not contributed to the MNPCEs reduction. Conclusions This research suggests absence of genotoxicity of THALS, dose-, time- and sex-independent, and its combination with DXR can reduce the genotoxic effects of DXR. POHALS and FOHALS also showed absence of genotoxicity, but their association with DXR showed no antigenotoxic effects. PMID:24694203

  7. Mechanism of an indirect effect of aqueous cigarette smoke extract on the adhesion of monocytic cells to endothelial cells in an in vitro assay revealed by transcriptomics analysis.

    PubMed

    Poussin, Carine; Gallitz, Inka; Schlage, Walter K; Steffen, Yvonne; Stolle, Katrin; Lebrun, Stefan; Hoeng, Julia; Peitsch, Manual C; Lietz, Michael

    2014-08-01

    The adhesion of monocytic cells to the "dysfunctional" endothelium constitutes a critical step in the initiation of atherosclerosis. Cigarette smoke (CS) has been shown to contribute to this process, the complex mechanism of which still needs to be unraveled. We developed an in vitro adhesion assay to investigate the CS-induced adhesion of monocytic MM6 cells to human umbilical vein endothelial cells (HUVECs) following exposure to an aqueous CS extract (smoke-bubbled phosphate buffered saline: sbPBS), reasoning that in vivo monocytes and endothelial cells are exposed primarily to soluble constituents from inhaled CS absorbed through the lung alveolar wall. MM6 cell adhesion was increased exclusively by the conditioned medium from sbPBS-exposed MM6 cells, not by direct sbPBS exposure of the HUVECs within a range of sbPBS doses. Using a transcriptomics approach followed by confirmation experiments, we identified different exposure effects on both cell types and a key mechanism by which sbPBS promoted the adhesion of MM6 cells to HUVECs. While sbPBS provoked a strong oxidative stress response in both cell types, the expression of E-selectin, VCAM-1 and ICAM-1, responsible for the adhesion of MM6 cells to HUVECs, was induced in the latter through a proinflammatory paracrine effect. We confirmed that this effect was driven mainly by TNFα produced by MM6 cells exposed to sbPBS. In conclusion, we have elucidated an indirect mechanism by which sbPBS increases the adhesion of monocytic cells to endothelial cells in this in vitro assay that was designed for tobacco product risk assessment while mimicking the in vivo exposure conditions as closely as possible.

  8. An in vitro peptide complementation assay for CYT-18-dependent group I intron splicing reveals a new role for the N-terminus.

    PubMed

    Geng, Chun; Paukstelis, Paul J

    2014-03-04

    The mitochondrial tyrosyl tRNA synthetase from Neurospora crassa (CYT-18 protein) is a bifunctional group I intron splicing cofactor. CYT-18 is capable of splicing multiple group I introns from a wide variety of sources by stabilizing the catalytically active intron structures. CYT-18 and mt TyrRSs from related fungal species have evolved to assist in group I intron splicing in part by the accumulation of three N-terminal domain insertions. Biochemical and structural analysis indicate that the N-terminal insertions serve primarily to create a structure-stabilizing scaffold for critical tertiary interactions between the two major RNA domains of group I introns. Previous studies concluded that the primarily α-helical N-terminal insertion, H0, contributes to protein stability and is necessary for splicing the N. crassa ND1 intron but is dispensable for splicing the N. crassa mitochondrial LSU intron. Here, we show that CYT-18 with a complete H0 deletion retains residual ND1 intron splicing activity and that addition of the missing N-terminus in trans is capable of restoring a significant portion of its splicing activity. The development of this peptide complementation assay has allowed us to explore important characteristics of the CYT-18/group I intron interaction including the stoichiometry of H0 in intron splicing and the importance of specific H0 residues. Evaluation of truncated H0 peptides in this assay and a re-examination of the CYT-18 crystal structure suggest a previously unknown structural role of the first five N-terminal residues of CYT-18. These residues interact directly with another splicing insertion, making H0 a central structural element responsible for connecting all three N-terminal splicing insertions.

  9. A simple screening assay for the most common JK*0 alleles revealed compound heterozygosity in Jk(a-b-) probands from Guam.

    PubMed

    Wester, E S; Gustafsson, J; Snell, B; Spruell, P; Hellberg, A; Olsson, M L; Storry, J R

    2009-01-01

    The Jk(a-b-) phenotype results from alterations in the JK gene and is characterized by absence of the RBC urea transporter in the cell membrane. The frequency of Jk(a-b-) varies among populations,but this phenotype is most commonly found in people of Polynesian and Finnish descent. Although rare, Jk(a-b-) individuals present a clinical challenge because anti-Jk3 is produced readily in response to transfusion and pregnancy, and Jk(a-b-) blood is not routinely available. Identification of Jk(a-b-) patients and donors is most often performed serologically. However, ten JK*0 alleles have been identified, and this information can be used in DNA-based typing. We selected five JK*0 alleles that had been encountered by our reference laboratory in two or more samples from unrelated individuals and designed an allele-specific primer PCR assay for use as an initial screening tool. After in-house validation,we tested genomic DNA from a family: a mother and her two sons referred to us for genetic investigation of their Jk(a-b-)phenotypes. Two different nucleotide substitutions, -1g>a in intron 5 (IVS5) and 956C>T in exon 10, originally associated with Polynesian and Indian/African populations respectively, were identified in the family. The mother and one son were compound heterozygotes, and the second son was homozygous for IVS5-1g>a. We conclude that the effort to design and validate such a screening assay was cost-efficient when compared with DNA sequencing costs. Furthermore, selection of the more common JK*0 mutations was a practical approach that resulted in rapid identification of the genetic bases behind the Jk(a-b-) phenotypes in this unusual family. Although an obvious target for eventual inclusion into high-throughput genotyping platforms for clinical diagnostic services, current systems are very limited. Our approach provides a simple and inexpensive method for the identification of these rare alleles.

  10. Catalytic contributions of key residues in the adenine glycosylase MutY revealed by pH dependent kinetics and cellular repair assays

    PubMed Central

    Brinkmeyer, Megan K.; Pope, Mary Ann Miles; David, Sheila S.

    2012-01-01

    Summary MutY enzymes prevent mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenines opposite OG. pH dependence analyses of the adenine glycosylase activity establish that Asp 138 of MutY must be deprotonated for maximal catalytic activity consistent with the role of this residue in stabilizing the oxacarbenium ion transition state in an SN1 mechanism. Use of a cellular OG:A repair assay allowed further validation of the critical role of Asp 138. Conservative substitutions of the catalytic residues Asp 138 and Glu 37 resulted in enzymes with a range of activity that were used to correlate the efficiency of adenine excision with overall OG:A repair and suppression of DNA mutations in vivo. The results indicate that MutY variations that reduce glycosylase activity as a consequence of reduced mismatch affinity result in more dramatic reductions in cellular OG:A repair than those that only compromise adenine excision catalysis. PMID:22365610

  11. An X-ray shielded irradiation assay reveals EMT transcription factors control pluripotent adult stem cell migration in vivo in planarians.

    PubMed

    Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A; Aboobaker, A Aziz

    2017-09-11

    Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infections. Inappropriate migration also underpins the formation of metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate that we can use this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions without local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum Our work establishes planarians as a suitable model for further in depth study of the processes controlling stem cell migration in vivo. © 2017. Published by The Company of Biologists Ltd.

  12. 2,3,7,8 tetrachlorodibenzodioxin in fish from the Pigeon river of Eastern Tennessee: Its toxicity and mutagenicity as revealed by the Ames Salmonella Assay

    SciTech Connect

    Blevins, R.D. )

    1990-04-01

    Levels of 2,3,7,8 tetrachlorodibenzodioxin (TCDD)were determined in both striated muscle (fillets) and whole body extracts of fish specimens harvested during a two year period (1987-1989) from the Pigeon River (between Hartford and Newport) of Eastern Tennessee. Whole body (wet weight) fish extract levels as high as 117 {mu}g/kg body weight and composite fish fillet (wet weight) extract levels as high 87 {mu}g/kg fillet weight were observed. Pure TCDD was found to be highly toxic to the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA1535 at TCDD dosages which exceeded 825 ng/ml in the top agar of the Ames Salmonella assay. An 825 ng/ml TCDD dosage was not mutagenic to any of the tested Salmonella strains, (both with and without metabolic activation (S9) mix). However, when both acidic and alcohol fish extracts from the Pigeon River were tested for mutagenicity, several of the fish extracts were found to be mutagenic to Salmonella strains TA97, TA98, and TA100 (having mutagenic ratios which greatly exceeded the 2.5 {times} spontaneous ratio). These mutagenic extracts also demonstrated mutagenic dose-response curves. Other chemicals within the extracts as well as synergistic effects may account for the mutagenicity.

  13. Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

    2014-11-01

    Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

  14. Tb3+-Cleavage Assays Reveal Specific Mg2+ Binding Sites Necessary to Pre-fold the btuB Riboswitch for AdoCbl Binding

    PubMed Central

    Choudhary, Pallavi K.; Gallo, Sofia; Sigel, Roland K. O.

    2017-01-01

    Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement. PMID:28377919

  15. Tb3+-cleavage assays reveal specific Mg2+ binding sites necessary to pre-fold the btuB riboswitch for AdoCbl binding

    NASA Astrophysics Data System (ADS)

    Choudhary, Pallavi K.; Gallo, Sofia; Sigel, Roland K. O.

    2017-03-01

    Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

  16. Topoisomerase Assays

    PubMed Central

    Nitiss, John L.; Soans, Eroica; Rogojina, Anna; Seth, Aman; Mishina, Margarita

    2012-01-01

    Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are of assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay for examining topoisomerase covalent complexes in vivo and an assay for measuring DNA cleavage in vitro. PMID:22684721

  17. Molecular assays reveal the presence of Theileria spp. and Babesia spp. in Asian water buffaloes (Bubalus bubalis, Linnaeus, 1758) in the Amazon region of Brazil.

    PubMed

    Silveira, Júlia A G; de Oliveira, Cairo H S; Silvestre, Bruna T; Albernaz, Tatiana T; Leite, Rômulo C; Barbosa, José D; Oliveira, Carlos M C; Ribeiro, Múcio F B

    2016-07-01

    Approximately 50% of buffalo herds in Brazil are located in Pará state in northern Brazil. There are several properties where cattle and buffalo live and graze together, and thus, buffalo pathogens may threaten the health of cattle and vice versa. Therefore, knowledge of infectious agents of buffalo is essential for maintaining healthy livestock. Clinical disease caused by Theileria and Babesia parasites in the Asian water buffalo is not common, although these animals may act as reservoir hosts, and the detection of these hemoparasites in buffaloes is as important as it is in cattle. Studies of the infection of buffaloes by hemoparasites in Brazil are scarce. The objective of the present study was to investigate the occurrence of Piroplasmida parasites in Asian water buffaloes in the state of Pará in the Amazon region of Brazil using nested PCR assays and phylogenetic analysis. The 18S rRNA gene and ITS complete region were amplified from DNA extracted from blood samples collected from 308 apparently healthy buffaloes bred on six properties in the state of Pará, Brazil. The prevalence of positive buffalo samples was 4.2% (13/308) for Theileria spp., 3.6% (11/308) for Babesia bovis and 1% (3/308) for Babesia bigemina. Animals infected with Theileria were detected in 50% (3/6) of the assessed properties. Phylogenetic analyses indicated that the Theileria species detected in this study were closely related to Theileria buffeli, Theileria orientalis and Theileria sinensis. To our knowledge, this is the first report of Theileria in Asian water buffaloes in the Americas. The majority of Theileria-positive buffaloes (11/13) belong to a property that has a history of animals presenting lymphoproliferative disease of unknown etiology. Therefore, the present research suggests that this disorder can be associated with Theileria infection in this property. Our results provide new insights on the distribution and biological aspects of hemoparasites transmissible from

  18. Please do not disturb: Destruction of chromatin structure by supravital nucleic acid probes revealed by a novel assay of DNA-histone interaction

    PubMed Central

    Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2008-01-01

    The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. Herein we comment on potential applications of diverse DNA binding probes in view of the recent article by Wojcik & Dobrucki on DRAQ 5 and SYTO 17. The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents. PMID:18671237

  19. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  20. Equilibrium binding assays reveal the elevated stoichiometry and salt dependence of the interaction between full-length human sex-determining region on the Y chromosome (SRY) and DNA.

    PubMed

    Baud, Stephanie; Margeat, Emmanuel; Lumbroso, Serge; Paris, Françoise; Sultan, Charles; Royer, Catherine; Poujol, Nicolas

    2002-05-24

    In an effort to better define the molecular mechanism of the functional specificity of human sex-determining region on the Y chromosome (SRY), we have carried out equilibrium binding assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3epsilon gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of the free oligonucleotide in solution increases substantially upon binding the protein to the labeled target DNA. Our results indicate that the full-length human wild-type SRY (SRY(WT)) forms a complex of high stoichiometry with its target DNA. Moreover, we have demonstrated a strong salt dependence of both the affinity and specificity of the interaction. We have also addressed the DNA bending properties of full-length human SRY(WT) in solution by fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher than the classical 1:1. Oligomerization thus appears, at least in vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the mutant protein SRY(Y129N), identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well.

  1. Angiogenesis Assays.

    PubMed

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

  2. Multiplexed Elispot Assay

    PubMed Central

    Harriman, William D.; Collarini, Ellen J.; Cromer, Remy G.; Dutta, April; Strandh, Magnus; Zhang, Fen; Kauvar, Lawrence M.

    2009-01-01

    Micron scale latex beads are well established as highly biocompatible reagents. Imbibing two fluorescent dyes into the interior of the beads enables the creation of a family of combinatorially colored labels. Previous use of such beads, in flow cytometry for example, has focused on beads of ~5μm diameter. We show here that 280 nm combinatorially labeled particles can be used to create ELISA-style assays in 200 μm scale virtual wells, using digital microscopy as the readout. The utility of this technique is illustrated by profiling the secreted cytokine footprints of peripheral blood mononuclear cells in a multiparametric version of the popular Elispot assay. Doing so reveals noncanonical classes of T lymphocytes. We further show that the secreting cell type can be concurrently identified by surface staining with a cell type specific antibody conjugated to the same multiplexed beads. PMID:19084532

  3. A non-invasive specimen collection method and a novel simian foamy virus (SFV) DNA quantification assay in New World primates reveal aspects of tissue tropism and improved SFV detection.

    PubMed

    Muniz, Cláudia P; Zheng, HaoQiang; Jia, Hongwei; Cavalcante, Liliane T F; Augusto, Anderson M; Fedullo, Luiz P; Pissinatti, Alcides; Soares, Marcelo A; Switzer, William M; Santos, André F

    2017-01-01

    Simian foamy viruses (SFVs) co-evolved with a wide range of Old World and New World primates (OWPs and NWPs, respectively) and occasionally transmit to humans. Previous studies of OWPs showed that the predominant site of SFV replication is the oral mucosa. However, very little is known about SFV viral loads (VLs) in the oral mucosa or blood of NWPs. NWPs have smaller body sizes, limiting collection of sufficient whole blood volumes to molecularly detect and quantify SFV. Our study evaluated the use of noninvasively collected buccal swabs to detect NWP SFV compared with detection in blood using a new NWP SFV quantitative PCR (qPCR) assay. Buccal and blood samples were collected from 107 captive NWPs in Brazil comprising eleven distinct genera at the Primate Center of Rio de Janeiro (n = 58) and at Fundação Jardim Zoológico da Cidade do Rio Janeiro (n = 49). NWP SFV western blot (WB) testing was performed on a subset of animals for comparison with PCR results. The qPCR assay was validated using distinct SFV polymerase sequences from seven NWP genera (Callithrix, Sapajus, Saimiri, Ateles, Alouatta, Cacajao and Pithecia). Assay sensitivity was 20 copies/106 cells, detectable in 90% of replicates. SFV DNA VLs were higher in buccal swabs (5 log copies/106 cells) compared to peripheral blood mononuclear cells (PBMCs) (3 log copies/106 cells). The qPCR assay was also more sensitive than nested PCR for detection of NWP SFV infection and identified an additional 27 SFV-infected monkeys of which 18 (90%) were WB-positive and three that were WB-negative. We show the utility of using both blood and buccal swabs and our new qPCR assay for detection and quantification of diverse NWP SFV, which will assist a better understanding of the epidemiology of SFV in NWPs and any potential zoonotic infection risk for humans exposed to NWPs.

  4. Binding of the Munc13-1 MUN domain to membrane-anchored SNARE complexes.

    PubMed

    Guan, Rong; Dai, Han; Rizo, Josep

    2008-02-12

    The core of the membrane fusion machinery that governs neurotransmitter release includes the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin, which form a tight "SNARE complex", and Munc18-1, which binds to the SNARE complex and to syntaxin-1 folded into a closed conformation. Release is also controlled by specialized proteins such as complexins, which also bind to the SNARE complex, and unc13/Munc13s, which are crucial for synaptic vesicle priming and were proposed to open syntaxin-1, promoting SNARE complex assembly. However, the biochemical basis for unc13/Munc13 function and its relationship to other SNARE interactions are unclear. To address this question, we have analyzed interactions of the MUN domain of Munc13-1, which is key for this priming function, using solution binding assays and cofloatation experiments with SNARE-containing proteoliposomes. Our results indicate that the Munc13-1 MUN domain binds to membrane-anchored SNARE complexes, even though binding is barely detectable in solution. The MUN domain appears to compete with Munc18-1 but not with complexin-1 for SNARE complex binding, although more quantitative assays will be required to verify these conclusions. Moreover, our data also uncover interactions of membrane-anchored syntaxin-1/SNAP-25 heterodimers with the MUN domain, Munc18-1 and complexin-1. The interaction with complexin-1 is surprising, as it was not observed in previous solution studies. Our results emphasize the importance of studying interactions within the neurotransmitter release machinery in a native membrane environment, and suggest that unc13/Munc13s may provide a template to assemble syntaxin-1/SNAP-25 heterodimers, leading to an acceptor complex for synaptobrevin.

  5. Digital Assays Part II: Digital Protein and Cell Assays.

    PubMed

    Basu, Amar S

    2017-08-01

    A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

  6. [PTH assay: whole PTH assay (new IRMA assay)].

    PubMed

    Tanno, Yudo; Shigematsu, Takashi

    2003-03-01

    The common intact-PTH assay detects not only PTH (1-84) but also PTH (7-84) fragment. Recently, it is reported that PTH (7-84) fragment is the antagonist to PTH (1-84) biological action. Thus, conventional intact-PTH assay might mislead the overestimation of parathyroid function in uremic patients. Whole PTH assay, which detect only PTH (1-84) fragments may be useful for more precise evaluation of PTH activity in uremic patient.

  7. Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

    PubMed Central

    Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R.; Hannah, Brian P.; Matsuda, Zene; Whitbeck, J. Charles; Cohen, Gary H.

    2013-01-01

    Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion. PMID:23946457

  8. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

    PubMed Central

    de Jong, Arthur P. H.; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F. G.; Sørensen, Jakob B.; Verhage, Matthijs

    2016-01-01

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1T112A), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1Δ109–116), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1. PMID:27091977

  9. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

    PubMed

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F G; Sørensen, Jakob B; Verhage, Matthijs

    2016-05-03

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

  10. Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans

    PubMed Central

    Park, Seungmee; Bin, Na-Ryum; Michael Rajah, Maaran; Kim, Byungjin; Chou, Ting-Chieh; Kang, Soo-young Ann; Sugita, Kyoko; Parsaud, Leon; Smith, Matthew; Monnier, Philippe P.; Ikura, Mitsuhiko; Zhen, Mei; Sugita, Shuzo

    2016-01-01

    Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (“closed” vs. “open”) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of “closed” syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1–depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the “open” syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between “closed” syntaxin-1A and Munc18-1, whereas the same mutation in the “open” syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane. PMID:26700321

  11. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    DOE PAGES

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; ...

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. Wemore » tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.« less

  12. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus.

    PubMed

    Lund, Christian H; Bromley, Jennifer R; Stenbæk, Anne; Rasmussen, Randi E; Scheller, Henrik V; Sakuragi, Yumiko

    2015-01-01

    A growing body of evidence suggests that protein-protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. Our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

  13. A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus

    SciTech Connect

    Lund, C. H.; Bromley, J. R.; Stenbaek, A.; Rasmussen, R. E.; Scheller, H. V.; Sakuragi, Y.

    2014-10-18

    A growing body of evidence suggests that protein–protein interactions (PPIs) occur amongst glycosyltransferases (GTs) required for plant glycan biosynthesis (e.g. cell wall polysaccharides and N-glycans) in the Golgi apparatus, and may control the functions of these enzymes. However, identification of PPIs in the endomembrane system in a relatively fast and simple fashion is technically challenging, hampering the progress in understanding the functional coordination of the enzymes in Golgi glycan biosynthesis. To solve the challenges, we adapted and streamlined a reversible Renilla luciferase protein complementation assay (Rluc-PCA), originally reported for use in human cells, for transient expression in Nicotiana benthamiana. We tested Rluc-PCA and successfully identified luminescence complementation amongst Golgi-localizing GTs known to form a heterodimer (GAUT1 and GAUT7) and those which homooligomerize (ARAD1). In contrast, no interaction was shown between negative controls (e.g. GAUT7, ARAD1, IRX9). Rluc-PCA was used to investigate PPIs amongst Golgi-localizing GTs involved in biosynthesis of hemicelluloses. Although no PPI was identified among six GTs involved in xylan biosynthesis, Rluc-PCA confirmed three previously proposed interactions and identified seven novel PPIs amongst GTs involved in xyloglucan biosynthesis. Notably, three of the novel PPIs were confirmed by a yeast-based split-ubiquitin assay. Finally, Gateway-enabled expression vectors were generated, allowing rapid construction of fusion proteins to the Rluc reporters and epitope tags. In conclusion, our results show that Rluc-PCA coupled with transient expression in N. benthamiana is a fast and versatile method suitable for analysis of PPIs between Golgi resident proteins in an easy and mid-throughput fashion in planta.

  14. The extended cell panel assay characterizes the relationship of prion strains RML, 79A, and 139A and reveals conversion of 139A to 79A-like prions in cell culture.

    PubMed

    Oelschlegel, Anja M; Fallahi, Mohammad; Ortiz-Umpierre, Shannon; Weissmann, Charles

    2012-05-01

    Three commonly used isolates of murine prions, 79A, 139A, and RML, were derived from the so-called Chandler isolate, which was obtained by propagating prions from scrapie-infected goat brain in mice. RML is widely believed to be identical with 139A; however, using the extended cell panel assay (ECPA), we here show that 139A and RML isolates are distinct, while 79A and RML could not be distinguished. We undertook to clone 79A and 139A prions by endpoint dilution in murine neuroblastoma-derived PK1 cells. Cloned 79A prions, when returned to mouse brain, were unchanged and indistinguishable from RML by ECPA. However, 139A-derived clones, when returned to brain, yielded prions distinct from 139A and similar to 79A and RML. Thus, when 139A prions were transferred to PK1 cells, 79A/RML-like prions, either present as a minor component in the brain 139A population or generated by mutation in the cells, were selected and, after being returned to brain, were the major if not only component of the population.

  15. RAS - Screens & Assays

    Cancer.gov

    A primary goal of the RAS Initiative is to develop assays for RAS activity, localization, and signaling and adapt those assays so they can be used for finding new drug candidates. Explore the work leading to highly validated screening protocols.

  16. Assays of Serum Testosterone.

    PubMed

    Herati, Amin S; Cengiz, Cenk; Lamb, Dolores J

    2016-05-01

    The diagnosis of male hypogonadism depends on an assessment of the clinical signs and symptoms of hypogonadism and serum testosterone level. Current clinical laboratory testosterone assay platforms include immunoassays and mass spectrometry. Despite significant advances to improve the accuracy and precision of the currently available assays, limited comparability exists between assays at the lower and upper extremes of the testosterone range. Because of this lack of comparability, there is no current gold standard assay for the assessment of total testosterone levels.

  17. A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae

    PubMed Central

    Watanabe, Kiyoshi; Yabe, Makoto; Kasahara, Koji; Kokubo, Tetsuro

    2015-01-01

    In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner. The purpose of the present study was to identify functional TATA sequences enriched in one particular core promoter and compare them with the TATA or TATA-like elements that serve as the pre-initiation complex (PIC) assembly sites on the yeast genome. For this purpose, we conducted a randomized screen of the TATA element in the CYC1 promoter by using a novel reporter assay system and identified several hundreds of unique sequences that were tentatively classified into nine groups. The results indicated that the 7 bp TATA element (i.e., TATAWAD) and several sets of TATA-like sequences are preferred specifically by this promoter. Furthermore, we find that the most frequently isolated TATA-like sequence, i.e., TATTTAAA, is actually utilized as a functional core promoter element for the endogenous genes, e.g., ADE5,7 and ADE6. Collectively, these results indicate that the sequence requirements for the functional TATA or TATA-like elements in one particular core promoter are not as stringent. However, the variation of these sequences differs significantly from that of the PIC assembly sites on the genome, presumably depending on promoter structures and reflecting the gene-specific function of these sequences. PMID:26046838

  18. A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae.

    PubMed

    Watanabe, Kiyoshi; Yabe, Makoto; Kasahara, Koji; Kokubo, Tetsuro

    2015-01-01

    In Saccharomyces cerevisiae, the core promoters of class II genes contain either TATA or TATA-like elements to direct accurate transcriptional initiation. Genome-wide analyses show that the consensus sequence of the TATA element is TATAWAWR (8 bp), whereas TATA-like elements carry one or two mismatches to this consensus. The fact that several functionally distinct TATA sequences have been identified indicates that these elements may function, at least to some extent, in a gene-specific manner. The purpose of the present study was to identify functional TATA sequences enriched in one particular core promoter and compare them with the TATA or TATA-like elements that serve as the pre-initiation complex (PIC) assembly sites on the yeast genome. For this purpose, we conducted a randomized screen of the TATA element in the CYC1 promoter by using a novel reporter assay system and identified several hundreds of unique sequences that were tentatively classified into nine groups. The results indicated that the 7 bp TATA element (i.e., TATAWAD) and several sets of TATA-like sequences are preferred specifically by this promoter. Furthermore, we find that the most frequently isolated TATA-like sequence, i.e., TATTTAAA, is actually utilized as a functional core promoter element for the endogenous genes, e.g., ADE5,7 and ADE6. Collectively, these results indicate that the sequence requirements for the functional TATA or TATA-like elements in one particular core promoter are not as stringent. However, the variation of these sequences differs significantly from that of the PIC assembly sites on the genome, presumably depending on promoter structures and reflecting the gene-specific function of these sequences.

  19. Colorimetric protein assay techniques.

    PubMed

    Sapan, C V; Lundblad, R L; Price, N C

    1999-04-01

    There has been an increase in the number of colorimetric assay techniques for the determination of protein concentration over the past 20 years. This has resulted in a perceived increase in sensitivity and accuracy with the advent of new techniques. The present review considers these advances with emphasis on the potential use of such technologies in the assay of biopharmaceuticals. The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in the literature, accuracy and reproducibility/coefficient of variation/laboratory-to-laboratory variation. A comparison of the use of several assays with the same sample population is presented. It is suggested that the most critical issue in the use of a chromogenic protein assay for the characterization of a biopharmaceutical is the selection of a standard for the calibration of the assay; it is crucial that the standard be representative of the sample. If it is not possible to match the standard with the sample from the perspective of protein composition, then it is preferable to use an assay that is not sensitive to the composition of the protein such as a micro-Kjeldahl technique, quantitative amino acid analysis or the biuret assay. In a complex mixture it might be inappropriate to focus on a general method of protein determination and much more informative to use specific methods relating to the protein(s) of particular interest, using either specific assays or antibody-based methods. The key point is that whatever method is adopted as the 'gold standard' for a given protein, this method needs to be used routinely for calibration.

  20. C-terminal truncation and histidine-tagging of cytochrome c oxidase subunit II reveals the native processing site, shows involvement of the C-terminus in cytochrome c binding, and improves the assay for proton pumping.

    PubMed

    Hiser, C; Mills, D A; Schall, M; Ferguson-Miller, S

    2001-02-13

    To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

  1. The integration of GC-MS and LC-MS to assay the metabolomics profiling in Panax ginseng and Panax quinquefolius reveals a tissue- and species-specific connectivity of primary metabolites and ginsenosides accumulation.

    PubMed

    Liu, Jia; Liu, Yang; Wang, Yu; Abozeid, Ann; Zu, Yuan-Gang; Tang, Zhong-Hua

    2017-02-20

    The traditional medicine Ginseng mainly including Panax ginseng and Panax quinquefolius is the most widely consumed herbal product in the world. Despite the extensive investigation of biosynthetic pathway of the active compounds ginsenosides, our current understanding of the metabolic interlink between ginsenosides synthesis and primary metabolism at the whole-plant level. In this study, the tissue-specific profiling of primary and the secondary metabolites in two different species of ginseng were investigated by gas chromatography- and liquid chromatography coupled to mass spectrometry. A complex continuous coordination of primary- and secondary-metabolic network was modulated by tissues and species factors during growth. The results showed that altogether 149 primary compounds and 10 ginsenosides were identified from main roots, lateral roots, stems, petioles and leaves in P. ginseng and P. quinquefolius. The partial least squares-discriminate analysis (PLS-DA) revealed obvious compounds distinction among tissue-specific districts relative to species. To survey the dedication of carbon and nitrogen metabolism in different tissues to the accumulation of ginsenosides, we inspected the tissue-specific metabolic changes. Our study testified that the ginsenosides content was dependent on main roots and lateral roots energy metabolism, whereas independent of leaves and petiole photosynthesis during ginsenosides accumulation. When tow species were compared, the results indicated that high rates of C assimilation to C accumulation are closely associated with ginsenosides accumulation in P. ginseng main roots and P. quinquefolius lateral roots, respectively. Taken together, our results suggest that tissue-specific metabolites profiling dynamically changed in process of ginsenosides biosynthesis, which may offer a new train of thoughts to the mechanisms of the ginsenosides biosynthesis at the metabolite level. Copyright © 2016 The Author(s). Published by Elsevier B.V. All

  2. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2012-05-15

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  3. Absolute nuclear material assay

    DOEpatents

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  4. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  5. A monoclonal antibody may show cross-reactivities in Ouchterlony assays but not in other assays.

    PubMed

    Molinaro, G A; Eby, W C; Reimer, C

    1987-02-11

    A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by Ouchterlony assays in the presence of polyethylene glycol, the antibody formed lines with all four IgG proteins. The line with IgG3 was sharp and stable, but the lines with the other three IgG subclasses tended to blur with time and with the lower PEG concentrations. These findings show that Ouchterlony assays can reveal cross-reactions of a monoclonal antibody that can be missed by more sensitive assays.

  6. CPTAC Assay Portal: a repository of targeted proteomic assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  7. Factor IX assay

    MedlinePlus

    Christmas factor assay; Serum factor IX; Hemophilic factor B; Plasma thromboplastin component; PTC ... chap 137. Chernecky CC, Berger BJ. Factor IX (Christmas factor, hemophilic factor B, plasma thromboplastin component, PTC) - ...

  8. Tube-Forming Assays.

    PubMed

    Brown, Ryan M; Meah, Christopher J; Heath, Victoria L; Styles, Iain B; Bicknell, Roy

    2016-01-01

    Angiogenesis involves the generation of new blood vessels from the existing vasculature and is dependent on many growth factors and signaling events. In vivo angiogenesis is dynamic and complex, meaning assays are commonly utilized to explore specific targets for research into this area. Tube-forming assays offer an excellent overview of the molecular processes in angiogenesis. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. A detailed experimental protocol on the implementation of the assay is described in conjunction with an in-depth review of methods that can be applied to the analysis of the tube formation. In addition, an ImageJ plug-in is presented which allows automatic quantification of tube images reducing analysis times while removing user bias and subjectivity.

  9. Cell viability assays: introduction.

    PubMed

    Stoddart, Martin J

    2011-01-01

    The measurement of cell viability plays a fundamental role in all forms of cell culture. Sometimes it is the main purpose of the experiment, such as in toxicity assays. Alternatively, cell viability can be used to -correlate cell behaviour to cell number, providing a more accurate picture of, for example, anabolic -activity. There are wide arrays of cell viability methods which range from the most routine trypan blue dye exclusion assay to highly complex analysis of individual cells, such as using RAMAN microscopy. The cost, speed, and complexity of equipment required will all play a role in determining the assay used. This chapter aims to provide an overview of many of the assays available today.

  10. Lateral flow assays

    PubMed Central

    Koczula, Katarzyna M.

    2016-01-01

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  11. Functional synergy between the Munc13 C-terminal C1 and C2 domains

    PubMed Central

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-01-01

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.001 PMID:27213521

  12. Functional synergy between the Munc13 C-terminal C1 and C2 domains.

    PubMed

    Liu, Xiaoxia; Seven, Alpay Burak; Camacho, Marcial; Esser, Victoria; Xu, Junjie; Trimbuch, Thorsten; Quade, Bradley; Su, Lijing; Ma, Cong; Rosenmund, Christian; Rizo, Josep

    2016-05-23

    Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca(2+)-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a 'primed' state that does not fuse but is ready for fast fusion upon Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.

  13. Multiple log potash assay

    NASA Astrophysics Data System (ADS)

    Hill, D. G.

    1993-10-01

    A five-mineral multiple-log potash assay technique has been successfully applied to evaluate potash-rich intervals in evaporite sequences. The technique is able to distinguish economic potash minerals from non-economic potash minerals and from other non-potash radioactive minerals. It can be applied on location, using a programmable calculator or microcomputer, providing near real-time logs of potash mineral concentrations. Log assay values show good agreement with core wet chemistry analyses.

  14. Doped colorimetric assay liposomes

    DOEpatents

    Charych, Deborah; Stevens, Raymond C.

    2001-01-01

    The present invention provides compositions comprising colorimetric assay liposomes. The present invention also provides methods for producing colorimetric liposomes and calorimetric liposome assay systems. In preferred embodiments, these calorimetric liposome systems provide high levels of sensitivity through the use of dopant molecules. As these dopants allow the controlled destabilization of the liposome structure, upon exposure of the doped liposomes to analyte(s) of interest, the indicator color change is facilitated and more easily recognized.

  15. Serum selenium assay following serum ferritin assay

    SciTech Connect

    Stevens, R.G.; Morris, J.S.; Hann, H.L.; Pulsipher, B.; Stahlhut, M.W.

    1986-08-01

    Stored serum samples can be an important research resource into the etiology of cancer. These sera cannot be replaced and should therefore be used to best advantage. In previous epidemiologic studies, only single serum constituents have been assayed in individual serum samples. For example, serum ferritin has been examined in samples stored for as long as 10 years at -20C for a possible relation with general mortality (1) and cancer death (2). Ferritin is the tissue iron-storage protein and is therefore subject to denaturation. Serum selenium has also been examined in relation to cancer risk in a prospective manner by using stored frozen serum samples (3, 4). The interactions of a variety of serum factors in relation to cancer risk would be a desirable research goal, except that the amounts of serum typically available in frozen serum banks are less than 1 ml. It was the purpose of this investigation to determine if a radioimmunoassay for ferritin affected a subsequent neutron activation assay for selenium on the same 0.1 ml serum sample.

  16. SNAP Assay Technology.

    PubMed

    O'Connor, Thomas P

    2015-12-01

    The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

  17. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  18. SIGMA RECEPTOR BINDING ASSAYS

    PubMed Central

    CHU, UYEN B.; RUOHO, ARNOLD E.

    2016-01-01

    Sigma receptors belong to a class of small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated receptors, of which there are two subtypes: the Sigma-1 receptor (S1R) and the Sigma-2 receptor (S2R). Both S1R and S2R bind to a number of drugs including antipsychotic, haloperidol, and the opioid analgesic, (+)-pentazocine. Sigma receptors are implicated in multiple disease pathologies associated with the nervous system including diseases affecting motor control such as Amyotrophic Lateral Sclerosis (ALS) and Alzeimher's disease. This unit describes methods for the pharmacological characterization of S1R and S2R using radioligand-binding assays. In the first section, radioligand saturation binding assay to determine receptor densities and competitive inhibition assays to characterize affinities of novel compounds are presented for S1R using the selective S1R ligand, [3H]-(+)-pentazocine. The second section describes radioligand saturation binding assay and competitive inhibition assays for the S2R using a non-selective S1R and S2R ligand, [3H]-1,3-di(2-tolyl)guanidine ([3H]-DTG). PMID:26646191

  19. Rover waste assay system

    SciTech Connect

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  20. Clonogenic Assay: Adherent Cells

    PubMed Central

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T.; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C.

    2011-01-01

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  1. Multiplex Flow Assays

    PubMed Central

    2016-01-01

    Lateral flow or dipstick assays (e.g., home pregnancy tests), where an analyte solution is drawn through a porous membrane and is detected by localization onto a capture probe residing at a specific site on the flow strip, are the most commonly and extensively used type of diagnostic assay. However, after over 30 years of use, these assays are constrained to measuring one or a few analytes at a time. Here, we describe a completely general method, in which any single-plex lateral flow assay is transformed into a multiplex assay capable of measuring an arbitrarily large number of analytes simultaneously. Instead of identifying the analyte by its localization onto a specific geometric location in the flow medium, the analyte-specific capture probe is identified by its association with a specific optically encoded region within the flow medium. The capture probes for nucleic acids, antigens, or antibodies are attached to highly porous agarose beads, which have been encoded using multiple lanthanide emitters to create a unique optical signature for each capture probe. The optically encoded capture probe-derivatized beads are placed in contact with the analyte-containing porous flow medium and the analytes are captured onto the encoded regions as the solution flows through the porous medium. To perform a multiplex diagnostic assay, a solution comprising multiple analytes is passed through the flow medium containing the capture probe-derivatized beads, and the captured analyte is treated with a suitable fluorescent reporter. We demonstrate this multiplex analysis technique by simultaneously measuring DNA samples, antigen–antibody pairs, and mixtures of multiple nucleic acids and antibodies. PMID:27819063

  2. Assays for calcitonin receptors

    SciTech Connect

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is /sup 125/I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed.

  3. CTL ELISPOT assay.

    PubMed

    Ranieri, Elena; Popescu, Iulia; Gigante, Margherita

    2014-01-01

    Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T cell (CTL) studies have taken advantage with this high-throughput technology by providing insights into quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T cells is of relevance for immune diagnostic. The most utilized Elispot assay is the interferon-gamma (IFN-γ) test, a marker for CD8(+) CTL activation, but Elispot can also be used to distinguish different subsets of activated T cells by using other cytokines such as T-helper (Th) 1-type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21, and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10, and IL-13), and Th17 (IL-17) cells. The reliability of Elispot-generated data, by the evaluation of T cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Miltenyi cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method

  4. Fluorometric assay for aflatoxins

    SciTech Connect

    Chakrabarti, A.G.

    1984-11-01

    The method that is now widely adopted by the government laboratories for the assay of individual aflatoxin components (B/sub 1/, B/sub 2/, G/sub 1/, and G/sub 2/) utilizes a TLC technique. The extraction and clean-up steps of this technique were further researched but the method is still time consuming. It is, therefore, very important to develop a rapid and accurate assay technique for aflatoxins. The current research proposes a technique which utilizes a Turner Fluorometer.

  5. Lateral flow strip assay

    DOEpatents

    Miles, Robin R [Danville, CA; Benett, William J [Livermore, CA; Coleman, Matthew A [Oakland, CA; Pearson, Francesca S [Livermore, CA; Nasarabadi, Shanavaz L [Livermore, CA

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  6. Kinetic tetrazolium microtiter assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L. (Inventor); Stowe, Raymond P. (Inventor); Koeing, David W. (Inventor)

    1992-01-01

    A method for conducting an in vitro cell assay using a tetrazolium indicator is disclosed. The indicator includes a nonionic detergent which solubilizes a tetrazolium reduction product in vitro and has low toxicity for the cells. The incubation of test cells in the presence of zolium bromide and octoxynol (TRITON X-100) permits kinetics of the cell metabolism to be determined.

  7. Sigma Receptor Binding Assays.

    PubMed

    Chu, Uyen B; Ruoho, Arnold E

    2015-12-08

    Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [³H]-(+)-pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [³H]-1,3-di(2-tolyl)guanidine ([³H]-DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors.

  8. Instrument for assaying radiation

    DOEpatents

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  9. New oligosaccharyltransferase assay method.

    PubMed

    Kohda, Daisuke; Yamada, Masaki; Igura, Mayumi; Kamishikiryo, Jun; Maenaka, Katsumi

    2007-11-01

    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.

  10. Interaction of Munc18 and Syntaxin in the regulation of insulin secretion

    SciTech Connect

    Dong, Yongming; Wan, Qunfang; Yang, Xiaofei; Bai, Li; Xu, Pingyong . E-mail: pyxu@moon.ibp.ac.cn

    2007-08-31

    Syntaxin1A and Munc18-1 play essential roles in exocytosis. However, the molecular mechanism and the functional roles of their interaction in insulin secretion remain to be explored. Using membrane capacitance measurement, we examine effect of overexpressing Munc18-1 on exocytosis in pancreatic {beta} cells. The results show that Munc18-1 negatively regulates vesicle fusion. To probe the interaction between Munc18-1 and Syntaxin1A, Munc18-1-Tdimer2 and EGFP-Syntaxin1A were co-transfected into INS-1 cells. FRET measurement confirmed that Munc18-1 interacted with wild type Syntaxin 1A, but not the constitutively open form (DM) of Syntaxin1A. Overexpressing DM in primary pancreatic {beta} cells augmented insulin secretion, and this effect can overcome the inhibitory effect of Munc18-1 overexpression. We propose that Munc18-1 inhibitis the SNARE complex assembly by stabilizing Syntaxin1A in a closed conformation in vesicle priming process, therefore negatively regulates insulin secretion.

  11. Fuller Revealed

    NASA Image and Video Library

    2015-03-16

    MESSENGER's low-altitude campaign has enabled imaging of Fuller crater (named after American architect Buckminster Fuller) in greater detail than previously possible. The top left panel shows an image of Fuller, with the crater rim outlined in pink and the edge of a low-altitude broadband MDIS image in green. The large panel applies a different stretch to the same MDIS broadband image in the first panel, revealing details of the shadowed surface inside Fuller! In particular, as highlighted with yellow arrows in the bottom left panel, the image reveals a region inside Fuller that is lower in reflectance. The edge of the low-reflectance region has a sharp and well-defined boundary, even when imaged at 46 m/pixel, suggesting that the low-reflectance material is sufficiently young to have preserved a sharp boundary against lateral mixing by impact cratering. Models for surface and near-surface temperature within Fuller crater predict a region that is sufficiently cold to host long-lived water ice beneath the surface but too hot to support water ice at the surface. The low-reflectance region revealed in the images matches the thermal characteristics expected for a lag deposit of volatile, organic-rich material that overlies the water ice. http://photojournal.jpl.nasa.gov/catalog/PIA19244

  12. Arabidopsis assay for mutagenicity.

    PubMed

    Gichner, T; Badayev, S A; Demchenko, S I; Relichová, J; Sandhu, S S; Usmanov, P D; Usmanova, O; Velemínský, J

    1994-10-16

    Four laboratories, two in the Czech Republic (Brno and Prague) and two in the CIS (Moscow and Duschanbe), participated in the International Programme on Chemical Safety's (IPCS) collaborative study to evaluate the utility of the most commonly used plant test systems, including the Arabidopsis thaliana assay, for assessing the mutagenic potential of environmental agents. Out of the five compounds evaluated in the Arabidopsis assay, three compounds, i.e., ethyl methanesulfonate, N-methyl-N-nitrosourea, and azidoglycerol, were reported to be mutagenic by all four participating laboratories. Sodium azide (NaN3) demonstrated a negative response in all four laboratories, whereas maleic hydrazide was reported to be weakly mutagenic by one laboratory and nonmutagenic by the other three laboratories.

  13. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  14. Kinetic Tetrazolium Microtiter Assay

    NASA Technical Reports Server (NTRS)

    Pierson, Duane L.; Stowe, Raymond; Koenig, David

    1993-01-01

    Kinetic tetrazolium microtiter assay (KTMA) involves use of tetrazolium salts and Triton X-100 (or equivalent), nontoxic, in vitro color developer solubilizing colored metabolite formazan without injuring or killing metabolizing cells. Provides for continuous measurement of metabolism and makes possible to determine rate of action of antimicrobial agent in real time as well as determines effective inhibitory concentrations. Used to monitor growth after addition of stimulatory compounds. Provides for kinetic determination of efficacy of biocide, greatly increasing reliability and precision of results. Also used to determine relative effectiveness of antimicrobial agent as function of time. Capability of generating results on day of test extremely important in treatment of water and waste, disinfection of hospital rooms, and in pharmaceutical, agricultural, and food-processing industries. Assay also used in many aspects of cell biology.

  15. The corneal pocket assay.

    PubMed

    Ziche, Marina; Morbidelli, Lucia

    2015-01-01

    The cornea in most species is physiologically avascular, and thus this assay allows the measurement of newly formed vessels. The continuous monitoring of neovascular growth in the same animal allows the evaluation of drugs acting as suppressors or stimulators of angiogenesis. Under anesthesia a micropocket is produced in the cornea thickness and the angiogenesis stimulus (tumor tissue, cell suspension, growth factor) is placed into the pocket in order to induce vascular outgrowth from the limbal capillaries. Neovascular development and progression can be modified by the presence of locally released or applied inhibitory factors or by systemic treatments. In this chapter the experimental details of the avascular cornea assay, the technical challenges, and advantages and disadvantages in different species are discussed. Protocols for local drug treatment and tissue sampling for histology and pharmacokinetic profile are reported.

  16. Cytotoxicity assay automation

    NASA Technical Reports Server (NTRS)

    Levinthal, E. C.; Payne, R. O.

    1971-01-01

    The design and construction of a system to automatically test HLP antigens are described. Major efforts were made to test and evaluate the performance of such a system, and compare its performance with nonautomatic tissue typing techniques. The system is based on the fluorochromatic cytotoxicity assay. Results show the system will work but is subject to malfunctions after a few samplings, and poses problems in showing correctly the necessary readings.

  17. B cell helper assays.

    PubMed

    Abrignani, Sergio; Tonti, Elena; Casorati, Giulia; Dellabona, Paolo

    2009-01-01

    Activation, proliferation and differentiation of naïve B lymphocytes into memory B cells and plasma cells requires engagement of the B cell receptor (BCR) coupled to T-cell help (1, 2). T cells deliver help in cognate fashion when they are activated upon recognition of specific MHC-peptide complexes presented by B cells. T cells can also deliver help in a non-cognate or bystander fashion, when they do not find specific MHC-peptide complexes on B cells and are activated by alternative mechanisms. T-cell dependent activation of B cells can be studied in vitro by experimental models called "B cell helper assays" that are based on the co-culture of B cells with activated T cells. These assays allow to decipher the molecular bases for productive T-dependent B cell responses. We show here examples of B cell helper assays in vitro, which can be reproduced with any subset of T lymphocytes that displays the appropriate helper signals.

  18. Open chromatin reveals the functional maize genome

    USDA-ARS?s Scientific Manuscript database

    Every cellular process mediated through nuclear DNA must contend with chromatin. As results from ENCODE show, open chromatin assays can efficiently integrate across diverse regulatory elements, revealing functional non-coding genome. In this study, we use a MNase hypersensitivity assay to discover o...

  19. Radon assay for SNO+

    SciTech Connect

    Rumleskie, Janet

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  20. Radon assay for SNO+

    NASA Astrophysics Data System (ADS)

    Rumleskie, Janet

    2015-12-01

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  1. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  2. Biosensors: Viruses for ultrasensitive assays

    NASA Astrophysics Data System (ADS)

    Donath, Edwin

    2009-04-01

    A three-dimensional assay based on genetically engineered viral nanoparticles and nickel nanohairs can detect much lower levels of protein markers associated with heart attacks than conventional assays.

  3. Revealing Rembrandt

    PubMed Central

    Parker, Andrew J.

    2014-01-01

    The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI). Our results emphasized the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt's portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings. PMID:24795552

  4. Research highlights: digital assays on chip.

    PubMed

    Kim, Donghyuk; Wei, Qingshan; Kong, Janay Elise; Ozcan, Aydogan; Di Carlo, Dino

    2015-01-07

    The ability to break up a volume of fluid into smaller pieces that are confined or separated to prevent molecular communication/transport is a key capability intrinsic to microfluidic systems. This capability has been used to develop or implement digital versions of traditional molecular analysis assays, including digital PCR and digital immunoassays/ELISA. In these digital versions, the concentration of the target analyte is in a range such that, when sampled into smaller fluid volumes, either a single molecule or no molecule may be present. Subsequent amplification is sensitive enough to obtain a digital readout of the presence of these target molecules. Advantages of such approaches that are claimed include quantification without calibration and robustness to variations in reaction conditions or times because the digital readout is less sensitive to absolute signal intensity levels. Weaknesses of digital approaches include a lower dynamic range of concentrations over which the assay is sensitive, which depends on the total volume that can be analyzed. We highlight recent efforts to expand the dynamic range of digital assays based on exploiting reaction/diffusion phenomena. A side-by-side study that evaluates the strengths of digital assays reveals that the majority of these claims are supported, with specific caveats. Finally, we highlight approaches to apply digital assays to analyze new types of reactions, including the active transport of protons across membranes by ATPases at the single protein level - perhaps opening up new biophysical understanding and screening opportunities, similar to widely deployed single-molecule ion channel analysis.

  5. TOTAL CULTURABLE VIRUS QUANTAL ASSAY

    EPA Science Inventory

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced ...

  6. Quantifiable Lateral Flow Assay Test Strips

    NASA Technical Reports Server (NTRS)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  7. Quantifiable Lateral Flow Assay Test Strips

    NASA Technical Reports Server (NTRS)

    2003-01-01

    As easy to read as a home pregnancy test, three Quantifiable Lateral Flow Assay (QLFA) strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the middle left and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations. The color intensity correlates with concentration of contamination.

  8. Chemotaxis: Under Agarose Assay.

    PubMed

    Brazill, Derrick

    2016-01-01

    The unicellular eukaryote Dictyostelium discoideum represents a superb model for examining chemotaxis. Under vegetative conditions, the amoebae are chemotactically responsive to pterins, such as folate. Under starved conditions, they lose their sensitivity to pterins, and become chemotactically responsive to cAMP. As an NIH model system, Dictyostelium offers a variety of advantages in studying chemotaxis, including its conservation of mammalian signaling pathways, its ease of growth, and its genetic tractability. In this chapter, we describe the use of the under agarose chemotaxis assay to identify proteins involved in controlling motility and directional sensing in Dictyostelium discoideum. Given the similarities between Dictyostelium and mammalian cells, this allows us to dissect the conserved pathways involved in eukaryotic chemotaxis.

  9. Survival assays using Caenorhabditis elegans

    PubMed Central

    Park, Hae-Eun H.; Jung, Yoonji; Lee, Seung-Jae V.

    2017-01-01

    Caenorhabditis elegans is an important model organism with many useful features, including rapid development and aging, easy cultivation, and genetic tractability. Survival assays using C. elegans are powerful methods for studying physiological processes. In this review, we describe diverse types of C. elegans survival assays and discuss the aims, uses, and advantages of specific assays. C. elegans survival assays have played key roles in identifying novel genetic factors that regulate many aspects of animal physiology, such as aging and lifespan, stress response, and immunity against pathogens. Because many genetic factors discovered using C. elegans are evolutionarily conserved, survival assays can provide insights into mechanisms underlying physiological processes in mammals, including humans. PMID:28241407

  10. Cell Proliferation and Cytotoxicity Assays.

    PubMed

    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

    Cell viability is defined as the number of healthy cells in a sample and proliferation of cells is a vital indicator for understanding the mechanisms in action of certain genes, proteins and pathways involved cell survival or death after exposing to toxic agents. Generally, methods used to determine viability are also common for the detection of cell proliferation. Cell cytotoxicity and proliferation assays are generally used for drug screening to detect whether the test molecules have effects on cell proliferation or display direct cytotoxic effects. Regardless of the type of cell-based assay being used, it is important to know how many viable cells are remaining at the end of the experiment. There are a variety of assay methods based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. These methods could be basically classified into different categories: (I) dye exclusion methods such as trypan blue dye exclusion assay, (II) methods based on metabolic activity, (III) ATP assay, (IV) sulforhodamine B assay, (V) protease viability marker assay, (VI) clonogenic cell survival assay, (VII) DNA synthesis cell proliferation assays and (V) raman micro-spectroscopy. In order to choose the optimal viability assay, the cell type, applied culture conditions, and the specific questions being asked should be considered in detail. This particular review aims to provide an overview of common cell proliferation and cytotoxicity assays together with their own advantages and disadvantages, their methodologies, comparisons and intended purposes.

  11. Coagulation assays and anticoagulant monitoring.

    PubMed

    Funk, Dorothy M Adcock

    2012-01-01

    Anticoagulant therapy, including conventional agents and a variety of new oral, fast-acting drugs, is prescribed for millions of patients annually. Each anticoagulant varies in its effect on routine and specialty coagulation assays and each drug may require distinct laboratory assay(s) to measure drug concentration or activity. This review provides an overview of the assorted assays that can measure anticoagulant drug concentration or activity and includes key assay interferences. The effect of these conventional and new anticoagulant agents on specialty coagulation assays used to evaluate for bleeding or clotting disorders, and whether this impact is physiological or factitious, is included. Also provided is a short review of superwarfarin poisoning and features distinguishing this from warfarin overdose. Knowledge of clinically significant pearls and pitfalls pertinent to coagulation assays in relation to anticoagulant therapy are important to optimize patient care.

  12. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.

    PubMed

    Fotakis, George; Timbrell, John A

    2006-01-05

    The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.

  13. Biomolecular Interaction Assay

    SciTech Connect

    Bruckner-Lea, Cindy J.; Brown, L; Holman, David A.; Olson, Lydia; Grate, Jay W.

    2000-12-29

    Understanding the binding interactions of complexes of multiple proteins is an important area of medical research since many biological signaling pathways involve multiple protein complexes. A number of sensor technologies have been adapted to monitoring biomolecular interactions. Acoustic wave devices such as flexural plate wave devices, surface transverse waves, and quartz crystal microbalances detect the mass increase observed upon binding of a solution biomolecule to a surface bound biomolecule. However, these devices will also respond to changes in viscosity, temperature, liquid density, and viscoelastic effects, which may confound the interpretation of observed signals. Nonspecific binding is indistinguishable from specific binding. Several techniques for refractive index sensing, such as planar wave guides and surface plasmon resonance (SPR), can also be used to observe biomolecular interactions localized at a surface. Again, nonspecific binding is indistinguishable from specific binding. In addition, the derivatized surface must be very thin and uniform to obtain adequate sensitivity and reproducibility, and the technique is not suited for monitoring large multiple protein complexes since the measurement sensitivity decreases rapidly with distance from the sensor surface. All of these techniques use planar surfaces that are difficult to prepare and characterize, and must be prepared fresh for each assay.

  14. Assay for calcium channels

    SciTech Connect

    Glossmann, H.; Ferry, D.R.

    1985-01-01

    This chapter focuses on biochemical assays for Ca/sup 2 +/-selective channels in electrically excitable membranes which are blocked in electrophysiological and pharmacological experiments by verapamil, 1,4-dihydropyridines, diltiazen (and various other drugs), as well as inorganic di- or trivalent cations. The strategy employed is to use radiolabeled 1,4-dihydropyridine derivatives which block calcium channels with ED/sub 50/ values in the nanomolar range. Although tritiated d-cis-diltiazem and verapamil can be used to label calcium channels, the 1,4-dihydropyridines offer numerous advantages. The various sections cover tissue specificity of channel labeling, the complex interactions of divalent cations with the (/sup 3/H)nimodipine-labeled calcium channels, and the allosteric regulation of (/sup 3/H)nimodipine binding by the optically pure enantiomers of phenylalkylamine and benzothiazepine calcium channel blockers. A comparison of the properties of different tritiated 1,4-dihydropyridine radioligands and the iodinated channel probe (/sup 125/I)iodipine is given.

  15. Cell migration and invasion assays.

    PubMed

    Moutasim, Karwan A; Nystrom, Maria L; Thomas, Gareth J

    2011-01-01

    A number of in vitro assays have been developed to study tumor cell motility. Historically, assays have been mainly monocellular, where carcinoma cells are studied in isolation. Scratch assays can be used to study the collective and directional movement of populations of cells, whereas two chamber assays lend themselves to the analysis of chemotactic/haptotactic migration and cell invasion. However, an inherent disadvantage of these assays is that they grossly oversimplify the complex process of invasion, lacking the tumor structural architecture and stromal components. Organotypic assays, where tumor cells are grown at an air/liquid interface on gels populated with stromal cells, are a more physiologically relevant method for studying 3-dimensional tumor invasion.

  16. Advanced analysis techniques for uranium assay

    SciTech Connect

    Geist, W. H.; Ensslin, Norbert; Carrillo, L. A.; Beard, C. A.

    2001-01-01

    Uranium has a negligible passive neutron emission rate making its assay practicable only with an active interrogation method. The active interrogation uses external neutron sources to induce fission events in the uranium in order to determine the mass. This technique requires careful calibration with standards that are representative of the items to be assayed. The samples to be measured are not always well represented by the available standards which often leads to large biases. A technique of active multiplicity counting is being developed to reduce some of these assay difficulties. Active multiplicity counting uses the measured doubles and triples count rates to determine the neutron multiplication (f4) and the product of the source-sample coupling ( C ) and the 235U mass (m). Since the 35U mass always appears in the multiplicity equations as the product of Cm, the coupling needs to be determined before the mass can be known. A relationship has been developed that relates the coupling to the neutron multiplication. The relationship is based on both an analytical derivation and also on empirical observations. To determine a scaling constant present in this relationship, known standards must be used. Evaluation of experimental data revealed an improvement over the traditional calibration curve analysis method of fitting the doubles count rate to the 235Um ass. Active multiplicity assay appears to relax the requirement that the calibration standards and unknown items have the same chemical form and geometry.

  17. High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay.

    PubMed

    Liu, Yichin; Gerber, Raphaele; Wu, John; Tsuruda, Trace; McCarter, John D

    2008-07-01

    Silent information regulator or sirtuin (SIRT) enzymes are beta-nicotinamide adenine dinucleotide (oxidized) (NAD(+))-dependent class III histone deacetylases. In this paper, two distinct assays to measure SIRT1 activity are described: a microfluidic mobility shift assay utilizing a fluorophore-labeled peptide substrate and a bioluminescence assay based upon quantitation of remaining NAD(+). The mobility shift assay involves the electrophoretic separation of an N-acetyl-lysine-containing peptide substrate from deacetylated product which bears an additional positive charge. Interference from fluorescent compounds is minimized during screening by direct visualization of separated fluorophore-labeled substrate and product. A preferred peptide substrate for SIRT1 was identified using this assay. The NAD(+) bioluminescence assay couples NAD(+) consumption to the bacterial luciferase-catalyzed oxidation of decanal. This assay does not require synthesis of a labeled peptide and is applicable to sirtuins of any specificity with respect to peptide substrate. The stoichiometry between NAD(+) consumption and peptide deacetylation was shown to be 1:1 by the NAD(+) bioluminescence assay. Kinetic parameters of peptide and NAD(+) cosubstrates and IC(50) values of standard reference inhibitors determined in either assay were similar. With robust Z' values (0.7), both assays are amenable to high-throughput screening.

  18. Label-free functional selectivity assays.

    PubMed

    Ferrie, Ann M; Goral, Vasiliy; Wang, Chaoming; Fang, Ye

    2015-01-01

    G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

  19. Assay of gentamicin in cerebrospinal fluid.

    PubMed Central

    Deacon, S

    1976-01-01

    A comparison of standard curves obtained from a conventional plate diffusion assay method revealed significant differences when gentamicin standards were made up in different media. Standards made up in distilled water resulted in a curve which differed from that of standards made up in pooled human cerebrospinal fluid by a factor of up to 4. When the assay medium was supplemented with 0-5% sodium chloride, the difference between the two standard curves was reduced to a factor of about 1-5. The curve obtained from standards made up in 150 mM sodium chloride/4-5 mM calcium chloride correlated well with that from standards made up in cerebrospinal fluid. There was no evidence of gentamicin being bound to protein in the cerebrospinal fluid. PMID:956457

  20. Doxylamine: a cause for false-positive gas chromatographic assay for phencyclidine.

    PubMed

    Schaldenbrand, J D; McClatchey, K D; Patel, J A; Muilenberg, M J

    1981-01-01

    A 25-year-old white woman ingested an unknown quantity of doxylamine succinate and flurazepam. Urine immunoassay screen (EMIT-dau) was positive for benzodiazopine and negative for phencyclidine. Subsequent gas chromatographic assay of the urine revealed a markedly positive assay for phencyclidine. Doxylamine was ultimately found to be the cause for the false-positive gas chromatographic assay for phencyclidine.

  1. ELISPOT assay on membrane microplates.

    PubMed

    Kalyuzhny, Alexander E

    2009-01-01

    Membranes used for western blotting can be also used for ELISPOT, an enzyme-linked immunospot assay, which allows determining frequencies of cytokine-secreting immune system cells. In addition to their high antibody-retaining capacity PVDF and NC membranes provide good support to immune system cells cultured in vitro and do not affect their physiology. ELISPOT assays utilizing membrane-backed microplates are used in many areas of research including vaccine development, HIV research, cancer and infection disease research, autoimmune disease, and allergy research.ELISPOT utilizes the same antibody "sandwich" technique as enzyme-linked immunosorbent assay, but unlike the latter ELISPOT belongs to state-of-the-art techniques when outcome of the assay depends on skills and accuracy of the operator, a thorough selection of matched pairs of capture and detection antibodies, and using appropriate staining reagents. This review covers basics of ELISPOT assay including its immunochemical design, selection of reagents and membrane microplates, and some troubleshooting recommendations.

  2. From Antenna to Assay

    PubMed Central

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  3. Transporter assays and assay ontologies: useful tools for drug discovery.

    PubMed

    Zdrazil, Barbara; Chichester, Christine; Zander Balderud, Linda; Engkvist, Ola; Gaulton, Anna; Overington, John P

    2014-06-01

    Transport proteins represent an eminent class of drug targets and ADMET (absorption, distribution, metabolism, excretion, toxicity) associated genes. There exists a large number of distinct activity assays for transport proteins, depending on not only the measurement needed (e.g. transport activity, strength of ligand–protein interaction), but also due to heterogeneous assay setups used by different research groups. Efforts to systematically organize this (divergent) bioassay data have large potential impact in Public-Private partnership and conventional commercial drug discovery. In this short review, we highlight some of the frequently used high-throughput assays for transport proteins, and we discuss emerging assay ontologies and their application to this field. Focusing on human P-glycoprotein (Multidrug resistance protein 1; gene name: ABCB1, MDR1), we exemplify how annotation of bioassay data per target class could improve and add to existing ontologies, and we propose to include an additional layer of metadata supporting data fusion across different bioassays.

  4. Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

    NASA Astrophysics Data System (ADS)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

    2015-01-01

    Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

  5. Oestradiol assays: fitness for purpose?

    PubMed

    Middle, Jonathan G; Kane, John W

    2009-11-01

    In this review we discuss the analytical inadequacies of oestradiol assays in relation to the clinical requirements for performing them, and make recommendations for their improvement. The measurement of oestradiol can be requested in a number of clinical scenarios (precocious puberty, infertility, assisted conception, hormone replacement therapy). The very wide dynamic range of oestradiol concentrations is a huge challenge for routine assays, which they are unlikely to meet on theoretical as well as practical grounds. The EQA performance of oestradiol assays in terms of trueness, comparability, recovery and analytical sensitivity leaves much to be desired and indicates that calibration is compromised by poor analytical specificity. To make oestradiol assays fit for purpose requires concerted action by all stakeholders to define analytical quality specifications for the various clinical scenarios involved, and then to encourage concerted action by the diagnostic industry to use the steroid reference measurement system to improve specificity, trueness and traceability.

  6. Protein tyrosine phosphatase: enzymatic assays.

    PubMed

    Montalibet, Jacqueline; Skorey, Kathryn I; Kennedy, Brian P

    2005-01-01

    Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

  7. Plaque assay for murine norovirus.

    PubMed

    Gonzalez-Hernandez, Mariam B; Bragazzi Cunha, Juliana; Wobus, Christiane E

    2012-08-22

    Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration. However, its limit of detection is higher compared to a plaque assay. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of

  8. Methods to assay Drosophila behavior.

    PubMed

    Nichols, Charles D; Becnel, Jaime; Pandey, Udai B

    2012-03-07

    Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases(1). We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials(2-4). The rapid iterative negative geotaxis (RING) assay(5) has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously

  9. Label-Free Receptor Assays

    PubMed Central

    Fang, Ye

    2010-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery. PMID:21221420

  10. Label-Free Receptor Assays.

    PubMed

    Fang, Ye

    2011-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery.

  11. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  12. Microbiological assay using bioluminescent organism

    SciTech Connect

    Stiffey, A.V.

    1987-12-21

    This invention relates to testing processes for toxicity involving microorganisms and, more particularly, to testing processes for toxicity involving bioluminescent organisms. The present known method of testing oil-well drilling fluids for toxicity employs the mysid shrimp (Mysidopsis bahia) as the assay organism. The shrimp are difficult to raise and handle as laboratory assay organisms. This method is labor-intensive, because it requires a assay time of about 96 hours. Summary of the Invention: A microbiological assay in which the assay organism is the dinoflagellate, Pyrocystis lunula. A sample of a substance to be assayed is added to known numbers of the bioluminescent dinoflagellate and the mixture is agitated to subject the organisms to a shear stress causing them to emit light. The amount of light emitted is measured and compared with the amount of light emitted by a known non-toxic control mixture to determine if there is diminution or non-diminution of light emitted by the sample under test which is an indication of the presence or absence of toxicity, respectively. Accordingly, an object of the present invention is the provision of an improved method of testing substances for toxicity. A further object of the invention is the provision of an improved method of testing oil-well drilling fluids for toxicity using bioluminescent dinoflagellate (Pyrocystis lunula).

  13. Synaptic vesicle chips to assay botulinum neurotoxins

    PubMed Central

    2005-01-01

    BoNTs (botulinum neurotoxins), considered to be the most toxic of all biological substances, inhibit neurotransmission through proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [VAMP (vesicle-associated membrane protein, or synaptobrevin), SNAP-25 (25 kDa synaptosome-associated protein) or syntaxin]. Expansion in the use of BoNTs as therapeutic and cosmetic agents, and the potential threat they constitute as biological weapons, underlines the need for rapid and sensitive in vitro assays. Here, we present new automatized bioassays to detect VAMP cleavage by BoNT/B and F. Western blotting and SPR (surface plasmon resonance) methods revealed that BoNT/B and F totally cleave their substrate on immunoisolated SVs (synaptic vesicles). Real-time monitoring of the immunocapture of native SVs from crude lysates on SPR sensor chips enabled the detection of picogram amounts of different SV proteins. Pre-incubation of a membrane fraction containing SVs with BoNT specifically inhibited capture by anti-VAMP antibodies, and amounts as low as 0.1 pg of BoNT/B were detected. This automated SPR assay is approx. 200 times more sensitive, and 25 times more rapid, than the in vivo BoNT/B test currently used. Moreover, the method can be performed using a few thousand cultured neurons and constitutes a new screening assay for inhibitors. Our data indicate that native VAMP is an optimal substrate for in vitro BoNT assays that can be monitored by SPR. PMID:16011482

  14. The neutral red release assay: a review.

    PubMed

    Zuang, V

    2001-01-01

    The neutral red release (NRR) assay is a cytotoxicity test that can be used to measure the immediate toxic effects of test substances on the cell membrane, resulting in the leaking of intracellular contents. The assay has already been used for several years to evaluate the cytotoxicities of various kinds of products, such as cosmetics, pharmaceuticals, industrial chemicals and household products. It has undergone in-house validation by many companies, and has been found to be particularly useful for identifying substances that are potentially capable of causing adverse reactions on coming into brief contact with the eye or the skin at relatively high concentrations, such as might occur in an adventitious splash into the eye or onto the skin, followed by a quick rinse. Because of the relatively long existence of the NRR assay, its practicality and its proven usefulness for particular purposes, ECVAM decided to review the status of the method, in order to decide whether prevalidation and formal validation studies on the test might be profitable. The review of the status of the test was carried out by performing a comprehensive review of the literature, and by conducting a survey involving companies and institutes with experience in using the test. Both the review and the survey revealed that the assay could provide extremely valuable information when it was used for particular purposes, such as for the evaluation and comparison of immediate toxic effects on the eye or the skin caused by certain products or chemicals such as surfactants. Most of those who responded in the survey favoured a prevalidation/validation study.

  15. Bacterial assay of contact lens wearers.

    PubMed

    Hart, D E; Hosmer, M; Georgescu, M; Farris, R L

    1996-03-01

    The goal of the project was to determine the quantity of bacteria on the contact lens and adjacent areas of the eye. This paper is a quantitative study of the contact lens and ocular aerobic microbiota in a mixed group of daily and extended wear disposable contact lens users. The contact lens, the lower fornix, tears collecting at the lower fornix, and edge of the lower lid at the Meibomian gland margin were assayed for the quantity of bacterial colony forming units (CFU). Eighteen patients wearing 49 disposable high water content hydrogel contact lenses were assayed and the mean lens age was 8.8 +/- 4.6 days. Three patients wore their lenses on a daily wear basis and 15 on an extended wear schedule. Tear samples were obtained with sterile microbial loops and the lens was macerated into small particles with a tissue grinder. The samples were poured onto the surface of chocolate agar plates and incubated at 35 degrees C for 48 h in 5% Co2. The lid margin revealed the greatest bacterial presence (mean = 9.7 CFU; median = 2 CFU; mode = 0 CFU). The lens showed the next greatest presence of CFU (mean = 4.5 CFU; median = 1 CFU; mode = 0). The fornix and tears revealed the least bacterial presence (fornix: mean = 2.6 CFU; median = 0 CFU; mode = 0 CFU). The bacteria were coagulase-negative staphylococci. The bacterial assay of disposable lens wearing contact lens subjects indicates that the lid margins are the greatest source of bacteria with the tears being the lowest. These studies support the concept that in the eye, the lens typically does not possess a large number of bacteria under normal conditions.

  16. Titanium dioxide nanoparticles: an in vitro study of DNA binding, chromosome aberration assay, and comet assay.

    PubMed

    Patel, Suhani; Patel, Palak; Bakshi, Sonal R

    2017-04-01

    Engineered titanium dioxide nanoparticles (TiO2 NPs) are extensively used in cosmetic, pharmaceutical and other industries globally due to their unique properties, which has raised concern for biosafety. Genotoxicity assessment is an important part of biosafety evaluation; we report in vitro cytogenetic assays for NPs considering their unique physicochemical characteristics to fill the gap of laboratory data regarding biological safety along with mechanistic study for mode of interaction of NP with genetic material. Comet and chromosome aberration assay (CA assay) using short-term human peripheral blood cultures following exposure to TiO2 NPs; along with physicochemical parameters for stability of nano form in cultures; and DNA binding activity were carried out. The dynamic light scattering and zeta potential measurements revealed mono dispersion in media. The fluorescence spectroscopy for binding affinity of TiO2 NPs and human genomic DNA showed binding constant (Kb), 4.158 × 10(6) M(-1) indicating strong binding affinity and negative ΔG(0) value suggesting spontaneous DNA binding supporting its genotoxic potential. Following in vitro exposure to TiO2 NPs for 24 h, the cultures were analyzed for comet and CA assays, which showed significant results (p < 0.05) for % DNA intensity in tail, Olive Tail Moment and frequency of Chromosomal aberrations (CA) at 75 and 125 μM but not at 25 μM.

  17. A fluorogenic assay for methylglyoxal.

    PubMed

    Shaheen, Fozia; Shmygol, Anatoly; Rabbani, Naila; Thornalley, Paul J

    2014-04-01

    MG (methylglyoxal) is a potent glycating agent and an endogenous reactive dicarbonyl metabolite formed in all live cells and organisms. It is an important precursor of AGEs (advanced glycation end-products) and is implicated in aging and disease. MG is assayed by derivatization by 1,2-diaminobenzene derivatives in cell extracts. Such assays are not applicable to high sample throughput, subcellular, live-cell and in vivo estimations. The use of fluorogenic probes designed for NO (nitric oxide) detection in biological samples and living cells has inadvertently provided probes for the detection of dicarbonyls such as MG. We describe the application of DAF-2 (4,5-diaminofluorescein) and DAR-1 (4,5-diaminorhodamine) for the detection of MG in cell-free systems and application for high-throughput assay of glyoxalase activity and assay of glucose degradation products in peritoneal dialysis fluids. DAF-2 and DAR-1, as for related BODIPY probes, do not have sufficient sensitivity to detect MG in live cells. Care will also be required to control for NO and dehydroascorbate co-detection and interference from peroxidase catalysing the degradation of probes to MG and glyoxal. Fluorogenic detection of MG, however, has great potential to facilitate the assay of MG and to advance towards that capability of imaging this product in live cells in vitro and small animals in vivo.

  18. An in situ assay for determination of benzodiazepine binding.

    PubMed

    Sher, P K; Schrier, B K; Van Putten, D

    1982-01-01

    Benzodiazepine (BDZ) receptors have been demonstrated recently in a variety of mammalian tissues. However, assay methods for such receptors have required that disrupted tissues be used. We have developed an in situ assay for this receptor utilizing intact cells cultured from the cerebral cortices of fetal mice which is more sensitive and physiologic than those used previously. Results obtained with this assay differ in the following ways from those in which disrupted tissues are used: (1) total and specific BDZ binding was as much as 10-fold higher in the in situ assays; (2) Scatchard analysis of the binding data is consistently nonlinear, revealing at least two binding sites with KD values of 5.5 and 303 nM, and (3) presumed nonneuronal receptors were found in abundance.

  19. A High-Throughput Screening Assay to Detect ...

    EPA Pesticide Factsheets

    In support of the Endocrine Disruption Screening Program (EDSP21), the US EPA ToxCast program is developing assays to enable screening for chemicals that may disrupt thyroid hormone synthesis. Thyroperoxidase (TPO) is critical for TH synthesis and is a known target of thyroid-disrupting chemicals that adversely impact neurodevelopment. The AUR-TPO assay was recently developed to screen >1,900 ToxCast chemicals for potential TPO inhibition activity. Parallel assays were used to determine which AUR-TPO actives were more selective for TPO inhibition. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO assay and an orthogonal peroxidase oxidation assay using guaiacol as substrate to confirm putative TPO inhibition profiles. Bioactivity results from the AUR-TPO assay were used to identify chemical substructures associated with in vitro TPO inhibition. Substructure profiles were generated for each chemical in the ToxCast test set using the publicly-available ToxPrint 2.0 chemotypes. Chemotypes enriched among the putative TPO inhibitors were identified using a cumulative hypergeometric probability (p < 0.01). Of the total 729 chemotypes evaluated, 44 were overrepresented among TPO inhibitors. Another 24 chemotypes were found to be significantly underrepresented among AUR-TPO actives. Examination of these chemotypes revealed four basic pharmacophores that accounted for 70% of the ToxCast chemicals active in the AUR-TPO assay:

  20. Biological detection of low radiation doses with integrated photothermal assay

    NASA Astrophysics Data System (ADS)

    Zharov, Vladimir P.; Viegas, Mark; Soderberg, Lee S. F.

    2005-04-01

    The goal of this paper was to evaluate the diagnostic value of integrated photothermal (PT) assay with additional fluorescent and photoacoustic (PA) modules to assess both the "safety limit" of exposure to ionizing γ-radiation and optimal therapeutic doses for cancer treatment. With this assay, the influences of γ irradiation on cancer cells (pancreatic-AR42J and hepatocytes-hepG2) and healthy cells (mouse lymphocytes and erythrocytes) was examined as a function of exposure dose (0.6-5 Gy) and time after irradiation, in vitro and in vivo. Independent verification of data obtained with conventional assays revealed that integrated PT assay allowed us to detect the different stages of radiation impact, including changes in cell metabolism at low dose, or stages related to cell death (apoptosis and necrosis) at high doses with a threshold sensitivity of at least three orders of magnitude better than existing assays. Also, PT assay was capable of quantitatively differentiating the biological action of γ irradiation alone and in combination with drug and nicotine impact. Finally, we demonstrated on an animal model that IPT assay has the potential for use in routine rapid evaluation of biological consequences of low-dose exposure a few days after irradiation.

  1. A radiometric kynurenine monooxygenase assay

    SciTech Connect

    Wiseman, J.S.; Nichols, J.S. )

    1990-01-01

    Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-(3H)kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.

  2. Barcoded microchips for biomolecular assays.

    PubMed

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  3. Logistic Proliferation of Cells in Scratch Assays is Delayed.

    PubMed

    Jin, Wang; Shah, Esha T; Penington, Catherine J; McCue, Scott W; Maini, Philip K; Simpson, Matthew J

    2017-03-23

    Scratch assays are used to study how a population of cells re-colonises a vacant region on a two-dimensional substrate after a cell monolayer is scratched. These experiments are used in many applications including drug design for the treatment of cancer and chronic wounds. To provide insights into the mechanisms that drive scratch assays, solutions of continuum reaction-diffusion models have been calibrated to data from scratch assays. These models typically include a logistic source term to describe carrying capacity-limited proliferation; however, the choice of using a logistic source term is often made without examining whether it is valid. Here we study the proliferation of PC-3 prostate cancer cells in a scratch assay. All experimental results for the scratch assay are compared with equivalent results from a proliferation assay where the cell monolayer is not scratched. Visual inspection of the time evolution of the cell density away from the location of the scratch reveals a series of sigmoid curves that could be naively calibrated to the solution of the logistic growth model. However, careful analysis of the per capita growth rate as a function of density reveals several key differences between the proliferation of cells in scratch and proliferation assays. Our findings suggest that the logistic growth model is valid for the entire duration of the proliferation assay. On the other hand, guided by data, we suggest that there are two phases of proliferation in a scratch assay; at short time, we have a disturbance phase where proliferation is not logistic, and this is followed by a growth phase where proliferation appears to be logistic. These two phases are observed across a large number of experiments performed at different initial cell densities. Overall our study shows that simply calibrating the solution of a continuum model to a scratch assay might produce misleading parameter estimates, and this issue can be resolved by making a distinction between the

  4. [Seizures revealing phosphocalcic metabolism abnormalities].

    PubMed

    Hmami, F; Chaouki, S; Benmiloud, S; Souilmi, F Z; Abourazzak, S; Idrissi, M; Atmani, S; Bouharrou, A; Hida, M

    2014-01-01

    Hypocalcemia due to hypoparathyroidism produces a broad spectrum of clinical manifestations, but overt symptoms may be sparse. One unusual presentation is onset or aggravation of epilepsy in adolescence revealing hypoparathyroidism. This situation can lead to delayed diagnosis, with inefficacity of the antiepileptic drugs. We report five cases of adolescence-onset epilepsy with unsuccessful antiepileptic therapy, even with gradually increasing dose. Physical examination revealed signs of hypocalcemia, confirmed biologically. Full testing disclosed the origin of the seizures: hypoparathyroidism in three patients and pseudohypoparathyroidism in the other two. In four of five patients, computed tomography showed calcification of the basal ganglia, defining Fahr's syndrome. The patients were treated with oral calcium and active vitamin D (1-alphahydroxy vitamin D3). Seizure frequency progressively decreased and serum calcium levels returned to normal. These cases illustrate the importance of the physical examination and of routine serum calcium assay in patients with new-onset epileptic seizures in order to detect hypocalcemia secondary to hypoparathyroidism.

  5. Antioxidant assays for plant and food components.

    PubMed

    Moon, Joon-Kwan; Shibamoto, Takayuki

    2009-03-11

    Recently, research on natural antioxidants has become increasingly active in various fields. Accordingly, numerous articles on natural antioxidants, including polyphenols, flavonoids, vitamins, and volatile chemicals, have been published. Assays developed to evaluate the antioxidant activity of plants and food constituents vary. Therefore, to investigate the antioxidant activity of chemical(s), choosing an adequate assay based on the chemical(s) of interest is critical. There are two general types of assays widely used for different antioxidant studies. One is an assay associated with lipid peroxidations, including the thiobarbituric acid assay (TBA), malonaldehyde/high-performance liquid chromatography (MA/HPLC) assay, malonaldehyde/gas chromatography (MA/GC) assay, beta-carotene bleaching assay, and conjugated diene assay. Other assays are associated with electron or radical scavenging, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, ferric reducing/antioxidant power (FRAP) assay, ferrous oxidation-xylenol orange (FOX) assay, ferric thiocyanate (FTC) assay, and aldehyde/carboxylic acid (ACA) assay. In this review, assays used recently were selected for extended discussion, including discussion of the mechanisms underlying each assay and its application to various plants and foods.

  6. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  7. Three dimensional colorimetric assay assemblies

    SciTech Connect

    Charych, D.; Reichart, A.

    2000-06-27

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  8. Three dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichart, Anke

    2000-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flu virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  9. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  10. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  11. Biochemical Assays of Cultured Cells

    NASA Technical Reports Server (NTRS)

    Barlow, G. H.

    1985-01-01

    Subpopulations of human embryonic kidney cells isolated from continuous flow electrophoresis experiments performed at McDonnell Douglas and on STS-8 have been analyzed. These analyses have included plasminogen activator assays involving indirect methodology on fibrin plated and direct methodology using chromogenic substrates. Immunological studies were performed and the conditioned media for erythropoietin activity and human granulocyte colony stimulating (HGCSF) activity was analyzed.

  12. Broad base biological assay using liquid based detection assays

    SciTech Connect

    Milanovich, F; Albala, J; Colston, B; Langlois, R; Venkateswaren, K

    2000-10-31

    The release of a biological agent by terrorists represents a serious threat to the safety of US citizens. At present there are over 50 pathogens and toxins on various agency threat lists. Most of these pathogens are rarely seen by public health personnel so the ability to rapidly identify their infection is limited. Since many pathogenic infections have symptomatic delays as long as several days, effective treatment is often compromised. This translates into two major deficiencies in our ability to counter biological terrorism (1) the lack of any credible technology to rapidly detect and identify all the pathogens or toxins on current threat lists and (2) the lack of a credible means to rapidly diagnose thousands of potential victims. In this SI we are developing a rapid, flexible, inexpensive, high throughput, and deeply multiplex-capable biological assay technology. The technology, which we call the Liquid Array (LA), utilizes optical encoding of small diameter beads which serve as the templates for biological capture assays. Once exposed to a fluid sample these beads can be identified and probed for target pathogens at rates of several thousand beads per second. Since each bead can be separately identified, one can perform parallel assays by assigning a different assay to each bead in the encoded set. The goal for this development is a detection technology capable of simultaneously identifying 100s of different bioagents and/or of rapidly diagnosing several thousand individuals. We are pursuing this research in three thrusts. In the first we are exploring the fundamental interactions of the beads with proteins and nucleic acids in complex mixtures. This will provide us with a complete understanding of the limits of the technology with respect to throughput and complex environment. A major spin-off of this activity is in the rapidly emerging field of proteomics where we may be able to rapidly assess the interactions responsible for cell metabolism, structural

  13. Assay strategies and methods for phospholipases

    SciTech Connect

    Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

    1991-01-01

    Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is {approximately} as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous.

  14. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State feed...

  15. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State feed...

  16. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State feed...

  17. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State feed...

  18. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  19. Comet Assay in Cancer Chemoprevention.

    PubMed

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks.

  20. Important Norwegian crude assays updated

    SciTech Connect

    Corbett, R.A

    1990-03-12

    New assays on two important Norwegian North Sea crude oils, Statfjord and Gullfaks, are presented. Both are high-quality, low-sulfur crudes that will yield a full range of good-quality products. All assay data came from industry-standard test procedures. The Statfjord field is the largest in the North Sea. Production started in 1979. Statfjord is a typical North Sea crude, produced from three separate platforms and three separate loading buoys with interconnecting lines. Current production is about 700,000 b/d. Gullfaks is produced from a large field in Block 34/10 of the Norwegian sector of the North Sea production area. Gullfaks crude oil is more biodegraded than other crudes from the region. Biodegradation has removed most of the waxy normal paraffins, resulting in a heavier, more naphthenic and aromatic crude.

  1. Microbiologic assay of space hardware.

    NASA Technical Reports Server (NTRS)

    Favero, M. S.

    1971-01-01

    Review of the procedures used in the microbiological examination of space hardware. The general procedure for enumerating aerobic and anaerobic microorganisms and spores is outlined. Culture media and temperature-time cycles used for incubation are reviewed, along with assay systems designed for the enumeration of aerobic and anaerobic spores. The special problems which are discussed are involved in the precise and accurate enumeration of microorganisms on surfaces and in the neutralization of viable organisms buried inside solid materials that could be released to a planet's surface if the solid should be fractured. Special attention is given to sampling procedures including also the indirect techniques of surface assays of space hardware such as those using detachable or fallout strips. Some data on comparative levels of microbial contamination on lunar and planetary spacecraft are presented.

  2. Protein binding assay for hyaluronate

    SciTech Connect

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  3. Two offshore Australian crudes assayed

    SciTech Connect

    Rhodes, A.K.

    1994-05-09

    Two light, sweet crudes from offshore Australia have been assayed. Gippsland crude, also called Bass Strait, is produced off the coast of Victoria, in southeastern Australia. The 47 API, 0.09% sulfur crude was analyzed in mid-1993. Skua, a 42 API, 0.06 wt % sulfur crude, is produced in the Timor Sea. Data are given on the whole crude and fractions for both deposits. Both chemical and physical properties are listed.

  4. Microbiological Assay Using Bioluminescent Organism.

    DTIC Science & Technology

    A microbiological assay based on bioluminesce employing the bioluminescent dinoflagellate Pyrocystis lunula. An oil well drilling fluid sample is... Pyrocystis lunula in suspension. The mixture is agitated to subject th Pyrocystis lunula to a shear stress. Light emitted as a result of the shear...stress on the Pyrocystic lunula is measure and compared with a control to determine if there is diminution of light produced by the Pyrocystis lunula in

  5. [Visible spectrophotometric assay of ranitidine].

    PubMed

    Apostu, M; Dorneanu, V; Bibire, Nela

    2003-01-01

    Ranitidine, belonging to H2-antagonist group, is a compound containing a furanic moiety and is used in peptic ulcer therapy. This paper debates the possibility of developing a new visible spectrophotometric assessment by using the reaction between ranitidine and eosine. We carried out our determinations at 505 nm, where the absorbency of ranitidine-eosine complex is maximal, and we have established the optimal reaction conditions. This method was successfully applied for ranitidine assay from pharmaceutical dosage forms.

  6. Bioavailable Ferric Iron (BAFelll) Assay

    DTIC Science & Technology

    2007-02-01

    Immobilization and Magnetite Formation via Ferric Oxide Reduction by Shewanella putrefaciens 200. Environ. Sci. Technol, 34:100-106. EA...bioavailable ferric iron BET Brunauer-Emmett-Teller bgs below ground surface BTEX benzene-toluene-ethylbenzene-xylenes BrY Shewanella alga BrY CDB...reducing bacterium Shewanella alga BrY, lactate as an electron donor, and a mineral salts medium supplemented with reagents that accelerate the assay

  7. Bioluminescence assay for cell viability.

    PubMed

    Lomakina, G Yu; Modestova, Yu A; Ugarova, N N

    2015-06-01

    Theoretical aspects of the adenosine triphosphate bioluminescence assay based on the use of the firefly luciferin-luciferase system are considered, as well as its application for assessing cell viability in microbiology, sanitation, medicine, and ecology. Various approaches for the analysis of individual or mixed cultures of microorganisms are presented, and capabilities of the method for investigation of biological processes in live cells including necrosis, apoptosis, as well as for investigation of the dynamics of metabolism are described.

  8. Optical fiber hybridization assay fluorosensor

    NASA Astrophysics Data System (ADS)

    Pilevar, Saeed; Davis, Christopher C.; Hodzic, Vildana; Portugal, Frank

    1999-04-01

    The present work describes an all-fiber hybridization assay sensor that relies on the evanescent field excitation of fluorescence from surface-bound fluorophores. The evanescent field is made accessible through the use of a long adiabatically tapered single-mode fiber probe. A semiconductor laser operating at 785 nm wavelength is used in a pulsed mode to excite fluorescence in the tapered region of a fiber probe using the near-infrared fluorophore IRD 41. We have carried out real-time hybridization tests for IRD 41-labeled oligonucleotide at various probe concentrations binding to complementary oligonucleotide cross-linked to the tapered fiber surface. Short oligonucleotides (20-mer) bound to the fiber surface have been used to detect near-infrared dye labeled complementary sequences at sub-nanomolar levels. Sandwich assays with total RNA were conducted to examine the capability of the biosensor for detecting bacterial cells using rRNA as the target. The results indicate that this fluorosensor is capable of detecting H. pylori in a sandwich assay at picomolar concentrations.

  9. Mitochondrial base excision repair assays

    PubMed Central

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

    2010-01-01

    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 glycosylases, AP endonuclease, DNA polymerase (POLγ in mitochondria), and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques, as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability. PMID:20188838

  10. The Rabbit Corneal Pocket Assay.

    PubMed

    Morbidelli, Lucia; Ziche, Marina

    2016-01-01

    The rabbit corneal micropocket angiogenesis assay uses the avascular cornea as a substrate canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 1-2 weeks and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor and a synthetic polymer to allow slow release. A micropocket is surgically created in the rabbit cornea under anesthesia and a pellet implanted. On the days later, the angiogenic response is measured and qualified using a slit lamp, as well as the concomitant vascular phenotype or inflammatory features. The results of the assay are used to assess the ability of potential therapeutic molecules to modulate angiogenesis in vivo, both when released locally or given by ocular formulations or through systemic treatment. In this chapter, the experimental details of the rabbit cornea assay and technical implementations to the original protocol are described.

  11. Assay of ribulose bisphosphate carboxylase

    SciTech Connect

    Pike, C.; Berry, J.

    1987-04-01

    Assays of ribulose bisphosphate carboxylase (rubisco) can be used to illustrate many properties of photosynthetic systems. Many different leaves have been assayed with this standard procedure. The tissue is ground with a mortar and pestle in extraction buffer. The supernatant after centrifugation is used as the source of enzyme. Buffer, RuBP, (/sup 14/C)-NaHCO/sub 3/, and enzyme are combined in a scintillation vial; the reaction is run for 1 min at 30/sup 0/. The acid-stable products are counted. Reproducibility in student experiments has been excellent. The assay data can be combined with analyses of leaf properties such as fresh and dry weight, chlorophyll and protein content, etc. Students have done projects such as the response of enzyme to temperature and to various inhibitors. They also report on the use of a transition state analog, carboxyarabinitol bisphosphate, to titrate the molar concentration of rubisco molecules (active sites) in an enzyme sample. Thus, using crude extracts the catalytic activity of a sample can be compared to the absolute quantity of enzyme or to the turnover number.

  12. Assay of Deoxyhypusine Synthase Activity

    PubMed Central

    Wolff, Edith C.; Lee, Seung Bum; Park, Myung Hee

    2011-01-01

    Deoxyhypusine synthase catalyzes an unusual protein modification reaction. A portion of spermidine is covalently added to one specific lysine residue of one eukaryotic protein, eIF5A (eukaryotic initiation factor 5A) to form a deoxyhypusine residue. The assay measures the incorporation of radioactivity from [1,8-3H]spermidine into the eIF5A protein. The enzyme is specific for the eIF5A precursor protein and does not work on short peptides (<50 amino acids). Optimum conditions for the reaction and four detection methods for the product, deoxyhypusine-containing eIF5A, are described in this chapter. The first, and most specific, method is the measurement of the amount of [3H]deoxyhypusine in the protein hydrolysate after its separation by ion exchange chromatography. However, this method requires some specialized equipment. The second method is counting the radioactivity in TCA-precipitated protein after thorough washing. The third method involves determining the radioactivity in the band of [3H] deoxyhypusine-containing eIF5A after separation by SDS-PAGE. The fourth method is a filter-binding assay. It is important to minimize nonspecific binding of [3H]spermidine to proteins in the assay mixture, especially for methods 2 and 4, as illustrated in a comparison figure in the chapter. PMID:21318875

  13. Experiences with the in vivo and in vitro comet assay in regulatory testing.

    PubMed

    Frötschl, Roland

    2015-01-01

    The in vivo comet assay has recently been implemented into regulatory genotoxicity testing of pharmaceuticals with inclusion into the ICH S2R1 guidance. Regulatory genotoxicity testing aims to detect DNA alterations in form of gene mutations, larger scale chromosomal damage and recombination and aneuploidy. The ICH S2R1 guideline offers two options of standard batteries of tests for the detection of these endpoints. Both options start with an AMES assay and option 1 includes an in vitro mammalian cell assay and an in vivo micronucleus assay in rodent, whereas option 2 includes an in vivo micronucleus assay in bone marrow in rodent and a second in vivo assay in a second tissue with a second endpoint. The test recommended as second in vivo test is the comet assay in rat liver. The in vivo comet assay is considered as mature enough to ensure reliable detection of relevant in vivo genotoxicants in combination with the micronucleus test in bone marrow and the AMES assay. Although lots of research papers have been published using the in vitro comet assay, the in vitro version has not been implemented into official regulatory testing guidelines. A survey of the years 1999-2014 revealed 27 in vivo comet assays submitted to BfArM with market authorisation procedures, European and national advice procedures and clinical trial applications. In three procedures, in vitro comet assays had been submitted within the genetic toxicology packages.

  14. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-05-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This unit describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the unit is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This unit also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  15. Assays for determination of protein concentration.

    PubMed

    Olson, Bradley J S C; Markwell, John

    2007-09-01

    Biochemical analysis of proteins relies on accurate quantitation of protein concentration. This appendix describes how to perform commonly used protein assays, e.g., Lowry, Bradford, BCA, and UV spectroscopic protein assays. The primary focus of the appendix is assay selection, emphasizing sample and buffer compatibility. Protein assay standard curves and data processing fundamentals are discussed in detail. This appendix also details high-throughput adaptations of the commonly used protein assays, and also contains a protocol for BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels, which is reliable, inexpensive, and quick.

  16. Radioenzymatic assay for quinolinic acid

    SciTech Connect

    Foster, A.C.; Okuno, E.; Brougher, D.S.; Schwarcz, R.

    1986-10-01

    A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing (/sup 3/H)ATP, further to (/sup 3/H) deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

  17. Rotor assembly and assay method

    DOEpatents

    Burtis, C.A.; Johnson, W.F.; Walker, W.A.

    1993-09-07

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor. 34 figures.

  18. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  19. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  20. Rotor assembly and assay method

    DOEpatents

    Burtis, Carl A.; Johnson, Wayne F.; Walker, William A.

    1993-01-01

    A rotor assembly for carrying out an assay includes a rotor body which is rotatable about an axis of rotation, and has a central chamber and first, second, third, fourth, fifth, and sixth chambers which are in communication with and radiate from the central chamber. The rotor assembly further includes a shuttle which is movable through the central chamber and insertable into any of the chambers, the shuttle including a reaction cup carrying an immobilized antigen or an antibody for transport among the chambers. A method for carrying out an assay using the rotor assembly includes moving the reaction cup among the six chambers by passing the cup through the central chamber between centrifugation steps in order to perform the steps of: separating plasma from blood cells, binding plasma antibody or antigen, washing, drying, binding enzyme conjugate, reacting with enzyme substrate and optically comparing the resulting reaction product with unreacted enzyme substrate solution. The movement of the reaction cup can be provided by attaching a magnet to the reaction cup and supplying a moving magnetic field to the rotor.

  1. Assay of potentially contaminated propellant

    SciTech Connect

    Koster, J.E.; Williams, H.E. III; Scott, W.S.

    1995-02-01

    One of the decontamination and decommissioning projects within DOD is demilitarization of an aging stockpile of munitions. A large portion of the stockpile contains depleted uranium (DU) as an armor piercing core and so these munitions must be assayed for the presence of uranium in other components. The assay method must be fast and preferably easy to implement. Presence of DU is indicated by its alpha decay. The alpha particles in turn produce ions in the ambient air. If a significant fraction of these ions can escape the quantity of propellant, the ions can be detected instead of the alpha particles. As a test of the feasibility of detecting alpha emissions from DU somewhere within a cartridge of propellant, the transmission of ions through layers of real propellant was measured. The propellant is in the form of graphite-coated cylindrical pellets. A 105nun cartridge was modified for use as a pellet chamber. A check source served as an ion source. The ion detector consisted of a grid held at 300V coupled to an ammeter. Results confirm that this is a promising technique for testing the propellant for the presence of DU quickly yet with sensitivity.

  2. Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

    PubMed

    Jose, D Amilan; Elstner, Martin; Schiller, Alexander

    2013-10-18

    Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme β-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. β-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for β-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.

  3. Widespread Nanoparticle-Assay Interference: Implications for Nanotoxicity Testing

    PubMed Central

    Ong, Kimberly J.; MacCormack, Tyson J.; Clark, Rhett J.; Ede, James D.; Ortega, Van A.; Felix, Lindsey C.; Dang, Michael K. M.; Ma, Guibin; Fenniri, Hicham; Veinot, Jonathan G. C.; Goss, Greg G.

    2014-01-01

    The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments. PMID:24618833

  4. Comet assay to assess the genotoxicity of Persian walnut (Juglans regia L.) husks with statistical evaluation.

    PubMed

    Petriccione, Milena; Ciniglia, Claudia

    2012-07-01

    The aim of this study was to confirm the utility of the Comet assay as a genotoxicity screening test for evaluating the impact of walnut husk aqueous extract. Phytotoxicity assays using diluted and undiluted walnut husk aqueous extracts were performed on young roots of Raphanus sativus (radish), and the Comet assay was used to evaluate DNA integrity in isolated radish radicle nuclei. The results reveal a dose-dependent accumulation of DNA damage in radish radicles treated with walnut husks water extract and that the Kolmogorov-Smirnov test combined with Johnson SB distribution was the best approach for describing Comet assay data.

  5. A colorimetric assay for cytokinin oxidase.

    PubMed

    Libreros-Minotta, C A; Tipton, P A

    1995-11-01

    A simple and rapid colorimetric assay for cytokinin oxidase is described. The assay is based on the formation of a Schiff base between the enzymatic reaction product 3-methyl-2-butenal and p-aminophenol. The assay is effective in the submicromolar concentration range and can be used in crude plant extracts as well as in more highly purified preparations.

  6. Data transformation methods for multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2013-07-23

    Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

  7. Sperm chromatin structure assay (SCSA®).

    PubMed

    Evenson, Donald P

    2013-01-01

    The SCSA(®) is the pioneering assay for the detection of damaged sperm DNA and altered proteins in sperm nuclei via flow cytometry of acridine orange (AO) stained sperm. The SCSA(®) is considered to be the most precise and repeatable test providing very unique, dual parameter data (red vs. green fluorescence) on a 1,024 × 1,024 channel scale, not only on DNA fragmentation but also on abnormal sperm characterized by lack of normal exchange of histones to protamines. Raw semen/sperm aliquots or purified sperm can be flash frozen, placed in a box with dry ice and shipped by overnight courier to an experienced SCSA(®) lab. The samples are individually thawed, prepared, and analyzed in ∼10 min. Of significance, data on 5,000 individual sperm are recorded on a 1,024 × 1,024 dot plot of green (native DNA) and red (broken DNA) fluorescence. Repeat measurements have virtually identical dot plot patterns demonstrating that the low pH treatment that opens up the DNA strands at the sites of breaks and staining by acridine orange (AO) are highly precise and repeatable (CVs of 1-3%) and the same between fresh and frozen samples. SCSAsoft(®) software transforms the X-Y data to total DNA stainability versus red/red + green fluoresence (DFI) providing a more accurate determination of % DFI as well as the more sensitive value of standard deviation of DFI (SD DFI) as demonstrated by animal fertility and dose-response toxicology studies. The current established clinical threshold is 25% DFI for placing a man into a statistical probability of the following: (a) longer time to natural pregnancy, (b) low odds of IUI pregnancy, (c) more miscarriages, or (d) no pregnancy. Changes in lifestyle as well as medical intervention can lower the %DFI to increase the probability of natural pregnancy. Couples of men with >25% DFI are counseled to try ICSI and when in the >50% range may consider TESE/ICSI. The SCSA(®) simultaneously determines the % of sperm with high DNA stainability (%HDS

  8. Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis.

    PubMed

    Saito, Kazutoshi; Nukada, Yuko; Takenouchi, Osamu; Miyazawa, Masaaki; Sakaguchi, Hitoshi; Nishiyama, Naohiro

    2013-12-01

    Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Human Arterial Ring Angiogenesis Assay.

    PubMed

    Seano, Giorgio; Primo, Luca

    2016-01-01

    In this chapter we describe a model of human angiogenesis where artery explants from umbilical cords are embedded in gel matrices and subsequently produce capillary-like structures. The human arterial ring (hAR) assay is an innovative system that enables three-dimensional (3D) and live studies of human angiogenesis. This ex vivo model has the advantage of recapitulating several steps of angiogenesis, including endothelial sprouting, migration, and differentiation into capillaries. Furthermore, it can be exploited for (1) identification of new genes regulating sprouting angiogenesis, (2) screening for pro- or anti-angiogenic drugs, (3) identification of biomarkers to monitor the efficacy of anti-angiogenic regimens, and (4) dynamic analysis of tumor microenvironmental effects on vessel formation.

  10. Steroid Assays in Paediatric Endocrinology

    PubMed Central

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation. Conflict of interest:None declared. PMID:21274330

  11. Optimized microturbidimetric assay for fibrinogen.

    PubMed

    Macart, M; Koffi, A; Henocque, G; Mathieu, J F; Guilbaud, J C

    1989-02-01

    In this assay we measure the turbidity produced by precipitation of plasma fibrinogen with a reagent composed of ammonium sulfate, EDTA, and guanidine hydrochloride. The two-step reagent addition, and use of fixed reaction times, eliminates interference from bilirubin, hemoglobin, and chylomicrons. We checked 135 monoclonal proteins for interference, finding the probability of encountering major interference in samples from adults to be very low, P = 0.0002. The method is calibrated with purified fibrinogen and the response is linear over the range 0-10 g/L. Within-run precision (CV) is less than 2% from 1 to 10 g/L. Correlations with the immunoturbidimetric (r = 0.99), chronometric (r = 0.99), and clotting (r = 0.97) methods were extremely high.

  12. Assay of cysteine dioxygenase activity

    SciTech Connect

    Bagley, P.J.; Stipanuk, M.H. )

    1990-02-26

    It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD{sup +} and has a pH optimum of 6.8 and one which is not stimulated by NAD{sup +} and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac{trademark} PA1 (4x250 mm)) was used to separate the ({sup 35}S)cysteinesulfinic acid produced from ({sup 35}S)cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine.

  13. Steroid assays in paediatric endocrinology.

    PubMed

    Honour, John W

    2010-01-01

    Most steroid disorders of the adrenal cortex come to clinical attention in childhood and in order to investigate these problems, there are many challenges to the laboratory which need to be appreciated to a certain extent by clinicians. The analysis of sex steroids in biological fluids from neonates, over adrenarche and puberty present challenges of specificities and concentrations often in small sample sizes. Different reference ranges are also needed for interpretations. For around 40 years, quantitative assays for the steroids and their regulatory peptide hormones have been possible using immunoassay techniques. Problems are recognised and this review aims to summarise the benefits and failings of immunoassays and introduce where tandem mass spectrometry is anticipated to meet the clinical needs for steroid analysis in paediatric endocrine investigations. It is important to keep a dialogue between clinicians and the laboratory, especially when any laboratory result does not make sense in the clinical investigation.

  14. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  15. Predictive assay for cancer targets

    NASA Astrophysics Data System (ADS)

    Suess, Amanda; Nguyen, Christine; Sorensen, Karen; Montgomery, Jennifer; Souza, Brian; Kulp, Kris; Dugan, Larry; Christian, Allen

    2005-11-01

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1- methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  16. Predictive Assay For Cancer Targets

    SciTech Connect

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  17. Comparison of Established and Emerging Biodosimetry Assays

    PubMed Central

    Rothkamm, K.; Beinke, C.; Romm, H.; Badie, C.; Balagurunathan, Y.; Barnard, S.; Bernard, N.; Boulay-Greene, H.; Brengues, M.; De Amicis, A.; De Sanctis, S.; Greither, R.; Herodin, F.; Jones, A.; Kabacik, S.; Knie, T.; Kulka, U.; Lista, F.; Martigne, P.; Missel, A.; Moquet, J.; Oestreicher, U.; Peinnequin, A.; Poyot, T.; Roessler, U.; Scherthan, H.; Terbrueggen, B.; Thierens, H.; Valente, M.; Vral, A.; Zenhausern, F.; Meineke, V.; Braselmann, H.; Abend, M.

    2014-01-01

    Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools. PMID:23862692

  18. Microdroplet chain array for cell migration assays.

    PubMed

    Ma, Yan; Pan, Jian-Zhang; Zhao, Shi-Ping; Lou, Qi; Zhu, Ying; Fang, Qun

    2016-11-29

    Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

  19. Development of forensic assay signatures for ebolaviruses.

    PubMed

    Song, Jian; Doggett, Norman; Wren, Melinda; Burr, Tom; Fenimore, P W; Hatcher, Eneida L; Bruno, William J; Li, Po-E; Stubben, Chris; Wolinsky, Murray

    2015-03-01

    Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

  20. Evaluating 6 ricin field detection assays.

    PubMed

    Slotved, Hans-Christian; Sparding, Nadja; Tanassi, Julia Tanas; Steenhard, Nina R; Heegaard, Niels H H

    2014-01-01

    This study presents data showing the performance of 6 commercial detection assays against ricin around concentrations specified as detection limits by the producers. A 2-fold dilution series of 20 ng/ml ricin was prepared and used for testing the lateral-flow kits: BADD, Pro Strips™, ENVI, RAID DX, Ricin BioThreat Alert, and IMASS™ device. Three of the 6 tested field assays (IMASS™ device, ENVI assay, and the BioThreat Alert assay) were able to detect ricin, although differences in the measured detection limits compared to the official detection limits and false-negative results were observed. We were not able to get the BADD, Pro Strips™, and RAID assays to function in our laboratory. We conclude that when purchasing a field responder assay, there is large variation in the specificity of the assays, and a number of in-house tests must be performed to ensure functionality.

  1. An immunoperoxidase plaque assay for reticuloendotheliosis virus and its application to a sensitive serum neutralization assay.

    PubMed

    Calvert, J G; Nazerian, K

    1994-01-01

    A rapid assay for the enumeration of reticuloendotheliosis virus (REV) is described. Chicken embryo fibroblast monolayer cultures were infected with REV and incubated 6 days under an agar overlay. After removal of the overlay, cells were fixed with acetone/ethanol. Foci of infection (hereafter referred to as plaques) were detected using either an anti-REV envelope monoclonal antibody or convalescent chicken serum as the primary antibody; the secondary antibody was either horseradish peroxidase-conjugated goat anti-mouse IgG (for use with monoclonals) or goat anti-chicken IgG (for use with chicken serum). Staining with a substrate solution containing diaminobenzidine, CoCl2, and H2O2 revealed individual dark plaques on a light gray background. This method worked equally well for the SNV, CSV, and REV-T strains of REV; further, it detected all six field isolates tested. Results indicate that this immunoperoxidase technique is a rapid and reliable method for detection and titration of REV as well as for the assay of neutralizing antibody in chicken serum.

  2. Evaluation of a research use only luminex based assay for measurement of procalcitonin in serum samples

    PubMed Central

    Garrigan, Charles; Han, Jennifer; Tolomeo, Pam; Johnson, Katherine J; Master, Stephen R; Lautenbach, Ebbing; Nachamkin, Irving

    2016-01-01

    Research use only (RUO) assays do not undergo a validation process similar to test kits used for clinical purposes. Several studies have suggested that RUO assays need to be validated prior to use in any research studies. We evaluated a research use only Luminex platform based assay for measuring serum procalcitonin levels (Bio-Plex ProTM Human Acute Phase Multiplex Assay, Bio-Rad Laboratories, Hercules, CA) for comparability with an FDA cleared assay for procalcitonin (VIDAS B.R.A.H.M.S. PCT Assay, bioMérieux, Durham, NC). We tested 1,072 serum samples collected from patients with suspected sepsis in an intensive care unit setting for the comparison. There was poor correlation of the luminex based assay (r=0.081) with the VIDAS PCT Assay in the clinically relevant measurement range (<10 ng/mL). Additionally the Bio-Plex assay showed poor precision. Mass-spectrometry analysis of material eluted from PCT beads did not reveal any identifiable procalcitonin. The results show that research use only assays need to be validated to determine their suitability for research studies. PMID:27830020

  3. Immunochromatographic assay of cadmium levels in oysters.

    PubMed

    Nishi, Kosuke; Kim, In-Hae; Itai, Takaaki; Sugahara, Takuya; Takeyama, Haruko; Ohkawa, Hideo

    2012-08-15

    Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.l mol L(-1) HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters.

  4. Sensitivity of two in vitro assays for evaluating plant activity against the infective stage of Haemonchus contortus strains.

    PubMed

    Al-Rofaai, A; Rahman, W A; Abdulghani, Mahfoudh

    2013-02-01

    The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.

  5. How Are Preferences Revealed?

    PubMed Central

    Beshears, John; Choi, James J.; Laibson, David; Madrian, Brigitte C.

    2009-01-01

    Revealed preferences are tastes that rationalize an economic agent’s observed actions. Normative preferences represent the agent’s actual interests. It sometimes makes sense to assume that revealed preferences are identical to normative preferences. But there are many cases where this assumption is violated. We identify five factors that increase the likelihood of a disparity between revealed preferences and normative preferences: passive choice, complexity, limited personal experience, third-party marketing, and intertemporal choice. We then discuss six approaches that jointly contribute to the identification of normative preferences: structural estimation, active decisions, asymptotic choice, aggregated revealed preferences, reported preferences, and informed preferences. Each of these approaches uses consumer behavior to infer some property of normative preferences without equating revealed and normative preferences. We illustrate these issues with evidence from savings and investment outcomes. PMID:24761048

  6. Improving techniques for clonogenic assays.

    PubMed

    Eliason, J F; Fekete, A; Odartchenko, N

    1984-01-01

    A serum-free medium has been developed which supports colony formation by cells from several human tumor cell lines, one colon adenocarcinoma (WiDr) and four melanoma (Me43, Me85, MP6, MeIuso). This medium consists of a 1:1 mixture of an enriched Dulbecco's modified Eagle's medium (EMED) and a modified Ham's F-12 nutrient mixture (FMED) supplemented with 0.9% methylcellulose, 1% bovine serum albumin, 80 micrograms/ml human transferrin, 3 micrograms/ml insulin, 2.8 micrograms/ml linoleic acid, 2.6 micrograms/ml cholesterol, 20 microM ethanolamine, and trace elements. Colony formation by WiDr cells is linear with the numbers of cells plated, having a plating efficiency (PE) of 34%, as compared to 26% in serum-containing medium. Two of the melanoma cell lines. MP6 and MeIuso, exhibit linear relationships between colony numbers and cell concentration with PEs of 21% and 70% respectively. Colony formation by the other two melanoma cell lines appears to be nonlinear. This work represents a step toward standardizing culture conditions for human tumor clonogenic cell assays.

  7. Assay development status report for total cyanide

    SciTech Connect

    Simpson, B.C.; Jones, T.E.; Pool, K.H.

    1993-02-01

    A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN{sup {minus}} ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation.

  8. Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

    PubMed

    Lieb, S; Littmann, T; Plank, N; Felixberger, J; Tanaka, M; Schäfer, T; Krief, S; Elz, S; Friedland, K; Bernhardt, G; Wegener, J; Ozawa, T; Buschauer, A

    2016-12-01

    A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

  9. A Quantitative High Throughput Assay for Identifying Gametocytocidal Compounds

    PubMed Central

    Tanaka, Takeshi Q.; Dehdashti, Seameen J.; Nguyen, Dac-Trung; McKew, John C.; Zheng, Wei; Williamson, Kim C.

    2013-01-01

    Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1,536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z′-factor for this assay were 3.2-fold and 0.68, respectively. The IC50 value of epoxomicin, the positive control compound, was 1.42 ± 0.09 nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the alamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1,280 known compounds, revealed two selective gametocytocidal compounds having 54 and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line. PMID:23454872

  10. Sensitive field assays for water analysis

    SciTech Connect

    Douglas, W.L.

    1984-08-01

    The goal of the project is to develop a rapid, simple, and inexpensive dry-film assay device for detection of environmental contaminants using the compound geosmin as a model. Phase I activities centered upon the immunochemical reagents necessary for the assay, development of an enzyme-cycling system that makes possible detection of substances in the parts per billion (PPB) range or lower, and demonstration of how the Immuno-Replacement-Assay can be used to detect geosmin.

  11. Magnetoresistive Sensors in Biological Assays

    NASA Astrophysics Data System (ADS)

    Tondra, Mark

    2010-03-01

    Magnetic beads or nanoparticles can be used as ``labels'' in biochemical assays by attaching the beads to the biospecies of interest using a bio-specific attachment. Once the labels are attached, they can be used to manipulate, capture, and detect the species to be analyzed. Magnetoresistive (MR) sensors may be used to detect and count these labels, and thus make an inference about the concentration of the species of interest. MR technology is especially promising for biosensor applications where making the detector small and integrated with related sample handling tools to form a ``lab-on-a-chip'' miniaturized system. The function of the MR sensors is to detect stray magnetic fields from the beads while they are exposed to a magnetic excitation field. Generally, the stray fields from beads and clusters of beads are complicated functions of geometry, so some care is required to relate the detected magnetic signal to the number and location of the bead labels. This presentation will begin with a broad overview of results from many groups working in this area. For convenience, the applications are divided into three categories, detection of: flowing magnetic beads, immobilized beads, and scanned samples. Next will be some discussion of how the choice of spintronic sensor technology might affect detection capabilities (AMR, GMR, TMR, Hall effect, etc). Then, challenges relating to integration of MR sensors into microfluidic products will be discussed. This is the focus of the presenter's current day-to-day work on developing and producing MR-based biosensors. And finally, a description of possible future avenues of study and development will be presented.

  12. Using the CPTAC Assay Portal to Identify and Implement Highly Characterized Targeted Proteomics Assays.

    PubMed

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, D R; Zimmerman, Lisa J; Meyer, Matthew R; Mesri, Mehdi; Boja, Emily; Carr, Steven A; Chan, Daniel W; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J C; Fenyö, David; Hiltke, Tara; Ketchum, Karen A; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D; Thomas, Stefani; Townsend, R Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

    2016-01-01

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and posttranslational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  13. Open chromatin reveals the functional maize genome

    PubMed Central

    Rodgers-Melnick, Eli; Vera, Daniel L.; Bass, Hank W.

    2016-01-01

    Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome. PMID:27185945

  14. Polycrystalline Silicon: a Biocompatibility Assay

    SciTech Connect

    Pecheva, E.; Fingarova, D.; Pramatarova, L.; Hikov, T.; Laquerriere, P.; Bouthors, Sylvie; Dimova-Malinovska, D.; Montgomery, P.

    2010-01-21

    Polycrystalline silicon (poly-Si) layers were functionalized through the growth of biomimetic hydroxyapatite (HA) on their surface. HA is the mineral component of bones and teeth and thus possesses excellent bioactivity and biocompatibility. MG-63 osteoblast-like cells were cultured on both HA-coated and un-coated poly-Si surfaces for 1, 3, 5 and 7 days and toxicity, proliferation and cell morphology were investigated. The results revealed that the poly-Si layers were bioactive and compatible with the osteoblast-like cells. Nevertheless, the HA coating improved the cell interactions with the poly-Si surfaces based on the cell affinity to the specific chemical composition of the bone-like HA and/or to the higher HA roughness.

  15. Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

    PubMed Central

    Goyette, Jesse; Salas, Citlali Solis; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel A.; Allard, Jun; Dushek, Omer

    2017-01-01

    Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling. PMID:28378014

  16. Diversity and genetic differentiation among subpopulations of Gliricidia sepium revealed by PCR-based assays.

    PubMed

    Dawson, I K; Simons, A J; Waugh, R; Powell, W

    1995-01-01

    Randomly amplified polymorphic DNA (RAPD), and a mitochondrial marker based on amplification of the V7 region of the mitochondrial small ribosomal RNA (srRNA) gene, were used to partition genetic variation within a single population of Gliricidia sepium sampled from Guatemala. Seventeen per cent of the variation detected with RAPDs was partitioned among subpopulations and indicated a greater level of discrimination than previously detected with isozymes. Cluster analysis indicated a direct relationship between this variation and the geographical distance between subpopulations. A polymorphism identified within the maternally inherited mitochondrial V7 srRNA product, which relied on digestion with restriction endonucleases, confirmed the genetic subdivision identified with RAPDs, and suggested a relatively limited role for seed in gene dispersal.

  17. Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1.

    PubMed

    Goyette, Jesse; Salas, Citlali Solis; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel A; Allard, Jun; Dushek, Omer

    2017-03-01

    Tethered enzymatic reactions are ubiquitous in signaling networks but are poorly understood. A previously unreported mathematical analysis is established for tethered signaling reactions in surface plasmon resonance (SPR). Applying the method to the phosphatase SHP-1 interacting with a phosphorylated tether corresponding to an immune receptor cytoplasmic tail provides five biophysical/biochemical constants from a single SPR experiment: two binding rates, two catalytic rates, and a reach parameter. Tether binding increases the activity of SHP-1 by 900-fold through a binding-induced allosteric activation (20-fold) and a more significant increase in local substrate concentration (45-fold). The reach parameter indicates that this local substrate concentration is exquisitely sensitive to receptor clustering. We further show that truncation of the tether leads not only to a lower reach but also to lower binding and catalysis. This work establishes a new framework for studying tethered signaling processes and highlights the tether as a control parameter in clustered receptor signaling.

  18. A Simple Cell-Based Assay Reveals That Diverse Neuropsychiatric Risk Genes Converge on Primary Cilia

    PubMed Central

    Marley, Aaron; von Zastrow, Mark

    2012-01-01

    Human genetic studies are beginning to identify a large number of genes linked to neuropsychiatric disorders. It is increasingly evident that different genes contribute to risk for similar syndromes and, conversely, the same genes or even the same alleles cross over traditional diagnostic categories. A current challenge is to understand the cellular biology of identified risk genes. However, most genes associated with complex neuropsychiatric phenotypes are not related through a known biochemical pathway, and many have an entirely unknown cellular function. One possibility is that diverse disease-linked genes converge at a higher-level cellular structure. The synapse is already known to be one such convergence, and emerging evidence suggests the primary cilium as another. Because many genes associated with neuropsychiatric illness are expressed also outside the nervous system, as are cilia, we tested the hypothesis that such genes affect conserved features of the primary cilium. Using RNA interference to test 41 broadly expressed candidate genes associated with schizophrenia, bipolar affective disorder, autism spectrum disorder and intellectual disability, we found 20 candidates that reduce ciliation in NIH3T3 cells when knocked down, and three whose manipulation increases cilia length. Three of the candidate genes were previously implicated in cilia formation and, altogether, approximately half of the candidates tested produced a ciliary phenotype. Our results support the hypothesis that primary cilia indeed represent a conserved cellular structure at which the effects of diverse neuropsychiatric risk genes converge. More broadly, they suggest a relatively simple cell-based approach that may be useful for exploring the complex biological underpinnings of neuropsychiatric disease. PMID:23056384

  19. A multidimensional blood stimulation assay reveals immune alterations underlying systemic juvenile idiopathic arthritis.

    PubMed

    Cepika, Alma-Martina; Banchereau, Romain; Segura, Elodie; Ohouo, Marina; Cantarel, Brandi; Goller, Kristina; Cantrell, Victoria; Ruchaud, Emily; Gatewood, Elizabeth; Nguyen, Phuong; Gu, Jinghua; Anguiano, Esperanza; Zurawski, Sandra; Baisch, Jeanine M; Punaro, Marilynn; Baldwin, Nicole; Obermoser, Gerlinde; Palucka, Karolina; Banchereau, Jacques; Amigorena, Sebastian; Pascual, Virginia

    2017-09-21

    The etiology of sporadic human chronic inflammatory diseases remains mostly unknown. To fill this gap, we developed a strategy that simultaneously integrates blood leukocyte responses to innate stimuli at the transcriptional, cellular, and secreted protein levels. When applied to systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory disease of unknown etiology, this approach identified gene sets associated with specific cytokine environments and activated leukocyte subsets. During disease remission and off treatment, sJIA patients displayed dysregulated responses to TLR4, TLR8, and TLR7 stimulation. Isolated sJIA monocytes underexpressed the IL-1 inhibitor aryl hydrocarbon receptor (AHR) at baseline and accumulated higher levels of intracellular IL-1β after stimulation. Supporting the demonstration that AHR down-regulation skews monocytes toward macrophage differentiation, sJIA monocytes differentiated in vitro toward macrophages, away from the dendritic cell phenotype. This might contribute to the increased incidence of macrophage activation syndrome in these patients. Integrated analysis of high-dimensional data can thus unravel immune alterations predisposing to complex inflammatory diseases. © 2017 Cepika et al.

  20. Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood.

    PubMed

    Kumar, R; Surendran, P K; Thampuran, N

    2008-02-01

    The study evaluated the efficiency of culture, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella-specific PCR assay was developed for 284 bp Salmonella-specific invA gene amplicon. PCR assay exhibited 31.6% seafood positive for Salmonella followed by ELISA (23.7%) and culture method (21.3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0.385 to 1.0) for different seafood samples. The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.

  1. Molecular Identification, Enzyme Assay, and Metabolic Profiling of Trichoderma spp.

    PubMed

    Bae, Soo-Jung; Park, Young-Hwan; Bae, Hyeun-Jong; Jeon, Junhyun; Bae, Hanhong

    2017-06-28

    The goal of this study was to identify and characterize selected Trichoderma isolates by metabolic profiling and enzyme assay for evaluation of their potential as biocontrol agents against plant pathogens. Trichoderma isolates were obtained from the Rural Development Administration Genebank Information Center (Wanju, Republic of Korea). Eleven Trichoderma isolates were re-identified using ribosomal DNA internal transcribed spacer (ITS) regions. ITS sequence results showed new identification of Trichoderma isolates. In addition, metabolic profiling of the ethyl acetate extracts of the liquid cultures of five Trichoderma isolates that showed the best anti-Phytophthora activities was conducted using gas chromatography-mass spectrometry. Metabolic profiling revealed that Trichoderma isolates shared common metabolites with well-known antifungal activities. Enzyme assays indicated strong cell walldegrading enzyme activities of Trichoderma isolates. Overall, our results indicated that the selected Trichoderma isolates have great potential for use as biocontrol agents against plant pathogens.

  2. Assay-dependent variability of serum insulin concentrations: a comparison of eight assays.

    PubMed

    Tohidi, Maryam; Arbab, Parvaneh; Ghasemi, Asghar

    2017-04-01

    Although insulin measurement is essential for both clinical and research purposes, there is currently no reference method for insulin assays. The aim of this study was to compare results of serum insulin determined by a number of commercially available assays. We compared eight insulin assays by analyzing 165 serum samples. Assays included two chemiluminescence (Roche and DiaSorin), four ELISA (Tosoh, Mercodia, Monobind, and Diametra), and two IRMA (Izotop and BioSource) methods. Each assay was compared with the mean of all assay methods and Bland-Altman difference plots were used to measure agreement between each assay and overall mean. Least squared perpendicular distance regression analysis (Deming's method) was used to calculate slope and intercept for bias and also for each assay vs. mean of eight assays. Findings showed that the lowest and highest median insulin concentrations varied by a factor of 1.8. Maximum and minimum correlations with mean of assays were observed for Roche (0.992) and BioSource (0.844), respectively. Significant bias was observed in six assays. In pairwise comparisons of different assays, the highest and least mean differences were 7.78 μU/mL and -0.14 μU/mL, respectively. In conclusion, serum insulin measurement with different assays showed a maximum of 1.8-fold difference, a point that should be taken into consideration in the interpretation of circulating insulin levels in both clinical and research fields.

  3. Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability.

    PubMed

    Wu, Xiang; Sills, Matthew A; Zhang, Ji-Hu

    2005-09-01

    High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

  4. A lateral electrophoretic flow diagnostic assay.

    PubMed

    Lin, Robert; Skandarajah, Arunan; Gerver, Rachel E; Neira, Hector D; Fletcher, Daniel A; Herr, Amy E

    2015-03-21

    Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings.

  5. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  6. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  7. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  8. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

  9. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  10. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  11. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  12. Statistical inference for serial dilution assay data.

    PubMed

    Lee, M L; Whitmore, G A

    1999-12-01

    Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.

  13. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  14. 21 CFR 866.3210 - Endotoxin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a...

  15. Determining Vitamin D Status: A Comparison between Commercially Available Assays

    PubMed Central

    Snellman, Greta; Melhus, Håkan; Gedeborg, Rolf; Byberg, Liisa; Berglund, Lars; Wernroth, Lisa; Michaëlsson, Karl

    2010-01-01

    Background Vitamin D is not only important for bone health but can also affect the development of several non-bone diseases. The definition of vitamin D insufficiency by serum levels of 25-hydroxyvitamin D depends on the clinical outcome but might also be a consequence of analytical methods used for the definition. Although numerous 25-hydroxyvitamin D assays are available, their comparability is uncertain. We therefore aim to investigate the precision, accuracy and clinical consequences of differences in performance between three common commercially available assays. Methodology/Principal Findings Serum 25-hydroxyvitamin D levels from 204 twins from the Swedish Twin Registry were determined with high-pressure liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), a radioimmunoassay (RIA) and a chemiluminescent immunoassay (CLIA). High inter-assay disagreement was found. Mean 25-hydroxyvitamin D levels were highest for the HPLC-APCI-MS technique (85 nmol/L, 95% CI 81–89), intermediate for RIA (70 nmol/L, 95% CI 66–74) and lowest with CLIA (60 nmol/L, 95% CI 56–64). Using a 50-nmol/L cut-off, 8% of the subjects were insufficient using HPLC-APCI-MS, 22% with RIA and 43% by CLIA. Because of the heritable component of 25-hydroxyvitamin D status, the accuracy of each method could indirectly be assessed by comparison of within-twin pair correlations. The strongest correlation was found for HPLC-APCI-MS (r = 0.7), intermediate for RIA (r = 0.5) and lowest for CLIA (r = 0.4). Regression analyses between the methods revealed a non-uniform variance (p<0.0001) depending on level of 25-hydroxyvitamin D. Conclusions/Significance There are substantial inter-assay differences in performance. The most valid method was HPLC-APCI-MS. Calibration between 25-hydroxyvitamin D assays is intricate. PMID:20644628

  16. Acellular comet assay: a tool for assessing variables influencing the alkaline comet assay.

    PubMed

    Kennedy, Erin K; McNamee, James P; Prud'homme Lalonde, Louise; Jones, Trevor; Wilkinson, Diana

    2012-01-01

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of (60)Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from (60)Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified.

  17. Paper disk assay for glycosaminoglycan sulfotransferases

    SciTech Connect

    Sugahara, K.; Ishii, T.; Yamashina, I.

    1987-11-01

    A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the (/sup 35/S)sulfate-labeled products are precipitated and then separated from a sulfate donor ((/sup 35/S)PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.

  18. Artificial membrane assays to assess permeability.

    PubMed

    Faller, Bernard

    2008-11-01

    This paper reviews the development of artificial membrane assays in the last decade. Reasons why parallel artificial membrane assays (PAMPA) became widely used are discussed and the various PAMPA assays targeting gastro-intestinal absorption, blood brain barrier and skin penetration are presented. Improvements in the assay technology like the introduction of a paracellular component and the critical factors to get quality data are reviewed. The question how does PAMPA compare with Caco-2 monolayer permeability is being addressed. New dimensions in artificial membrane assays like octanol/water logP measurement, influence of excipients on solubility/permeability and binding constants measurements are introduced. Finally, similarity and differences between partition coefficients and permeability values are discussed.

  19. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-06-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  20. Performance Evaluation of the Serum Thyroglobulin Assays With Immunochemiluminometric Assay and Immunoradiometric Assay for Differentiated Thyroid Cancer.

    PubMed

    Cho, Yoon Young; Chun, Sejong; Lee, Soo Youn; Chung, Jae Hoon; Park, Hyung Doo; Kim, Sun Wook

    2016-09-01

    Measurement of postoperative serum thyroglobulin (Tg) is important for detecting persistent or recurrent differentiated thyroid cancer. We evaluated the analytic performance of the DxI 800 assay (Beckman Coulter, USA) for serum Tg and anti-thyroglobulin antibodies (TgAbs) in comparison with that of the GAMMA-10 assay (Shinjin Medics Inc., Korea) for serum Tg and RIA-MAT 280 assay (Stratec, Germany) for TgAb. We prospectively collected blood samples from 99 patients thyroidectomized for thyroid cancer. The functional sensitivity was investigated in standards and human serum. Precision and linearity were evaluated according to the guidelines of the Clinical and Laboratory Standards Institute. The correlation between the two assays was assessed in samples with different Tg ranges. The functional sensitivity of the DxI 800 assay for serum Tg was between 0.0313 and 0.0625 ng/mL. The total CV was 3.9-5.6% for serum Tg and 5.3-6.9% for serum TgAb. The coefficient of determination (R²) was 1.0 and 0.99 for serum Tg and TgAb, respectively. The cut-offs for serum TgAb were 4.0 IU/mL (DxI 800) and 60.0 IU/mL (RIA-MAT 280), and the overall agreement was 68.7%. The correlation between the two assays was excellent; the correlation coefficient was 0.99 and 0.88 for serum Tg and TgAb, respectively. The DxI 800 is a sensitive assay for serum Tg and TgAb, and the results correlated well with those from the immunoradiometric assays (IRMA). This assay has several advantages over the IRMA and could be considered an alternative test for Tg measurement.

  1. US weapons secrets revealed

    SciTech Connect

    Norris, R.S.; Arkin, W.M.

    1993-03-01

    Extraordinary details have only recently been revealed about the struggle over the control of early U.S. nuclear weapons and their initial deployments abroad. The information comes from a newly declassified top secret report, part of a larger study, The History of the Strategic Arms Competition, 1945-1972, commissioned by Defense Secretary James R. Schlisinger in summer 1974.

  2. DNA Methyltransferase Activity Assays: Advances and Challenges.

    PubMed

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice.

  3. DNA Methyltransferase Activity Assays: Advances and Challenges

    PubMed Central

    Poh, Wan Jun; Wee, Cayden Pang Pee; Gao, Zhiqiang

    2016-01-01

    DNA methyltransferases (MTases), a family of enzymes that catalyse the methylation of DNA, have a profound effect on gene regulation. A large body of evidence has indicated that DNA MTase is potentially a predictive biomarker closely associated with genetic disorders and genetic diseases like cancer. Given the attention bestowed onto DNA MTases in molecular biology and medicine, highly sensitive detection of DNA MTase activity is essential in determining gene regulation, epigenetic modification, clinical diagnosis and therapeutics. Conventional techniques such as isotope labelling are effective, but they often require laborious sample preparation, isotope labelling, sophisticated equipment and large amounts of DNA, rendering them unsuitable for uses at point-of-care. Simple, portable, highly sensitive and low-cost assays are urgently needed for DNA MTase activity screening. In most recent technological advances, many alternative DNA MTase activity assays such as fluorescent, electrochemical, colorimetric and chemiluminescent assays have been proposed. In addition, many of them are coupled with nanomaterials and/or enzymes to significantly enhance their sensitivity. Herein we review the progress in the development of DNA MTase activity assays with an emphasis on assay mechanism and performance with some discussion on challenges and perspectives. It is hoped that this article will provide a broad coverage of DNA MTase activity assays and their latest developments and open new perspectives toward the development of DNA MTase activity assays with much improved performance for uses in molecular biology and clinical practice. PMID:26909112

  4. Global haemostasis assays, from bench to bedside.

    PubMed

    van Geffen, Mark; van Heerde, Waander L

    2012-06-01

    Bleeding and thrombosis are the ultimate clinical outcomes of aberrations in the haemostatic process. Haemostasis prevents excessive blood loss due to the effort of various compartments like the vasculature, blood cells, coagulation and fibrinolysis. The complexity of all processes involved makes the diagnosis of aberrations difficult, cumbersome and expensive. A single assay to detect any factor disturbing this haemostatic balance with high sensitivity and specificity would be of great value, especially if the outcome of this assay correlates well with clinical outcome. Despite years of research, such an assay is not yet available; however, some interesting candidates are under development and combine the effects of various compartments. This review describes the development of global haemostasis assays and summarizes the current state of the art of these haemostasis assays covering thrombin and plasmin generation, turbidity and thromboelastography/thromboelastometry. Finally, we discuss the applicability of global assays in clinical practice and we provide a future perspective on the ongoing development of automation and miniaturisation as it is our belief that these developments will benefit the standardization of global haemostasis assays. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Zhang, BinBin; Zheng, Dan; Al-Rubeaan, Khalid; Luong, John H T; Sheu, Fwu-Shan

    2011-10-01

    This study revealed a major interference from sulfo-N-hydroxysuccinimide (sulfo-NHS) in the bicinchoninic acid (BCA) protein assay. Sulfo-NHS, a common reagent used in bioconjugation and analytical biochemistry, exhibited absorbance signals and absorbance peaks at 562 nm, comparable to bovine serum albumin (BSA). However, the combined absorbance of sulfo-NHS and BSA was not strictly additive. The sulfo-NHS interference was suggested to be caused by the reduction of Cu(2+) in the BCA Kit's reagent B (4% cupric sulfate) in a manner similar to that of the protein.

  6. Development of Plaque Assay Systems for Poliovirus.

    DTIC Science & Technology

    1982-04-01

    from as little as 25 mg/mL (1/10th of that used in the actual plaque assay) to 1000 mg/mL, and to inhibit poliovirus cytopathic effect In...1067 DEVELOPMENT OF PLAQUE ASSAY SYSTEMS FOR POLIOVIRUS (U) by R.E. Fulton and K. Munroe Abstract During the summer months of 1978, Ms. Krista Munroe...quantitation of infectious poliovirus type 1. .Two different plaque assay techniques were developed and compared. The results of this work are presented

  7. Assuring reliable performance of antibiotic assay media.

    PubMed

    Freeman, K A; Johnson, D P; Garth, M A

    1977-11-01

    The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.

  8. Nondestructive assay confirmatory assessment experiments: mixed oxide

    SciTech Connect

    Lemming, J.F.

    1980-04-30

    The confirmatory assessment experiments demonstrate traceable nondestructive assay (NDA) measurements of plutonium in mixed oxide powder using commercially available spontaneous-fission assay systems. The experiments illustrate two major concepts: the production of calibration materials using calorimetric assay, and the use of paired measurements for measurement assurance. Two batches of well-characterized mixed oxide powder were used to establish the random and systematic error components. The major components of an NDA measurement assurance technique to establish and maintain traceability are identified and their functions are demonstrated. 20 refs., 10 figs., 10 tabs.

  9. Methods for threshold determination in multiplexed assays

    DOEpatents

    Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

    2014-06-24

    Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.

  10. Comparison of antigen assay and reverse transcriptase assay for detecting human immunodeficiency virus in culture.

    PubMed Central

    Feorino, P; Forrester, B; Schable, C; Warfield, D; Schochetman, G

    1987-01-01

    We compared an antigen capture assay (Abbott Laboratories, North Chicago, Ill.) with a reverse transcriptase assay to identify and quantify human immunodeficiency virus (HIV) in culture. In direct comparisons of serial dilutions of lymphadenopathy-associated virus type 1, the antigen assay was 100-fold more sensitive than the reverse transcriptase assay in detecting the virus. The antigen assay reacted strongly with 60 different HIV isolates but did not cross-react with human T-cell lymphotropic virus type I, human T-cell lymphotropic virus type II, cytomegalovirus, varicella-zoster virus, herpes simplex virus type 1, Epstein-Barr virus, adenovirus type 5, or poliovirus type 1 or with extracts from four different control human cell lines and eight different phytohemagglutinin-stimulated normal human lymphocytes. Peripheral blood lymphocyte samples from 50 individuals were evaluated by both the antigen assay and the reverse transcriptase assay. The cells from the 34 seropositive individuals were all positive by the antigen assay (range, 3 to 9 days; average time, 5.9 days) and the reverse transcriptase assay (range, 7 to 16 days; average time, 9.6 days). Cells from the 16 seronegative individuals were negative by both assays. These results indicate that the antigen assay is an important addition to the monitoring of HIV production in the lymphocytes of infected patients. PMID:2448334

  11. Kit-On-A-Lid-Assays for accessible self-contained cell assays.

    PubMed

    Berthier, Erwin; Guckenberger, David J; Cavnar, Peter; Huttenlocher, Anna; Keller, Nancy P; Beebe, David J

    2013-02-07

    Microscale methods for cell-based assays typically rely on macroscopic reagent handling and fluidic loading protocols that are technically challenging and do not scale with the number of assays favorably. Here, we demonstrate a microfluidic platform technology called "Kit-On-A-Lid-Assay" (KOALA), that enables the creation of self-contained microfluidic cell-based assays, integrating all the steps required to perform cell-based assays. The KOALA platform allows the pre-packaging of reagents, cryopreservation of cell suspensions, thawing of cell suspensions, culture of cells, and operation of whole cell-based assays. The operation of the KOALA platform is user-friendly and consists of bringing together a lid containing the microchannels, and a base containing the pre-packaged reagents, thereby causing fluidic exchange in all the channels simultaneously. We demonstrate that the KOALA cell-based assays can be simply operated from start to finish without any external laboratory equipment.

  12. A single-islet microplate assay to measure mouse and human islet insulin secretion.

    PubMed

    Truchan, Nathan A; Brar, Harpreet K; Gallagher, Shannon J; Neuman, Joshua C; Kimple, Michelle E

    2015-01-01

    One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

  13. Magneto immunofluorescence assay for diagnosis of celiac disease.

    PubMed

    Kergaravat, Silvina V; Beltramino, Luis; Garnero, Nidia; Trotta, Liliana; Wagener, Marta; Fabiano, Silvia N; Pividori, Maria Isabel; Hernandez, Silvia R

    2013-10-10

    A magneto immunofluorescence assay for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The ATG2 were recognized by transglutaminase enzyme immobilized on the magnetic beads and then the immunological reaction was revealed by antibodies labeled with peroxidase. The fluorescent response of the enzymatic reaction with o-phenylenediamine and H2O2 as substrates was correlated with anti-transglutaminase titer, showing EC50 and LOD values of 1:11,600 and 1:74,500 of antibody titers, respectively. A total number of 29 sera samples from clinically confirmed cases of celiac disease and 19 negative control samples were tested by the novel magneto immunofluorescence assay. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 8.1 U was the most effective cut-off value to discriminate correctly between celiac and non-celiac patients. The immunofluorescence assay exhibited a sensitivity of 96.6%, a specificity of 89.5% and an efficiency 93.8% compared with the commercial optical ELISA kit.

  14. A quantitative assay for telomere protection in Saccharomyces cerevisiae.

    PubMed Central

    DuBois, Michelle L; Haimberger, Zara W; McIntosh, Martin W; Gottschling, Daniel E

    2002-01-01

    Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This "chromosomal cap" assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected. PMID:12136006

  15. Recommendations for increasing alkaline comet assay reliability in plants.

    PubMed

    Pourrut, Bertrand; Pinelli, Eric; Celiz Mendiola, Vanessa; Silvestre, Jérôme; Douay, Francis

    2015-01-01

    In plants, an increasing interest for the comet assay was shown in the last decade. This versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, the lack of a standardised protocol and the low throughput of the assay limit its use in plants. The aims of this paper are to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. We examined the effect of varying several parameters on four different plant species: broad bean (Vicia faba), white clover (Trifolium repens), English ryegrass (Lolium perenne) and miscanthus (Miscanthus x giganteus). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated. Results clearly indicate that short chopping is more efficient to isolate nuclei than the standard slicing method. Filtration and lysis steps were shown to be unnecessary and thus should be skipped. Data also demonstrate that high room temperatures and light could induce DNA damage in isolated nuclei. Calibration tests with H2O2 or ethyl methanesulfonate revealed that a special attention should be paid to plant growing stage, leaf position and exposure duration. © The Author 2014. Published by Oxford University Press on behalf of the Mutagenesis Society. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Linearization of the bradford protein assay.

    PubMed

    Ernst, Orna; Zor, Tsaffrir

    2010-04-12

    Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

  17. FACS binding assay for analysing GDNF interactions.

    PubMed

    Quintino, Luís; Baudet, Aurélie; Larsson, Jonas; Lundberg, Cecilia

    2013-08-15

    Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and...

  19. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and...

  20. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and...

  1. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This assay provides diagnostic information for the evaluation of erythrocytosis (increased total red cell mass) and...

  2. BIOMARKER ASSAYS IN NIPPLE APIRATE FLUID

    EPA Science Inventory

    ABSTRACT

    The noninvasive technique of nipple aspiration as a potential source of biomarkers of breast cancer risk was evaluated. The feasibility of performing mutagenesis assays, amplifying DNA and performing protein electrophoresis on nipple aspirate fluid was explored. ...

  3. Developmental Toxicity Assays Using the Drosophila Model

    PubMed Central

    Rand, Matthew D.; Montgomery, Sara L.; Prince, Lisa; Vorojeikina, Daria

    2014-01-01

    The fruit fly (Drosophila melanogaster) has long been a premier model for developmental biologists and geneticists. The utility of Drosophila for toxicology studies has only recently gained broader recognition as a tool to elaborate molecular genetic mechanisms of toxic substances. In this article two practical applications of Drosophila for developmental toxicity assays are described. The first assay takes advantage of newly developed methods to render the fly embryo accessible to small molecules, toxicants and drugs. The second assay engages straightforward exposures to developing larvae and easy to score outcomes of adult development. With the extensive collections of flies that are publicly available and the ease with which to create transgenic flies, these two assays have a unique power for identifying and characterizing molecular mechanisms and cellular pathways specific to the mode of action of a number of toxicants and drugs. PMID:24789363

  4. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  5. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  6. Optical assay for biotechnology and clinical diagnosis.

    PubMed

    Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

    2011-08-01

    In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

  7. Electrochemical Assay of Gold-Plating Solutions

    NASA Technical Reports Server (NTRS)

    Chiodo, R.

    1982-01-01

    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  8. Displacement Assay on a Porous Membrane.

    DTIC Science & Technology

    1996-01-11

    Inventor’s Name: Ligler et al. 1 2 3 DISPLACEMENT ASSAY ON A POROUS MEMBRANE 4 5 Background of the Invention 6 1. Field of the Invention 7 The...immobilization of the antibody and labelled antigen on a porous membrane would not provide a suitable matrix for the displacement assay since this...14 may be a hollow fiber configured so the sample flows along the 15 hollow center before passing through the membrane. In any 16 embodiment of

  9. Analytical characterization of the APTIMA HPV Assay.

    PubMed

    Dockter, Janel; Schroder, Astrid; Eaton, Barbara; Wang, Ann; Sikhamsay, Nathan; Morales, Liezel; Giachetti, Cristina

    2009-07-01

    Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. To determine the analytical performance characteristics of the APTIMA HPV Assay. Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems. The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or low-risk HPV types. Spermicides, anti-fungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-lot, and inter-run signal variability were <10% for >99% of the data. Intra-run variability was <15%, except for those samples with concentrations at or below the 95% detection limit of the assay. Based upon the analytical sensitivity, analytical specificity, and low variability, the APTIMA HPV Assay showed excellent performance and robustness.

  10. Electrochemical Assay of Gold-Plating Solutions

    NASA Technical Reports Server (NTRS)

    Chiodo, R.

    1982-01-01

    Gold content of plating solution is assayed by simple method that required only ordinary electrochemical laboratory equipment and materials. Technique involves electrodeposition of gold from solution onto electrode, the weight gain of which is measured. Suitable fast assay methods are economically and practically necessary in electronics and decorative-plating industries. If gold content in plating bath is too low, poor plating may result, with consequent economic loss to user.

  11. Radioimmune assay of human platelet prostaglandin synthetase

    SciTech Connect

    Roth, G.J.; Machuga, E.T.

    1982-02-01

    Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH/sub 2/ from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and (/sup 125/I)-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the (/sup 125/I)antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10/sup 9/ platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency.

  12. Protein immobilization techniques for microfluidic assays

    PubMed Central

    Kim, Dohyun; Herr, Amy E.

    2013-01-01

    Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization. PMID:24003344

  13. In vitro cell migration and invasion assays.

    PubMed

    Kramer, Nina; Walzl, Angelika; Unger, Christine; Rosner, Margit; Krupitza, Georg; Hengstschläger, Markus; Dolznig, Helmut

    2013-01-01

    Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.

  14. Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

    PubMed

    Prince, J A; Feuk, L; Howell, W M; Jobs, M; Emahazion, T; Blennow, K; Brookes, A J

    2001-01-01

    We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

  15. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  16. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout.

    PubMed

    Gläsker, Sabine; Lulla, Aleksei; Lulla, Valeria; Couderc, Therese; Drexler, Jan Felix; Liljeström, Peter; Lecuit, Marc; Drosten, Christian; Merits, Andres; Kümmerer, Beate Mareike

    2013-07-15

    Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. A VRP based CHIKV neutralization assay using Gluc as readout represents a fast and useful method to

  17. Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays *

    PubMed Central

    Karakosta, Theano D.; Soosaipillai, Antoninus; Batruch, Ihor

    2016-01-01

    Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples

  18. Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

    PubMed

    Maeda, Akihisa; Takahashi, Kei; Tsuchiyama, Hiromi; Oshida, Keiyu

    2016-11-15

    The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Evaluation of the reliability of maize reference assays for GMO quantification.

    PubMed

    Papazova, Nina; Zhang, David; Gruden, Kristina; Vojvoda, Jana; Yang, Litao; Buh Gasparic, Meti; Blejec, Andrej; Fouilloux, Stephane; De Loose, Marc; Taverniers, Isabel

    2010-03-01

    A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb

  20. Revealing power in truth

    PubMed Central

    Lee, Kelley

    2015-01-01

    Jeremy Shiffman’s editorial appropriately calls on making all forms of power more apparent and accountable, notably productive power derived from expertise and claims to moral authority. This commentary argues that relationships based on productive power can be especially difficult to reveal in global health policy because of embedded notions about the nature of power and politics. Yet, it is essential to recognize that global health is shot through with power relationships, that they can take many forms, and that their explicit acknowledgement should be part of, rather than factored out of, any reform of global health governance. PMID:25844390

  1. The Universe Revealed

    NASA Astrophysics Data System (ADS)

    Spence, Pam

    1998-10-01

    The Universe is a bewildering place to the uninitiated. The concepts and theories that govern space seem complex and often contradictory. The Universe Revealed provides the keys to unlocking the wonders of the cosmos. Elegantly written and lavishly illustrated, it begins with the Sun and stretches through our solar system into deepest space. Lucid prose, written by many of the people who have shaped our current thinking on space, and spectacular photographs make the physics of the Universe accessible and provide a solid background for understanding the most recent astronomical discoveries. Covering the most intriguing features of the cosmos, the topics discussed range from the Earth and global warming to cosmic collisions and the size of the Universe. Major sections examine the Solar System, stars, galaxies, cosmology, and the observational techniques used by astronomers, both amateur and professional. The Universe Revealed represents the collaboration of internationally renowned experts in astronomy and cosmology, with contributions from authors including David Malin, F. Duccio Macchetto, Iain Nicholson, Neil Bone, Ian Ridpath, Seth Shostak, Mike Lancaster, Steve Miller, Ken Croswell, Geoff McNamara, and Steven Young. This extraordinary blend of astronomy, astrophysics, and cosmology, will appeal to amateur and armchair astronomers alike.

  2. Mysterious Blob Galaxies Revealed

    NASA Image and Video Library

    2005-01-11

    This image composite shows a giant galactic blob (red) and the three merging galaxies NASA's Spitzer Space Telescope discovered within it (yellow). Blobs are intensely glowing clouds of hot hydrogen gas that envelop faraway galaxies. They are about 10 times as large as the galaxies they surround. Visible-light images reveal the vast extent of blobs, but don't provide much information about their host galaxies. Using its heat-seeking infrared eyes, Spitzer was able to see the dusty galaxies tucked inside one well-known blob located 11 billion light-years away. The findings reveal three monstrously bright galaxies, trillions of times brighter than the Sun, in the process of merging together. Spitzer also observed three other blobs located in the same cosmic neighborhood, all of which were found to be glaringly bright. One of these blobs is also known to be a galactic merger, only between two galaxies instead of three. It remains to be seen whether the final two blobs studied also contain mergers. The Spitzer data were acquired by its multiband imaging photometer. The visible-light image was taken by the Blanco Telescope at the Cerro Tololo Inter-American Observatory, Chile. http://photojournal.jpl.nasa.gov/catalog/PIA07220

  3. Controlling variation in the comet assay

    PubMed Central

    Collins, Andrew R.; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation. PMID:25368630

  4. Cell Culture Assay for Human Noroviruses [response

    SciTech Connect

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  5. The root causes of pharmacodynamic assay failure.

    PubMed

    Ferry-Galow, Katherine V; Makhlouf, Hala R; Wilsker, Deborah F; Lawrence, Scott M; Pfister, Thomas D; Marrero, Allison M; Bigelow, Kristina M; Yutzy, William H; Ji, Jiuping J; Butcher, Donna O; Gouker, Brad A; Kummar, Shivaani; Chen, Alice P; Kinders, Robert J; Parchment, Ralph E; Doroshow, James H

    2016-08-01

    Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents. Copyright © 2016. Published by Elsevier Inc.

  6. The EpiDerm™ 3D human reconstructed skin micronucleus (RSMN) assay: Historical control data and proof of principle studies for mechanistic assay adaptations.

    PubMed

    Roy, Shambhu; Kulkarni, Rohan; Hewitt, Nicola J; Aardema, Marilyn J

    2016-07-01

    The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm™ is a promising novel animal alternative for evaluating genotoxicity of topically applied chemicals. It is particularly useful for assessing cosmetic ingredients that can no longer be tested using in vivo assays. To advance the use of this test especially for regulatory decision-making, we have established the RSMN assay in our laboratory according to Good Laboratory Practice and following the principles of the OECD test guideline 487 in vitro mammalian cell micronucleus test. Proficiency with the assay was established by correctly identifying direct-acting genotoxins and genotoxins requiring metabolism, as well as non-genotoxic/non-carcinogenic chemicals. We also report the analysis of our historical control data that demonstrate vehicle control and positive control values for %micronuclei in binucleated cells are in the ranges reported previously. Technical issues including evaluating various solvents with both 48h and 72h treatment regimens were investigated. For the first time, mechanistic studies using CREST analysis revealed that the RSMN assay is suitable for distinguishing aneugens and clastogens. Moreover, the assay is also suitable for measuring cytokines as markers for proliferative and toxic effects of chemicals.

  7. Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays

    PubMed Central

    Sozhamannan, Shanmuga; Holland, Mitchell Y.; Hall, Adrienne T.; Negrón, Daniel A.; Ivancich, Mychal; Koehler, Jeffrey W.; Minogue, Timothy D.; Campbell, Catherine E.; Berger, Walter J.; Christopher, George W.; Goodwin, Bruce G.; Smith, Michael A.

    2015-01-01

    Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes. PMID:26090727

  8. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    SciTech Connect

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  9. Measurement of dabigatran in standardly used clinical assays, whole blood viscoelastic coagulation, and thrombin generation assays.

    PubMed

    van Ryn, Joanne; Grottke, Oliver; Spronk, Henri

    2014-09-01

    Dabigatran, a direct thrombin inhibitor, is increasingly used clinically as one of the new oral anticoagulants. This review summarizes the assays available to measure its activity and includes the relative sensitivity of the different assays for this agent. In addition to plasma-based clotting tests, assays commonly used in surgical/emergency settings, such as activated clotting time and thromboelastometry/thromboelastography, are reviewed. In addition, the thrombin generation assay is discussed as an important method to determine the potential risk of thrombosis or bleeding and its relevance to the measurement of direct thrombin inhibitors. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Application of the Attagene FACTORIAL™ assay to ...

    EPA Pesticide Factsheets

    Bioassays can be used to evaluate the integrated effects of complex mixtures from both known and unidentified contaminants present in environmental samples. However, such bio-monitoring approaches have typically focused only on one or a few pathways (e.g. estrogen receptor, androgen receptor) despite the fact that the chemicals in a mixture may exhibit a range of biological activities. High-throughput screening approaches that can rapidly assess samples for a broad diversity of biological activities offer a means to provide a more comprehensive characterization of complex mixtures. The Attagene FactorialTM platform is a high-throughput, cell based assay utilized by US EPA’s ToxCast Program, which provides high-content assessment of over 90 different gene regulatory pathways and all 48 human nuclear receptors (NRs). This assay has previously been used in a preliminary screening of surface water extracts from sites across the Great Lakes. In the current study, surface waters samples from 38 sites were collected, extracted, and screened through the Factorial assay as part of a USGS nationwide stream assessment. All samples were evaluated in a six point, 3-fold dilution series and analyzed using the ToxCast Data Pipeline (TCPL) to generate dose-response curves and corresponding half-maximal activity concentration (AC50) estimates. A total of 27 assay endpoints responded to extracts from one or more sites, with up to 14 assays active for a single extract. The four

  11. Gusev's Rim Revealed

    NASA Technical Reports Server (NTRS)

    2004-01-01

    NASA's Mars Exploration Rover Spirit took this panoramic camera image on sol 91 (April 5, 2004). Spirit is looking to the southeast, and through the martian haze has captured the rim of Gusev Crater approximately 80 kilometers (49.7 miles) away on the horizon.

    The right side of this image reveals the portion of the crater edge that descends into the mouth of Ma'adim Vallis, a channel that opens into Gusev Crater. Spirit is currently traveling toward the informally named 'Columbia Hills,' which lie to the left of the region pictured here.

    This image is similar to a panoramic camera image taken on sol 68, but Gusev's ridge is more visible here because the atmospheric dust caused by winter dust storms has settled. Scientists expect to get even clearer images than this one in upcoming sols.

    This image has been modified to make the crater rim more visible.

  12. Ancient River revealed

    NASA Astrophysics Data System (ADS)

    Recent flights of the Spaceborne Imaging Radar-C/X-band Synthetic Aperture Radar (SIR-C/X-SAR) mission aboard the space shuttle Endeavour discovered a previously unknown branch of an ancient river. The images, released at AGU's Spring Meeting, show the river channel buried under thousands of years worth of windblown sand in a region of North Africa's Sahara Desert near the Kufra Oasis in southeast Libya, centered at 23.3°N latitude, 22.9°E longitude. The image from the flight last October reveals a system of old, now inactive stream valleys, called “paleodrainage systems,” which carried running water northward across the Sahara during periods of wetter climate.

  13. The Climate Revealed

    NASA Astrophysics Data System (ADS)

    Burroughs, William

    1999-10-01

    El Niño, La Niña, global warming--terms that crop up frequently in current media coverage of anomalous weather conditions: a spring thaw in January in New York City...a snowstorm in Bakersfield, California...winterlike temperatures in Miami. Such phenomena as these and reports of devastating droughts, floods, and storms around the world bring home the fact of how deeply climate affects our daily lives--and of our inability to control the consequences of climatic events. Extraordinarily timely, The Climate Revealed explores the human-climate "relationship" in all its fascinating complexity. Packed with 250 beautiful, full-color photographs, the volume travels the globe to provide a detailed portrait of individual climate zones from the polar icecaps to the fiercest deserts. The expert and highly accessible text uncovers the essential elements--earth, air, fire and water--that make up the world's various climates. William Burroughs reveals the dramatic discoveries and techniques of historians and archaeologists in their search to understand climates of the past. In the book's conclusion he considers the future and presents every facet of the current environmental debate. With its detailed coverage of the past, present, and future, this marvelous work is essential reading for all those who want to understand one of the most critical facets of life, climate. William Burroughs is a well known and successful science author who has written four books on the weather including Does the Weather Really Matter? (1997), Weather Cycles: Real or Imaginary (1992), and Watching the World's Weather (1991), all published by Cambridge University Press.

  14. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

    PubMed

    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

  15. Genomic fingerprinting of "Haemophilus somnus" isolates by using a random-amplified polymorphic DNA assay.

    PubMed Central

    Myers, L E; Silva, S V; Procunier, J D; Little, P B

    1993-01-01

    The random-amplified polymorphic DNA (RAPD) assay was used to generate DNA fingerprints for 16 isolates of "Haemophilus somnus," and one isolate each of "Haemophilus agni," "Histophilus ovis," "Actinobacillus seminis," Pasteurella haemolytica, and Escherichia coli. The RAPD assay differentiated among "H. somnus" isolates, which shared similarity coefficients of 0.46 to 1.00 on the basis of pairwise comparisons of RAPD markers produced with nine random decamer primers. Three virulent encephalitic "H. somnus" isolates exhibited identical banding patterns, suggesting a common clonal ancestry. The RAPD assay clearly distinguished between the "H. somnus"-"H. agni"-"H. ovis" group and the other bacterial species tested. The results of the present study suggest that DNA fingerprinting of "H. somnus" isolates by the RAPD assay could be valuable in revealing subspecific divisions within this largely unexplored species. Images PMID:8458944

  16. Design and Construction of a Two-Temperature Preference Behavioral Assay for Undergraduate Neuroscience Laboratories

    PubMed Central

    Daniels, Richard L.; McKemy, David D.

    2010-01-01

    Behavioral assays in the undergraduate neuroscience laboratory are useful for illustrating a variety of physiological concepts. An example is homeostatic temperature regulation (thermoregulation). Many model organisms, from flies to mice, regulate internal temperatures in part by moving to suitable climates (thermotaxis). A particularly reliable method of quantifying temperature-dependent thermotactic behaviors is the two-temperature preference behavioral assay. In this preparation, an organism is free to move between two temperature-controlled surfaces, thus revealing its preferred thermal environment. Here we present the design and construction of a two-temperature preference assay chamber. The device uses Peltier-based thermoelectric modules (TECs) for heating and cooling, and is capable of precision control of temperatures from −5ºC to 60ºC. Our approach can be easily adapted for use in a variety of physiological and behavioral assays that require precise temperature control over a wide range of temperatures. PMID:23494724

  17. Ultramicro forward-mutation assay and it's application to the survey of indoor air pollution

    SciTech Connect

    Takagi, Y.; Goto, S.; Kuo, C.T.; Sugita, S.; Murata, M.

    1988-01-01

    A highly sensitive ultramicro forward-mutation assay using Salmonella typhimurium TM677 was achieved by the introduction of a micro-vessel in the preincubation. The assay was about 10 times higher in sensitivity than the micro forward-mutation assay and was able to measure mutagenicity of extracts from airborne particulates obtained by only 3 cu m air sampling. Repeatability of the assay was nearly same as that of the micro forward-mutation assay, that is, the coefficient of variation of the mutation frequency for airborne particular extracts was 12.3% in the test condition without S9 mix and 13.7% with S9 mix. The assay was applied to the measurement of hourly variation mutagenic activity of airborne particulates indoors and outdoors, and revealed the mutagenic activity of indoor air was generally higher than that of outdoor air and that the mutagenic activity indoors in the absence of S9 mix was well correlated with human activity. It was also found by the assay and PAH analysis that indoor pollution by carcinogens and mutagens was largely affected by cigarette smoking and an air cleaner was useful for reduction of indoor pollution by mutagens and PAHs.

  18. SPR-based assays enable the full functional analysis of bispecific molecules.

    PubMed

    Meschendoerfer, W; Gassner, C; Lipsmeier, F; Regula, J T; Moelleken, J

    2017-01-05

    The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins raises new challenges for functional characterization. When compared to standard antibodies, two individual interactions and the inter-dependency of binding events need to be considered for bispecific antibodies. We have previously described an SPR-based assay setup, which enables us to assess the binding activity of a bivalent-bispecific molecule to both targets simultaneously and - in addition to one individual target - in a single setup. However, there might be some pitfalls when applying the bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an alternative SPR-based assay principle, which allows the individual assessment of both targets in solution. Comparison of data between the assays showed that simultaneous binding can be calculated based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay principles allow a "full" functional analysis of a bispecific CrossMab in only one assay. The assay principles can be qualified and enable an efficient drug development.

  19. Simultaneous multi-analyte urinary protein assay for bladder cancer detection

    PubMed Central

    2014-01-01

    Background The ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of a myriad of clinical assays. Here, we tested the performance of a new, multiplex protein array platform to quantitate three bladder cancer-associated proteins in urine samples. The following analytes, interleukin 8 (IL8), matrix metallopeptidase 9 (MMP9), and vascular endothelial growth factor A (VEGFA) were monitored using Q-plex, a customized multiplex ELISA system from Quansys Biosciences, and individual target commercial ELISA kits. The performance of the two approaches was compared by evaluating the diagnostic accuracy of the biomarker assays in samples from a cohort of 73 subjects of known bladder cancer status. Results The combination biomarker panel analyses revealed an AUROC value of 0.9476 for the Q-plex assay, and 0.9119 for the combination of the single-target ELISA assays. The Q-plex assay achieved an overall diagnostic sensitivity of 0.93 and specificity of 0.81, and the individual target ELISA assays achieved an overall sensitivity of 0.77 and specificity of 0.91. Conclusion Based on these encouraging preliminary data, we believe that the Q-Plex technology is a viable platform that can be exploited as an efficient, highly accurate tool to quantitate multiplex panels of diagnostic proteins in biologic specimens. PMID:24684904

  20. Analytical and clinical performance of a new molecular assay for Epstein-Barr virus DNA quantitation.

    PubMed

    Hübner, Margit; Bozic, Michael; Konrad, Petra M; Grohs, Katharina; Santner, Brigitte I; Kessler, Harald H

    2015-02-01

    Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable.

  1. Unveiling new biological relationships using shared hits of chemical screening assay pairs

    PubMed Central

    Liu, Xueping; Campillos, Monica

    2014-01-01

    Motivation: Although the integration and analysis of the activity of small molecules across multiple chemical screens is a common approach to determine the specificity and toxicity of hits, the suitability of these approaches to reveal novel biological information is less explored. Here, we test the hypothesis that assays sharing selective hits are biologically related. Results: We annotated the biological activities (i.e. biological processes or molecular activities) measured in assays and constructed chemical hit profiles with sets of compounds differing on their selectivity level for 1640 assays of ChemBank repository. We compared the similarity of chemical hit profiles of pairs of assays with their biological relationships and observed that assay pairs sharing non-promiscuous chemical hits tend to be biologically related. A detailed analysis of a network containing assay pairs with the highest hit similarity confirmed biological meaningful relationships. Furthermore, the biological roles of predicted molecular targets of the shared hits reinforced the biological associations between assay pairs. Contact: monica.campillos@helmholtz-muenchen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25161250

  2. Assay Portal | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

  3. Assay Characterization Guidance Documents | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    CPTAC characterized assays are defined as those that meet the criteria described in the Assay Characterization Guidance Document. This guidance document aligns with recommendations by the research community as “fit-for-purpose” validation requirements of targeted proteomics assays.

  4. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian J.; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-29

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  5. Bacillus spore inactivation methods affect detection assays.

    PubMed

    Dang, J L; Heroux, K; Kearney, J; Arasteh, A; Gostomski, M; Emanuel, P A

    2001-08-01

    Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.

  6. Enzyme immunometric assay for leukotriene C4.

    PubMed

    Volland, H; Vulliez Le Normand, B; Mamas, S; Grassi, J; Créminon, C; Ezan, E; Pradelles, P

    1994-09-30

    An enzyme immunometric assay of LTC4 named SPIE-IA is described. The assay involves different sequential steps: (1) immunocapture of LTC4 by monoclonal anti-LTC4 antibodies coated on 96-well microtiter plates; (2) cross-linking of LTC4 via its amino group to the wells using glutaraldehyde; (3) treatment with HCl; (4) measurement of linked LTC4 using the same monoclonal anti-LTC4 antibodies labeled with acetylcholinesterase. A minimal detectable concentration of 2 pg/ml after 60 min of enzymatic reaction was obtained. Cross-reactivity was less than 15% with LTD4 or LTE4. The coefficient of variation was less than 6% in the 20-1000 pg/ml range. Good correlation was observed between SPIE-IA and a competitive enzyme immunoassay for biological samples. The different sequential steps of the assay are investigated.

  7. A high-throughput radiometric kinase assay

    PubMed Central

    Duong-Ly, Krisna C.; Peterson, Jeffrey R.

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  8. Fungicide resistance assays for fungal plant pathogens.

    PubMed

    Secor, Gary A; Rivera, Viviana V

    2012-01-01

    Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol demethylation inhibitor; DMI) fungicides, and inhibition of spore germination for quinone outside inhibitor (QoI) fungicides.

  9. Robot speeds assays and enhances safeguards

    SciTech Connect

    Phelan, P.F.; Powell, W.D.; Blankenship, R.W.

    1990-01-01

    At the Los Alamos National Laboratory Plutonium Facility, a robotics system utilizing a gantry robot and an automated inventory system operates five calorimeters and two gamma isotopic assay instruments. This system has significantly improved safeguards, because the opportunity for diversion has been greatly reduced. Not only is the accountability much more timely because throughput has doubled but the special nuclear material has been made physically more secure in several ways. First, items awaiting assay are kept in the inventory system, whose doors remain locked whenever the robot is unattended. An alarm sounds if the doors are unlocked without authorization. Second, light curtains surround the robot's work envelope and pressure-sensitive pads cover the floor to detect entry into the assay area. Third, the robot weighs each item whenever it is moved, and the result is compared with the weight that was measured when the item was first put into inventory. 2 refs., 3 figs.

  10. [Radioenzymatic assay for catecholamines (author's transl)].

    PubMed

    Oshima, T; Maruyama, Y

    1978-11-01

    Catecholamines are neurotransmitters produced and secreted by the central and autonomic nervous systems. In addition to being neurotransmitters, amines produced mainly by the adrenal medulla also act as hormones. Fluorometric measurements of amines yield variable results because of the poor sensitivity of the techniques, and the low concentration of these amines in tissues and biological fluids. The lack of specific and sensitive analytical methods has been an obstacle to resolving the mechanism of action of these neurotransmitters and hormones. The possibility of achieving qualitative and quantitative determination of picomole or femtomole amounts of these amines is a major need. Recently, radioenzymatic procedures for catecholamine assay have been developed and there has been a significant improvement in both sensitivity and accuracy of catecholamine assays. In this article, details of these radioenzymatic assay methods are reviewed.

  11. EDTA interference in electrochemiluminescence ACTH assay.

    PubMed

    Toprak, Burak; Yalcin, Hulya; Arı, Elif; Colak, Ayfer

    2016-11-01

    Background As plasma is the recommended sample type for Roche adrenocorticotropic hormone (ACTH) assay, we evaluated the effect of EDTA concentration on Cobas ACTH assay. Methods Samples containing twofold and fourfold higher concentrations of EDTA were prepared by adding plasma to empty K2EDTA tubes and by making under-filled EDTA tubes. All measurements were performed with four replicates. Results Increased EDTA concentration resulted in a significant decrease in ACTH concentration. Fifty-per cent-filled EDTA tube showed 19% decrease in ACTH concentration and 25% filled EDTA tube showed 50% decrease in ACTH concentration. Conclusion We recommend that inadequately filled EDTA specimens should be rejected when using Cobas ACTH assay.

  12. Angiogenesis assays using chick chorioallantoic membrane.

    PubMed

    West, D C; Thompson, W D; Sells, P G; Burbridge, M F

    2001-01-01

    The study of the angiogenic process and the search for novel therapeutic agents to inhibit, or stimulate, angiogenesis has employed a wide range of in vivo 'angiogenesis' assays (reviewed in 1-3). These differ greatly in their difficulty, quantitative nature, rapidity, and cost. The classical in vivo models include the rabbit ear chamber, hamster cheek pouch, dorsal skin chamber, dorsal skin and air-sac model, anterior chamber/iris and avascular corneal pocket assay, and the chick embryo chorioallantoic membrane (CAM) assay. More recent methods involve the implantation of preloaded Matrigel or alginate plugs, or collagen or poly vinyl sponges (1). Largely owing to its simplicity and low cost, the CAM is the most widely used in vivo model for the study of both angiogenesis and antiangiogenesis (1,4).

  13. Bacillus Spore Inactivation Methods Affect Detection Assays

    PubMed Central

    Dang, Jessica L.; Heroux, Karen; Kearney, John; Arasteh, Ameneh; Gostomski, Mark; Emanuel, Peter A.

    2001-01-01

    Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid- and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment. PMID:11472945

  14. Development of an objective comedogenicity assay.

    PubMed

    Tucker, S B; Flannigan, S A; Dunbar, M; Drotman, R B

    1986-06-01

    The rabbit ear comedogenicity assay is useful as a screening procedure for evaluating agents that come in contact with human skin. Controversy exists regarding the reliability of this assay because of differences in results from various laboratories. The subjective nature of the standard method of grading may also contribute to this variation. We use a more objective comedogenicity assay that utilizes increasing follicular orifice size on the rabbit ear as a measure of comedogenic activity. A generally linear increase in the degree of follicular orifice area was noted with several agents evaluated over a four-week application period. Further, a noninvasive Silastic elastomer mold was used to allow measurement of the same follicular orifice areas over time.

  15. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  16. Nuclear Resonance Fluorescence for Materials Assay

    SciTech Connect

    Quiter, Brian; Ludewigt, Bernhard; Mozin, Vladimir; Prussin, Stanley

    2009-06-05

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has ben performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX's photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  17. Flow cytometer measurement of binding assays

    DOEpatents

    Saunders, George C.

    1987-01-01

    A method of measuring the result of a binding assay that does not require separation of fluorescent smaller particles is disclosed. In a competitive binding assay the smaller fluorescent particles coated with antigen compete with antigen in the sample being analyzed for available binding sites on larger particles. In a sandwich assay, the smaller, fluorescent spheres coated with antibody attach themselves to molecules containing antigen that are attached to larger spheres coated with the same antibody. The separation of unattached, fluorescent smaller particles is made unnecessary by only counting the fluorescent events triggered by the laser of a flow cytometer when the event is caused by a particle with a light scatter measurement within a certain range corresponding to the presence of larger particles.

  18. Paraspinal abscess secondary to tuberculous spondylitis diagnosed by Xpert MTB/RIF assay in rural Tanzania.

    PubMed

    Hella, Jerry Joseph; Hiza, Hellen Charles; Mfinanga, Elirehema; Reither, Klaus

    2013-04-10

    A 31-year-old HIV-negative man presented to our clinic with a 6-month history of back pain and a swelling at the back. Radiological studies revealed lumbar vertebral destruction. Ultrasound of the mass showed a septated cystic mass with turbid fluid. Diagnostic aspiration revealed thick pus and smear microscopy detected acid-fast bacilli. Xpert MTB/RIF assay detected Mycobacterium tuberculosis with no rifampicin resistance.

  19. Bicinchoninic acid (BCA) assay in low volume.

    PubMed

    Bainor, Anthony; Chang, Lyra; McQuade, Thomas J; Webb, Brian; Gestwicki, Jason E

    2011-03-15

    The BCA assay is a colorimetric method for estimating protein concentration. In 96-well plates, the relationship between protein content and absorbance is nearly linear over a wide range; however, performance is reduced in lower volume. To overcome this limitation, we performed the BCA assays in opaque, white 384-well plates. These plates emit fluorescence between 450-600 nm when excited at 430 nm; thus, their fluorescence is quenched by the BCA chromophore (λ(max) 562 nm). This arrangement allowed accurate determination of protein content using only 2 μL of sample. Moreover, soluble flourescein could replace the white plates, creating a homogenous format.

  20. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  1. Continuous Fluorescence Assay for Peptidoglycan Glycosyltransferases.

    PubMed

    Egan, Alexander J F; Vollmer, Waldemar

    2016-01-01

    Bacterial cell wall peptidoglycan is synthesized from its precursor lipid II by two enzymatic reactions. First, glycosyltransferases polymerize the glycan strands and second, DD-transpeptidases form cross-links between peptides of neighboring strands. Most bacteria possess bifunctional peptidoglycan synthesis enzymes capable of catalyzing both reactions. Here, we describe a continuous fluorescence glycosyltransferase assay using Dansyl-labeled lipid II as substrate. Progression of the reaction is monitored by the reduction in fluorescence over time. The assay is suitable to investigate the effect of protein interaction partners on the glycan strand synthesis activity of peptidoglycan polymerases.

  2. The comet assay: a heavenly method!

    PubMed

    Collins, Andrew R

    2015-01-01

    The contributions to this special issue of Mutagenesis have been selected to cover the main research areas served by the comet assay, namely genotoxicology, environmental toxicology, human biomonitoring and fundamental investigations into mechanisms of DNA damage and repair. Innovative methods are described, technical issues are explored, and guidelines are given for venturing into relatively new or unexploited areas of research. The popularity of the comet assay in a historical context is illustrated by a bibliometric survey. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Sensitive radioenzymatic assay for catechol drugs

    SciTech Connect

    Durrett, L.R.; Ziegler, M.G.

    1980-01-01

    This assay measures picogram quantities of catechol drugs and endogenous catecholamines in body tissues and fluids. The catechols are converted to their 3H-O-methyl metabolites during incubation with 3H-S-adenosylmethionine then separated by solvent extraction and thin-layer chromatography. Most drugs containing the catechol structure can be radiolabeled and separated from norepinephrine and epinephrine by this technique to provide simultaneous measurement of endogenous and exogenously administered catechols. The disposition of isoproterenol in tissues and fluids of man and experimental animals is measured to illustrate the utility of this assay. The reactivity of several commonly administered catechol drugs with COMT is described and the possible implications discussed.

  4. Neutron Assay System for Confinement Vessel Disposition

    SciTech Connect

    Frame, Katherine C; Bourne, Mark M; Crooks, William J; Evans, Louise; Mayo, Douglas R; Miko, David K; Salazar, William R; Stange, Sy; Valdez, Jose I; Vigil, Georgiana M

    2012-07-13

    Waste will be removed from confinement vessels remaining from 1970s-era experiments. Los Alamos has 9+ spherical confinement vessels remaining from experiments. Each vessel contains {approx} 500 lbs of radioactive debris such as actinide metals and oxides, metals, powdered silica, graphite, and wires and hardware. In order to dispose of the vessels, debris and contamination must be removed. Neutron assay system was designed to assay vessels before and after cleanout. System requirements are: (1) Modular and moveable; (2) Capable of detecting {approx}100g {sup 239}Pu equivalent in a 2-inch thick steel sphere with 6 foot diameter; and (3) Capable of safeguards-quality assays. Initial design parameters arethe use of 4-atm {sup 3}He tubes with length of 6 feet, and {sup 3}He tubes embedded in polyethelene for moderation. This paper describes the calibration of the Confinement Vessel Assay System (CVAS) and quantification of its uncertainties. Assay uncertainty depends on five factors: (1) Statistical uncertainty in the assay measurement; (2) Statistical uncertainty in the background measurement; (3) Statistical uncertainty in the isotopics determination - This should be much smaller than the other uncertainties; (4) Systematic uncertainty due to position bias; and (5) Systematic uncertainty due to fluctuations in cosmic ray spallation. This one can be virtually eliminated by performing the background measurement with an empty vessel - but that may not be possible. We used modeling and experiments to quantify the systematic uncertainties. The calibration assumes a uniform distribution of material, but reality will be different. MCNPX modeling was used to quantify the positional bias. The model was benchmarked to build confidence in its results. Material at top of vessel is 44% greater than amount assayed, according to singles. Material near 19-tube detector is 38% less than amount assayed, according to singles. Cosmic ray spallation contributes significantly to the

  5. Instructions for Uploading Data to the Assay Portal - Instructions for Uploading Data to the Assay Portal

    Cancer.gov

    This document provides instructions for configuring and uploading data files to the CPTAC Assay Portal. It is divided into sections, with an overview checklist provided at the end. If help is needed at any stage of the process, please use the support page: https://assays.cancer.gov/support/

  6. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform.

    PubMed

    Filomena, Angela; Guenther, Anna; Planatscher, Hannes; Topin, Francois; She, Joseph; Formichella, Luca; Terradot, Laurent; Gerhard, Markus; Joos, Thomas O; Meyer, Hannelore; Schneiderhan-Marra, Nicole

    2017-10-03

    Infection with Helicobacter pylori (H. pylori) occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231) and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99%) and specificity (100%). Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2%) demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  7. Evaluation of the ARCHITECT urine NGAL assay: assay performance, specimen handling requirements and biological variability.

    PubMed

    Grenier, Frank C; Ali, Salman; Syed, Hina; Workman, Ryan; Martens, Frans; Liao, Ming; Wang, Y; Wong, Pui-Yuen

    2010-04-01

    NGAL (Neutrophil Gelatinase-Associated Lipocalin) has emerged as a new biomarker for the identification of acute kidney injury. Reliable clinical evaluations require a simple, robust test method for NGAL, and knowledge of specimen handling and specimen stability characteristics. We evaluated the performance of a new urine NGAL assay on the ARCHITECT analyzer. Assay performance characteristics were evaluated using standard protocols. Urine specimen storage requirements were determined and biological variability was assessed in a self-declared apparently healthy population. Assay performance data showed good precision, sensitivity and lot-to-lot reproducibility. There was good short term 2-8 degrees C sample stability, however, long term storage samples must be kept at -70 degrees C or colder. The largest variance component in a biological variance study was within-day. The ARCHITECT NGAL assay proved to be a precise and reproducible assay for the determination of urine NGAL. Copyright 2009 The Canadian Society of Clinical Chemists. All rights reserved.

  8. Evaluation of a new random-access HBV DNA molecular assay: The VERIS HBV assay.

    PubMed

    Fourati, Slim; Challine, Dominique; Poveda, Jean-Dominique; Laperche, Syria; Rallier, Sandrine; Pawlotsky, Jean-Michel; Chevaliez, Stéphane

    2017-07-01

    Detection and quantification of HBV DNA are essential to diagnose chronic HBV infection, monitor the virological response to treatment and the possible selection of resistant viruses in order to tailor therapy. The VERIS/MDx System HBV Assay is a random-access system that quantifies HBV DNA in clinical samples using unique single sample and reagent access during the workflow process without the need to reload other tests and delivers results within 1.2h following sampling. The goal of this study was to evaluate the analytical performance of the VERIS HBV assay for HBV DNA detection and quantification in clinical samples from a series of patients chronically infected with different HBV genotypes. The specificity of the VERIS HBV assay was estimated to be over 99.5%. The limit of detection (LOD) was estimated to be 4.1IU/mL (95%CI: 3.20-5.90IU/mL). Using an HBV linearity panel and controls (Seracare LifeScience), intra-assay and inter-assay coefficients of variation ranged from 0.12% to 3.64% and from 1.05% to 7.35%, respectively. The influence of the HBV genotype was evaluated from 120 clinical specimens containing HBV genotypes A to G tested in parallel with the VERIS HBV assay and the COBAS AmpliPrep/COBAS TaqMan HBV v2.0 assay. A linear relationship between the HBV DNA levels measured with both assays was found. A modest bias of HBV DNA levels was observed in the VERIS assay as compared to CAP/CTM HBV v2.0 in most of the samples tested (mean VERIS minus CAP/CTM difference: -0.395 log IU/mL). Overall, the VERIS HBV assay is well suited to monitoring clinical HBV DNA levels in infected patients according to current clinical practice guidelines. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Performance Characteristics of the Cavidi ExaVir Viral Load Assay and the Ultra-Sensitive P24 Assay Relative to the Roche Monitor HIV-1 RNA Assay

    PubMed Central

    Stewart, Paul; Cachafeiro, Ada; Napravnik, Sonia; Eron, Joseph J.; Frank, Ian; van der Horst, Charles; Bosch, Ronald J.; Bettendorf, Daniel; Bohlin, Peter; Fiscus, Susan A.

    2010-01-01

    Background The Cavidi viral load assay and the ultra-sensitive p24 antigen assay (Up24 Ag) have been suggested as more feasible alternatives to PCR-based HIV viral load assays for use in monitoring patients infected with HIV-1 in resource-limited settings. Objectives To describe the performance of the Cavidi ExaVir Load™ assay (version 2.0) and two versions of the Up24 antigen assay and to characterize their agreement with the Roche Monitor HIV-1 RNA assay (version 1.5). Study Design Observational study using a convenience sample of 342 plasma specimens from 108 patients enrolled in two ACTG clinical trials to evaluate the performance characteristics of the Up24 Ag assay using two different lysis buffers and the Cavidi ExaVir Load™ assay. Results In analysis of agreement with the Roche assay, the Cavidi assay demonstrated superiority to the Up24 Ag assays in accuracy and precision, as well as sensitivity, specificity, and positive and negative predictive values for HIV-1 RNA ≥400, ≥1000 and ≥5000 copies/mL. Logistic performance curves indicated that the Cavidi assay was superior to the Up24 assays for viral loads greater than 650 copies/mL. Conclusions The results suggest that the Cavidi ExaVir Load assay could be used for monitoring HIV-1 viral load in resource-limited settings. PMID:20832356

  10. Automated filtration capture immunoelectrochemical assay of bacteria

    NASA Astrophysics Data System (ADS)

    Brewster, Jeffrey D.

    1999-01-01

    An automated system for filtration capture and immunoelectrochemical detection of bactria in liquid samples is described. The detector incorporates a porous electrode in contact with the filter, rather than the solid electrode used previously, to allow sample and reagent solutions to be delivered in a flowing stream. This eliminated the need for manual assembly of the electrode and filter for each assay and allowed repetitive assays on a single filter/electrode. The electrochemical response of the novel gold grid electrode under static and flow conditions was found to be consistent with theory for a planar electrode operating in laminar flow conditions. A computer-controlled fluid handling system was coupled to the detector for delivery of samples and reagents at controlled flow rates and times. The combination of flow detector and fluid handling system allows for automation of the previous assay protocol as well as providing new operating modes with enhanced background rejection and improved sensitivity. The use of these operating modes is demonstrated by a simple assay for Escherichia coli O157:H7 with virtually no background current.

  11. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425...

  12. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490...

  13. 21 CFR 864.7250 - Erythropoietin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7250...

  14. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  15. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  16. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  17. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  18. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7490 Sulfhemoglobin...

  19. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  20. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  1. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7425 Carboxyhemoglobin...

  2. TOTAL CULTURABLE VIRUS QUANTAL ASSAY | Science ...

    EPA Pesticide Factsheets

    This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced cytopathic effects. The quantal method can be used to assay viruses concentrated from sewages, effluents and waters using the methods described in Chapters 5, 6 and 14 (February 1999), Dahling and Wright (1986b), Fout et al. (1996) or from sludges and other solids (Chapter 7, September 1989 Revision; Fout, 1999). Develop sensitive techniques to detect and identify emerging human waterborne pathogenic viruses and viruses on the CCL.Determine effectiveness of viral indicators to measure microbial quality in water matrices.Support activities: (a) culture and distribution of mammalian cells for Agency and scientific community research needs, (b) provide operator expertise for research requiring confocal and electron microscopy, (c) glassware cleaning, sterilization and biological waste disposal for the Cincinnati EPA facility, (d) operation of infectious pathogenic suite, (e) maintenance of walk-in constant temperature rooms and (f) provide Giardia cysts.

  3. Functionalized Nanofiber Meshes Enhance Immunosorbent Assays.

    PubMed

    Hersey, Joseph S; Meller, Amit; Grinstaff, Mark W

    2015-12-01

    Three-dimensional substrates with high surface-to-volume ratios and subsequently large protein binding capacities are of interest for advanced immunosorbent assays utilizing integrated microfluidics and nanosensing elements. A library of bioactive and antifouling electrospun nanofiber substrates, which are composed of high-molecular-weight poly(oxanorbornene) derivatives, is described. Specifically, a set of copolymers are synthesized from three 7-oxanorbornene monomers to create a set of water insoluble copolymers with both biotin (bioactive) and triethylene glycol (TEG) (antifouling) functionality. Porous three-dimensional nanofiber meshes are electrospun from these copolymers with the ability to specifically bind streptavidin while minimizing the nonspecific binding of other proteins. Fluorescently labeled streptavidin is used to quantify the streptavidin binding capacity of each mesh type through confocal microscopy. A simplified enzyme-linked immunosorbent assay (ELISA) is presented to assess the protein binding capabilities and detection limits of these nanofiber meshes under both static conditions (26 h) and flow conditions (1 h) for a model target protein (i.e., mouse IgG) using a horseradish peroxidase (HRP) colorimetric assay. Bioactive and antifouling nanofiber meshes outperform traditional streptavidin-coated polystyrene plates under flow, validating their use in future advanced immunosorbent assays and their compatibility with microfluidic-based biosensors.

  4. A Rapid and Quantitative Recombinase Activity Assay

    USDA-ARS?s Scientific Manuscript database

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  5. Three-dimensional colorimetric assay assemblies

    DOEpatents

    Charych, Deborah; Reichert, Anke

    2001-01-01

    A direct assay is described using novel three-dimensional polymeric assemblies which change from a blue to red color when exposed to an analyte, in one case a flue virus. The assemblies are typically in the form of liposomes which can be maintained in a suspension, and show great intensity in their color changes. Their method of production is also described.

  6. In vitro solubility assays in drug discovery.

    PubMed

    Kerns, Edward H; Di, Li; Carter, Guy T

    2008-11-01

    The solubility of a compound depends on its structure and solution conditions. Structure determines the lipophilicity, hydrogen bonding, molecular volume, crystal energy and ionizability, which determine solubility. Solution conditions are affected by pH, co-solvents, additives, ionic strength, time and temperature. Many drug discovery experiments are conducted under "kinetic" solubility conditions. In drug discovery, solubility has a major impact on bioassays, formulation for in vivo dosing, and intestinal absorption. A good goal for the solubility of drug discovery compounds is >60 ug/mL. Equilibrium solubility assays can be conducted in moderate throughput, by incubating excess solid with buffer and agitating for several days, prior to filtration and HPLC quantitation. Kinetic solubility assays are performed in high throughput with shorter incubation times and high throughput analyses using plate readers. The most frequently used of these are the nephelometric assay and direct UV assay, which begin by adding a small volume of DMSO stock solution of each test compound to buffer. In nephelometry, this solution is serially diluted across a microtitre plate and undissolved particles are detected via light scattering. In direct UV, undissolved particles are separated by filtration, after which the dissolved material is quantitated using UV absorption. Equilibrium solubility is useful for preformulation. Kinetic solubility is useful for rapid compound assessment, guiding optimization via structure modification, and diagnosing bioassays. It is often useful to customize solubility experiments using conditions that answer specific research questions of drug discovery teams, such as compound selection and vehicle development for pharmacology and PK studies.

  7. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  8. Semiautomated Method for Microbiological Vitamin Assays

    PubMed Central

    Berg, T. M.; Behagel, H. A.

    1972-01-01

    A semiautomated method for microbiological vitamin assays is described, which includes separate automated systems for the preparation of the cultures and for the measurement of turbidity. In the dilution and dosage unit based on the continuous-flow principle, vitamin samples were diluted to two different dose levels at a rate of 40 per hr, mixed with the inoculated test broth, and dispensed into culture tubes. After incubation, racks with culture tubes were placed on the sampler of an automatic turbidimeter. This unit, based on the discrete-sample system, measured the turbidity and printed the extinction values at a rate of 300 per hr. Calculations were computerized and the results, including statistical data, are presented in an easily readable form. The automated method is in routine use for the assays of thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, pantothenol, and folic acid. Identical vitamin solutions assayed on different days gave variation coefficients for the various vitamin assays of less than 10%. Images PMID:4553802

  9. Real-time protein kinase assay.

    PubMed

    Sun, Hongye; Low, Karen E; Woo, Sam; Noble, Richard L; Graham, Ronald J; Connaughton, Sonia S; Gee, Melissa A; Lee, Linda G

    2005-04-01

    We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

  10. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  11. PCR Assay Specific for Chicken Feces

    PubMed Central

    Cisar, Cindy R.; Akiyama, Tatsuya; Hatley, Jonathan; Arney, Lori; Kezunovic, Nebojsa; Owen, Daniel

    2011-01-01

    Bacteroidales are fecal anaerobic bacteria that are common in the digestive systems and feces of warm-blooded animals. Some strains of Bacteroidales have been reported to be host-specific. In this study, Bacteroidales strains from chicken feces were examined for their potential use as indicators of chicken fecal contamination. Bacteroidales 16S rRNA gene sequences from chicken feces were amplified, cloned and sequenced. Phylogenetic analysis was performed using these sequences and published Bacteroidales 16S rRNA gene sequences from human and bovine feces. Primers were designed based on putative chicken feces-specific 16S rRNA gene sequences and the primer pairs were tested for specificity in PCR assays. One set of primers, chBact F1 and chBact R16, specifically amplified DNA from chicken feces in a PCR assay, but did not amplify wild turkey, cat, bovine, or deer fecal DNAs. In addition, DNA from feces contaminated straw-based chicken litter produced a product in the PCR assay. However, DNA from feces contaminated wood shavings-based chicken litter was not amplified. The PCR assay described here may prove a useful tool for the detection of chicken feces and for source tracking in watersheds with fecal contamination. PMID:24839330

  12. PCR Assay Specific for Chicken Feces.

    PubMed

    Cisar, Cindy R; Akiyama, Tatsuya; Hatley, Jonathan; Arney, Lori; Kezunovic, Nebojsa; Owen, Daniel

    2010-01-01

    Bacteroidales are fecal anaerobic bacteria that are common in the digestive systems and feces of warm-blooded animals. Some strains of Bacteroidales have been reported to be host-specific. In this study, Bacteroidales strains from chicken feces were examined for their potential use as indicators of chicken fecal contamination. Bacteroidales 16S rRNA gene sequences from chicken feces were amplified, cloned and sequenced. Phylogenetic analysis was performed using these sequences and published Bacteroidales 16S rRNA gene sequences from human and bovine feces. Primers were designed based on putative chicken feces-specific 16S rRNA gene sequences and the primer pairs were tested for specificity in PCR assays. One set of primers, chBact F1 and chBact R16, specifically amplified DNA from chicken feces in a PCR assay, but did not amplify wild turkey, cat, bovine, or deer fecal DNAs. In addition, DNA from feces contaminated straw-based chicken litter produced a product in the PCR assay. However, DNA from feces contaminated wood shavings-based chicken litter was not amplified. The PCR assay described here may prove a useful tool for the detection of chicken feces and for source tracking in watersheds with fecal contamination.

  13. Assay for Angiotensin-Converting Enzyme.

    ERIC Educational Resources Information Center

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  14. Implementation of a radioreceptor assay for dexmedetomidine.

    PubMed

    Salonen, M; Maze, M

    1993-11-01

    We have implemented a radioreceptor assay for dexmedetomidine, a novel alpha 2-adrenoceptor agonist. Receptor-bearing membranes were prepared from rat cerebral cortex and 3H-clonidine, 4 nM, was used as the labeled ligand. Dexmedetomidine displaced 3H-clonidine in a linear fashion over a concentration of 2 x 10(-10) to 2 x 10(-8)M. The detection limit of dexmedetomidine (i.e. 10% of radiolabeled ligand displaced) in this assay was 50 pg.ml-1 which is comparable to that seen with the reference method which utilizes gas chromotography with mass spectrometer (GC/MS) in series (Vuorilehto et al. 1989). Endogenous catecholamines, which can displace the radiolabeled ligand from its binding site, could easily be eliminated with a one-step extraction procedure. A comparison was made with the reference method (GC/MS) in 47 human plasma samples; the correlation coefficient (r2) was 0.61 (P < 0.001). The radioreceptor assay was also successfully applied for determining dexmedetomidine concentration in rabbit samples. These data indicate that the radioreceptor assay can be utilized for characterizing the pharmacokinetics of novel alpha 2 agonists which are now being introduced into the clinical practice of anaesthesia.

  15. 21 CFR 225.158 - Laboratory assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Laboratory assays. 225.158 Section 225.158 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Product Quality Assurance § 225.158...

  16. Assay for Angiotensin-Converting Enzyme.

    ERIC Educational Resources Information Center

    Russo, Salvatore F.

    1983-01-01

    Describes a three-hour experiment designed to introduce students to chemistry of the angiotensis-converting enzyme, illustrate design of a quenched fluorescence substrate, and examine considerations necessary in designing a clinical assay. Includes background information on the biochemistry of hypertension, reagents/materials needed, procedures…

  17. Benzodiazepine Synthesis and Rapid Toxicity Assay

    ERIC Educational Resources Information Center

    Fletcher, James T.; Boriraj, Grit

    2010-01-01

    A second-year organic chemistry laboratory experiment to introduce students to general concepts of medicinal chemistry is described. Within a single three-hour time window, students experience the synthesis of a biologically active small molecule and the assaying of its biological toxicity. Benzodiazepine rings are commonly found in antidepressant…

  18. Sensitive radioenzymatic assay for epinephrine forming enzymes

    SciTech Connect

    Ziegler, M.G.; Kennedy, B.; Elayan, H.

    1988-01-01

    Epinephrine (E) is formed in the adrenal medulla by phenylethanolamine-N-methyltransferase (PNMT), and in other tissues. Enzymes other than PNMT may be able to synthesize E, but this has been difficult to investigate because most assays do not have E as their final product. This assay produces /sup 3/H-E from norepinephrine (NE) and /sup 3/H-S-adenosylmethionine. The /sup 3/H-E is isolated on alumina, /sup 3/H-S-adenosylmethionine is precipitated and the /sup 3/H-E is suspended in diethylhexyl phosphoric acid in toluene for scintillation counting. The assay is sensitive and linear over a wide range. E was formed by most tissues tested. Brain and adrenal contained an enzyme specific for NE, but cardiac ventricle contained an enzyme that methylated both NE and dopamine. Denervated tissues in adrenal medullectomized rats contained very little NE, but still had E and E forming enzyme present. This assay detects a non-neuronal E forming enzyme with activity in vitro and in vivo.

  19. 21 CFR 864.7525 - Heparin assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... coagulation factor X (Stuart factor) or procedures based on the neutralization of heparin by protamine sulfate...

  20. Analysis of Gold Ores by Fire Assay

    ERIC Educational Resources Information Center

    Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

    2004-01-01

    Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

  1. Assays for mammalian tyrosinase: a comparative study

    SciTech Connect

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.

  2. Production and assay of forskolin antibodies

    SciTech Connect

    Ho, L.T.; Ho, R.J.

    1986-05-01

    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  3. Paraprotein interference with turbidimetric gentamicin assay

    PubMed Central

    Bassett, Kendra; Brown, Nigel

    2015-01-01

    Introduction Gentamicin due to its low level of resistance and rapid bactericidal activity is commonly used to treat gram-negative bacteria. However, due to its toxic effects it needs to be monitored. To date, no interference has been reported with gentamicin assays. Materials and methods A patient with leg cellulitis and sepsis received a single dose of gentamicin and a sample was sent for gentamicin analysis. The sample showed high blank absorbance readings on Beckman DxC800 and DC800 analysers with various dilutions. A second sample was received and analysed on a Roche Cobas system to obtain a result. A third sample was received 107 hours later with the same results and this sample was then analysed neat and post ethanol precipitation on all the turbidimetric assays available on the DxC800 analyser. Results The high blank absorbance was observed upon addition of the reactive reagents due to protein precipitation. Although not obvious from the patient protein results, it was shown the presence of high IgM paraprotein, 18.9 g/L (reference range 0.4-2.3 g/L) was the cause of precipitation, giving high blank readings. Of all the other turbidimetric assays, only vancomicin and valproate showed similar high blank absorbance readings. To be able to provide more rapid results it was shown ethanol could be used as a precipitant of proteins in both calibrators and patient samples with acceptable recovery. Conclusion IgM paraprotein was identified as the cause of interference with the gentamicin, vancomicin and valproate assays. Protein interference in these assays can be overcome by precipitation with ethanol. PMID:25672475

  4. Single-step colony assay for screening antibody libraries.

    PubMed

    Kato, Mieko; Hanyu, Yoshiro

    2017-08-10

    We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10(3) scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

    NASA Astrophysics Data System (ADS)

    Sugiura, Kanako; Kaji, Noritada; Okamoto, Yukihiro; Tokeshi, Manabu; Baba, Yoshinobu

    High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc.

  6. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

    PubMed

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

    2015-03-30

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

  7. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

    PubMed Central

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

    2015-01-01

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

  8. Propagation and assay of hepatitis A virus in vitro.

    PubMed

    Siegl, G; deChastonay, J; Kronauer, G

    1984-08-01

    Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37 degrees C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.

  9. Enzyme-Linked Immunosorbent-Assay for Deoxynivalenol (DON)

    PubMed Central

    Ji, Fang; Li, Hua; Xu, Jianhong; Shi, Jianrong

    2011-01-01

    Deoxynivalenol (DON), one of the trichothecene mycotoxins, is a worldwide contaminant of wheat and barley, especially when infected by Fusarium graminearum, the causative agent of an epidemic wheat disease called Fusarium Head Blight. Because of the high risk of DON ingestion and the possibility of frequent exposure, it is important to develop a rapid and highly sensitive method for easy identification and quantification of DON in grain samples. In this study, we have developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) to detect DON in wheat. We conjugated 3-O-Hemisuccinyl-DON (3HS-DON) to Bovine serum albumin (BSA) and Ovalbumin (OVA), and obtained DON-specific mice antisera. The indirect competitive ELISA revealed that the optimal concentration of mice serum and the coated antigen was 1/1600 and 1/1500, respectively. The antiserum cross-reacted with the trichothecenes 3-acetyl-DON and T-2 toxin, reaching about 55.2% and 6.3%, respectively, as compared with DON. Results showed that the assay could be performed satisfactorily using an extraction buffer containing less than 15% methanol. Recovery from DON was 82–93% in grains. The linear detection range of DON in grains was between 0.01 and 100 μg/mL. PMID:22069751

  10. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  11. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  12. Variola Virus-Specific Diagnostic Assays: Characterization, Sensitivity, and Specificity

    PubMed Central

    Kondas, Ashley V.; Olson, Victoria A.; Li, Yu; Abel, Jason; Laker, Miriam; Rose, Laura; Wilkins, Kimberly; Turner, Jonathan; Kline, Richard

    2015-01-01

    A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified. PMID:25673790

  13. The assay of pyruvate kinase activity in gastric mucosa.

    PubMed

    Orwell, R L; Thornton, H C; Piper, D W

    1975-05-01

    Pyruvate kinase activity in gastric mucosal supernatant preparations shows large responses to exogenous effectors. At pH 7.5, fructose 1,6-diphosphate can cause large stimulations in activity. Alanine inhibits the reaction but this effect is partially reversed by fructose 1,6-diphosphate. In the absence of these compounds, 4.0-5.0 mM phosphoenolpyruvate (PEP) is required to generate maximal activity in the assay system used. Electrophoresis reveals an isoenzyme pattern containing only one form of pyruvate kinase, an M isoenzyme, in both fundic and antral mucosa. The pyruvate kinase of gastric mucosa thus resembles the M-type enzymes of leucocytes and liver. Measurements of activity at both 1.0 mM PEP and 4.0--5.0 mM PEP, with and without additions of fructose 1,6-diphosphate, are recommended for the reliable estimation of pyruvate kinase activity in this tissue.

  14. Assessment of immunoglobulin concentrates on thrombogenic activity by thrombin generation assay, prekallikrein activator assay, and size-exclusion chromatography.

    PubMed

    Seifner, Alexandra; Beck, Gerhard; Bayer, Patrick; Eichmeir, Stephanie; Lackner, Friedrich; Rögelsperger, Olga; Weber, Katharina; Wollein, Gabriele

    2014-02-01

    Immunoglobulin G (IgG) concentrates have recently been found to be contaminated with procoagulant impurities causing thromboembolic events (TEEs) in vivo. In this study the question was raised whether a thrombin generation assay (TGA) will be able to characterize IgG samples from the Austrian market with regard to their thrombogenic potential. A total of 44 IgG concentrates have been assayed by TGA employing pooled normal plasma and Factor (F)XI-deficient plasma (FXIdp). Furthermore, the prekallikrein activator assay including determination of blank values, size-exclusion chromatography, and further test systems required for batch release testing of IgG concentrates according to the European Pharmacopeia (Pharm. Eur.) were carried out. All samples complied with acceptance criteria stated in the Plarm. Eur. and/or prescribed by the marketing approval. One intravenous immunoglobulin (IVIG) involved in TEEs exceeded a threshold level of 350 nmol peak thrombin, which was not exceeded after change of manufacture and by all the other IVIGs tested. Two hyperimmune globulins revealed elevated peak thrombin levels of up to 810 nmol in FXI and up to 285 nmol in FXIdp. The study indicates that the TGA is able to reliably predict procoagulant activities probably associated with the presence of FXIa and potential thrombogenicity. Comparison of thrombin generation with product-specific acceptance criteria as well as variables from other test systems as amidolytic activity and molecular size can help to monitor IgG quality and manufacturing changes with regard to thrombogenicity. © 2013 American Association of Blood Banks.

  15. Evaluation of the behavior of murine and human embryonic stem cells in in vitro migration and invasion assays.

    PubMed

    T'Joen, V; Somers, P; Declercq, H; Cornelissen, M

    2013-04-01

    Cell migration and invasion are essential processes in a variety of physiological events in the body, but also in several patho-physiological events. In this paper, the behavior of murine and human embryonic stem cells is examined in in vitro migration and invasion models. mESC and hESC were applied as spheroids, also known as patches, and as single cells, to mimic possible cell therapy application strategies. Two known in vitro migration assays, the ECM (extracellular matrix) assay and the Boyden chamber migration assay were selected. These assays revealed that mESC are statistically significantly more infiltrative than hESC. Application as spheroid results in a slightly higher infiltrative capacity compared single cells. The PHF (precultured chick heart fragment) assay was selected as an invasion assay. In the PHF assay a more 3D examination of the infiltrative nature of the ESC can be observed. The mESC showed infiltrative behavior, as spheroids and as single cells. The hESC were infiltrative as single cells but not as spheroids. The results of these assays are mostly complementary and prove the applicability of these assays, which were originally applied in tumor biology, in migratory behavior studies regarding stem cells and their progeny in basic and other conditions.

  16. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    SciTech Connect

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  17. Bead-Based Assays for Biodetection: From Flow-Cytometry to Microfluidics

    SciTech Connect

    Ozanich, Richard M.; Antolick, Kathryn C.; Bruckner-Lea, Cindy J.; Bunch, Kyle J.; Dockendorff, Brian P.; Grate, Jay W.; Nash, Michael A.; Tyler, Abby J.

    2009-05-04

    ABSTRACT The potential for the use of biological agents by terrorists is a real threat. Two approaches for detection of biological species will be described: 1) The use of microbead arrays for multiplexed flow cytometry detection of cytokines and botulinum neurotoxin simulant, and 2) a microfluidic platform for capture and separation of different size superparamagnetic nanoparticles followed by on-chip fluorescence detection of the sandwich complex. The methods and automated fluidic systems used for trapping functionalized microbeads will be described. This approach allows sample, assay reagents, and wash solutions to be perfused over a micro-column of beads, resulting in faster and more sensitive assays. The automated fluidic approach resulted in up to five-fold improvements in assay sensitivity/speed as compared to identical assays performed in a typical manual batch mode. A second approach for implementing multiplexed bead-based assays without using flow cytometry detection is currently under development. The goal of the microfluidic-based approach is to achieve rapid (<20 minutes), multiplexed (> 3 bioagents) detection using a simple and low-cost, integrated microfluidic/optical detection platform. Using fiber-optic guided laser-induced fluorescence, assay detection limits were shown to be in the 100’s of picomolar range (10’s of micrograms per liter) for botulinum neurotoxin simulant without any optimization of the microfluidic device or optical detection approach. Video taping magnetic nanoparticle capture and release was used to improve understanding of the process and revealed interesting behavior.

  18. SNP genotyping using TaqMan technology: the CYP2D6*17 assay conundrum.

    PubMed

    Gaedigk, Andrea; Freeman, Natalie; Hartshorne, Toinette; Riffel, Amanda K; Irwin, David; Bishop, Jeffrey R; Stein, Mark A; Newcorn, Jeffrey H; Jaime, Lazara Karelia Montané; Cherner, Mariana; Leeder, J Steven

    2015-03-19

    CYP2D6 contributes to the metabolism of many clinically used drugs and is increasingly tested to individualize drug therapy. The CYP2D6 gene is challenging to genotype due to the highly complex nature of its gene locus. TaqMan technology is widely used in the clinical and research settings for genotype analysis due to assay reliability, low cost, and the availability of commercially available assays. The assay identifying 1023C>T (rs28371706) defining a reduced function (CYP2D6*17) and several nonfunctional alleles, produced a small number of unexpected diplotype calls in three independent sets of samples, i.e. calls suggested the presence of a CYP2D6*4 subvariant containing 1023C>T. Gene resequencing did not reveal any unknown SNPs in the primer or probe binding sites in any of the samples, but all affected samples featured a trio of SNPs on their CYP2D6*4 allele between one of the PCR primer and probe binding sites. While the phenomenon was ultimately overcome by an alternate assay utilizing a PCR primer excluding the SNP trio, the mechanism causing this phenomenon remains elusive. This rare and unexpected event underscores the importance of assay validation in samples representing a variety of genotypes, but also vigilance of assay performance in highly polymorphic genes such as CYP2D6.

  19. An immunochromatography assay for rapid antemortem diagnosis of dogs suspected to have canine distemper.

    PubMed

    An, Dong-Jun; Kim, Tae-Young; Song, Dae-Sub; Kang, Bo-Kyu; Park, Bong-Kyun

    2008-02-01

    A new assay was developed for rapid and antemortem diagnosis of canine distemper (CD). This immunochromatography (IC)-based assay, which employs two monoclonal anti-CDV antibodies, was compared with nested PCR. When serial dilutions of purified CDV were tested, the CDV detection limits of the nested PCR and IC assays were 2 x 10(2)TCID(50)/ml and 5 x 10(2)TCID(50)/ml, respectively. Nasal irrigation fluid, conjunctival swabs, and blood lymphocytes from 66 dogs suspected to have CD were tested. Preliminary IC experiments revealed that the optimal sample volume and reaction time were 100 microl and 5 min, respectively. Relative to nested PCR, the sensitivity and specificity of the IC assay was maximal (100% and 100%, respectively) when conjunctival swabs were tested. This is significant because conjunctival swab specimens are easy to obtain in the early phase of CD infection. However, with blood lymphocytes and nasal samples, the IC assay was slightly less sensitive (89.7% and 85.7%, respectively) and specific (94.6% and 100%, respectively) than nested PCR. Since this novel IC assay does not require special instruments, it is a simple enough for dog owners to use. Since early detection of CD would allow appropriate treatment and quarantine to be instituted quickly, such a test would help reduce the morbidity and mortality associated with CD help to prevent its spread to other animals.

  20. In vitro cell-based assays for evaluation of antioxidant potential of plant-derived products.

    PubMed

    Nascimento da Silva, Luís Cláudio; Bezerra Filho, Clovis Macêdo; Paula, Raiana Apolinário de; Silva E Silva, Cristiane Santos; Oliveira de Souza, Larissa Isabela; Silva, Márcia Vanusa da; Correia, Maria Tereza Dos Santos; Figueiredo, Regina Célia Bressan Queiroz de

    2016-08-01

    Several plant-derived compounds have been screened by antioxidant assays, but many of these results are questionable, since they do not evaluate the pharmacologic parameters. In fact, the development of better antioxidants stills a great challenge. In vitro cell-based assays have been employed to assess the antioxidant effect of various compounds at subcellular level. Cell-based assays can also reveal compounds able to enhance the antioxidant pathways, but without direct radical scavenging action (which could not be detected by traditional assays). These methodologies are general of easy implementation and reproducible making them suitable for the early stages of drug discovery. Hydrogen peroxide, a nonradical derivative of oxygen, can be employed as an oxidative agent in these assays due its biochemical properties (presence of all biological systems, solubility) and capacity to induce cell death. Truthfully, if their limitations are understood (such as difference on cell metabolism when in in vitro conditions), these cell-based assays can provide useful information about the pathways involved in the protective effects of phytochemicals against cell death induced by oxidative stress, which can be exploited to develop new therapeutic approaches.