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Sample records for acid bca assay

  1. 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. PMID:21078286

  2. Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.

    PubMed

    Chen, Lei; Yu, Zhi; Lee, Youngju; Wang, Xu; Zhao, Bing; Jung, Young Mee

    2012-12-21

    A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu(+), which is reduced by protein in an alkaline environment, is exploited to create a BCA-Cu(+) complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL(-1) and 10 pg mL(-1). There are many advantages of using RR and SERRS-based assays. These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques. PMID:23099478

  3. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample. PMID:21762678

  4. Cloning and Expression of the γ-Polyglutamic Acid Synthetase Gene pgsBCA in Bacillus subtilis WB600

    PubMed Central

    Lin, Biaosheng; Li, Zhijuan; Zhang, Huixia; Wu, Jiangwen; Luo, Maochun

    2016-01-01

    To clone and express the γ-polyglutamic acid (γ-PGA) synthetase gene pgsBCA in Bacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA into Bacillus subtilis WB600. PgsBCA was expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesize γ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher. γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates of γ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of the γ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing the γ-PGA synthetase gene pgsBCA in B. subtilis system has potential industrial applications. PMID:27073802

  5. EFFECT OF GESTATIONAL EXPOSURE TO ETHANE DIMETHANESULFONATE (EDS), BROMOCHLOROACETIC ACID (BCA) AND MOLINATE ON REPRODUCTIVE FUNCTION IN CD-1 MALE MICE

    EPA Science Inventory

    EFFECT OF GESTATIONAL EXPOSURE TO ETHANE DIMETHANESULFONATE (EDS), BROMOCHLOROACETIC ACID (BCA) AND MOLINATE ON REPRODUCTIVE FUNCTION IN CD-1 MALE MICE. D.K. Tarka1,2 , G.R. Klinefelter2, J.C. Rockett2, J.D. Suarez2, N.L. Roberts2 and J.M. Rogers1,2. 1 University of North Carol...

  6. DELAYED PREPUTIAL SEPARATION (PPS) AND SP22 MEASUREMENT IN RATS ADMINISTERED BROMOCHLOROACETIC ACID (BCA) IN DRINKING WATER

    EPA Science Inventory

    Reproductive effects of BCA were determined in a dose range finding study (DRFS) and definitive two-generational study. Adult male and female CD� (SD) rats were administered BCA in drinking water for two weeks in the DRFS (10/sex/group) and ten weeks in the definitive study (25/s...

  7. The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

    PubMed

    Sampson, D L; Chng, Y L; Upton, Z; Hurst, C P; Parker, A W; Parker, T J

    2013-11-01

    Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead. PMID:23911526

  8. Bioprocess monitoring: minimizing sample matrix effects for total protein quantification with bicinchoninic acid assay.

    PubMed

    Reichelt, Wieland N; Waldschitz, Daniel; Herwig, Christoph; Neutsch, Lukas

    2016-09-01

    Determining total protein content is a routine operation in many laboratories. Despite substantial work on assay optimization interferences, the widely used bicinchoninic acid (BCA) assay remains widely recognized for its robustness. Especially in the field of bioprocess engineering the inaccuracy caused by interfering substances remains hardly predictable and not well understood. Since the introduction of the assay, sample pre-treatment by trichloroacetic acid (TCA) precipitation has been indicated as necessary and sufficient to minimize interferences. However, the sample matrix in cultivation media is not only highly complex but also dynamically changing over process time in terms of qualitative and quantitative composition. A significant misestimation of the total protein concentration of bioprocess samples is often observed when following standard work-up schemes such as TCA precipitation, indicating that this step alone is not an adequate means to avoid measurement bias. Here, we propose a modification of the BCA assay, which is less influenced by sample complexity. The dynamically changing sample matrix composition of bioprocessing samples impairs the conventional approach of compensating for interfering substances via a static offset. Hence, we evaluated the use of a correction factor based on an internal spike measurement for the respective samples. Using protein spikes, the accuracy of the BCA protein quantification could be improved fivefold, taking the BCA protein quantification to a level of accuracy comparable to other, more expensive methods. This will allow reducing expensive iterations in bioprocess development to due inaccurate total protein analytics. PMID:27314233

  9. Nucleic acid detection assays

    DOEpatents

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann; Dahlberg, James E.

    2005-04-05

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  10. AUTOUBIQUITINATION OF BCA2 RING E3 LIGASE REGULATES ITS OWN STABILITY AND AFFECTS CELL MIGRATION

    PubMed Central

    Amemiya, Yutaka; Azmi, Peter; Seth, Arun

    2009-01-01

    Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2 binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, majority of those were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitinA-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the N-terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared to wild-type BCA2. On the basis of these findings, we propose that BCA2 maybe an important factor regulating breast cancer cell migration/metastasis. We put-forward a novel model for BCA2 E3 ligase mediated cell regulation. PMID:18819927

  11. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

  12. Coccolith B/Ca ratios using SIMS ion probe analysis

    NASA Astrophysics Data System (ADS)

    Stoll, H.; Shimizu, N.; Langer, G.

    2009-04-01

    B/Ca ratios are proposed as a paleo-carbonate ion or paleo-pH proxy due to the preferential incorporation of borate ion into the calcite lattice, relative to boric acid which is the dominant species of B at lower pH. The relative importance of cellular regulation vs external pH on the carbonate B/Ca remains to be characterized for most organisms. Here we describe initial results of B/Ca analyses of coccoliths produced in laboratory culture under variable carbonate ion concentrations. Due to the impossibility of physically separating the micron-sized coccoliths from non-coccolith sediment material in quantities large enough for TIMS or ICP-MS analysis of B/Ca, eventual analysis of coccolith B/Ca from the fossil record will need to be conducted on individually picked coccoliths on the ion probe as is currently done for other trace element (eg. Sr/Ca) ratios. Hence, we employ the CAMECA IMS 1280 ion probe at the Northeast National Ion Microprobe Facility at Woods Hole Oceanographic Institution to measure B/Ca in coccoliths from cultures. We evaluated cleaning methods using a synthetic cleaning target (crushed marble) contaminated with noncalcifying algae. Cleaning is crucial for obtaining accurate B/Ca ratios and precluding sample charging. B/Ca ratios of different genera of modern coccoliths range from 5 to 25 umol/mol, 3 to 10fold lower than planktic foraminifera or abiogenic calcite precipitated in seawater in the same pH range. These low ratios suggest much more restricted uptake of B into the algae cell in the vesicle calcification used by coccolithophores, compared with the seawater vacuole calcification typical of foraminifera. Different coccolith species grown at the same pH exhibit different B/Ca ratios. One species, Coccolithus pelagicus, cultured at a range of pH conditions from 7.7 to 8.4, exhibits no significant change in B/Ca ratios across the range of pH. One explanation is pH homeostasis at the calcification site. In possible support of pH homeostasis

  13. Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription.

    PubMed Central

    Chugani, S A; Parsek, M R; Hershberger, C D; Murakami, K; Ishihama, A; Chakrabarty, A M

    1997-01-01

    The soil bacterium Pseudomonas putida is capable of degrading many aromatic compounds, including benzoate, through catechol as an intermediate. The catabolism of catechol is mediated by the catBCA operon, whose induction requires the pathway intermediate cis,cis-muconate as an inducer and the regulatory protein, CatR. CatR also regulates the plasmid-borne pheBA operon of P. putida PaW85, which is involved in phenol catabolism. We have used an in vitro transcription system to study the roles of CatR, cis,cis-muconate, Escherichia coli RNA polymerase, and promoter sequences in expression of the cat and phe operons. The assay confirmed the requirement of both CatR and cis,cis-muconate for transcript formation. We also examined the in vitro transcription of three site-directed mutants of the catBCA promoter; the results obtained compared favorably with previous in vivo data. The requirement of the alpha subunit of RNA polymerase for expression of the catBCA and the pheBA transcripts was also examined. The C-terminal region of the alpha subunit of RNA polymerase has been implicated in direct protein-protein contact with transcriptional regulatory proteins and/or direct contact with the DNA. We show that the carboxyl terminus of the alpha subunit is required for the expression of the catBCA and the pheBA operons because RNA polymerases with truncated alpha subunits were deficient in activation. Further experiments demonstrated the arginine at position 265 and the asparagine at position 268 of the alpha subunit as possible amino acids involved in activation. On the basis of these and previous results, we propose a model to explain the interaction of the different regulatory components leading to CatR-dependent activation of the catBCA operon. PMID:9079907

  14. Do Coccolith B/Ca Ratios Elucidate the Response of the Smallest Calcifiers to Ocean Acidification?

    NASA Astrophysics Data System (ADS)

    Stoll, H. M.; Langer, G.; Shimizu, N.; Kanamaru-Shinn, K.

    2009-12-01

    Coccolithophorid algae are microscopic but prolific calcifiers in modern and ancient oceans. Different species and strains have exhibited diverse calcification responses to laboratory ocean acidification experiments. This hampers our ability to predict future alteration of marine biogeochemical cycles. We used SIMS ion probe to measure B/Ca ratios of coccoliths from three different strains of Emiliania huxleyi and one strain of Coccolithus pelagicus braarudi under different pH conditions to ascertain if B/Ca in fossil coccoliths might be an indicator of calcification stress to past events. B/Ca in abiogenic calcites increases at higher pH because of the preferential incorporation of borate ion into the calcite lattice, relative to boric acid which is the dominant species of B at lower pH. We find, however, that the behavior of B/Ca in coccoliths differs substantially from that of abiogenic calcites. First, B/Ca ratios of coccoliths are generally lower than those of abiogenic calcites precipitated in a comparable pH range, suggesting that the transport of ions into the cell reduces the ratio of B to bicarbonate in the calcifying vesicle compared to seawater. The slowest growing strain of E. huxleyi and one strain of C. braarudi exhibited low B/Ca ratios (<10 μmol/mol) which were constant as a function of culture pH; the calcite/cell of this E. huxleyi strain decreased with decreasing pH whereas that of the C. braarudi was constant. Two other more rapidly growing strains of E. huxleyi exhibited a large range in B/Ca ratio (55 to 25 μmol/mol), inversely correlated with pH which is opposite to the relationship observed in abiogenic calcites. Calcite/cell in both of these strains was constant or increased slightly with decreasing pH. B/Ca ratios therefore do not show a clear relationship with calcification stress. The variation in B/Ca ratios is most plausibly explained by changes in transport of B into the cell. B intake may be controlled by passive boric acid uptake

  15. Assays for Determination of Protein Concentration.

    PubMed

    Olson, Bradley J S C

    2016-01-01

    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. PMID:27248579

  16. Investigating B/Ca of coccoliths using SIMS ion probe analysis

    NASA Astrophysics Data System (ADS)

    Stoll, H. M.; Shimizu, N.; Langer, G.

    2008-12-01

    B/Ca ratios are proposed as a paleo-carbonate ion or paleo-pH proxy due to the preferential incorporation of borate ion into the calcite lattice, relative to boric acid which is the dominant species of B at lower pH. The relative importance of cellular regulation vs external pH on the carbonate B/Ca remains to be characterized for most organisms. Here we describe initial results of B/Ca analyses of coccoliths produced in laboratory culture under variable carbonate ion concentrations. Due to the impossibility of physically separating the micron-sized coccoliths from non-coccolith sediment material in quantities large enough for TIMS or ICP-MS analysis of B/Ca, eventual analysis of coccolith B/Ca from the fossil record will need to be conducted on individually picked coccoliths on the ion probe as is currently done for other trace element (eg. Sr/Ca) ratios. Hence, we employ the CAMECA IMS 1280 ion probe at the Northeast National Ion Microprobe Facility at Woods Hole Oceanographic Institution to measure B/Ca in coccoliths from cultures. We evaluated cleaning methods using a synthetic cleaning target (crushed marble) contaminated with noncalcifying algae. Cleaning is crucial for obtaining accurate B/Ca ratios and precluding sample charging. B/Ca ratios of different genera of modern coccoliths range from 5 to 25 umol/mol, 3 to 10fold lower than planktic foraminifera or abiogenic calcite precipitated in seawater in the same pH range. These low ratios suggest much more restricted uptake of B into the algae cell in the vesicle calcification used by coccolithophores, compared with the seawater vacuole calcification typical of foraminifera. Different coccolith species grown at the same pH exhibit different B/Ca ratios. A single species, Coccolithus pelagicus, cultured at a range of pH conditions from 7.7 to 8.4, exhibits no significant change in B/Ca ratios across the range of pH. One explanation is pH homeostasis at the calcification site. In possible support of p

  17. Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

    PubMed

    Brady, Pamlea N; Macnaughtan, Megan A

    2015-12-15

    Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

  18. Prognostic Value of Preoperative Serum Levels of Periostin (PN) in Early Breast Cancer (BCa).

    PubMed

    Nuzzo, Pier Vitale; Rubagotti, Alessandra; Argellati, Francesca; Di Meglio, Antonio; Zanardi, Elisa; Zinoli, Linda; Comite, Paola; Mussap, Michele; Boccardo, Francesco

    2015-01-01

    PN is a secreted cell adhesion protein critical for carcinogenesis. Elevated serum levels of PN have been implicated as playing an important role in different types of cancer, and a few reports suggest a potential role as a prognostic marker. We evaluated the prognostic significance of preoperative serum PN concentration in patients with BCa receiving curative surgery. Enzyme-Linked Immunosorbent Assay (ELISA) was performed to determine the preoperative serum PN level in 182 patients. The correlations between serum PN concentration with clinical pathological features and PN expression in primary tumor samples were analyzed. The prognostic impact of serum PN levels with all-cause and BCa-specific mortality was also investigated. Appropriate statistics were used. Elevated serum PN levels were significantly associated with patient age (p = 0.005), adjuvant systemic therapy (p = 0.04) and progesterone receptor (PgR) status (p = 0.02). No correlation between PN preoperative serum levels and other clinical-pathological parameters, including either the epithelial or the stromal PN expression of primary tumor or the combination of the two, was found. Similarly, no association between serum PN levels and either all-cause or BCa-specific mortality was found. However, subgroup analysis revealed a correlation between higher PN serum levels and all-cause mortality in patients with node-negative disease (p = 0.05) and in those with a low PgR expression (p = 0.03). Higher levels of serum PN were also found to correlate with BCa-specific mortality in the subgroup of patients who did not receive any adjuvant systemic therapy (p = 0.04). Our findings suggest that PN was detectable in the serum of early BCa patients before surgery and increased base-line serum levels predicted worse long-term survival outcomes in specific subgroups of patients. PMID:26225965

  19. B/Ca in coccoliths and relationship to calcification vesicle pH and dissolved inorganic carbon concentrations

    NASA Astrophysics Data System (ADS)

    Stoll, Heather; Langer, Gerald; Shimizu, Nobumichi; Kanamaru, Kinuyo

    2012-03-01

    Coccolithophorid algae are microscopic but prolific calcifiers in modern and ancient oceans. When the pH of seawater is modified, as may occur in the future due to ocean acidification, different species and strains of coccolithophorids have exhibited diverse calcification responses in laboratory culture. Since their biomineralization is a completely intracellular process, it is unclear why their response should be affected by extracellular seawater pH. Variations in the B/Ca in coccoliths are potential indicators of pH shifts in the intracellular coccolith vesicle where calcification occurs, because B/Ca in abiogenic calcites increases at higher pH due to the greater abundance of borate ions, the only B species incorporated into calcite. We used a SIMS ion probe to measure B/Ca of coccoliths from three different strains of Emiliania huxleyi and one strain of Coccolithus braarudii braarudii cultured under different seawater pH conditions to ascertain if the B/Ca can be used to elucidate how coccolithophorids respond to changing ocean pH. These data are interpreted with the aid of a conceptual model of cellular boron acquisition by coccolithophorids. Based on uptake in other plants, we infer that boron uptake by coccolithophorid cells is dominated by passive uptake of boric acid across the lipid bilayer. Subsequently, in the alkaline coccolith vesicle (C.V.), boron speciates according to the C.V. pH, and borate is incorporated into the coccolith. At increasing seawater pH, the relative abundance of the neutral boric acid in seawater decreases, lowering the potential B flux into the cell. Homeostasis or constant pH of the coccolith vesicle results in a decrease of the B/Ca in the coccolith with increasing seawater pH. In contrast, if coccolith vesicle pH increases with increasing seawater pH, then the B/Ca will increase as the fraction of borate in the coccolith vesicle increases. The coccolith B/Ca is also expected to depend inversely on the dissolved inorganic

  20. Simple Bulk Readout of Digital Nucleic Acid Quantification Assays.

    PubMed

    Morinishi, Leanna S; Blainey, Paul

    2015-01-01

    Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays. We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout. Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology. PMID:26436576

  1. Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS).

    PubMed

    Schlegel, Benjamin J; Nowell, Victoria J; Parreira, Valeria R; Soltes, Glenn; Prescott, John F

    2012-10-01

    This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature. PMID:23543949

  2. Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS)

    PubMed Central

    Schlegel, Benjamin J.; Nowell, Victoria J.; Parreira, Valeria R.; Soltes, Glenn; Prescott, John F.

    2012-01-01

    This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature. PMID:23543949

  3. Determination of protein levels in soy and peanut oils by colorimetric assay and ELISA.

    PubMed

    Jablonski, Joseph E; Fu, Tong-Jen; Jackson, Lauren S; Gendel, Steven M

    2010-01-01

    Analytical methods are needed for measuring the levels of protein from allergenic food transferred into cooking oil. A simple method for determination of total protein in cooking oils was developed. Oil was extracted with phosphate-buffered saline with 0.05% Tween (PBST) and the extracts were partitioned with hexane to remove residual oil. Total protein in the PBST extracts was assayed with bicinchoninic acid (BCA), micro-BCA, reducing-agent compatible BCA and CB-XT kits. These methods were used to measure recovery of protein from peanut butter spikes of soy and peanut oil in the range of 50-1000 ppm. Recoveries were generally above 70%. However, the BCA and micro-BCA assays were subject to interference and enhanced color formation which were probably due to co-extracted antioxidants present in oil. The reducing agent-compatible BCA and CB-X protein assays reduced interference and gave lower protein values in crude, cold-pressed, and refined peanut oils. Heating oil to 180 degrees C before extraction also reduced interference-induced color enhancement. A commercial ELISA test kit was also used to measure peanut protein in oil spiked with peanut butter. Recovery of peanut residues measured by ELISA was significantly decreased when the peanut butter-spiked oil was heated to 180 degrees C compared to unheated oil. Recovery of spiked peanut butter protein measured by the buffer extraction-colorimetric method was not decreased in heated oil. The method developed here could be used to determine protein levels in crude and refined oil, and to assess the potential for allergen cross-contact from reused cooking oil. PMID:20334183

  4. 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 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  5. 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 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  6. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  7. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  8. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enterovirus nucleic acid assay. 866.3225 Section 866.3225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3225...

  9. Development of a stable isotope dilution assay for tenuazonic acid.

    PubMed

    Asam, Stefan; Liu, Yang; Konitzer, Katharina; Rychlik, Michael

    2011-04-13

    A stable isotope dilution assay (SIDA) for the Alternaria mycotoxin tenuazonic acid was developed. Therefore, [(13)C(6),(15)N]-tenuazonic acid was synthesized from [(13)C(6),(15)N]-isoleucine by Dieckmann intramolecular cyclization after acetoacetylation with diketene. The synthesized [(13)C(6),(15)N]-tenuazonic acid was used as the internal standard for determination of tenuazonic acid in tomato products by liquid chromatography tandem mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Method validation revealed a limit of detection of 0.1 μg/kg and a limit of quantitation of 0.3 μg/kg. Recovery was close to 100% in the range of 3-300 μg/kg. Determination of tenuazonic acid in two samples of different tomato ketchups (naturally contaminated) was achieved with a coefficient of variation of 2.3% and 4.7%. Different tomato products (n = 16) were analyzed for their content of tenuazonic acid using the developed SIDA. Values were between 15 and 195 μg/kg (tomato ketchup, n = 9), 363 and 909 μg/kg (tomato paste, n = 2), and 8 and 247 μg/kg (pureed tomatoes and comparable products, n = 5). PMID:21370870

  10. Automated Protein Assay Using Flow Injection Analysis

    NASA Astrophysics Data System (ADS)

    Wolfe, Carrie A. C.; Oates, Matthew R.; Hage, David S.

    1998-08-01

    The technique of flow injection analysis (FIA) is a common instrumental method used in detecting a variety of chemical and biological agents. This paper describes an undergraduate laboratory that uses FIA to perform a bicinchoninic acid (BCA) colorimetric assay for quantitating protein samples. The method requires less than 2 min per sample injection and gives a response over a broad range of protein concentrations. This method can be used in instrumental analysis labs to illustrate the principles and use of FIA, or as a means for introducing students to common methods employed in the analysis of biological agents.

  11. Comparison of amino acid digestibility of feedstuffs determined with the precision-fed cecectomized rooster assay and the standardized ileal amino acid digestibility assay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate and compare amino acid digestibility of several feedstuffs using 2 commonly accepted methods: the precision-fed cecectomized rooster assay (PFR) and the standardized ileal amino acid assay (SIAAD). Six corn, 6 corn distillers dried grains with or without s...

  12. Bioluminescence regenerative cycle (BRC) system for nucleic acid quantification assays

    NASA Astrophysics Data System (ADS)

    Hassibi, Arjang; Lee, Thomas H.; Davis, Ronald W.; Pourmand, Nader

    2003-07-01

    A new label-free methodology for nucleic acid quantification has been developed where the number of pyrophosphate molecules (PPi) released during polymerization of the target nucleic acid is counted and correlated to DNA copy number. The technique uses the enzymatic complex of ATP-sulfurylase and firefly luciferase to generate photons from PPi. An enzymatic unity gain positive feedback is also implemented to regenerate the photon generation process and compensate any decay in light intensity by self regulation. Due to this positive feedback, the total number of photons generated by the bioluminescence regenerative cycle (BRC) can potentially be orders of magnitude higher than typical chemiluminescent processes. A system level kinetic model that incorporates the effects of contaminations and detector noise was used to show that the photon generation process is in fact steady and also proportional to the nucleic acid quantity. Here we show that BRC is capable of detecting quantities of DNA as low as 1 amol (10-18 mole) in 40μlit aqueous solutions, and this enzymatic assay has a controllable dynamic range of 5 orders of magnitude. The sensitivity of this technology, due to the excess number of photons generated by the regenerative cycle, is not constrained by detector performance, but rather by possible PPi or ATP (adenosine triphosphate) contamination, or background bioluminescence of the enzymatic complex.

  13. Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

    PubMed

    Carroll, Karen C; Buchan, Blake W; Tan, Sokha; Stamper, Paul D; Riebe, Katherine M; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V; Trevino, Ernest A; Weissfeld, Alice S; Ledeboer, Nathan A

    2013-12-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype

  14. Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

    PubMed Central

    Buchan, Blake W.; Tan, Sokha; Stamper, Paul D.; Riebe, Katherine M.; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V.; Trevino, Ernest A.; Weissfeld, Alice S.; Ledeboer, Nathan A.

    2013-01-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as

  15. Standard in vitro assays for protein-nucleic acid interactions--gel shift assays for RNA and DNA binding.

    PubMed

    Mitchell, Sarah F; Lorsch, Jon R

    2014-01-01

    The characterization of protein-nucleic acid interactions is necessary for the study of a wide variety of biological processes. One straightforward and widely used approach to this problem is the electrophoretic mobility shift assay (EMSA), in which the binding of a nucleic acid to one or more proteins changes its mobility through a nondenaturing gel matrix. Usually, the mobility of the nucleic acid is reduced, but examples of increased mobility do exist. This type of assay can be used to investigate the affinity of the interaction between the protein and nucleic acid, the specificity of the interaction, the minimal binding site, and the kinetics of the interaction. One particular advantage of EMSA is the ability to analyze multiple proteins, or protein complexes, binding to nucleic acids. This assay is relatively quick and easy and utilizes equipment available in most laboratories; however, there are many variables that can only be determined empirically; therefore, optimization is necessary and can be highly dependent upon the system. The protocol described here is for the poly(A)-binding protein (PABP) binding to an unstructured RNA probe of 43 bases. While this may be a useful protocol for some additional assays, it is recommended that both reaction conditions and gel running conditions be tailored to the individual interaction to be probed. PMID:24674072

  16. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  17. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  18. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  19. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  20. 21 CFR 866.5910 - Quality control material for cystic fibrosis nucleic acid assays.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Test Systems § 866.5910 Quality control material for cystic fibrosis nucleic acid assays. (a... cystic fibrosis nucleic acid assays is a device intended to help monitor reliability of a test system by... testing. This type of device includes recombinant, synthetic, and cell line-based DNA controls....

  1. A Fluorescent Assay for Plant Caffeic Acid O-methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed a facile, sensitive and continuous assay to measure the activities of plant COMTs using s-adenosyl homocysteine hydrolase as a coupling enzyme and and adeonsine a thiol-specific fluor, Thioglo1, as the detecting reagent. This assay was validated using recombinant sorghum COMT (BMR-...

  2. Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates.

    PubMed

    Horne, D W; Patterson, D

    1988-11-01

    Microbiological assay is still widely used for estimating folic acid derivatives in serum and other biological samples. We describe here a modification of this procedure involving use of 96-well microtiter plates. This procedure, used with modern, computer-interfaced microtiter-plate readers and data-reduction software, greatly shortens the time and minimizes reagent costs for this assay. Under the conditions of our assay procedures, all folic acid derivatives tested gave equal growth response for Lactobacillus casei. Results for assays of rat liver extracts showed excellent agreement between the standard bioassay and the 96-well procedure. PMID:3141087

  3. 76 FR 65769 - Airport Improvement Program: Modifications to Benefit Cost Analysis (BCA) Threshold

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-24

    ... benefit cost analyses (BCA) for capacity projects, which was published in the Federal Register. (78 FR... cost analyses (BCA) for capacity projects (78 FR 78798-02, December 16, 2010). This Notice requested.... Issuance of Executive Order No. 12893, ``Principles for Federal Infrastructure Investments,'' 59 FR...

  4. Distinct responses of planktonic foraminiferal B/Ca to dissolution on seafloor

    NASA Astrophysics Data System (ADS)

    Dai, Yuhao; Yu, Jimin; Johnstone, Heather J. H.

    2016-04-01

    We have measured B/Ca in four core-top planktonic foraminiferal species (Globigerinoides ruber (white), Globigerinoides sacculifer (without final sac-like chamber), Neogloboquadrina dutertrei, and Pulleniatina obliquiloculata) from three depth transects (the Caribbean Sea, the southwestern Indian Ocean, and the Ontong Java Plateau) to evaluate the effect of dissolution on planktonic foraminiferal B/Ca. At each transect, G. ruber (w) and G. sacculifer (w/o sac) show decreasing B/Ca with increasing water depth. This decrease in B/Ca is accompanied with decreases in shell weights, Mg/Ca, and bottom water calcite saturation state. This indicates a postdepositional dissolution effect on B/Ca in these two species. The strong correlation observed between changes in B/Ca and bottom water calcite saturation state offers an approach to correcting for the dissolution bias. By contrast, B/Ca in N. dutertrei and P. obliquiloculata remains unchanged along depth transects, although shell weights and Mg/Ca display significant declines. Overall, our core-top results suggest species-specific dissolution effects on B/Ca in different planktonic foraminiferal species.

  5. Development of an assay to simultaneously measure orotic acid, amino acids, and acylcarnitines in dried blood spots

    PubMed Central

    Held, Patrice K.; Haynes, Christopher A.; De Jesús, Víctor R.; Baker, Mei W.

    2016-01-01

    Background Orotic aciduria in the presence of hyperammonemia is a key indicator for a defect in the urea cycle, specifically ornithine transcarbamylase (OTC) deficiency. Current newborn screening (NBS) protocols can detect several defects of the urea cycle, but screening for OTC deficiency remains a challenge due to the lack of a suitable assay. The purpose of this study was to develop a high-throughput assay to measure orotic acid in dried blood spot (DBS) specimens as an indicator for urea cycle dysfunction, which can be readily incorporated into routine NBS. Methods Orotic acid was extracted from DBS punches and analyzed using flow-injection analysis tandem mass spectrometry (FIA–MS/MS) with negative-mode ionization, requiring <2 min/sample run time. This method was then multiplexed into a conventional newborn screening assay for analysis of amino acids, acylcarnitines, and orotic acid. Results We describe 2 assays which can quantify orotic acid in DBS: a stand-alone method and a combined method for analysis of orotic acid, amino acids, and acylcarnitines. Both methods demonstrated orotic acid recovery of 75–85% at multiple levels of enrichment. Precision was also comparable to traditional FIA–MS/MS methods. Analysis of residual presumptively normal NBS specimens demonstrated a 5:1 signal to noise ratio and the average concentration of orotic acid was approximately 1.2 μmol/l. The concentration of amino acids and acylcarnitines as measured by the combined method showed no significant differences when compared to the conventional newborn screening assay. In addition, retrospective analysis of confirmed patients and presumptively normal newborn screening specimens suggests potential for the methods to identify patients with OTC deficiency, as well as other urea cycle defects. Conclusion The assays described here quantify orotic acid in DBS using a simple extraction and FIA–MS/MS analysis procedures that can be implemented into current NBS protocols. PMID

  6. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

    PubMed Central

    Hellman, Lance M.; Fried, Michael G.

    2009-01-01

    The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

  7. Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

    PubMed Central

    Campos, Cristine G.; Borst, Luke

    2013-01-01

    Burkholderia pseudomallei is a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) in B. pseudomallei strain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ to bcaA is Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations of bcaA and bcaB were constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaA and Bp340ΔbcaB mutants to wild-type B. pseudomallei in vitro demonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen. PMID:23340315

  8. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    PubMed

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs. PMID:12517876

  9. Highly accurate boronimeter assay of concentrated boric acid solutions

    SciTech Connect

    Ball, R.M. )

    1992-01-01

    The Random-Walk Boronimeter has successfully been used as an on-line indicator of boric acid concentration in an operating commercial pressurized water reactor. The principle has been adapted for measurement of discrete samples to high accuracy and to concentrations up to 6000 ppm natural boron in light water. Boric acid concentration in an aqueous solution is a necessary measurement in many nuclear power plants, particularly those that use boric acid dissolved in the reactor coolant as a reactivity control system. Other nuclear plants use a high-concentration boric acid solution as a backup shutdown system. Such a shutdown system depends on rapid injection of the solution and frequent surveillance of the fluid to ensure the presence of the neutron absorber. The two methods typically used to measure boric acid are the chemical and the physical methods. The chemical method uses titration to determine the ionic concentration of the BO[sub 3] ions and infers the boron concentration. The physical method uses the attenuation of neutrons by the solution and infers the boron concentration from the neutron absorption properties. This paper describes the Random-Walk Boronimeter configured to measure discrete samples to high accuracy and high concentration.

  10. Seasonal variability in B speciation and B/Ca in planktonic foraminifera from the Cariaco Basin, Venezuela

    NASA Astrophysics Data System (ADS)

    Wejnert, K. E.; Thunell, R.; Bizimis, M.; Pellechia, P. J.; Astor, Y.

    2011-12-01

    We determined B/Ca on four planktonic foraminiferal species (Globigerinoides ruber, Globigerinoides sacculifer, Orbulina universa, and Globorotalia menardii) on a Thermo Element 2 high resolution ICP-MS. The material is from biweekly sediment trap samples collected in Cariaco Basin, Venezuela (10o30' N, 65o31' W) over a three year period between May 2003 and May 2006. The data are compared to local hydrography and water column chemistry to evaluate environmental controls on B incorporation into foraminiferal calcite. In addition, seasonal variability of B speciation in the foraminiferal calcite is assessed using 11B magic angle spinning (MAS) nuclear magnetic resonance (NMR) on samples of O. universa from January, February, and April 2007 and of G. menardii from December 2006. The B/Ca (μmol/mol) data displays clear depth stratification with the surface dwelling G. ruber having the highest B/Ca (202 - 455), followed by G. sacculifer (137 - 335) and the deeper dwelling G. menardii and O. universa having the lowest (102 - 281; 109 - 222 respectively), consistent with a decrease in pH with depth. The data also show a repeatable seasonal pattern with the highest values occurring when the water column is stratified in June and July and the lowest occurring during upwelling in December and January. However, none of the environmental variables have a strong correlation with B/Ca. The 11B MAS NMR O. universa data show a seasonal change in the speciation of B within the foraminiferal calcite. During January the boron is almost entirely incorporated (~ 90%) in a previously unrecognized trigonal form. However, in April only ~75% of the boron is in this trigonal form, whereas the rest of the boron is divided evenly between borate and boric acid. The observed trigonal form has a Cq of 3.0, which is similar to the theoretical value of 3.15 for the corner-sharing borate carbonate complex, B(OH2)CO3-. It is hypothesized that during calcification boron is converted to a borate

  11. Apoptosis Inducing Effects of Kuding Tea Polyphenols in Human Buccal Squamous Cell Carcinoma Cell Line BcaCD885

    PubMed Central

    Zhao, Xin; Pang, Liang; Li, Jing; Song, Jia-Le; Qiu, Li-Hua

    2014-01-01

    Tea polyphenols are functional substances present in tea. Kuding tea as a traditional drink also contains these compounds. After 25, 50 and 100 μg/mL of Kuding tea polyphenol treatment for 48 h, cell proliferation of human buccal squamous cell carcinoma cell line BcaCD885 was inhibited, and the 100 μg/mL of Kuding tea polyphenol showed the highest inhibitory rate at 72.3%. Compared to the lower concentration, the 100 μg/mL of Kuding tea polyphenols significantly (p < 0.05) induced apoptosis as determined by flow cytometry analysis, the content of sub-G1 cancer cells was 32.7%. By RT-PCR and western blot assays, Kuding tea polyphenol significantly induced apoptosis in BcaCD885 cancer cells (p < 0.05) by upregulating caspase-3, caspase-8, caspase-9, Fas/FasL, Bax, p53, p21, E2F1, p73 and downregulating Bcl-2, Bcl-xL, HIAP-1, and HIAP-2 mRNA and protein expressions. Kuding tea polyphenols thus present apoptosis inducing effects in vitro. PMID:25100434

  12. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    PubMed Central

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives. PMID:20228949

  13. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

  14. Identification of new quinic acid derivatives as histone deacetylase inhibitors by fluorescence-based cellular assay.

    PubMed

    Son, Dohyun; Kim, Chung Sub; Lee, Kang Ro; Park, Hyun-Ju

    2016-05-01

    A fluorescence-based cellular assay system was established to identify potential epigenetic modulator ligands. This assay method is to detect the de-repression of an EGFP reporter in cancer cells by the treatment of HDAC (histone deacetylase) or DNMT (DNA methyltransferase) inhibitor. Using this system, we conducted a preliminary screening of in-house natural product library containing extracts and pure compounds, and identified several active compounds. Among them, novel quinic acid derivatives were recognized as excellent HDAC inhibitors by both enzymatic and cell-based HDAC assays. PMID:26996372

  15. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids.

    PubMed

    Wang, Yuhong; Nugen, Sam R

    2013-10-01

    The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)3 (2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)3 (2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used. PMID:23525961

  16. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate.

    PubMed

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F; Zhang, Kate

    2015-06-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann-Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann-Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  17. Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

    PubMed Central

    Chuang, Wei-Lien; Pacheco, Joshua; Cooper, Samantha; Kingsbury, Jonathan S.; Hinds, John; Wolf, Pavlina; Oliva, Petra; Keutzer, Joan; Cox, Gerald F.; Zhang, Kate

    2015-01-01

    Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls. PMID:26937397

  18. Development and Evaluation of a Calibrator Material for Nucleic Acid-Based Assays for Diagnosing Aspergillosis

    PubMed Central

    Abdul-Ali, Deborah; Loeffler, Juergen; White, P. Lewis; Wickes, Brian; Herrera, Monica L.; Alexander, Barbara D.; Baden, Lindsey R.; Clancy, Cornelius; Denning, David; Nguyen, M. Hong; Sugrue, Michele; Wheat, L. Joseph; Wingard, John R.; Donnelly, J. Peter; Barnes, Rosemary; Patterson, Thomas F.; Caliendo, Angela M.

    2013-01-01

    Twelve laboratories evaluated candidate material for an Aspergillus DNA calibrator. The DNA material was quantified using limiting-dilution analysis; the mean concentration was determined to be 1.73 × 1010 units/ml. The calibrator can be used to standardize aspergillosis diagnostic assays which detect and/or quantify nucleic acid. PMID:23616459

  19. Analysis of Citric Acid in Beverages: Use of an Indicator Displacement Assay

    ERIC Educational Resources Information Center

    Umali, Alona P.; Anslyn, Eric V.; Wright, Aaron T.; Blieden, Clifford R.; Smith, Carolyne K.; Tian, Tian; Truong, Jennifer A.; Crumm, Caitlin E.; Garcia, Jorge E.; Lee, Soal; Mosier, Meredith; Nguyen, Chester P.

    2010-01-01

    The use of an indicator displacement assay permits the visualization of binding events between host and guest molecules. An undergraduate laboratory experiment is described to demonstrate the technique in the determination of citric acid content in commercially available beverages such as soda pop and fruit juices. Through the technique, students…

  20. Biological Monitoring of 3-Phenoxybenzoic Acid in Urine by an Enzyme -Linked Immunosorbent Assay

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6...

  1. Response to oxalic acid as a resistance assay for Sclerotinia minor in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Response to oxalic acid was evaluated as a potential assay for screening peanut breeding lines for resistance to Sclerotinia blight caused by Sclerotinia minor. Detached stems of seven Spanish- and six runner-type peanut cultivars and advanced breeding lines, varying in resistance to Sclerotinia bl...

  2. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  3. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  4. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  5. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  6. 21 CFR 866.3980 - Respiratory viral panel multiplex nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... detection and identification of a combination of the following viruses: (1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory... Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates...

  7. Molecular Cloning and Characterization of the srdBCA Operon, Encoding the Respiratory Selenate Reductase Complex, from the Selenate-Reducing Bacterium Bacillus selenatarsenatis SF-1▿†

    PubMed Central

    Kuroda, Masashi; Yamashita, Mitsuo; Miwa, Emiko; Imao, Kanako; Fujimoto, Noriyuki; Ono, Hisayo; Nagano, Kouta; Sei, Kazunari; Ike, Michihiko

    2011-01-01

    Previously, we isolated a selenate- and arsenate-reducing bacterium, designated strain SF-1, from selenium-contaminated sediment and identified it as a novel species, Bacillus selenatarsenatis. B. selenatarsenatis strain SF-1 independently reduces selenate to selenite, arsenate to arsenite, and nitrate to nitrite by anaerobic respiration. To identify the genes involved in selenate reduction, 17 selenate reduction-defective mutant strains were isolated from a mutant library generated by random insertion of transposon Tn916. Tn916 was inserted into the same genome position in eight mutants, and the representative strain SF-1AM4 did not reduce selenate but did reduce nitrate and arsenate to the same extent as the wild-type strain. The disrupted gene was located in an operon composed of three genes designated srdBCA, which were predicted to encode a putative oxidoreductase complex by the BLASTX program. The plasmid vector pGEMsrdBCA, containing the srdBCA operon with its own promoter, conferred the phenotype of selenate reduction in Escherichia coli DH5α, although E. coli strains containing plasmids lacking any one or two of the open reading frames from srdBCA did not exhibit the selenate-reducing phenotype. Domain structure analysis of the deduced amino acid sequence revealed that SrdBCA had typical features of membrane-bound and molybdopterin-containing oxidoreductases. It was therefore proposed that the srdBCA operon encoded a respiratory selenate reductase complex. This is the first report of genes encoding selenate reductase in Gram-positive bacteria. PMID:21357486

  8. Real-time quantification of fatty acid uptake using a novel fluorescence assay.

    PubMed

    Liao, Jinfang; Sportsman, Richard; Harris, Jeff; Stahl, Andreas

    2005-03-01

    Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery. PMID:15547301

  9. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification

    PubMed Central

    Saulnier-Blache, Jean Sébastien; Girard, Alexia; Simon, Marie-Françoise; Lafontan, Max; Valet, Philippe

    2000-01-01

    The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat lysophosphatidic acid acyl transferase (LPAAT) produced in Escherichia coli was used. In the presence of [14C]oleoyl CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [14C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmoles with a minimal detection of 0.2 pmoles. This method was used to quantify LPA in butanol- extracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids. PMID:11108727

  10. The uronic acids assay: a method for the determination of chemical activity on biofilm EPS.

    PubMed

    Mojica, Kristina D A; Cooney, Michael J

    2010-01-01

    In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS. PMID:20087802

  11. A facile assay to monitor secretory phospholipase A₂ using 8-anilino-1-naphthalenesulfonic acid.

    PubMed

    Vivek, Hamse K; Swamy, Supritha G; Priya, Babu S; Sethi, Gautam; Rangappa, Kanchugarakoppal S; Swamy, S Nanjunda

    2014-09-15

    Secretory phospholipases A2 (sPLA2s) are present in snake venoms, serum, and biological fluids of patients with various inflammatory, autoimmune and allergic disorders. Lipid mediators in the inflammatory processes have potential value for controlling phospholipid metabolism through sPLA2 inhibition. Thus, it demands the need for screening of potential leads for sPLA2 inhibition. To date, sPLA2 activity has been assayed using expensive radioactive or chromogenic substrates, thereby limiting a large number of assays. In this study, a simple and sensitive NanoDrop assay was developed using non-fluorogenic and non-chromogenic phospholipid substrate 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 8-anilino-1-naphthalenesulfonic acid (ANS) as interfacial hydrophobic probe. The modified assay required a 10ng concentration of sPLA2. ANS, as a strong anion, binds predominantly to cationic group of choline head of DMPC through ion pair formation, imparting hydrophobicity and lipophilicity and resulting in an increase in fluorescence. Triton X-100 imparts correct geometrical space during sPLA2 catalyzing DMPC, releasing lysophospholipid and acidic myristoyl acid, which in turn alters the hydrophobic environment prevailing around ANS-DMPC, which leads to weakening of the electrostatic ion pair interaction between DMPC and ANS ensuing decrease in fluorescence. These characteristic fluorescence changes between DMPC and ANS in response to sPLA2 catalysis are well documented and validated in this study. PMID:24915638

  12. Integrated sample-to-detection chip for nucleic acid test assays.

    PubMed

    Prakash, R; Pabbaraju, K; Wong, S; Tellier, R; Kaler, K V I S

    2016-06-01

    Nucleic acid based diagnostic techniques are routinely used for the detection of infectious agents. Most of these assays rely on nucleic acid extraction platforms for the extraction and purification of nucleic acids and a separate real-time PCR platform for quantitative nucleic acid amplification tests (NATs). Several microfluidic lab on chip (LOC) technologies have been developed, where mechanical and chemical methods are used for the extraction and purification of nucleic acids. Microfluidic technologies have also been effectively utilized for chip based real-time PCR assays. However, there are few examples of microfluidic systems which have successfully integrated these two key processes. In this study, we have implemented an electro-actuation based LOC micro-device that leverages multi-frequency actuation of samples and reagents droplets for chip based nucleic acid extraction and real-time, reverse transcription (RT) PCR (qRT-PCR) amplification from clinical samples. Our prototype micro-device combines chemical lysis with electric field assisted isolation of nucleic acid in a four channel parallel processing scheme. Furthermore, a four channel parallel qRT-PCR amplification and detection assay is integrated to deliver the sample-to-detection NAT chip. The NAT chip combines dielectrophoresis and electrostatic/electrowetting actuation methods with resistive micro-heaters and temperature sensors to perform chip based integrated NATs. The two chip modules have been validated using different panels of clinical samples and their performance compared with standard platforms. This study has established that our integrated NAT chip system has a sensitivity and specificity comparable to that of the standard platforms while providing up to 10 fold reduction in sample/reagent volumes. PMID:27165104

  13. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  14. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  15. Rapid Sensitive Assay for Interferons Based on the Inhibition of MM Virus Nucleic Acid Synthesis

    PubMed Central

    Allen, Patton T.; Giron, David J.

    1970-01-01

    A method for assaying mouse interferon based on the inhibition of viral ribonucleic acid (RNA) synthesis was devised. The amount of MM virus and RNA synthesized in interferon-treated L-cell cultures was determined by measuring the amount of 3H-uridine converted into a trichloroacetic acid-insoluble form after treatment of the infected cultures with 2.5 μg of actinomycin D per ml. The amount of RNA synthesized was inversely related to the concentration of interferon used for treatment. A linear dose-response regression curve was obtained by plotting the log of the amount of RNA made, expressed as a percentage of the control, versus the log of the reciprocal of the interferon dilution. A unit of interferon was defined as that concentration which inhibited nucleic acid synthesis by 50% (INAS50). The concentration of mouse interferon could be determined within 24 hr. This assay method, on the average, was approximately half as sensitive as the method which measured the 50% reduction of MM virus plaque number (PDD50-MM method), but was, on the average, almost 1.7 times as sensitive as the PDD50-VSV method. It averaged approximately 20 times the sensitivity of the methods which used as end points the 70% reduction in yield of MM virus or the complete inhibition of cytopathic effect by MM virus. The reproducibility of the INAS50 technique was tested in two ways. (i) Four independent assays of an interferon specimen were performed with replicate cultures. The standard deviation was 11.2% of the mean titer. (ii) On different dates, one interferon specimen was assayed seven times and another was assayed four times. The standard deviations were 21.5 and 26.6% of the respective mean titers. PMID:4320919

  16. Final Report Nucleic Acid System - PCR, Multiplex Assays and Sample Preparation Project

    SciTech Connect

    Koopman, R.P.; Langlois, R.G.; Nasarabadi, S.; Benett, W.J.; Richards, J.B.; Hadley, D.R.; Miles, R.R.; Brown, S.B.; Stratton, P.L.; Milanovich, F.P.

    2001-04-20

    The objective of this project was to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction). This entailed not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This project had two principal deliverables: (1) design, construct, test and deliver a 24 chamber, multiplex capable suitcase sized PCR instrument, and (2) develop and reduce to practice a multiplex assay for the detection of PCR product by flow cytometry. In addition, significant resources were allocated to test and evaluation of the Hand-held Advanced Nucleic Acid Analyzer (HANAA). This project helps provide the signature and intelligence gathering community the ability to perform, on-site or remote, rapid analysis of environmental or like samples for the presence of a suite of biological warfare pathogens.

  17. A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

    PubMed Central

    Eggleston, A K; Rahim, N A; Kowalczykowski, S C

    1996-01-01

    We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID:8614617

  18. In situ screening assay for cell viability using a dimeric cyanine nucleic acid stain.

    PubMed

    Becker, B; Clapper, J; Harkins, K R; Olson, J A

    1994-08-15

    A rapid and sensitive assay is described for the determination of cell viability of adherent and nonadherent cells that can be performed in situ in 96-well microtiter plates using fluorescence plate scanners. The assay, based on dye exclusion, utilizes a plasma membrane-impermeable, dimeric cyanine dye (YOYO-1). YOYO-1 fluoresces brightly only when bound to nucleic acids. Cells are incubated with YOYO-1, and fluorescence is measured before and after the addition of detergent, which allows the dye to enter the cells. The fluorescence before detergent treatment originates from nonviable cells that have membrane damage and take up YOYO-1. The fluorescence after detergent treatment originates from all cells in the sample. The ratio of the two fluorescence values is used as an indicator of cell viability. The cell viability results of this microplate assay closely resemble those of dye exclusion studies by flow cytometry and are similar but not identical to those of the thiazolyl blue assay, which uses a metabolic indicator of cell death. Because the assay can be performed in situ, without removing the medium, disintegrated cells, cell aggregates, and cells that stick to culture vessel walls are all included in the measurement. PMID:7527190

  19. Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.

    PubMed

    Batchelor, Robert H; Sarkez, Adam; Cox, W Gregory; Johnson, Iain

    2007-10-01

    As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9. PMID:18019342

  20. Determination of boron in produced water using the carminic acid assay.

    PubMed

    Floquet, Cedric F A; Sieben, Vincent J; MacKay, Bruce A; Mostowfi, Farshid

    2016-04-01

    Using the carminic acid assay, we determined the concentration of boron in oilfield waters. We investigated the effect of high concentrations of salts and dissolved metals on the assay performance. The influence of temperature, development time, reagent concentration, and water volume was studied. Ten produced and flowback water samples of different origins were measured, and the method was successfully validated against ICP-MS measurements. In water-stressed regions, produced water is a potential source of fresh water for irrigation, industrial applications, or consumption. Therefore, boron concentration must be determined and controlled to match the envisaged waste water reuse. Fast, precise, and onsite measurements are needed to minimize errors introduced by sample transportation to laboratories. We found that the optimum conditions for our application were a 5:1 mixing volume ratio (reagent to sample), a 1 g L(-1) carminic acid concentration in 99.99% sulfuric acid, and a 30 min reaction time at ambient temperature (20 °C to 23 °C). Absorption values were best measured at 610 nm and 630 nm and baseline corrected at 865 nm. Under these conditions, the sensitivity of the assay to boron was maximized while its cross-sensitivity to dissolved titanium, iron, barium and zirconium was minimized, alleviating the need for masking agents and extraction methods. PMID:26838405

  1. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR ISOCUPRESSIC ACID AND SERUM METABOLITES OF ISOCUPRESSIC ACID

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid as the primary abortifacient compound in these ...

  2. Quantification of proteins using enhanced etching of Ag coated Au nanorods by the Cu2+/bicinchoninic acid pair with improved sensitivity

    NASA Astrophysics Data System (ADS)

    Liu, Wenqi; Hou, Shuai; Yan, Jiao; Zhang, Hui; Ji, Yinglu; Wu, Xiaochun

    2015-12-01

    Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical simulation shows that Au nanorods with large aspect ratios and small core sizes show high detection sensitivity. Via optimized sensor design, we achieved an increased sensitivity (the limit of detection was 3.4 ng ml-1) and a wide working range (0.5 to 1000 μg ml-1) compared with the traditional BCA assay. The universal applicability of our method to various proteins further proves its potential in practical applications.Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu2+. The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu2+/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu2+ and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical

  3. A fluorometric assay platform for caffeic acid detection based on the G-quadruplex/hemin DNAzyme.

    PubMed

    Cai, Nan; Li, Yan; Chen, Shufan; Su, Xingguang

    2016-07-21

    In this paper, a fluorometric assay platform for fluorescence detection of caffeic acid was designed based on the peroxidase-mimicking activities of G-quadruplex/hemin DNAzyme. Under the catalysis of the formed G-quadruplex/hemin complex, H2O2 could be decomposed into hydroxyl radicals with strong oxidation properties. Then caffeic acid would be oxidized by the released hydroxyl radicals, resulting in the product caffeic acid-quinone. Normally, caffeic acid has no influence on the fluorescence of graphene quantum dots. But when mixed with the G-quadruplex/hemin complex and H2O2, the fluorescence of graphene quantum dots was obviously quenched by the oxidized caffeic acid. Under the optimized experimental conditions, the quenched fluorescence intensity was linearly correlated with the concentration of caffeic acid, ranging from 2 μM to 350 μM with a detection limit of 200 nM. The proposed method was applied to the determination of caffeic acid in human serum samples with satisfactory results. PMID:27220084

  4. Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

    SciTech Connect

    Koopman, R P; Langlois, R G; Nasarabadi, S; Benett, W J; Colston, B W; Johnson, D C; Brown, S B; Stratton, P L; Milanovich, F P

    2002-04-17

    This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flow cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.

  5. Identification and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

    PubMed Central

    Mukherjee, Sourav; Hanson, Alicia M.; Shadrick, William R.; Ndjomou, Jean; Sweeney, Noreena L.; Hernandez, John J.; Bartczak, Diana; Li, Kelin; Frankowski, Kevin J.; Heck, Julie A.; Arnold, Leggy A.; Schoenen, Frank J.; Frick, David N.

    2012-01-01

    Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC50 = 1.4 μM), suramin sodium salt (IC50 = 3.6 μM), NF 023 hydrate (IC50 = 6.2 μM) and tyrphostin AG 538 (IC50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors. PMID:22740655

  6. Rapid turbidimetric assay for quantification of fusidic acid in a dermatological cream.

    PubMed

    Curbete, Mariane Machado; Salgado, Hérida Regina Nunes

    2016-06-01

    Fusidic acid is an antibiotic steroid widely used for the treatment of serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains. Microbiological methods are indispensable to determine the mean percentage of antimicrobial in medicaments during manufacturing and quality control processes. The aim of this study was to develop and validate a microbiological method for the quantification of fusidic acid in dermatological cream by turbidimetry, using Staphylococcus epidermidis (ATCC 12228) and casoy broth as the culture medium. The validation parameters were in accordance with ICH specifications and demonstrated accuracy, precision, selectivity, and robustness, with linear ranges from 0.25 to 2.25μgmL(-1). This method is an alternative to the diffusion agar assay currently employed to quantify fusidic acid in dermatological cream, since it is sensitive, fast, and more economical. PMID:27130089

  7. Degradation of caffeic acid in subcritical water and online HPLC-DPPH assay of degradation products.

    PubMed

    Khuwijitjaru, Pramote; Suaylam, Boonyanuch; Adachi, Shuji

    2014-02-26

    Caffeic acid was subjected to degradation under subcritical water conditions within 160-240 °C and at a constant pressure of 5 MPa in a continuous tubular reactor. Caffeic acid degraded quickly at these temperatures; the main products identified by liquid chromatography-diode array detection/mass spectrometry were hydroxytyrosol, protocatechuic aldehyde, and 4-vinylcatechol. The reaction rates for the degradation of caffeic acid and the formation of products were evaluated. Online high-performance liquid chromatography/2,2-diphenyl-1-picryhydrazyl assay was used to determine the antioxidant activity of each product in the solution. It was found that the overall antioxidant activity of the treated solution did not change during the degradation process. This study showed a potential of formation of antioxidants from natural phenolic compounds under these subcritical water conditions, and this may lead to a discovering of novel antioxidants compounds during the extraction by this technique. PMID:24483598

  8. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    PubMed

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. PMID:27544648

  9. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum

    NASA Astrophysics Data System (ADS)

    Das, Jagotamoy; Ivanov, Ivaylo; Montermini, Laura; Rak, Janusz; Sargent, Edward H.; Kelley, Shana O.

    2015-07-01

    The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

  10. Norms and Standards for Computer Education (MCA, BCA) through Distance Mode.

    ERIC Educational Resources Information Center

    Rausaria, R.R., Ed.; Lele, Nalini A., Ed.; Bhushan, Bharat, Ed.

    This document presents the norms and standards for computer education in India through distance mode, including the Masters in Computer Applications (MCA) and Bachelor in Computer Applications (BCA) programs. These norms and standards were considered and approved by the Distance Education Council, Indira Gandhi National Open University (India), at…

  11. B/Ca in planktonic foraminifera as a proxy for surface seawater pH

    NASA Astrophysics Data System (ADS)

    Yu, Jimin; Elderfield, Henry; HöNisch, BäRbel

    2007-06-01

    Boron isotope systematics indicate that boron incorporation into foraminiferal CaCO3 is determined by the partition coefficient, KD (= ?), and [B(OH)4-/HCO3-]seawater, providing, in principle, a method to estimate seawater pH and PCO2. We have measured B/Ca ratios in Globigerina bulloides and Globorotaliainflata for a series of core tops from the North Atlantic and the Southern Ocean and in Globigerinoides ruber (white) from Ocean Drilling Program (ODP) site 668B on the Sierra Leone Rise in the eastern equatorial Atlantic. B/Ca ratios in these species of planktonic foraminifera seem unaffected by dissolution on the seafloor. KD shows a strong species-specific dependence on calcification temperature, which can be corrected for using the Mg/Ca temperature proxy. A preliminary study of G. inflata from Southern Ocean sediment core CHAT 16K suggests that temperature-corrected B/Ca was ˜30% higher during the last glacial. Correspondingly, pH was 0.15 units higher and aqueous PCO2 was 95 μatm lower at this site at the Last Glacial Maximum. The covariation between reconstructed PCO2 and the atmospheric pCO2 from the Vostok ice core demonstrates the feasibility of using B/Ca in planktonic foraminifera for reconstructing past variations in pH and PCO2.

  12. Gamma aminobutyric acid radioreceptor-assay a possible biomarker for human exposure to certain agrochemicals.

    PubMed

    Saleh, M A; Abou Zied, M; el-Baroty, G; Abdel-Reheim, E; Abdel-Rahman, F; Wallace, C; el-Sebae, A H; Blancato, J N

    1993-12-01

    Cyclodiene insecticides, hexachlorocyclohexanes, pyrethroids, bicyclophosphates, the bicycloorthocarboxylate insecticides and some of their metabolites and environmental degradation products are central nervous system toxicants with high specific binding affinity to the chloride channel of the gamma-aminobutyric acid (GABA)A receptor-ionophore sites. [35S] tertiary-butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABAA receptor for the development of a radioreceptor assay technique. The GABA receptor was prepared by ultra centrifugation and dialysis of brain homogenates of either cow, goat, rat or catfish. The receptor was then labeled with [35S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with samples containing the analytes. A radioreceptor assay protocol was developed to measure the amount of the alpha-endosulfan in blood samples. The assay was extremely sensitive, and can detect 0.2 nM of endosulfan at a level equivalent to 0.08 ppb or 8 x 10(-11) gm of endosulfan in each ml of the blood samples. PMID:8270763

  13. Antioxidant assay-guided purification and LC determination of ellagic acid in pomegranate peel.

    PubMed

    Panichayupakarananta, Pharkphoom; Issuriya, Atcharaporn; Sirikatitham, Anusak; Wang, Wei

    2010-07-01

    On the basis of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay-guided purification, ellagic acid was isolated from the methanol extract of pomegranate fruit peel by liquid-liquid extraction and chromatographic techniques. A reversed-phase high-performance liquid chromatography was described for determination of ellagic acid in pomegranate fruit peel extract. The method involved the use of a TSK-gel ODS-80Tm column with a mixture of 2% aqueous acetic acid and methanol (gradient elution mode: 0-15 min, 40-60% v/v methanol and 15-20 min, 60% v/v methanol) as the mobile phase and detection at 254 nm. The parameters of linearity, repeatability, reproducibility, accuracy, and specificity of the method were evaluated. The recovery of the method was 98.5% and linearity (r(2) > 0.9995) was obtained for ellagic acid. A high degree of specificity as well as repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved. The limits of detection and quantification were 1.00 and 2.50 microg/mL, respectively. The solvent for extraction of ellagic acid from pomegranate fruit peel was examined in order to maximize the ellagic acid content of the extract. A solution of 10% v/v water in methanol was capable of increasing the ellagic acid content in the extract up to 7.66% w/w. The ellagic acid content and antioxidant activity of the ethyl acetate fraction separated from the crude extract using water and ethyl acetate partition was higher than that of the crude extract. PMID:20822660

  14. QUANTIFICATION OF GLIAL FIBRILLARY ACIDIC PROTEIN: COMPARISON OF SLOT-IMMUNOBINDING ASSAYS WITH A NOVEL SANDWICH ELISA

    EPA Science Inventory

    Detailed protocols are presented for assaying glial fibrillary acidic protein (GFAP), an astrocyte localized protein rich serves as a quantitative marker of toxicant- induced injury to the central nervous system. wo different solid-phase assay procedures are described: 1) a nitro...

  15. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae.

    PubMed

    Griffiths, M J; van Hille, R P; Harrison, S T L

    2010-11-01

    Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae. PMID:20820931

  16. Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses.

    PubMed

    Lanciotti, R S; Kerst, A J

    2001-12-01

    The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States. PMID:11724870

  17. Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C.

    PubMed Central

    Barth, P T; Grinter, N J

    1975-01-01

    We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA. The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates. We also investigated various parameters in making this measurement. Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected. The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible. PMID:234416

  18. Validation of a UV Spectrometric Method for the Assay of Tolfenamic Acid in Organic Solvents

    PubMed Central

    Ahmed, Sofia; Mustaan, Nafeesa; Sheraz, Muhammad Ali; Nabi, Syeda Ayesha Ahmed un; Ahmad, Iqbal

    2015-01-01

    The present study has been carried out to validate a UV spectrometric method for the assay of tolfenamic acid (TA) in organic solvents. TA is insoluble in water; therefore, a total of thirteen commonly used organic solvents have been selected in which the drug is soluble. Fresh stock solutions of TA in each solvent in a concentration of 1 × 10−4 M (2.62 mg%) were prepared for the assay. The method has been validated according to the guideline of International Conference on Harmonization and parameters like linearity, range, accuracy, precision, sensitivity, and robustness have been studied. Although the method was found to be efficient for the determination of TA in all solvents on the basis of statistical data 1-octanol, followed by ethanol and methanol, was found to be comparatively better than the other studied solvents. No change in the stock solution stability of TA has been observed in each solvent for 24 hours stored either at room (25 ± 1°C) or at refrigerated temperature (2–8°C). A shift in the absorption maxima has been observed for TA in various solvents indicating drug-solvent interactions. The studied method is simple, rapid, economical, accurate, and precise for the assay of TA in different organic solvents. PMID:26783497

  19. Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins.

    PubMed

    Cao, Ya; Chen, Weiwei; Han, Peng; Wang, Zhuxin; Li, Genxi

    2015-08-26

    In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500 pM with a detection limit of 0.57 pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis. PMID:26347164

  20. Paper-based fluorescence resonance energy transfer assay for directly detecting nucleic acids and proteins.

    PubMed

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

    2016-06-15

    Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis. PMID:26807518

  1. A rapid assay for affinity and kinetics of molecular interactions with nucleic acids.

    PubMed

    Donaldson, Gregory P; Roelofs, Kevin G; Luo, Yiling; Sintim, Herman O; Lee, Vincent T

    2012-04-01

    The Differential Radial Capillary Action of Ligand Assay (DRaCALA) allows detection of protein interactions with low-molecular weight ligands based on separation of the protein-ligand complex by differential capillary action. Here, we present an application of DRaCALA to the study of nucleic acid-protein interactions using the Escherichia coli cyclic AMP receptor protein (CRP). CRP bound in DRaCALA specifically to (32)P-labeled oligonucleotides containing the consensus CRP binding site, but not to oligonucleotides with point mutations known to abrogate binding. Affinity and kinetic studies using DRaCALA yielded a dissociation constant and dissociation rate similar to previously reported values. Because DRaCALA is not subject to ligand size restrictions, whole plasmids with a single CRP-binding site were used as probes, yielding similar results. DNA can also function as an easily labeled carrier molecule for a conjugated ligand. Sequestration of biotinylated nucleic acids by streptavidin allowed nucleic acids to take the place of the protein as the immobile binding partner. Therefore, any molecular interactions involving nucleic acids can be tested. We demonstrate this principle utilizing a bacterial riboswitch that binds cyclic-di-guanosine monophosphate. DRaCALA is a flexible and complementary approach to other biochemical methods for rapid and accurate measurements of affinity and kinetics at near-equilibrium conditions. PMID:22210888

  2. Properties of kojic acid and curcumin: Assay on cell B16-F1

    NASA Astrophysics Data System (ADS)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  3. An enzymic assay for uric acid in serum and urine compared with HPLC.

    PubMed

    Dubois, H; Delvoux, B; Ehrhardt, V; Greiling, H

    1989-03-01

    We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively. PMID:2708944

  4. Continuous colorimetric screening assays for the detection of specific L- or D-α-amino acid transaminases in enzyme libraries.

    PubMed

    Heuson, Egon; Petit, Jean-Louis; Debard, Adrien; Job, Aurélie; Charmantray, Franck; de Berardinis, Véronique; Gefflaut, Thierry

    2016-01-01

    In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of L- or D-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays' quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since L-CSA and D-CSA allowed the exclusive detection of L-TA and D-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable L- or D-homophenylalanine. PMID:26452497

  5. Uric acid photo-oxidation assay: in vitro comparison of sunscreening agents.

    PubMed

    Dunlap, W C; Yamamoto, Y; Inoue, M; Kashiba-Iwatsuki, M; Yamaguchi, M; Tomita, K

    1998-02-01

    We present a new method to evaluate the photo-oxidative activity of sunscreening agents based on the photodynamic oxidation of uric acid. Uric acid was selected as the oxidant probe for its high reactivity to singlet oxygen and oxygen radicals, high sensitivity of detection using electrochemical (EC) techniques, low light absorptivity and high photochemical stability in the UVA/B region of interest, and stability to autoxidation. The method is demonstrated by the photodynamic oxidation of uric acid on co-irradiation with Rose Bengal, a highly efficient photosensitizing dye for the production of singlet oxygen (1O2). Using this assay we found that the relative photodynamic oxidation rates of UVB-absorbing sunscreens in 80% methanol on irradiation with >290 nm light decreased in the order 2-ethylhexyl 4-dimethylaminobenzoate (DMABA-2EH) > 2-ethylhexyl 4-methoxycinnamate (MCA-2EH) and the experimental sunscreens, 1-(1,1-dimethylethyl)-3-octanoyl-4,4-dimethyl- 1,4,5,6,-tetrahydropyridine (ICI-319) and 1-(2-methylpropyl)-3-propionyl-4,4-dimethyl-1,4,5,6-tetrahydropyridine (ICI-855). The relative photodynamic oxidation rates of UVA-absorbing sunscreens decreased in the order 4-tert-butyl-4'-methoxydibenzoylmethane (BMDBM) and 4-(2-propyl)benzophenone (PB) > 2-hydroxy-4'-methoxy-benzophenone (HMB) and 2,2'-dihydroxy-4-methoxybenzophenone (DHMB). We have confirmed the photodynamic activity of DMABA-2EH for the production of singlet oxygen (1O2) using electron paramagnetic resonance (EPR) spectroscopy and the reagent 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP). We failed to detect the photodynamic production of the oxyradicals, superoxide (O2.-) and hydroxyl radical (HO.) using N-tert-butyl-a-phenylnitrone (PBN) and 5,5-dimethyl-1-pyrrolidine-1-oxide (DMPO) as a result of photochemical interference caused by these spin-trapping reagents. The uric acid photo-oxidation assay was also used to compare the photodynamic reactivity of light-reflective, microfine oxides TiO2, Zn

  6. Pantothenic acid quantification by a stable isotope dilution assay based on liquid chromatography-tandem mass spectrometry.

    PubMed

    Rychlik, Michael

    2003-07-01

    A stable isotope dilution assay for the quantification of free and total pantothenic acid has been developed by using [13C3,15N]-pantothenic acid as the internal standard. The three-dimensional specificity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the vitamin. Due to the very simple extraction and clean-up procedure, free pantothenic acid could be analysed within 2 h, which is much faster than by microbiological or gas chromatographic assays. For quantification of total pantothenic acid, the vitamin was liberated from its conjugates by an overnight incubation with pigeon liver pantetheinase and alkaline phosphatase. In analyses of corn flour, the intra-assay coefficient of variation was 8.5% (n = 5) and 15.3% (n = 4) for free and total pantothenic acid, respectively. When pantothenic acid was added to corn starch at a level of 6 mg kg(-1), a recovery of 97.5% was found. Application of the stable isotope dilution assay to whole egg powder, hazel nuts and corn revealed similar data compared to those listed in nutrition data bases, whereas the content in mushrooms and porcine liver determined by the newly developed assay appeared to be lower and that of cocoa higher than reported in the literature. PMID:12894818

  7. Toward better understanding of B/Ca and δ11B proxies: An experimental approach

    NASA Astrophysics Data System (ADS)

    Uchikawa, J.; Penman, D. E.; Harper, D. T.; Farmer, J. R.; Zachos, J. C.; Hoenisch, B.; Zeebe, R. E.

    2015-12-01

    The abundance and isotopic composition of boron (B/Ca and δ11B) in marine biogenic carbonates is an important paleoceanographic tool to probe carbon cycling in the ocean-atmosphere system. These B-based proxies rely on a fundamental assumption that boron incorporation into carbonates occur via B(OH)4- adsorption with little isotopic fractionation, which is based on key results from the classic inorganic experiments performed in the late 1990s (e.g., Hemming et al., 1995, GCA, v59, 371-379; Sanyal et al., 2000, GCA, v64, 1551-1555). However, a collection of new experimental data published in recent years consistently suggests a more complicated picture for fluid-crystal element and isotope partitioning of B into inorganic carbonates. For instance, we performed novel inorganic calcite precipitation experiments by systematically adjusting solution pH as well as total B, total DIC and Ca concentrations (Uchikawa et al., 2015, GCA, v150, 171-191), and the results showcased apparent kinetic effects related to precipitation rate on B/Ca. Moreover, the results also indicated a dependence of B/Ca on the concentration ratio of total B to total DIC, which was interpreted as indirect evidence for potential B(OH)3 incorporation into synthetic calcite. Notably, relatively simple solutions of NaCl-CaCl2-B(OH)3 system were used for our previous experiments. This presentation features our latest results from similar experiments but using artificial seawater in order to close the gap between simplified experimental conditions to in-situ marine settings. Our preliminary results reveal a precipitation rate control even when artificial seawater is used for the experiments, making a strong case that kinetic effects on B/Ca are universal in inorganic carbonates. With the aid of new isotopic results, we also attempt to discuss possible scenarios of B incorporation pathway in inorganic systems.

  8. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay.

    PubMed

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  9. Urinary Excretion of Phenolic Acids by Infants and Children: A Randomised Double-Blind Clinical Assay

    PubMed Central

    Uberos, J.; Fernández-Puentes, V.; Molina-Oya, M.; Rodríguez-Belmonte, R.; Ruíz-López, A.; Tortosa-Pinto, P.; Molina-Carballo, A.; Muñoz-Hoyos, A.

    2012-01-01

    Objectives: The present study, which is part of the ISRCTN16968287 clinical assay, is aimed at determining the effects of cranberry syrup or trimethoprim treatment for UTI. Methods: This Phase III randomised clinical trial was conducted at the San Cecilio Clinical Hospital (Granada, Spain) with a study population of 192 patients, aged between 1 month and 13 years. Criteria for inclusion were a background of recurrent UTI, associated or otherwise with vesico-ureteral reflux of any degree, or renal pelvic dilatation associated with urinary infection. Each child was randomly given 0.2 mL/Kg/day of either cranberry syrup or trimethoprim (8 mg/mL). The primary and secondary objectives, respectively, were to determine the risk of UTI and the levels of phenolic acids in urine associated with each intervention. Results: With respect to UTI, the cranberry treatment was non-inferior to trimethoprim. Increased urinary excretion of ferulic acid was associated with a greater risk of UTI developing in infants aged under 1 year (RR 1.06; CI 95% 1.024–1.1; P = 0.001). Conclusions: The results obtained show the excretion of ferulic acid is higher in infants aged under 1 year, giving rise to an increased risk of UTI, for both treatment options. PMID:23641168

  10. Genotoxic effects of boric acid and borax in zebrafish, Danio rerio using alkaline comet assay

    PubMed Central

    Gülsoy, Nagihan; Yavas, Cüneyd; Mutlu, Özal

    2015-01-01

    The present study is conducted to determine the potential mechanisms of Boron compounds, boric acid (BA) and borax (BX), on genotoxicity of zebrafish Danio rerio for 24, 48, 72 and 96-hours acute exposure (level:1, 4, 16, 64 mg/l BA and BX) in semi-static bioassay experiment. For that purpose, peripheral erythrocytes were drawn from caudal vein and Comet assay was applied to assess genotoxicity. Acute (96 hours) exposure and high concentrations of boric acid and borax increases % tail DNA and Olive tail moment. Genotoxicity was found for BA as concentration-dependent and BX as concentration and time dependent manner. In general, significant effects (P < 0,05) on both concentrations and exposure times were observed in experimental groups. DNA damage was highest at 96 h and 24 h for all BX and BA concentrations, respectively in peripheral blood of D. rerio. For the first time, our study demonstrates the effect of waterborne BA and BX exposure on genotoxicity at the molecular level, which may contribute to understanding the mechanism of boric acid and borax-induced genotoxicity in fish. PMID:26862320

  11. Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer

    PubMed Central

    Kanyong, Prosper; Rawlinson, Sean; Davis, James

    2016-01-01

    Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa. PMID:26958088

  12. Role of BCA in TIGER grant reviews: common errors and influence on the selection process.

    PubMed

    Homan, Anthony C

    2014-07-01

    Abstract As directed by the American Recovery and Reinvestment Act of 2009, the US Department of Transportation (DOT) created the Transportation Investment Generating Economic Recovery (TIGER) discretionary grant program for surface transportation infrastructure projects. Through 2013, there have been five rounds of the grant program. TIGER uses a multi-step competitive application process to award surface transportation funds. TIGER applications are initially screened by US DOT's staff of technical experts. For projects forwarded by the review team, US DOT economic experts then review the applicant's benefit-cost analysis (BCA) and attempt to determine the likelihood that the benefits exceeded costs (i.e. not the applicant's self-determination). The final awardees are then selected by a Review Team of Modal Administrators and DOT Office of the Secretary level officials. The purpose of this paper is to discuss many of the common errors in preparing, and issues in reviewing the applicant's BCA and in making a net benefit determination. A secondary purpose is to determine if the most deserving projects, based on an applicant's BCA and the likelihood that benefits exceeded costs, are more likely to receive grant funding. We do so for the second through the fifth rounds of the program. PMID:24995402

  13. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

    PubMed

    Shah, Adnan; Eikmanns, Bernhard J

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide "A" of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an "A" residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene. PMID:27119954

  14. Evaluation of the biogeochemical controls on B/Ca of Globigerinoides ruber white from the Oceanic Flux Program, Bermuda

    NASA Astrophysics Data System (ADS)

    Babila, Tali L.; Rosenthal, Yair; Conte, Maureen H.

    2014-10-01

    Boron to calcium (B/Ca) ratios in planktonic foraminifera is suggested to be a proxy for the surface oceanic carbonate system. The reliability of the proxy has been questioned due to conflicting reports from culture and sediment calibrations on the influence of temperature on B/Ca. To assess this issue, B/Ca and Mg/Ca ratios and δO18 were measured in tests of the symbiont-bearing surface dwelling planktonic foraminifer Globigerinoides ruber white collected by the Oceanic Flux Program (OFP) time series sediment traps, located approximately 75 km SE of Bermuda. B/Ca ratios in the 300-400 μm size fraction of G. ruber were approximately 10-20% higher than those in the 200-300 μm size fraction. In contrast, Mg/Ca ratios and δO18 values do not exhibit any relationship to test size, which indicates that the size effect observed for B/Ca is not due to differences in depth habitat but to vital effects. Seasonal variation in the B/Ca ratio was similar in both size fractions and ranged by ∼20-30 μmol/mol. This range is larger than that predicted by seasonal variations in seawater [B(OH)-4/HCO-3], the proposed boron species incorporated into planktonic foraminiferal calcite, and indicates that B/Ca in G. ruber is influenced by an additional variable (s). The seasonal cycle of B/Ca in G. ruber white was more strongly correlated with light intensity than with temperature. Both observations suggest that the presence of symbionts in G. ruber and seasonal variability in their photosynthetic activity act to modify the internal pH during calcification, by up to ∼0.2 units relative to ambient seawater.

  15. Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum

    PubMed Central

    Shah, Adnan; Eikmanns, Bernhard J.

    2016-01-01

    Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene. PMID:27119954

  16. Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate.

    PubMed

    Katano, Hajime; Takakuwa, Masahiro; Hayakawa, Hajime; Kimoto, Hisashi

    2016-01-01

    A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be hydrolyzed to produce GlcN. A 10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the mixture was kept at 100°C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to produce GlcN, even when the sample was a crab shell. A 20-μL aliquot of the hydrolysate was mixed with 20 μL of a 5 mol/L NaOH aqueous solution and 200 μL of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and 30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70°C for 30 min. In the mixture, GlcN reduced the Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN concentration in the hydrolysate was determined to be 0.97 ± 0.02 g/L (as hydrochloride salt), indicating that the sample contained 10.0 ± 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample. PMID:27302593

  17. Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin-Producing Escherichia coli.

    PubMed

    Terao, Yoshitaka; Takeshita, Kana; Nishiyama, Yasutaka; Morishita, Naoki; Matsumoto, Takashi; Morimatsu, Fumiki

    2015-08-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non-E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non-E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant. PMID:26219371

  18. Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages.

    PubMed

    Mazzotti, Fabio; Benabdelkamel, Hicham; Di Donna, Leonardo; Athanassopoulos, Constantinos M; Napoli, Anna; Sindona, Giovanni

    2012-07-01

    5-Dimethylamino-1-sulfonyl naphthalene (DNS, commonly referred as dansyl) is a functionality, bearing well-established properties in directing the fragmentation, by mass spectrometry (MS), of the corresponding ionized sulfonylated derivatives. This property is shared also by its labeled analogs. The use of d(0)/d(6) DNS derivatives is now exploited in the application of the well-established isotope dilution mass spectrometric approach in the assay of complex mixtures. A new method for the quantitation of amino acids (AAs) in beverages is therefore presented, which relies on liquid chromatographic separation of their N-dansylated derivatives followed by comparative electrospray tandem MS/MS of the d(0)/d(6) isobaric mixtures. Labeled and unlabeled DNS derivatives of the selected AAs are readily available by microwave-assisted synthetic protocols. The novelty of the method is represented by the use of heavy and light DNS-isotopologue providing suitable reporter groups. Multiple-reaction monitoring has been applied in the assay of AAs in wine, pineapple juice and bergamot juice with good-to-excellent results as proved by both relative standard deviation, lower than 15%, and by the accuracy values in the range 90-110%. PMID:22791261

  19. Unsaturated fatty acids show clear elicitation responses in a modified local lymph node assay with an elicitation phase, and test positive in the direct peptide reactivity assay.

    PubMed

    Yamashita, Kunihiko; Shinoda, Shinsuke; Hagiwara, Saori; Miyazaki, Hiroshi; Itagaki, Hiroshi

    2015-12-01

    The Organisation for Economic Co-operation and Development (OECD) Test Guidelines (TG) adopted the murine local lymph node assay (LLNA) and guinea pig maximization test (GPMT) as stand-alone skin sensitization test methods. However, unsaturated carbon-carbon double-bond and/or lipid acids afforded false-positive results more frequently in the LLNA compared to those in the GPMT and/or in human subjects. In the current study, oleic, linoleic, linolenic, undecylenic, fumaric, maleic, and succinic acid and squalene were tested in a modified LLNA with an elicitation phase (LLNA:DAE), and in a direct peptide reactivity assay (DPRA) to evaluate their skin-sensitizing potential. Oleic, linoleic, linolenic, undecylenic and maleic acid were positive in the LLNA:DAE, of which three, linoleic, linolenic, and maleic acid were positive in the DPRA. Furthermore, the results of the cross-sensitizing tests using four LLNA:DAE-positive chemicals were negative, indicating a chemical-specific elicitation response. In a previous report, the estimated concentration needed to produce a stimulation index of 3 (EC3) of linolenic acid, squalene, and maleic acid in the LLNA was < 10%. Therefore, these chemicals were classified as moderate skin sensitizers in the LLNA. However, the skin-sensitizing potential of all LLNA:DAE-positive chemicals was estimated as weak. These results suggested that oleic, linoleic, linolenic, undecylenic, and maleic acid had skin-sensitizing potential, and that the LLNA overestimated the skin-sensitizing potential compared to that estimated by the LLNA:DAE. PMID:26558466

  20. Quantification of proteins using enhanced etching of Ag coated Au nanorods by the Cu(2+)/bicinchoninic acid pair with improved sensitivity.

    PubMed

    Liu, Wenqi; Hou, Shuai; Yan, Jiao; Zhang, Hui; Ji, Yinglu; Wu, Xiaochun

    2016-01-14

    Plasmonic nanosensors show great potential in ultrasensitive detection, especially with the plasmon peak position as the detection modality. Herein, a new sensitive but simple total protein quantification method termed the SPR-BCA assay is demonstrated by combining plasmonic nanosensors with protein oxidation by Cu(2+). The easy tuning of localized surface plasmon resonance (LSPR) features of plasmonic nanostructures makes them ideal sensing platforms. We found that the Cu(2+)/bicinchoninic acid (BCA) pair exhibits accelerated etching of Au@Ag nanorods and results in the LSPR peak shift. A linear relationship between Cu(2+) and the LSPR shift is found in a double logarithmic coordinate. Such double logarithm relationship is transferred to the concentration of proteins. Theoretical simulation shows that Au nanorods with large aspect ratios and small core sizes show high detection sensitivity. Via optimized sensor design, we achieved an increased sensitivity (the limit of detection was 3.4 ng ml(-1)) and a wide working range (0.5 to 1000 μg ml(-1)) compared with the traditional BCA assay. The universal applicability of our method to various proteins further proves its potential in practical applications. PMID:26669539

  1. Direct Assay of δ-Aminolevulinic Acid Dehydratase in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry

    PubMed Central

    Choiniere, John R.; Scott, C. Ronald; Gelb, Michael H.; Tureček, František

    2010-01-01

    We report a new assay of human δ-aminolevulinic acid dehydratase (ALAD), an enzyme converting δ-aminolevulinic acid (ALA) into porphobilinogen. The assay is developed for use in the clinical diagnosis of δ-aminolevulinic acid dehydratase-deficient porphyria, a rare enzymatic deficiency of the heme biosynthetic pathway. The assay involves the incubation of erythrocyte lysate with the natural substrate, ALA, followed by quantitative in situ conversion of porphobilinogen to its butyramide, and liquid-liquid extraction into a mass spectrometer-friendly solvent. Quantitation of the butyrylated porphobilinogen is done by electrospray ionization tandem mass spectrometry, using a deuterium labeled internal standard. The assay stays well within the range wherein ALAD activity is linear with time. The Km of ALAD for ALA was measured as 333 μM, and the Vmax was 19.3 μM/hr. Average enzyme activity among a random sample of 36 anonymous individuals was 277 μmol/L erythrocyte lysate/hour with a standard deviation of 90 μmol/L erythrocyte lysate/hour. The tandem mass spectrometric assay should easily detect the enzyme deficiency, which causes a reduction of activity by 95–99%. The assay shows good reproducibility, low background, requires a simple workup, and uses a commercially available substrate. PMID:20583792

  2. [Development of direct competitive enzyme-linked immunosorbent assay for the determination of domoic acid].

    PubMed

    Wang, Qian; Cheng, Jin-Ping; Gao, Li-Li; Dong, Yu; Xi, Lei

    2012-02-01

    To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid detection of domoic acid concentrations, HRP (horse radish peroxidase) was successfully linked to DA using EDC. The concentration of DA was quantitatively analyzed on the basic of the specific immune responses between the DA- HRP and the monoclonal antibodies made in advance. Calibration curve were established after the optimization of reaction conditions such as the type of blocking solution, the blocking time and the incubation temperature. The results show that, the best reaction condition of the direct competitive ELISA is 1% gelatin, blocking 1 h at 37 degrees C, incubating 1 h at 37 degrees C after the monoclonal antibodies added. The detect limit is 3.58 ng x mL(-1), the coefficient of variation between the holes is below 15%, and the recovery is 80% - 120%. The whole analysis process could be completed within 1.5 h. It meets the requirements of rapid and batch detection of domoic acid. The method will have broad development prospects. PMID:22509610

  3. Medical devices; immunology and microbiology devices; classification of quality control material for cystic fibrosis nucleic acid assays. Final rule.

    PubMed

    2007-01-10

    The Food and Drug Administration (FDA) is classifying quality control material for cystic fibrosis nucleic acid assays into class II (special controls). The special control that will apply to the device is the guidance document entitled "Class II Special Controls Guidance Document: Quality Control Material for Cystic Fibrosis Nucleic Acid Assays." The agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. Elsewhere in this issue of the Federal Register, FDA is announcing the availability of the guidance document that will serve as the special control for this device. PMID:17294552

  4. Optimization of pH values to formulate the bireagent kit for serum uric acid assay.

    PubMed

    Huang, Ya; Chen, Yuanxiang; Yang, Xiaolan; Zhao, Hua; Hu, Xiaolei; Pu, Jun; Liao, Juan; Long, Gaobo; Liao, Fei

    2015-01-01

    A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous. PMID:24673428

  5. Isotope-dilution assay for urinary methylmalonic acid in the diagnosis of vitamin B12 deficiency. A prospective clinical evaluation

    SciTech Connect

    Matchar, D.B.; Feussner, J.R.; Millington, D.S.; Wilkinson, R.H. Jr.; Watson, D.J.; Gale, D.

    1987-05-01

    Vitamin B12 deficiency is a frequently considered diagnosis for which there is no single, commonly available and accurate test. A urinary methylmalonic acid assay using gas chromatography-mass spectrometry has been proposed as the preferred test. We reviewed vitamin B12 assays on 1599 consecutive patients and prospectively studied all patients with low serum B12 levels (n = 75) and a random sample of patients with normal levels (n = 68). Of 96 evaluable patients, 7 had clinical deficiency. All 7 deficient patients had urinary methylmalonic acid levels greater than 5 micrograms/mg creatine (sensitivity, 100%; confidence interval, 65% to 100%). Of the 89 patients who were not clinically deficient, 88 had urinary methylmalonic acid levels less than or equal to 5 micrograms/mg creatinine (specificity, 99%). The overall test accuracy in this population was 99%. If the high sensitivity and specificity of the gas chromatography-mass spectrometry assay for urinary methylmalonic acid is supported by other clinical studies, the methylmalonic acid assay may become the reference standard for the diagnosis of vitamin B12 deficiency.

  6. Use of submitochondrial particle (SMP) assays for assessing wetlands constructed for sequestering acid mine runoff

    SciTech Connect

    Bettermann, A.D.; Haahr, J.E.; Lazorchak, J.M.

    1995-12-31

    Use of constructed wetlands to sequester metals from acid mine drainage is part of a USEPA SITE demonstration at Burleigh Tunnel near Silverplume, Colorado. Samples are collected on a seasonal basis for toxicity evaluation of two different pilot treatment systems. Water samples were obtained from the outflow of two experimental wetland cells utilizing either upflow and downflow treatment, as well as upstream and downstream of the discharge of Burleigh Tunnel to Clear Creek. Submitochondrial Particle (SMP), Ceriodaphnia dubia and Pimephales promelas acute bioassays were used to evaluate the water quality. The SMP bioassay is based on the electron transfer complex derived from mitochondria. Toxic responses result from subcellular perturbations of various subsets of enzyme systems contained in the complex. In prior work, a 0.79 r{sup 2} was reported between the SMP bioassay and P. promelas for 11 inorganics on the EPA Priority Pollutant list. The SMP bioassay provided data consistent with the whole organism results. The two most toxic samples: the Burleigh outflow, and the Clear Creek Upstream sample, gave C. dubia LC50s of 1.01% and 8.41%, respectively. The Burleigh outflow P. promelas LC50 was 1.55%. SMP EC50s for the Burleigh outflow and the Clear Creek Upstream sample were 0.63% and 1.63%, respectively. As the SMP bioassay requires 1 hour to run and costs approximately 1/10th of whole organism assays, it was feasible to determine EC50 values for 7 samples vs. the two sample LC50s determined using whole organism assays. The SMP bioassays can provide sufficient sampling density, at low cost, allowing effective delineation of wetland performance over time.

  7. Should South Africa Be Performing Nucleic Acid Testing on HIV Enzyme-Linked Immunosorbent Assay-Negative Samples?▿

    PubMed Central

    Gous, Natasha; Scott, Lesley; Perovic, Olga; Venter, Francios; Stevens, Wendy

    2010-01-01

    The frequency of acute HIV infection (AHI) among HIV-1 enzyme-linked immunosorbent assay (ELISA)-negative samples received from general hospital patient admissions was assessed. Of 3,005 samples pooled for nucleic acid testing, a prevalence of 0.13% was found. Pooled nucleic acid testing may be feasible for low-cost identification of AHI in high-prevalence settings. PMID:20610671

  8. REPRODUCTIVE AND GENOMIC EFFECTS IN TESTES FROM MICE EXPOSED TO THE WATER DISINFECTANT BYPRODUCT BROMOCHLOROACETIC ACID

    EPA Science Inventory

    ABSTRACT

    A byproduct of drinking water disinfection, bromochloroacetic acid (BCA), acts as a reproductive toxicant in rats. To determine if BCA produces similar reproductive toxicity in mice, juvenile and adult C57BL/6 males were exposed to 0, 8, 24, 72 or 216 mg/kg of BC...

  9. Determination of low B/Ca ratios in carbonates using ICP-QQQ

    NASA Astrophysics Data System (ADS)

    Diez Fernández, Silvia; Ruiz Encinar, Jorge; Sanz-Medel, Alfredo; Isensee, Kirsten; Stoll, Heather M.

    2015-06-01

    The very low B/Ca ratios characteristic of some natural biogenic carbonates, are of interest for research in ocean acidification but represent an analytical challenge. We describe a method using a novel instrument configuration (ICP-QQQ), for which we are not aware of any previously published geological applications, and for coccoliths, a sample type unique in its low B content and organic phases. Detection limits as low as 0.41 µmol/mol were achieved. Isobaric interferences, out of the reach even for SF-ICP-MS, can be solved using this instrument, which permits the safe measurement of the lowest abundance Ca isotope (46Ca). This allows maximizing the B concentration measured (matrix concentration up to 800 ppm Ca) while maintaining both B and Ca signals in counting mode. More significantly for low B samples, the ICP-QQQ is also able to overcome the interference of the ubiquitous 12C tail on the 11B mass, which otherwise leads to significant overestimates at very low B concentrations. This could be a reason for the significantly lower B/Ca ratios observed for the low B content interlaboratory calibration standards (Carrara and OKA), while matching for the high B content standards was good. Finally, results obtained in the analysis of coccoliths grown in laboratory culture seems to corroborate that SIMS analysis of the samples mounted in Indium leads also to B/Ca overestimates due to porosity effects, as previously observed using LA-ICP-MS. This approach also permits the interference-free measurement of P/Ca and S/Ca ratios, which could be used as indicators of the complete removal of the organic matter from the samples.

  10. Efficacy of reducing sugar and phenol-sulfuric acid assays for analysis of soluble carbohydrates in feedstuffs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Reducing sugar (RSA) and phenol–sulfuric acid (PSA) assays are commonly used to analyze water-soluble carbohydrates. However, questions have arisen as to their accuracy for measurement of feedstuffs with diverse carbohydrate profiles. This study evaluated the efficacy of RSA and PSA as they would co...

  11. SENSITIVE ASSAY, BASED ON HYDROXY FATTY ACIDS FROM LIPOPOLYSACCHARIDE LIPID A, FOR GRAM-NEGATIVE BACTERIA IN SEDIMENTS

    EPA Science Inventory

    Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the li...

  12. Molecular Assay for Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae Isolates from Cultures and Clinical Nucleic Acid Amplification Test Specimens

    PubMed Central

    Peterson, S. W.; Martin, I.; Demczuk, W.; Bharat, A.; Hoang, L.; Wylie, J.; Allen, V.; Lefebvre, B.; Tyrrell, G.; Horsman, G.; Haldane, D.; Garceau, R.; Wong, T.

    2015-01-01

    We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism. PMID:26292300

  13. Development of a precision-fed ileal amino acid digestibility assay using 3-week-old broiler chicks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of these studies was to develop a precision-fed ileal digestibility assay, primarily for amino acids (AA), using 3-wk-old broiler chicks. For all experiments, day-old Ross × Ross 708 broiler chicks were fed a standard corn-soybean meal starter diet until 21 d of age. In experiment 1, f...

  14. Whole-cell microtiter plate screening assay for terminal hydroxylation of fatty acids by P450s.

    PubMed

    Weissenborn, Martin J; Notonier, Sandra; Lang, Sarah-Luise; Otte, Konrad B; Herter, Susanne; Turner, Nicholas J; Flitsch, Sabine L; Hauer, Bernhard

    2016-05-01

    A readily available galactose oxidase (GOase) variant was used to develop a whole cell screening assay. This endpoint detection system was applied in a proof-of-concept approach by screening a focussed mutant library. This led to the discovery of the thus far most active P450 Marinobacter aquaeolei mutant catalysing the terminal hydroxylation of fatty acids. PMID:27074906

  15. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    PubMed

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  16. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection. PMID:26202109

  17. Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

    PubMed

    Selvaraju, Suresh B; Bambach, Adrienne V; Leber, Amy L; Patru, Maria-Magdalena; Patel, Anami; Menegus, Marilyn A

    2014-09-01

    The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar. PMID:25209363

  18. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    PubMed

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings. PMID:22459802

  19. Ileal amino acid digestibility assay for the growing meat chicken--comparison of ileal and excreta amino acid digestibility in the chicken.

    PubMed

    Kadim, I T; Moughan, P J; Ravindran, V

    2002-09-01

    1. The apparent and true amino acid digestibilities in sorghum, wheat, soyabean meal, meat-and-bone meal, fish meal and blood meal for growing meat chickens were determined using an assay based on the collection of digesta from the terminal ileum and comparison was made with digestibility values determined using an excreta-based assay. 2. Five-week-old meat chickens were given maize-soyabean meal basal diet or mixtures of the basal diet and test diets containing the 6 ingredients as the sole source of dietary protein (50:50 on weight basis). Apparent amino acid digestibility values of assay diets at ileal and excreta levels were calculated using chromic oxide as the indigestible marker. True digestibility values were calculated using endogenous outputs determined by feeding a protein-free diet. Amino acid digestibilities of the ingredients were calculated by difference. 3. The site of measurement had no influence on endogenous amino acid output, the exceptions being aspartic acid and glutamic acid. The output of these two amino acids was higher in the excreta. 4. Significant differences were found between ileal and excreta-based digestibility of certain amino acids in some ingredients, with excreta values being usually higher than the ileal values, indicating a net catabolism of amino acids in the large intestine. The degree of net amino acid disappearance was found to be variable among amino acids and ingredients. In general, threonine had the lowest digestibility at the ileal level and, compared with other amino acids, the highest degredation during passage through the hindgut. 5. The results showed that digestibility determination based on excreta collection will overestimate the uptake for some amino acids in some feeds. The degree of overestimation was often considerable, ranging from 8.9% (apparent digestibility of threonine in soyabean meal) to 56% (apparent digestibility of aspartic acid in wheat). It is concluded that digestibility values measured at the

  20. BCA-kMC Hybrid Simulation for Hydrogen and Helium Implantation in Material under Plasma Irradiation

    NASA Astrophysics Data System (ADS)

    Kato, Shuichi; Ito, Atsushi; Sasao, Mamiko; Nakamura, Hiroaki; Wada, Motoi

    2015-09-01

    Ion implantation by plasma irradiation into materials achieves the very high concentration of impurity. The high concentration of impurity causes the deformation and the destruction of the material. This is the peculiar phenomena in the plasma-material interaction (PMI). The injection process of plasma particles are generally simulated by using the binary collision approximation (BCA) and the molecular dynamics (MD), while the diffusion of implanted atoms have been traditionally solved by the diffusion equation, in which the implanted atoms is replaced by the continuous concentration field. However, the diffusion equation has insufficient accuracy in the case of low concentration, and in the case of local high concentration such as the hydrogen blistering and the helium bubble. The above problem is overcome by kinetic Monte Carlo (kMC) which represents the diffusion of the implanted atoms as jumps on interstitial sites in a material. In this paper, we propose the new approach ``BCA-kMC hybrid simulation'' for the hydrogen and helium implantation under the plasma irradiation.

  1. Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements.

    PubMed

    Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant'Ana; Ferreira-Leitão, Viridiana Santana; da Silva Bon, Elba Pinto

    2012-12-01

    This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used. PMID:23103512

  2. Reconfigurable microfluidic dilution for high-throughput quantitative assays.

    PubMed

    Fan, Jinzhen; Li, Baoqing; Xing, Siyuan; Pan, Tingrui

    2015-06-21

    This paper reports a reconfigurable microfluidic dilution device for high-throughput quantitative assays, which can easily produce discrete logarithmic/binary concentration profiles ranging from 1 to 100-fold dilution in parallel from a fixed sample volume (e.g., 10 μL) without any assistance of continuous fluidic pump or robotic automation. The integrated dilution generation chip consists of switchable distribution and collection channels, metering reservoirs, reaction chambers, and pressure-activatable Laplace valves. Following the sequential loading of a sample, a diluent, and a detection reagent into their individual metering chambers, the top microfluidic layer can be reconfigured to collect the metered chemicals into the reaction chambers in parallel, where detection will be conducted. To facilitate mixing and reaction in the microchambers, two acoustic microstreaming actuation mechanisms have been investigated for easy integrability and accessibility. Furthermore, the microfluidic dilution generator has been characterized by both colorimetric and fluorescent means. A further demonstration of the generic usage of the quantitative dilution chip has utilized the commonly available bicinchoninic acid (BCA) assay to analyse the protein concentrations of human tissue extracts. In brief, the microfluidic dilution generator offers a high-throughput high-efficiency quantitative analytical alternative to conventional quantitative assay platforms, by simple manipulation of a minute amount of chemicals in a compact microfluidic device with minimal equipment requirement, which can serve as a facile tool for biochemical and biological analyses in regular laboratories, point-of-care settings and low-resource environments. PMID:25994379

  3. Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

    PubMed Central

    Lo, Shih-Hsiang; Cheng, Kai-Chung; Li, Ying-Xiao; Chang, Chin-Hong; Cheng, Juei-Tang; Lee, Kung-Shing

    2016-01-01

    Background G-protein-coupled bile acid receptor 1, also known as TGR5 is known to be involved in glucose homeostasis. In animal models, treatment with a TGR5 agonist induces incretin secretion to reduce hyperglycemia. Betulinic acid, a triterpenoid present in the leaves of white birch, has been introduced as a selective TGR5 agonist. However, direct activation of TGR5 by betulinic acid has not yet been reported. Methods Transfection of TGR5 into cultured Chinese hamster ovary (CHO-K1) cells was performed to establish the presence of TGR5. Additionally, TGR5-specific small interfering RNA was employed to silence TGR5 in cells (NCI-H716 cells) that secreted incretins. Uptake of glucose by CHO-K1 cells was evaluated using a fluorescent indicator. Amounts of cyclic adenosine monophosphate and glucagon-like peptide were quantified using enzyme-linked immunosorbent assay kits. Results Betulinic acid dose-dependently increases glucose uptake by CHO-K1 cells transfected with TGR5 only, which can be considered an alternative method instead of radioligand binding assay. Additionally, signals coupled to TGR5 activation are also increased by betulinic acid in cells transfected with TGR5. In NCI-H716 cells, which endogenously express TGR5, betulinic acid induces glucagon-like peptide secretion via increasing calcium levels. However, the actions of betulinic acid were markedly reduced in NCI-H716 cells that received TGR5-silencing treatment. Therefore, the present study demonstrates the activation of TGR5 by betulinic acid for the first time. Conclusion Similar to the positive control lithocholic acid, which is the established agonist of TGR5, betulinic acid has been characterized as a useful agonist of TGR5 and can be used to activate TGR5 in the future. PMID:27578964

  4. Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions.

    PubMed

    De Silva, Channa R; Vagner, Josef; Lynch, Ronald; Gillies, Robert J; Hruby, Victor J

    2010-03-01

    Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries. PMID:19852924

  5. Automated assay of methylmalonic acid in serum and urine by derivatization with 1-pyrenyldiazomethane, liquid chromatography, and fluorescence detection.

    PubMed

    Schneede, J; Ueland, P M

    1993-03-01

    Determination of methylmalonic acid (MMA) in serum has been established as an accurate test for the diagnosis of cobalamin deficiency. We describe here the development and performance of a liquid-chromatographic assay of MMA in blood and urine. The assay is based on our recent finding that one of the carboxylic acid moieties of some short-chain dicarboxylic acids reacts with the fluorogenic reagent 1-pyrenyldiazomethane in an aqueous medium, whereas the other remains underivatized (Anal Chem 1992; 63:315-9). The pH-dependent ionization of the free carboxylic acid group of 1-pyrenylmethyl monoesters permits retention on anion-exchange columns, which are used for solid-phase extraction. The analysis is done with a cyanopropyl column coupled in series with an octadecyldimethylsilyl column. Solid-phase extraction and sample injection are carried out automatically by a Gilson ASPEC sample processor. The assay response varies linearly with MMA concentration in the range 0.1-1000 mumol/L in serum. The within-day and between-day CVs are 2.8-10.9%, and the detection limit of 5 fmol injected (approximately 20 nmol/L in serum) is sufficiently low to determine MMA in serum (mean 0.187 mumol/L, SD 0.084, range 0.044-0.431, n = 44) and urine from healthy subjects. PMID:8448848

  6. Ni2+-Dependent and PsaR-Mediated Regulation of the Virulence Genes pcpA, psaBCA, and prtA in Streptococcus pneumoniae

    PubMed Central

    Manzoor, Irfan; Shafeeq, Sulman; Kuipers, Oscar P.

    2015-01-01

    Previous studies have shown that the transcriptional regulator PsaR regulates the expression of the PsaR regulon consisting of genes encoding choline binding protein (PcpA), the extracellular serine protease (PrtA), and the Mn2+-uptake system (PsaBCA), in the presence of manganese (Mn2+), zinc (Zn2+), and cobalt (Co2+). In this study, we explore the Ni2+-dependent regulation of the PsaR regulon. We have demonstrated by qRT-PCR analysis, metal accumulation assays, β-galactosidase assays, and electrophoretic mobility shift assays that an elevated concentration of Ni2+ leads to strong induction of the PsaR regulon. Our ICP-MS data show that the Ni2+-dependent expression of the PsaR regulon is directly linked to high, cell-associated, concentration of Ni2+, which reduces the cell-associated concentration of Mn2+. In vitro studies with the purified PsaR protein showed that Ni2+ diminishes the Mn2+-dependent interaction of PsaR to the promoter regions of its target genes, confirming an opposite effect of Mn2+ and Ni2+ in the regulation of the PsaR regulon. Additionally, the Ni2+-dependent role of PsaR in the regulation of the PsaR regulon was studied by transcriptome analysis. PMID:26562538

  7. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

    Lewis, Christina L.; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  8. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  9. Point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification.

    PubMed

    Zhang, Yi; Zhang, Lu; Sun, Jiashu; Liu, Yulei; Ma, Xingjie; Cui, Shangjin; Ma, Liying; Xi, Jianzhong Jeff; Jiang, Xingyu

    2014-07-15

    This report demonstrates a straightforward, robust, multiplexed and point-of-care microcapillary-based loop-mediated isothermal amplification (cLAMP) for assaying nucleic acids. This assay integrates capillaries (glass or plastic) to introduce and house sample/reagents, segments of water droplets to prevent contamination, pocket warmers to provide heat, and a hand-held flashlight for a visual readout of the fluorescent signal. The cLAMP system allows the simultaneous detection of two RNA targets of human immunodeficiency virus (HIV) from multiple plasma samples, and achieves a high sensitivity of two copies of standard plasmid. As few nucleic acid detection methods can be wholly independent of external power supply and equipment, our cLAMP holds great promise for point-of-care applications in resource-poor settings. PMID:24937125

  10. Exploring the structural requirements for inhibition of the ubiquitin E3 ligase breast cancer associated protein 2 (BCA2) as a treatment for breast cancer.

    PubMed

    Brahemi, Ghali; Kona, Fathima R; Fiasella, Annalisa; Buac, Daniela; Soukupová, Jitka; Brancale, Andrea; Burger, Angelika M; Westwell, Andrew D

    2010-04-01

    The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC(50)) in BCA2 positive MCF-7 and T47D cells but were inactive (IC(50) > 10 microM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development. PMID:20222671

  11. Exploring the Structural Requirements for Inhibition of the Ubiquitin E3 Ligase Breast Cancer Associated Protein 2 (BCA2)a as a Treatment for Breast Cancer

    PubMed Central

    Brahemi, Ghali; Kona, Fathima R.; Fiasella, Annalisa; Buac, Daniela; Soukupová, Jitka; Brancale, Andrea; Burger, Angelika M.; Westwell, Andrew D.

    2010-01-01

    The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C=S)S-S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC50) in BCA2 positive MCF-7 and T47D cells but were inactive (IC50 >10 μM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 negative MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development. PMID:20222671

  12. Dansylation metabolite assay: a simple and rapid method for sample amount normalization in metabolomics.

    PubMed

    Wu, Yiman; Li, Liang

    2014-10-01

    Metabolomics involves the comparison of the metabolomes of individual samples from two or more groups to reveal the metabolic differences. In order to measure the metabolite concentration differences accurately, using the same amount of starting materials is essential. In this work, we describe a simple and rapid method for sample amount normalization. It is based on dansylation labeling of the amine and phenol submetabolome of an individual sample, followed by solvent extraction of the labeled metabolites and ultraviolet (UV) absorbance measurement using a microplate reader. A calibration curve of a mixture of 17 dansyl-labeled amino acid standards is used to determine the total concentration of the labeled metabolites in a sample. According to the measured concentrations of individual samples, the volume of an aliquot taken from each sample is adjusted so that the same sample amount is taken for subsequent metabolome comparison. As an example of applications, this dansylation metabolite assay method is shown to be useful in comparative metabolome analysis of two different E. coli strains using a differential chemical isotope labeling LC-MS platform. Because of the low cost of equipment and reagents and the simple procedure used in the assay, this method can be readily implemented. We envisage that, this assay, which is analogous to the bicinchoninic acid (BCA) protein assay widely used in proteomics, will be applicable to many types of samples for quantitative metabolomics. PMID:25215550

  13. Multicenter evaluation of the Quidel Lyra Direct C. difficile nucleic acid amplification assay.

    PubMed

    Beck, Eric T; Buchan, Blake W; Riebe, Katherine M; Alkins, Brenda R; Pancholi, Preeti; Granato, Paul A; Ledeboer, Nathan A

    2014-06-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. PMID:24671790

  14. Multicenter Evaluation of the Quidel Lyra Direct C. difficile Nucleic Acid Amplification Assay

    PubMed Central

    Beck, Eric T.; Buchan, Blake W.; Riebe, Katherine M.; Alkins, Brenda R.; Pancholi, Preeti; Granato, Paul A.

    2014-01-01

    Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods. PMID:24671790

  15. Use of agar diffusion assay to measure bactericidal activity of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of caproic, caprylic, capric, lauric, and myristic acids. A 0.5M concentration of each fatty acid was dissolved in 1.0 M potassium hydroxide (KOH), and pH of the mixtures was adjusted to 10.5 with citric acid. Solu...

  16. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

    PubMed Central

    Kopp, Markus; Rychlik, Michael

    2015-01-01

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status. PMID:26605791

  17. Sensitive assay, based on hydroxy fatty acids from lipopolysaccharide lipid A, for Gram-negative bacteria in sediments.

    PubMed Central

    Parker, J H; Smith, G A; Fredrickson, H L; Vestal, J R; White, D C

    1982-01-01

    Biochemical measures have provided insight into the biomass and community structure of sedimentary microbiota without the requirement of selection by growth or quantitative removal from the sediment grains. This study used the assay of the hydroxy fatty acids released from the lipid A of the lipopolysaccharide in sediments to provide an estimate of the gram-negative bacteria. The method was sensitive to picomolar amounts of hydroxy fatty acids. The recovery of lipopolysaccharide hydroxy fatty acids from organisms added to sediments was quantitative. The lipids were extracted from the sediments with single-phase chloroform-methanol extraction. The lipid-extraction residue was hydrolyzed in 1 N HCl, and the hydroxy fatty acids of the lipopolysaccharide were recovered in chloroform for analysis by gas-liquid chromatography. This method proved to be about fivefold more sensitive than the classical phenol-water or trichloroacetic acid methods when applied to marine sediments. By examination of the patterns of hydroxy fatty acids, it was also possible to help define the community structure of the sedimentary gram-negative bacteria. PMID:6817712

  18. Validation and application of a liquid-chromatographic/enzymatic assay for individual bile acids in the serum of rats.

    PubMed

    Thompson, M B; Blair, P C; Morris, R W; Neptun, D A; Deyo, D F; Popp, J A

    1987-10-01

    A liquid-chromatographic technique with a post-column enzymatic reaction and fluorescence detection was validated for analysis of individual bile acids in the serum of rats. Extraction recoveries averaged 91.1% (SD 6.9%) for all bile acids. The assay was sensitive (minimum detection of 16.8 pmol per 100-microL injection), linear (r greater than 0.999 for concentrations ranging between 45 and 112,500 pmol per 100-microL injection), and reproducible (mean CVs for three different concentrations of standards and a serum pool ranged from 4.4% to 12.2%). In rats treated for three days with either neomycin, carbon tetrachloride, alpha-naphthylisothiocyanate, or total bile-duct ligation (five animals per group), total concentrations of bile acids were significantly increased (P less than 0.004). Concentrations of 16 of 17 individual bile acids differed significantly between groups (P less than 0.04). Examination of the relative concentrations (percent of total) of individual bile acids by canonical discriminant analysis placed each animal into the appropriate treatment or control group. Use of this technique in toxicological studies can help detect and identify specific types of disruptions in the enterohepatic circulation of bile acids. PMID:3665040

  19. Screening metagenomic data for viruses using the E-Probe Diagnostic Nucleic Acid Assay (EDNA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are many plant pathogen-specific diagnostic assays, based on PCR and immune-detection. However, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitat...

  20. High-throughput and functional SNP detection assays for oleic and linolenic acids in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean is a primary source of vegetable oil, accounting for 53% of the total vegetable oil consumption in the USA in 2013. Soybean oil with high oleic acid and low linolenic acid content is desired, because it not only improves the oxidative stability of the oil, but also reduces the amount of unde...

  1. Evaluating the utility of B/Ca ratios in planktic foraminifera as a proxy for the carbonate system: A case study of Globigerinoides ruber

    NASA Astrophysics Data System (ADS)

    Henehan, Michael J.; Foster, Gavin L.; Rae, James W. B.; Prentice, Katy C.; Erez, Jonathan; Bostock, Helen C.; Marshall, Brittney J.; Wilson, Paul A.

    2015-04-01

    B/Ca ratios in foraminifera have attracted considerable scientific attention as a proxy for past ocean carbonate system. However, the carbonate system controls on B/Ca ratios are not straightforward, with Δ[CO32-] ([CO32-]in situ - [CO32-]at saturation) correlating best with B/Ca ratios in benthic foraminifera, rather than pH, B>(OH>)/4-HCO3-, or B>(OH>)/4-DIC (as a simple model of boron speciation in seawater and incorporation into CaCO3 would predict). Furthermore, culture experiments have shown that in planktic foraminifera properties such as salinity and [B]sw can have profound effects on B/Ca ratios beyond those predicted by simple partition coefficients. Here, we investigate the controls on B/Ca ratios in G. ruber via a combination of culture experiments and core-top measurements, and add to a growing body of evidence that suggests B/Ca ratios in symbiont-bearing foraminiferal carbonate are not a straightforward proxy for past seawater carbonate system conditions. We find that while B/Ca ratios in culture experiments covary with pH, in open ocean sediments this relationship is not seen. In fact, our B/Ca data correlate best with [PO43-] (a previously undocumented association) and in most regions, salinity. These findings might suggest a precipitation rate or crystallographic control on boron incorporation into foraminiferal calcite. Regardless, our results underscore the need for caution when attempting to interpret B/Ca records in terms of the ocean carbonate system, at the very least in the case of mixed-layer planktic foraminifera.

  2. Using benthic foraminiferal B/Ca to constrain the effect of dissolution on key Pliocene Mg/Ca temperature records

    NASA Astrophysics Data System (ADS)

    White, S. M.; Ravelo, A. C.

    2015-12-01

    The state of the Pliocene tropical Pacific is currently the subject of heated debate. The debate hinges on the veracity of planktic foraminiferal Mg/Ca temperatures from the west Pacific warm pool (WPWP) and the eastern equatorial Pacific (EEP) that show Pliocene WPWP temperatures similar to today but a warmer Pliocene EEP, resulting in a much reduced east-west gradient [Wara et al., 2005]. These findings form the basis of the "permanent El Niño-like state" paradigm of Pliocene climate. However, recent studies using organic biomarker proxies produce temperature records that indicate a WPWP cooling trend since the Pliocene that differs markedly from Mg/Ca-temperature records [O'Brien et al., 2014; Zhang et al., 2014]. Though much of the debate has focused on changes in seawater Mg/Ca, spatial variations in proxy agreement point to dissolution as a key factor. Dissolution, which imparts a cool bias to Mg/Ca temperatures, varies across ocean basins depending on Δ[CO32-], the difference from the carbonate ion concentration needed for calcite saturation. By necessity, dissolution corrections use the modern value of Δ[CO32-] for the entire record, so it is possible that Pliocene proxy discrepancies could stem from varying Δ[CO32-] over time. Here we present benthic foraminiferal B/Ca data (a proxy for Δ[CO32-]) from the EEP and WEP spanning the past 5 Myr, to constrain the effect of dissolution on Pliocene Mg/Ca records. To account for possible changes in seawater B/Ca, we present paired epifaunal-infaunal B/Ca data. Infaunal species are much less sensitive to Δ[CO32-] than epifaunal species, but would still record long-term changes in seawater B/Ca. The true Δ[CO32-] can thus be calculated from the epifaunal-infaunal B/Ca difference [Brown et al., 2011]. Our study is the first to apply this approach downcore; by accounting for long-term changes in seawater, it greatly expands use of the B/Ca proxy and enables a first attempt at correcting for time

  3. Stratospheric Sulfuric Acid and Black Carbon Aerosol Measured During POLARIS and its Role in Ozone Chemistry

    NASA Technical Reports Server (NTRS)

    Strawa, Anthony W.; Pueschel, R. F.; Drdla, K.; Verma, S.; Gore, Warren J. (Technical Monitor)

    1998-01-01

    Stratospheric aerosol can affect the environment in three ways. Sulfuric acid aerosol have been shown to act as sites for the reduction of reactive nitrogen and chlorine and as condensation sites to form Polar Stratospheric Clouds, under very cold conditions, which facilitate ozone depletion. Recently, modeling studies have suggested a link between BCA (Black Carbon Aerosol) and ozone chemistry. These studies suggest that HNO3, NO2, and O3 may be reduced heterogeneously on BCA particles. The ozone reaction converts ozone to oxygen molecules, while HNO3 and NO2 react to form NOx. Finally, a buildup of BCA could reduce the single-scatter albedo of aerosol below a value of 0.98, a critical value that has been postulated to change the effect of stratospheric aerosol from cooling to warming. Correlations between measured BCA amounts and aircraft usage have been reported. Attempts to link BCA to ozone chemistry and other stratospheric processes have been hindered by questions concerning the amount of BCA that exists in the stratosphere, the magnitude of reaction probabilities, and the scarcity of BCA measurements. The Ames Wire Impactors (AWI) participated in POLARIS as part of the complement of experiments on the NASA ER-2. One of our main objectives was to determine the amount of aerosol surface area, particularly BCA, available for reaction with stratospheric constituents and assess if possible, the importance of these reactions. The AWI collects aerosol and BCA particles on thin Palladium wires that are exposed to the ambient air in a controlled manner. The samples are returned to the laboratory for subsequent analysis. The product of the AWI analysis is the size, surface area, and volume distributions, morphology and elemental composition of aerosol and BCA. This paper presents results from our experiments during POLARIS and puts these measurements in the context of POLARIS and other missions in which we have participated. It describes modifications to the AWI data

  4. Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

    PubMed Central

    Ha, T; Morgan, S L; Vaughn, W H; Eto, I; Baggott, J E

    1990-01-01

    The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylamino-imidazolecarboxamide formyltransferase; EC 2.1.2.3) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The Ki values of tIMP and azathioprine for the chicken liver enzyme are 39 +/- 4 microM and 120 +/- 10 microM, whereas the Ki values for the enzyme from PBMCs of the MRL/lpr mouse are 110 +/- 20 microM and 90 +/- 14 microM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase. PMID:2268263

  5. Evaluation of Surfactants-Assisted Folic Acid-Loaded Pectin Submicrospheres: Characterization and Hemocompatibility Assay.

    PubMed

    Varuna Kumara, J B; Ravikumara, N R; Madhusudhan, Basavaraj

    2016-10-01

    Folic acid is used for preventing and treating multiple diseases and disorders, administered in the form of oral supplements. The present research work was aimed to study the influence of two non-ionic surfactants Poloxamer and Tween 80 (Polysorbate 80) on pectin submicrospheres formulations. Typical natural polymer pectin was used to encapsulate folic acid by cross linking method. The resultant submicrospheres contributed to improve the aqueous solubility to enhance the bioavailability of folic acid. During investigation, it was observed that pectin polymers influenced kinetics of the rate of reaction more intensively than the surfactants. The physical phenomenon caused the change in their size, shape and chemistry of pectin polymers transforming into submicrospheres in aqueous condition. The characteristic differences of submicrospheres were assessed by scanning electron microscopy, differential scanning calorimetry and Fourier-transform infrared spectroscopy. The average diameters of the submicrospheres ranged between 250 and 500 nm. The encapsulation efficiency of submicrospheres ranged between 80 and 96 %. The characteristic swelling behavior of lyophilized submicrospheres was influenced by the ratio of pectin polymers and folic acid used in the formulations. The submicrospheres systems exhibited controlled release of folic acid due to the pH-dependent solubility of pectin polymers in aqueous medium. The submicrospheres showed good haemocompatibility suggesting them to be promising candidates for oral delivery. PMID:27605736

  6. Analysis prediction of Indonesian banks (BCA, BNI, MANDIRI) using adaptive neuro-fuzzy inference system (ANFIS) and investment strategies

    NASA Astrophysics Data System (ADS)

    Trianto, Andriantama Budi; Hadi, I. M.; Liong, The Houw; Purqon, Acep

    2015-09-01

    Indonesian economical development is growing well. It has effect for their invesment in Banks and the stock market. In this study, we perform prediction for the three blue chips of Indonesian bank i.e. BCA, BNI, and MANDIRI by using the method of Adaptive Neuro-Fuzzy Inference System (ANFIS) with Takagi-Sugeno rules and Generalized bell (Gbell) as the membership function. Our results show that ANFIS perform good prediction with RMSE for BCA of 27, BNI of 5.29, and MANDIRI of 13.41, respectively. Furthermore, we develop an active strategy to gain more benefit. We compare between passive strategy versus active strategy. Our results shows that for the passive strategy gains 13 million rupiah, while for the active strategy gains 47 million rupiah in one year. The active investment strategy significantly shows gaining multiple benefit than the passive one.

  7. Neutron characterization study for D-T, p-7Li neutron sources with new BCA based direct collision coupling method

    NASA Astrophysics Data System (ADS)

    Wang, Guan-bo; Yang, Xin; Qian, Da-zhi; Li, Run-dong; Tang, Bin

    2014-09-01

    The T(D,n)4He and 7Li(p,n)7Be neutron sources have been used for decades in nuclear physics research, stellar nucleosynthesis research and neutron therapy research. In this work, the neutron characterization including neutron yield, spectra, and angular distribution for D-T and p-7Li sources have been studied with our new binary collision approximation (BCA) based direct collision coupling method. Distinguished from the traditional path integration method for getting the neutron weight, the new model establishes a relationship between the scattering cross section and the impact parameter, which allows the secondary neutron generation carrying out jointly with ions BCA tracking. The experimental measurements of neutron characterizations have been employed for these two reactions, and the new algorithm is validated.

  8. Nucleic Acid Sandwich Hybridization Assay with Quantum Dot-Induced Fluorescence Resonance Energy Transfer for Pathogen Detection

    PubMed Central

    Chou, Cheng-Chung; Huang, Yi-Han

    2012-01-01

    This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection. PMID:23211753

  9. Expansion and Diversification of BTL Ring-H2 Ubiquitin Ligases in Angiosperms: Putative Rabring7/BCA2 Orthologs

    PubMed Central

    Aguilar-Henonin, Laura; Guzmán, Plinio

    2013-01-01

    RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes. PMID:23951330

  10. Expansion and diversification of BTL ring-H2 ubiquitin ligases in angiosperms: putative Rabring7/BCA2 orthologs.

    PubMed

    Aguilar-Hernández, Victor; Medina, Juliana; Aguilar-Henonin, Laura; Guzmán, Plinio

    2013-01-01

    RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes. PMID:23951330

  11. All0809/8/7 is a DevBCA-like ABC-type efflux pump required for diazotrophic growth in Anabaena sp. PCC 7120.

    PubMed

    Staron, Peter; Maldener, Iris

    2012-10-01

    Efflux pumps export a wide variety of proteinaceous and non-proteinaceous substrates across the Gram-negative cell wall. For the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the ATP-driven glycolipid efflux pump DevBCA-TolC has been shown to be crucial for the differentiation of N(2)-fixing heterocysts from photosynthetically active vegetative cells. In this study, a homologous system was described. All0809/8/7-TolC form a typical ATP-driven efflux pump as shown by surface plasmon resonance. This putative exporter is also involved in diazotrophic growth of Anabaena sp. PCC 7120. A mutant in all0809 encoding the periplasmic membrane fusion protein of the pump was not able to grow without combined nitrogen. Although heterocysts of this mutant were not distinguishable from those of the wild-type in light and electron micrographs, they were impaired in providing the microoxic environment necessary for N(2) fixation. RT-PCR of all0809 transcripts and localization studies on All0807-GFP revealed that All0809/8/7 was initially downregulated during heterocyst maturation and upregulated at later stages of heterocyst formation in all cells of the filament. A substrate of the efflux pump could not be identified in ATP hydrolysis assays. We discuss a role for All0809/8/7-TolC in maintaining the continuous periplasm and how this would be of special importance for heterocyst differentiation. PMID:22859614

  12. Polyacrylic acid-coated cerium oxide nanoparticles: An oxidase mimic applied for colorimetric assay to organophosphorus pesticides.

    PubMed

    Zhang, Shi-Xiang; Xue, Shi-Fan; Deng, Jingjing; Zhang, Min; Shi, Guoyue; Zhou, Tianshu

    2016-11-15

    It is important and urgent to develop reliable and highly sensitive methods that can provide on-site and rapid detection of extensively used organophosphorus pesticides (OPs) for their neurotoxicity. In this study, we developed a novel colorimetric assay for the detection of OPs based on polyacrylic acid-coated cerium oxide nanoparticles (PAA-CeO2) as an oxidase mimic and OPs as inhibitors to suppress the activity of acetylcholinesterase (AChE). Firstly, highly dispersed PAA-CeO2 was prepared in aqueous solution, which could catalyze the oxidation of TMB to produce a color reaction from colorless to blue. And the enzyme of AChE was used to catalyze the substrate of acetylthiocholine (ATCh) to produce thiocholine (TCh). As a thiol-containing compound with reducibility, TCh can decrease the oxidation of TMB catalyzed by PAA-CeO2. Upon incubated with OPs, the enzymatic activity of AChE was inhibited to produce less TCh, resulting in more TMB catalytically oxidized by PAA-CeO2 to show an increasing blue color. The two representative OPs, dichlorvos and methyl-paraoxon, were tested using our proposed assay. The novel assay showed notable color change in a concentration-dependent manner, and as low as 8.62 ppb dichlorvos and 26.73 ppb methyl-paraoxon can be readily detected. Therefore, taking advantage of such oxidase-like activity of PAA-CeO2, our proposed colorimetric assay can potentially be a screening tool for the precise and rapid evaluation of the neurotoxicity of a wealth of OPs. PMID:27208478

  13. A Continuous, Quantitative Fluorescent Assay for Plant Caffeic acid O-Methyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant caffeic acid O-methyltransferases (COMTs) use s-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in-vivo, and will generally utilize a range of phenolic compounds in-vitro. Collazo et al. (2005; Analytical Biochemistry 342: 86-92) have pu...

  14. A SIMPLE ASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID USING COATED TEST-STRIPS

    EPA Science Inventory

    Immunoassay test strips utilizing ascending chromatography has been devised for the detection of 2.4-dichlorophenoxyacetic acid (2,4-D). This test requires no instrumentation, inexpensive reagents and relies on the application of antibodies to 2,4-D adsorbed onto colloidal gol...

  15. An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

    PubMed

    Bansal, Sumit; Chai, Swee Fen; Lau, Aik Jiang

    2016-06-15

    Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation. PMID:27153105

  16. /sup 57/Co-soap assay for plasma total non-esterified fatty acids compared with a gas-liquid chromatographic method

    SciTech Connect

    Turnell, D.C.; Price, C.P.; France, M.M.

    1980-12-01

    A relatively rapid radiochemical assay for determining plasma non-esterified fatty acids is described. Results correlated well with those by gas-liquid chromatography. The between-batch CV for 1.12 mmol of non-esterified fatty acids per liter was 7%.

  17. A simple and highly sensitive assay of perfluorooctanoic acid based on resonance light scattering technique.

    PubMed

    Zhang, Fang; Zheng, Yonghong; Liang, Jiaman; Long, Sha; Chen, Xianping; Tan, Kejun

    2016-04-15

    A simple, highly sensitive resonance light scattering (RLS) method for the detection of perfluorooctanoic acid (PFOA) has been developed based on the interaction with crystal violet (CV). It was found that PFOA can form complexes with CV in acid medium resulting in remarkable enhancement of the RLS intensity of the system. And the enhanced RLS intensities are in proportion to the concentration of PFOA in the range of 0.1-25.0μmol/L (R(2)=0.9998), with a detection limit of 11.0nmol/L (S/N=3). In this work, the optimum reaction conditions and the interferences of foreign substances were investigated. The reaction mechanism between CV and PFOA was also studied by the absorption spectrum and scanning electron microscope (SEM). This method is successfully applied to the determination of PFOA in tap water and Jialing river water samples with RSD≤4.04%. PMID:26824483

  18. A simple and highly sensitive assay of perfluorooctanoic acid based on resonance light scattering technique

    NASA Astrophysics Data System (ADS)

    Zhang, Fang; Zheng, Yonghong; Liang, Jiaman; Long, Sha; Chen, Xianping; Tan, Kejun

    2016-04-01

    A simple, highly sensitive resonance light scattering (RLS) method for the detection of perfluorooctanoic acid (PFOA) has been developed based on the interaction with crystal violet (CV). It was found that PFOA can form complexes with CV in acid medium resulting in remarkable enhancement of the RLS intensity of the system. And the enhanced RLS intensities are in proportion to the concentration of PFOA in the range of 0.1-25.0 μmol/L (R2 = 0.9998), with a detection limit of 11.0 nmol/L (S/N = 3). In this work, the optimum reaction conditions and the interferences of foreign substances were investigated. The reaction mechanism between CV and PFOA was also studied by the absorption spectrum and scanning electron microscope (SEM). This method is successfully applied to the determination of PFOA in tap water and Jialing river water samples with RSD ≤ 4.04%.

  19. Evaluation of experimental teat dip containing sodium chlorite and lactic acid by excised teat assay.

    PubMed

    Schmidt, A L; Oliver, S P; Fydenkevez, M E

    1984-12-01

    An experimental teat dip containing sodium chlorite and lactic acid, diluted in water, was evaluated by excised teat protocol. The teat dip was tested against 21 microorganisms. Included were: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Numerous strains were tested for strain differences. Environmental bacteria were included because of their increasing importance as a cause of bovine mastitis. All excised teats were dipped in a bacterial suspension containing about 1 X 10(8) cfu/ml. Negative control teats were not dipped in a germicidal compound. Positive controls were dipped in 1% iodophor. Effectiveness of the experimental teat dip was expressed as the percent reduction in mean log of bacteria recovered from dipped teats as compared to numbers recovered from control teats. The sodium chlorite - lactic acid dip caused a greater percent log reduction than iodophor for 14 of 21 strains tested. However, differences were generally slight. The experimental teat dip appeared effective against Gram-negative bacteria. Some differences in percent log reduction were observed between strains of the same species. Lowest effectiveness and greatest strain variation were observed with Staphylococcus aureus for both dips tested. PMID:6530497

  20. Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay.

    PubMed

    Renko, Kostja; Hoefig, Carolin S; Hiller, Franziska; Schomburg, Lutz; Köhrle, Josef

    2012-05-01

    Enzymatic 5'- and 5-deiodination are key reactions for local and systemic activation and inactivation of iodothyronines and thyronamines. Expression of the three deiodinase (DIO) isoenzymes is regulated by a number of parameters, including thyroid status, genotype, micronutrient availability, and disease-related signaling. In addition, DIO are potential targets of pharmacological as well as environmentally derived substances, which might affect their enzymatic activity (endocrine disruptors). With the classical DIO activity assay, testing depends on the availability of radioactively labeled substrates (e.g. (125)I-rT(3)) to monitor the release of radioactive iodide. Recently, liquid chromatography-tandem mass spectrometry was described as an alternative method apparently resolving this limitation. However, it has a high demand in technical equipment and analytical routine and is limited in sample number by considerable measuring time. We therefore combined the classical deiodination assay with an easily accessible photometric method taking advantage of the Sandell-Kolthoff reaction for measuring iodide release. In brief, iodine works as a catalyst within this redox reaction between Ce(4+) and As(3+) leading to an acceleration of destaining. Furthermore, the protocol was adapted to minimize handling effort and time consumption. Because this method is not dependent on radioactivity, it expands the substrate spectrum of the classical method. Suitability of this assay was tested with tissue samples from animal experiments (hepatic Dio1 activity in hypo- and hyperthyroid mice) and established DIO inhibitors. As a new but not unexpected finding, the alleged inhibitor iopanoic acid turned out to be a DIO substrate. This finding was confirmed by liquid chromatography-tandem mass spectrometry, and its potential clinical impact requires further studies. PMID:22434082

  1. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays

    PubMed Central

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S.; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E.; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D.; Page, Mark

    2015-01-01

    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA. PMID:26562415

  2. Single Laboratory Validation of A Ready-to-Use Phosphatase Inhibition Assay for Detection of Okadaic Acid Toxins

    PubMed Central

    Smienk, Henry G. F.; Calvo, Dolores; Razquin, Pedro; Domínguez, Elena; Mata, Luis

    2012-01-01

    A phosphatase inhibition assay for detection of okadaic acid (OA) toxins in shellfish, OkaTest, was single laboratory validated according to international recognized guidelines (AOAC, EURACHEM). Special emphasis was placed on the ruggedness of the method and stability of the components. All reagents were stable for more than 6 months and the method was highly robust under normal laboratory conditions. The limit of detection and quantification were 44 and 56 µg/kg, respectively; both below the European legal limit of 160 µg/kg. The repeatability was evaluated with 2 naturally contaminated samples. The relative standard deviation (RSD) calculated was 1.4% at a level of 276 µg/kg and 3.9% at 124 µg/kg. Intermediate precision was estimated by testing 10 different samples (mussel and scallop) on three different days and ranged between 2.4 and 9.5%. The IC50 values of the phosphatase used in this assay were determined for OA (1.2 nM), DTX-1 (1.6 nM) and DTX-2 (1.2 nM). The accuracy of the method was estimated by recovery testing for OA (mussel, 78–101%; king scallop, 98–114%), DTX-1 (king scallop, 79–102%) and DTX-2 (king scallop, 93%). Finally, the method was qualitatively compared to the mouse bioassay and LC-MS/MS. PMID:22778904

  3. An enzyme-linked immunosorbent assay for detecting 3-phenoxybenzoic acid in plasma and its application in farmers and consumers

    PubMed Central

    Thiphom, Sarunya; Prapamontol, Tippawan; Chantara, Somporn; Mangklabruks, Ampica; Suphavilai, Chaisuree; Ahn, Ki Chang; Gee, Shirley J.; Hammock, Bruce D.

    2013-01-01

    The aim of this study was to identify a plasma biomarker of exposure to pyrethroid insecticides. A major metabolite, 3-phenoxybenzoic acid (3-PBA), can be detected in urine but urinary 3-PBA cannot be used to assess the active dose. The 3-PBA-adduct represents a much more persistent class of biomarkers than metabolites excreted into urine, having half lives up to several weeks or months. We developed an enzyme-linked immunosorbent assay (ELISA) for total 3-PBA including adduct formed after alkaline hydrolysis, liquid-liquid extraction (LLE) and solid phase extraction (SPE) of the sample. The developed ELISA had an IC50 value of 26.7 ng/mL. The intra- and inter-assay coefficients of variation (%CV) were lower than 5% and were within the optimum condition variance (OCV) range. The LLE cleanup technique satisfactorily eliminated the matrix effect from plasma samples before SPE and ELISA analysis yielding good recoveries (85.9–99.4%) with a limit of quantitation (LOQ, 5 ng/mL) that was 30- to 47-fold more sensitive than previous studies. Moreover, the developed method could separate more than 80% of 3-PBA from adduct form. The method was successfully applied to the detection of the target in real samples obtained from consumers (n=50) and farmers (n=50). To our knowledge, this is the first ELISA method for detecting 3-PBA in human plasma and applied to a field study. PMID:23667388

  4. Polydimethylsiloxane-Paper Hybrid Lateral Flow Assay for Highly Sensitive Point-of-Care Nucleic Acid Testing.

    PubMed

    Choi, Jane Ru; Liu, Zhi; Hu, Jie; Tang, Ruihua; Gong, Yan; Feng, Shangsheng; Ren, Hui; Wen, Ting; Yang, Hui; Qu, Zhiguo; Pingguan-Murphy, Belinda; Xu, Feng

    2016-06-21

    In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future. PMID:27012657

  5. Fibrinolytic Activity and Dose-Dependent Effect of Incubating Human Blood Clots in Caffeic Acid Phenethyl Ester: In Vitro Assays

    PubMed Central

    Elnager, Abuzar; Hassan, Rosline; Idris, Zamzuri; Mustafa, Zulkifli; Wan-Arfah, Nadiah; Sulaiman, S. A.; Gan, Siew Hua; Abdullah, Wan Zaidah

    2015-01-01

    Background. Caffeic acid phenethyl ester (CAPE) has been reported to possess time-dependent fibrinolytic activity by in vitro assay. This study is aimed at investigating fibrinolytic dose-dependent activity of CAPE using in vitro assays. Methods. Standardized human whole blood (WB) clots were incubated in either blank controls or different concentrations of CAPE (3.75, 7.50, 15.00, 22.50, and 30.00 mM). After 3 hours, D-dimer (DD) levels and WB clot weights were measured for each concentration. Thromboelastography (TEG) parameters were recorded following CAPE incubation, and fibrin morphology was examined under a confocal microscope. Results. Overall, mean DD (μg/mL) levels were significantly different across samples incubated with different CAPE concentrations, and the median pre- and postincubation WB clot weights (grams) were significantly decreased for each CAPE concentration. Fibrin removal was observed microscopically and indicated dose-dependent effects. Based on the TEG test, the Ly30 fibrinolytic parameter was significantly different between samples incubated with two different CAPE concentrations (15.0 and 22.50 mM). The 50% effective dose (ED50) of CAPE (based on DD) was 1.99 mg/mL. Conclusions. This study suggests that CAPE possesses fibrinolytic activity following in vitro incubation and that it has dose-dependent activities. Therefore, further investigation into CAPE as a potential alternative thrombolytic agent should be conducted. PMID:25664321

  6. Novel Conjugates of 1,3-Diacylglycerol and Lipoic Acid: Synthesis, DPPH Assay, and RP-LC-MS-APCI Analysis

    PubMed Central

    Madawala, Samanthi R. P.; Andersson, Rolf E.; Jastrebova, Jelena A.; Almeida, Maria; Dutta, Paresh C.

    2011-01-01

    1,3-Diacylglycerol is known to reduce body weight and fat deposits in humans. α-Lipoic acid is a potent antioxidant and effective against many pathological conditions, including obesity and related metabolic syndromes. The present work is based on the hypothesis that the hybrid molecules of 1,3-diacylglycerol and lipoic acid possess synergistic and/or additive effects compared with the parent compounds against obesity, overweight, and related metabolic syndromes. Laboratory scale synthesis of 1,3-dioleoyl-2-lipoyl-sn-glycerol (yield 80%) and 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (yield 70%) was performed for the first time and supported by NMR and MS data. Free radical scavenging capacity of the conjugates was assayed using DPPH test. A remarkably high in vitro free radical scavenging capacity was demonstrated for the 1,3-dioleoyl-2-dihydrolipoyl-sn-glycerol (EC50 value 0.21). RP-HPLC-MS-APCI analysis showed satisfactory separation between the conjugates (R~1). Protonated molecular ion of the conjugates at m/z 809 and m/z at 811, respectively, and their characteristic fragment ions were abundant. PMID:21966595

  7. Square-wave voltammetry assays for glycoproteins on nanoporous gold.

    PubMed

    Pandey, Binod; Bhattarai, Jay K; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V; Stine, Keith J

    2014-03-15

    Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A - ALP (or soybean agglutinin - ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A-ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL(-1) BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

  8. Square-wave voltammetry assays for glycoproteins on nanoporous gold

    PubMed Central

    Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.

    2014-01-01

    Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

  9. Development of a monoclonal antibody against domoic acid and its application in enzyme-linked immunosorbent assay and colloidal gold immunostrip.

    PubMed

    Tsao, Zih-Jay; Liao, Yi-Chun; Liu, Biing-Hui; Su, Ching-Chyuan; Yu, Feng-Yih

    2007-06-27

    A monoclonal antibody (mAb) specific to domoic acid was produced from a stable hybridoma cell line, 9F1F11, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with domoic acid--keyhole limpet hemocyanin. The 9F1F11 mAb belongs to the immunoglobulin G1 (kappa-chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. In the cdELISA, the concentration causing 50% inhibition (IC50) of binding of domoic acid-horseradish peroxidase to the antibody by domoic acid was found to be 0.58 ng/mL. A sensitive and rapid mAb-based colloidal gold immunostrip was also developed. The immunostrip assay, which has a detection limit of 5 ng/mL for domoic acid, can be completed in 10 min. Analysis of domoic acid in blue mussel samples revealed that data obtained from immunostrip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunostrip assay established in this study were sensitive and accurate for rapid screening of domoic acid in shellfish samples. PMID:17542614

  10. A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

    PubMed Central

    Wilton, D C

    1990-01-01

    1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase. PMID:2317197

  11. Calibration of the B/Ca proxy in symbiont-bearing planktonic foraminifera for application to the Paleocene-Eocene Thermal Maximum

    NASA Astrophysics Data System (ADS)

    Haynes, L.; Hoenisch, B.; Eggins, S.; Holland, K.; Rosenthal, Y.

    2015-12-01

    During the Paleocene-Eocene Thermal Maximum (PETM), rapid surface ocean acidification is indicated by a large decrease in the B/Ca ratios of planktic foraminiferal calcite, which is a proxy for the surface ocean carbonate system [1]. However, due to uncertainty in the effects of past seawater chemistry (e.g, different [Mg], [Ca], and [B]) on B/Ca, modern calibrations cannot be used to estimate the magnitude of acidification during this critical period. In addition, recent inorganic and sediment trap studies have respectively documented the controls of growth rate and light levels on B/Ca [2,3]. To extend the application of the B/Ca proxy to the PETM, we have conducted culturing experiments in O. universa, G. ruber, and G. sacculifer in which we simulated changes in pH and total DIC under Paleogene seawater conditions- high [Ca], low [Mg], and low [B]. We have further investigated the effects of variable light intensity (a control on symbiont activity), [Ca]seawater, and [B]seawater on the proxy. Results from O. universa confirm that B/Ca decreases with increasing DIC, decreasing pH, and decreasing [B]seawater, supporting a [B(OH)4-]/DIC control on the proxy [4]. In contrast, neither low light nor [Ca]seawater have a measurable effect on B/Ca, implying that influences of these parameters over the PETM were likely negligible. Critically, B/Ca appears to be more sensitive to pH at very low [B(OH)4-]/DIC in comparison to modern calibrations. Using estimates of surface ocean pH from boron isotopes, new calibrations can explain a larger proportion of the observed B/Ca excursion over the PETM. However, simulation of a large DIC pulse is necessary to explain the full excursion. New data will be presented from species that are more sensitive to pH, such as G. ruber and G. sacculifer, which will illuminate the range of responses of B/Ca to ocean acidification during the Paleogene. [1] Penman et al. 2014. Paleoceanography 29. [2] Uchikawa et al. 2015. GCA 150. [3] Babila et

  12. The dissolution effect on the B/Ca ratio of planktic foraminifers: a potential bias for paleo-pH reconstructions

    NASA Astrophysics Data System (ADS)

    Coadic, R.; Bassinot, F.; Dissard, D.; Douville, E.; Michel, E.; Greaves, M.

    2012-12-01

    The B/Ca ratio of planktonic foraminifers is a new paleoceanographic tool to reconstruct past sea-surface pH. However, we show that the dissolution of foraminifer shells may result in the preferential removal of B and represents, therefore, a potential bias for pH reconstructions, similar to that observed for Mg/Ca and paleo-temperature estimates. We determined B/Ca and Mg/Ca of the planktonic foraminifer species Globigerinoides sacculifer in seven core-tops from a depth transect along the Sierra Leone Rise, in the Eastern Equatorial Atlantic. Samples were analyzed independently at two laboratories, the LSCE at Gif-sur-Yvette and the Godwin Laboratory at Cambridge. Both sets of results show a systematic decrease of B/Ca and Mg/Ca along the depth transect, with an overall loss of ˜14 μmol/mol (˜15%) for B/Ca and of ˜0.7 mmol/mol (˜21%) for Mg/Ca between the shallowest (2640 m) and the deepest (4950 m) sites. Because of this dissolution bias, surface water pH reconstructed from B/Ca of G. sacculifer is ˜0.11 unit lower at the shallow water site than at the deep site. This dissolution-driven difference in pH has a similar magnitude than the expected glacial/interglacial surface water pH change. Because planktonic foraminifer dissolution is related to the saturation state of bottom-water with respect to calcite, we propose a correction approach based on benthic foraminifer B/Ca ratios, a proxy of carbonate ion saturation (ΔCO32-). Along our Sierra Leone Rise transect, we found a positive relationship between the B/Ca of the benthic species Cibicides wuellerstorfi (and hence ΔCO32-) and the relative loss of B/Ca and Mg/Ca. This empirical relationship makes it possible to correct estimates of temperature (Mg/Ca) and pH (B/Ca) for dissolution effects, and may be applied to paleoceanographic reconstructions to correct for the effect of past changes in bottom water carbonate ion saturation on these paleoceanographic proxies.

  13. High-Speed Microdialysis-Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells.

    PubMed

    Harstad, Rachel K; Bowser, Michael T

    2016-08-16

    We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay. PMID:27398773

  14. Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure.

    PubMed

    Schulpen, Sjors H W; Theunissen, Peter T; Pennings, Jeroen L A; Piersma, Aldert H

    2015-08-15

    Embryonic stem cell tests (EST) are considered promising alternative assays for developmental toxicity testing. Classical mouse derived assays (mEST) are being replaced by human derived assays (hEST), in view of their relevance for human hazard assessment. We have compared mouse and human neural ESTn assays for neurodevelopmental toxicity as to regulation of gene expression during cell differentiation in both assays. Commonalities were observed in a range of neurodevelopmental genes and gene ontology (GO) terms. The mESTn showed a higher specificity in neurodevelopment than the hESTn, which may in part be caused by necessary differences in test protocols. Moreover, gene expression responses to the anticonvulsant and human teratogen valproic acid were compared. Both assays detected pharmacological and neurodevelopmental gene sets regulated by valproic acid. Common significant expression changes were observed in a subset of homologous neurodevelopmental genes. We suggest that these genes and related GO terms may provide good candidates for robust biomarkers of neurodevelopmental toxicity in hESTn. PMID:26072468

  15. Development of a scintillation proximity assay for the Mycobacterium tuberculosis KasA and KasB enzymes involved in mycolic acid biosynthesis.

    PubMed

    Schaeffer, M L Merrill L; Carson, J D Jeffrey D; Kallender, Howard; Lonsdale, J T John T

    2004-01-01

    Tuberculosis remains a global health problem, and programs dedicated to discovery of novel compounds against Mycobacterium tuberculosis require robust assays for high-throughput screening of chemical and natural product libraries. Enzymes involved in the biosynthesis of mycolic acids, vital components of the mycobacterial cell wall, have received much attention as potential drug targets. KasA and KasB, examples of the beta-ketoacyl-acyl carrier protein synthase I/II (KASI/II) class of condensing enzymes of the M. tuberculosis fatty acid synthase II system have been the focus of several studies designed to biochemically characterize these enzymes. Whilst robust methods have been developed for FabH-like proteins, fast and sensitive assays for high-throughput screening of KASI/II enzymes have not been available. Here we report the development of a direct scintillation proximity assay (SPA) for the KASI/II enzymes, KasA and KasB. The SPA was more sensitive than existing assays, as shown by its ability to measure activity using less enzyme than other assay formats, and the SPA was validated using the known KAS inhibitor thiolactomycin. In addition, the KasA and KasB SPA was adapted for use with Staphylococcus aureus FabF to show the versatility of this assay format to KAS enzymes from other pathogenic organisms. PMID:15525558

  16. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

  17. New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

    PubMed Central

    Decousser, Jean-Winoc; Methlouthi, Imen; Pina, Patrick; Collignon, Anne; Allouch, Pierre

    2006-01-01

    A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys. PMID:16569894

  18. Use of agar diffusion assay to evaluate bactericidal activity of formulations of alkaline salts of fatty acids against bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine antibacterial activity of alkaline salts of fatty acids (FA). Wells in agar media seeded with bacteria were filled with FA-potassium hydroxide (KOH) solutions, plates were incubated, and zones of inhibition were measured. The relationship between bacteric...

  19. Helicase Assays

    PubMed Central

    Wang, Xin; Li, Jing; Diaz, Jason; You, Jianxin

    2016-01-01

    Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.

  20. Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

    PubMed Central

    Horn, T; Chang, C A; Urdea, M S

    1997-01-01

    The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology. PMID:9365266

  1. EVALUATING EFFECTS OF NEPTUNIUM ON THE SRS METHOD FOR CONTROLLED POTENTIAL COULOMETRIC ASSAY OF PLUTONIUM IN SULFURIC ACID SUPPORTING ELECTROLYTE

    SciTech Connect

    Holland, M; Sheldon Nichols, S

    2008-05-09

    A study of the impact of neptunium on the coulometric assay of plutonium in dilute sulfuric acid was performed. Weight aliquots of plutonium standard solutions were spiked with purified neptunium solution to evaluate plutonium measurement performance for aliquots with Pu:Np ratios of 50:1, 30:1, 20:1, 15:1, and 10:1. Weight aliquots of the pure plutonium standard solution were measured as controls. Routine plutonium instrument control standards were also measured. The presence of neptunium in plutonium aliquots significantly increases the random uncertainty associated with the plutonium coulometric measurement performed in accordance with ISO12183:2005.7 However, the presence of neptunium does not appear to degrade electrode performance and conditioning as aliquots of pure plutonium that were interspersed during the measurement of the mixed Pu:Np aliquots continued to achieve the historical short-term random uncertainty for the method. Lack of adequate control of the neptunium oxidation state is suspected to be the primary cause of the elevated measurement uncertainty and will be pursued in a future study.

  2. Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

    2014-06-01

    The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

  3. Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

    PubMed Central

    Modak, Sayli S.; Barber, Cheryl A.; Geva, Eran; Abrams, William R.; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

    2016-01-01

    Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject’s blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200–2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation. PMID:26819557

  4. Comparative Study of the One-step Nucleic Acid Amplification Assay and Conventional Histological Examination for the Detection of Breast Cancer Sentinel Lymph Node Metastases.

    PubMed

    Terada, Mizuho; Niikura, Naoki; Tsuda, Banri; Masuda, Shinobu; Kumaki, Nobue; Tang, Xiaoyan; Okamura, Takuho; Saito, Yuki; Suzuki, Yasuhiro; Tokuda, Yutaka

    2014-09-01

    Intraoperative sentinel lymph node (SLN) biopsy is widely used in patients with early-stage breast cancer and is conventionally performed using hematoxylin and eosin-based histological examination. The one-step nucleic acid amplification (OSNA) assay is a molecular diagnostic tool and a semi-automated lymph node examination method. The purpose of this study was to compare the performance of the OSNA assay and conventional histological examination with frozen sections (FSs) by using 111 SLN biopsy samples from 89 patients at the Tokai University Hospital. The SLN samples were split into 3 slices: the middle slice was used for FS histological examination and the other slices were used for the OSNA assay. The McNemar test was used to compare the differences in the sensitivity and specificity between the OSNA assay and FS histological examination. The sensitivity of the OSNA assay (97.1%) was less than that of FS histological examination (100%), but this difference was not statistically significant (P = 0.125). The specificity of both the methods was identical (96.9%). Despite previously published results suggesting that the OSNA assay is as reliable as histological examinations, our results indicate that this assay often fails to detect micrometastases or isolated tumor cells in SLNs. PMID:25248427

  5. A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

    PubMed Central

    Marron, Alan O.; Akam, Michael; Walker, Giselle

    2013-01-01

    Background Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Principal Findings Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. Conclusions The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop q

  6. Comparative quantitative analysis of hepatitis C mutations at amino acids 70 and 91 in the core region by the Q-Invader assay.

    PubMed

    Tadokoro, Kenichi; Kobayashi, Mariko; Suzuki, Fumitaka; Tanaka, Chie; Yamaguchi, Toshikazu; Nagano, Makoto; Egashira, Toru; Kumada, Hiromitsu

    2013-04-01

    Hepatitis C virus (HCV) is a major worldwide public health problem, and mutations at amino acids 70 and 91 in the genotype 1b core region predict the effectiveness of combination therapy with peginterferon and ribavirin. An assay based on the Q-Invader technology was developed to determine the relative ratios of the mutant to wild-type virus with high sensitivity. The assay detected a minor type plasmid that constituted only 1% of a mixture of plasmids containing wild-type and mutant sequences. The calculated ratios agreed with those of the template DNA. A total of 123 serum samples of HCV in Japan were examined with the Q-Invader assay. The Q-Invader assay detected all of the mutations that were detected by direct sequencing and even some mutants that direct sequencing could not. PCR with mutant specific primers confirmed those mutations found by the Q-Invader assay and not by direct sequencing. The Q-Invader assay, thus, is a useful tool for detecting mutations at positions 70 and 91 in the HCV-1b core region. PMID:23124003

  7. Library screening by means of mass spectrometry (MS) binding assays-exemplarily demonstrated for a pseudostatic library addressing γ-aminobutyric acid (GABA) transporter 1 (GAT1).

    PubMed

    Sindelar, Miriam; Wanner, Klaus T

    2012-09-01

    In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including

  8. A comprehensive review of the published assays for the quantitation of the immunosuppressant drug mycophenolic acid and its glucuronidated metabolites in biological fluids.

    PubMed

    Syed, Muzeeb; Srinivas, Nuggehally R

    2016-05-01

    Therapeutic use of mycophenolic acid (MPA) is steadily on the rise in combination with other immunosuppressant drugs in transplantation patients. The biotransformation of MPA resulted in the formation of glucuronide metabolites, MPAG and AcMPAG. There are a plethora of assays validated for the analysis of MPA alone or with MPAG/AcMPAG in various biological specimens including plasma/serum, urine, ultrafiltrate, saliva, PBMC, dried blood spots, tissue extract, tumor biopsies and vitreous humor. Based on the need for experimental work, a proper choice of the assay and internal standard may be made using the choices in the literature. While the chemical methods involving high-performance liquid chromatography (HPLC) or LC coupled with triple quadrupole mass spectrometry (LC-MS/MS) are popular, enzymatic assays, in spite of their higher bias, have been used for the routine drug monitoring of MPA. The objectives of the present review are: (a) to provide a focused systematic compilation of the HPLC or LC-MS/MS methods for MPA, MPAG and/or AcMPAG published in the last decade (2005 to current) to enable visual comparison of the methods; (b) to compare and contrast a few enzymatic assays with those of the chemical methods; and (c) to discuss relevant issues/limitations and perspectives on select assays under various subheadings. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26766308

  9. Nonamplification Sandwich Assay Platform for Sensitive Nucleic Acid Detection Based on AuNPs Enumeration with the Dark-Field Microscope.

    PubMed

    Li, Tian; Xu, Xiao; Zhang, Guoqing; Lin, Ruoyun; Chen, Yang; Li, Chenxi; Liu, Feng; Li, Na

    2016-04-19

    A simple and efficient assay platform with high sensitivity, convenient implementation, and moderate cost in reagents and instrumentation is most appropriate for routine applications. On the basis of the gold nanoparticle (AuNP) enumeration signal readout mode established in our laboratory, we have developed a nonamplification sandwich assay for nucleic acid detection with the 3 fM limit of detection for a sequence related to Alzheimer's disease. This AuNP counting based method takes advantages of the distinctive and strong localized surface plasmon resonance light scattering with the dark-field microscope and magnetic separation. It is shown that the presence of 20 nM random DNA sequence or calf thymus DNA with a mass up to 10(6)-fold of the targets do not significantly interfere with the assay signal. The spike recoveries of Hela cell lysate sample at 109.3% for 20 pM target and 110.5% for 100 pM target indicate the potential of this proposed method in practical sample applications. This nonamplification sandwich assay platform in principle is applicable to other assays such as the immunoassay and thus would be expected to find a breadth of applications that can make the best use of the simplicity and sensitivity. PMID:27023372

  10. Study on effect of peptide-conjugated near-infrared fluorescent quantum dots on the clone formation, proliferation, apoptosis, and tumorigenicity ability of human buccal squamous cell carcinoma cell line BcaCD885

    PubMed Central

    Sun, Deping; Yang, Kai; Zheng, Gang; Li, Zhigang; Cao, Yuan

    2010-01-01

    Quantum dots (QDs) have shown great development potential in noninvasive imaging and monitoring of cancer cells in vivo because of their unique optical properties. However, the key issue of whether or not QDs-labeled cancer cells affect the proliferation, apoptosis and in vivo tumorigenicity ability has not been reported. The primary issue is if the results obtained from the noninvasive visualization of QDs-labeled tumors are scientific. Here, we applied peptide-linked near-conjugated fluorescent QDs to label human buccal squamous cell carcinoma cell line (BcaCD885). We performed in vivo tumorigenicity ability assays, tumorigenic cells proliferation, and apoptotic capability assays detected by flow cytometry and plate clone formation experiment, and found that peptide-linked near-conjugated fluorescent QDs labeling did not affect the growth, proliferation, apoptosis, and tumorigenicity ability of those cancer cells. Our study provides scientific foundation to support the application of near-infrared fluorescent QDs in noninvasive imaging and monitoring of cancer cells in vivo. PMID:20957161

  11. Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15.

    PubMed

    Huzly, Daniela; Korn, Klaus; Bierbaum, Sibylle; Eberle, Björn; Falcone, Valeria; Knöll, Antje; Steininger, Philipp; Panning, Marcus

    2016-09-01

    The influenza season 2014/15 was dominated by drift variants of influenza A(H3N2), which resulted in a reduced vaccine effectiveness. It was not clear if the performance of commercial nucleic-acid-based amplification (NAT) assays for the detection of influenza was affected. The purpose of this study was to perform a real-life evaluation of two commercial NAT assays. During January-April 2015, we tested a total of 665 samples from patients with influenza-like illness using the Fast Track Diagnostics Respiratory pathogens 21, a commercial multiplex kit, (cohorts 1 and 2, n = 563 patients) and the Xpert Flu/RSV XC assay (cohort 3, n = 102 patients), a single-use cartridge system. An in-house influenza real-time RT-PCR (cohort 1) and the RealStar Influenza RT-PCR 1.0 Kit (cohort 2 and 3) served as reference tests. Compared to the reference assay, an overall agreement of 95.9 % (cohort 1), 95 % (cohort 2), and 98 % (cohort 3) was achieved. A total of 24 false-negative results were observed using the Fast Track Diagnostics Respiratory pathogens 21 kit. No false-negative results occurred using the Xpert Flu/RSV XC assay. The Fast Track Diagnostics Respiratory pathogens 21 kit and the Xpert Flu/RSV XC assay had sensitivities of 90.7 % and 100 % and specificities of 100 % and 94.1 %, respectively, compared to the RealStar 1.0 kit. Upon modification of the Fast Track Diagnostics Respiratory pathogens 21 kit, the sensitivity increased to 97.3 %. Influenza virus strains circulating during the 2014/15 season reduced the detection sensitivity of a commercial NAT assay, and continuous monitoring of test performance is therefore necessary. PMID:27316440

  12. Expression of Serum Sialic Acid, Early Antigen-IgA, and Viral Capsid Antigen-IgA in Nasopharynx Cancer Patients: The Diagnostic Implication of Combined Assays.

    PubMed

    Sun, Yuning; Sun, Caibo; Zhang, Endong

    2015-01-01

    BACKGROUND Ebstein-Barr virus (EBV) plays a critical role in nasopharynx cancer, which can be effectively monitored by serum levels of early antigen antibody (EA-IgA) and viral capsid antigen antibody (VCA-IgA). This study explored the diagnostic value of combined assays of sialic acid (SA), EA-IgA, and VCA-IgA via the expressional assay. MATERIAL AND METHODS A total of 42 nasopharynx cancer patients and 42 benign rhinitis and healthy controls were recruited in this study. Serum EA-IgA and VCA-IgA were tested by enzyme-linked immunosorbent assay (ELISA) and enzymatic assay of serum SA. Specificity and sensitivity of those 3 assays were compared. The diagnostic value of each parameter was evaluated by ROC curves. RESULTS All 3 indexes (SA, EA-IgA and VCA-IgA) showed elevated serum levels in nasopharynx cancer patients when compared to those with rhinitis, who had higher levels than healthy individuals. Concentrations of these factors were also positively correlated with the TNM staging of cancer. The sensitivity and specificity were 30.95% and 83.33% (in SA), 57.14% and 95.24% (in EA-IgA), and 76.19% and 92.86% (in VCA-IgA), respectively. VCA-IgA had the highest sensitivity among all 3 indexes. The combined assay increased the diagnostic sensitivity to 92.86% without compromising specificity. CONCLUSIONS SA, EA-IgA, and VCA-IgA levels were significantly elevated in nasopharynx patients' serum. The combined assay may have clinical value in diagnosis and monitoring. PMID:26709095

  13. Expression of Serum Sialic Acid, Early Antigen-IgA, and Viral Capsid Antigen-IgA in Nasopharynx Cancer Patients: The Diagnostic Implication of Combined Assays

    PubMed Central

    Sun, Yuning; Sun, Caibo; Zhang, Endong

    2015-01-01

    Background Ebstein-Barr virus (EBV) plays a critical role in nasopharynx cancer, which can be effectively monitored by serum levels of early antigen antibody (EA-IgA) and viral capsid antigen antibody (VCA-IgA). This study explored the diagnostic value of combined assays of sialic acid (SA), EA-IgA, and VCA-IgA via the expressional assay. Material/Methods A total of 42 nasopharynx cancer patients and 42 benign rhinitis and healthy controls were recruited in this study. Serum EA-IgA and VCA-IgA were tested by enzyme-linked immunosorbent assay (ELISA) and enzymatic assay of serum SA. Specificity and sensitivity of those 3 assays were compared. The diagnostic value of each parameter was evaluated by ROC curves. Results All 3 indexes (SA, EA-IgA and VCA-IgA) showed elevated serum levels in nasopharynx cancer patients when compared to those with rhinitis, who had higher levels than healthy individuals. Concentrations of these factors were also positively correlated with the TNM staging of cancer. The sensitivity and specificity were 30.95% and 83.33% (in SA), 57.14% and 95.24% (in EA-IgA), and 76.19% and 92.86% (in VCA-IgA), respectively. VCA-IgA had the highest sensitivity among all 3 indexes. The combined assay increased the diagnostic sensitivity to 92.86% without compromising specificity. Conclusions SA, EA-IgA, and VCA-IgA levels were significantly elevated in nasopharynx patients’ serum. The combined assay may have clinical value in diagnosis and monitoring. PMID:26709095

  14. Spectroscopy characterization of the interaction between brevifolin carboxylic acid and bovine serum albumin.

    PubMed

    Tian, Jianniao; Xie, Yuhuan; Zhao, Yanchun; Li, Caifeng; Zhao, Shulin

    2011-01-01

    Themechanism of binding of the antivirus drug, brevifolin carboxylic acid (BCA) with bovine serum albumin (BSA) was investigated by steady-state and time-resolved fluorescence, circular dichroism (CD), Fourier transform infrared (FT-IR) and Raman spectroscopy under pseudo-physiological conditions for the first time. A strong fluorescence quenching was observed and the quenching mechanism was considered as static quenching. Various binding parameters were evaluated. The quantitative analysis of CD spectral data revealed that the a-helical content of BSA increased from 48.91% (in free BSA) to 52.46% (in bound form) in the presence of BCA. Based on the Förster's theory of non-radiation energy transfer, the relation of the binding average distance r between the donor (BSA) and acceptor (BCA) and acceptor concentration was determined. The changes in association constants of BCA-BSA in the presence of the common ions are also discussed. From the CD, FT-IR, time-resolved fluorescence and Raman spectroscopic results, it is apparent that the interaction of BCA with BSA causes a conformational change in the protein, and the Trp and Tyr residues are buried in more hydrophobic surroundings. BCA mainly binds to residue Trp 212 located in domain II of BSA by hydrophobic interaction and hydrogen bond. PMID:20737652

  15. Using phospholipid fatty acid technique to study short-term effects of the biological control agent Pseudomonas fluorescens DR54 on the microbial microbiota in barley rhizosphere.

    PubMed

    Johansen, A; Olsson, S

    2005-02-01

    The biological control agent (BCA) Pseudomonas fluorescens DR54 was applied to seeds (experiment 1) or roots (experiment 2) of barley growing in microcosms, while noninoculated plants served as controls. The fate of the BCA and its effects on the rhizosphere microbial community was evaluated in microcosms destructively sampled at days 2, 4, 6, and 9 after inoculation. In both experiments the number of P. fluorescens DR54 cells decreased immediately after application as enumerated by immunostaining and microscope direct counting. Substrate-induced respiration (SIR) was taken as a measurement of the active microbial biomass, while indicators of the total microbiota (and main taxonomic groups) were obtained using the phospholipid fatty acid (PLFA) technique. In experiment 1, these parameters were unaffected by the relatively small number of BCA cells applied, whereas in experiment 2, the larger BCA input resulted in an enhanced level of both SIR and PLFAs from Gram-negative bacteria (which included the BCA itself). However, at day 9 after inoculation, treatments with P. fluorescens DR54 and controls were similar in all measured parameters in both experiments. This was also illustrated very clearly by principal component analysis of the PLFA data, which in both experiments were able to discriminate between treatments in the first days after BCA inoculation, thus confirming the sensitivity of this method. Laccase activity has a potential as an indicator of fungal stress, e.g., when challenged with an antifungal BCA. This seemed to be supported in experiment 2, where the activity of this enzyme was enhanced four-fold in the BCA treatment at day 2. Our study shows that under the present conditions, P. fluorescens DR54 disappears from the soil and causes only transient effects on the soil microbiota. It also shows that the PLFA technique is a sensitive and reliable monitoring tool in in situ assessment of BCA nontarget effect on indigenous microorganisms in soil. PMID

  16. Development of a real-time PCR genotyping assay to identify high oleic acid peanuts (Arachis hypogaea L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oleic acid, a monounsaturated, omega-9 fatty acid found in peanut (Arachis hypogaea L.) oil is an important seed quality trait because it provides increased shelf life, improved flavor, enhanced fatty acid composition, and has a beneficial effect on human health. Hence, a concentrated effort has be...

  17. A core-top study of dissolution effect on B/Ca in Globigerinoides sacculifer from the tropical Atlantic: Potential bias for paleo-reconstruction of seawater carbonate chemistry

    NASA Astrophysics Data System (ADS)

    Coadic, R.; Bassinot, F.; Dissard, D.; Douville, E.; Greaves, M.; Michel, E.

    2013-04-01

    has been recently shown that B/Ca in planktonic foraminiferal calcite can be used as a proxy for seawater pH. Based on the study of surface sediments (multi-cores) retrieved along a depth transect on the Sierra Leone Rise (Eastern Equatorial Atlantic), we document the decrease of B/Ca and Mg/Ca of Globigerinoides sacculifer shells with increasing water depth and dissolution. This effect of dissolution on B/Ca may potentially represent a severe bias for paleo-pH reconstructions using this species. Samples of G. sacculifer were analyzed independently at two laboratories for B/Ca and Mg/Ca. Both sets of results show a systematic decrease of B/Ca and Mg/Ca along the depth transect, with an overall loss of ~14 µmol/mol (~15%) for B/Ca and of ~0.7 mmol/mol (~21%) for Mg/Ca between the shallowest (2640 m) and the deepest (4950 m) sites. Because of this dissolution effect, surface water pH reconstructed from B/Ca of G. sacculifer decreases by ~0.11 units between the shallowest site and the deepest site, a magnitude similar to the expected glacial/interglacial surface water pH changes.

  18. Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

    PubMed

    Kugimiya, Akimitsu; Konishi, Hidenori; Fukada, Rie

    2016-03-01

    Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 μM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0). PMID:26554858

  19. Genotoxicity evaluation of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate using a combined rat comet/micronucleus assays.

    PubMed

    Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Kimura, Juki; Funabashi, Hitoshi; Saito, Koichi

    2015-07-01

    As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo alkaline comet assay (comet assay), we examined DNA damage in the liver, stomach, and bone marrow of rats dosed orally three times with up to 2000 mg/kg of benzene, di(2-ethylhexyl) phthalate, and trisodium ethylenediamine tetraacetic acid monohydrate. All three compounds gave negative results in the liver and stomach. In addition, a bone marrow comet and micronucleus analysis revealed that benzene, but not di(2-ethylhexyl) phthalate or trisodium ethylenediamine tetraacetic acid monohydrate induced a significant increase in the median % tail DNA and micronucleated polychromatic erythrocytes, compared with the respective concurrent vehicle control. These results were in good agreement with the previously reported genotoxicity findings for each compound. The present study has shown that combining the micronucleus test with the comet assay and carrying out these analyses simultaneously is effective in clarifying the mechanism of action of genotoxic compounds such as benzene. PMID:26212304

  20. Calcification and growth processes in planktonic foraminifera complicate the use of B/Ca and U/Ca as carbonate chemistry proxies

    NASA Astrophysics Data System (ADS)

    Salmon, Kate H.; Anand, Pallavi; Sexton, Philip F.; Conte, Maureen

    2016-09-01

    Although boron and uranium to calcium ratios (B/Ca, U/Ca) in planktonic foraminifera have recently received much attention as potential proxies for ocean carbonate chemistry, the extent of a carbonate chemistry control on these ratios remains contentious. Here, we use bi-weekly sediment trap samples collected from the subtropical North Atlantic in combination with measured oceanographic data from the same location to evaluate the dominant oceanographic controls on B/Ca and U/Ca in three depth-stratified species of planktonic foraminifera. We also test the control of biological, growth-related, processes on planktonic foraminiferal B and U incorporation by using foraminifer test area density (μ g /μm2) (a monitor of test thickness) and test size from the same samples. B/Ca and U/Ca show little or no significant correlation with carbonate system parameters both within this study and in comparison with other published works. We provide the first evidence for a strong positive relationship between area density (test thickness) and B/Ca, and reveal that this is consistent in all species studied, suggesting a likely role for calcification in controlling boron partitioning into foraminiferal calcite. This finding is consistent with previous observations of less efficient discrimination against trace element 'impurities' (such as B), at higher calcification rates. We observe little or no dependency of B/Ca on test size. In marked contrast, we find that U/Ca displays a strong species-specific dependency on test size in all species, but no relationship with test thickness, implicating some other biological control (possibly related to growth), rather than a calcification control, on U incorporation into foraminiferal calcite. Our results caution against the use of B/Ca and U/Ca in planktonic foraminifera as reliable proxies for the ocean carbonate system and recommend that future work should concentrate on improving the mechanistic understanding of how planktonic

  1. Kinetic Effects on B/Ca in Synthetic Calcite: Implications for B(OH)4- and B(OH)3 Incorporation.

    NASA Astrophysics Data System (ADS)

    Uchikawa, J.; Penman, D. E.; Zachos, J. C.; Zeebe, R. E.

    2014-12-01

    In this experimental study, we investigated the influence of solution chemistry on the boron abundance in synthetic calcite using a pH-stat system. We systematically varied solution pH as well as the concentration of total dissolved boron (BT), inorganic carbon (DIC) and calcium ion. We found robust positive correlations between boron abundance in calcite (measured as the boron to calcium ratio, B/Ca) and solution pH, [BT], [DIC] as well as [Ca2+], when a given parameter was solely manipulated while keeping the others constant. Except for [BT], raising these parameters also caused simultaneous increase in calcite saturation and precipitation rate. We found that much of the B/Ca variability as a results of the chemical manipulations tested here can be essentially explained by just precipitation rate and the [BT]/[DIC] ratio in the solution, which was particularly the case for relatively rapidly precipitated calcite samples. On the contrary, for relatively slowly precipitated samples, the [B(OH)4-]/[DIC] and [BT]/[DIC] ratios are equally effective in explaining the B/Ca variability (along with precipitation rate). This observation suggests the possibility of a hitherto unrecognized and apparently kinetically-controlled mechanism that promotes B(OH)3 incorporation for rapidly forming calcite. In recent years both the abundance and isotopic composition of boron in marine biogenic CaCO3 (B/Ca and δ11B, respectively) has been increasingly utilized to constrain past ocean carbonate chemistry. But these boron-based proxies crucially rely on the first order assumption that B(OH)4- is predominantly incorporated. Thus, the possibility of kinetically-controlled B(OH)3 incorporation presented here raises a concern for the reliability of the B/Ca and δ11B proxy. If it is similarly applicable to foraminifers, shell B/Ca and δ11B may be prone to significant uncertainties due to long-term changes in seawater chemistry. Our results further suggest that future calibrations of

  2. HPLC-PDA method for quinovic acid glycosides assay in Cat's claw (Uncaria tomentosa) associated with UPLC/Q-TOF-MS analysis.

    PubMed

    Pavei, Cabral; Kaiser, Samuel; Verza, Simone Gasparin; Borre, Gustavo Luis; Ortega, George Gonzalez

    2012-03-25

    Uncaria tomentosa (Willd.) is a medicinal plant largely used in folk medicine due to its wide range of biological activities, many of which are usually ascribed to the two main classes of secondary metabolites, namely, alkaloids and quinovic acid glycosides. In this work, a reversed phase HPLC-PDA method was developed and validated for the assay of quinovic acid glycosides in crude and dried extracts of Uncaria tomentosa (Cat's claw) bark. The validation comprised tests of specificity, accuracy, linearity, intermediate precision, repeatability and limits of detection and of quantification. Alpha-hederin was used as the external standard. High coefficients of determination with lower R.S.D. were achieved for both external standard and crude extract. The structural characterization of the main quinovic acid glycosides presented in the crude extract was carried out through UPLC/Q-TOF-MS. The identities of the compounds were obtained through the comparison of their fragmentation patterns with those reported in the literature. The analytical method was successfully applied for quantifying quinovic acid glycosides in two different dried extracts from U. tomentosa and in one quinovic acid glycosides purified fraction. PMID:22296654

  3. Assay of calcium borogluconate veterinary medicines for calcium gluconate, boric acid, phosphorus, and magnesium by using inductively coupled plasma emission spectrometry

    SciTech Connect

    Lyons, D.J.; Spann, K.P.

    1985-03-01

    An inductively coupled plasma spectrometric method is described for the determination of 4 elements (Ca, B, P, and Mg) in calcium borogluconate veterinary medicines. Samples are diluted, acidified, and sprayed directly into the plasma. Reproducibility relative confidence intervals for a single sample assay are +/- 1.4% (calcium), +/- 1.8% (boron), +/- 2.6% (phosphorus), and +/- 1.4% (magnesium). The total element concentrations for each of 4 elements compared favorably with concentrations determined by alternative methods. Formulation estimates of levels of calcium gluconate, boric acid, phosphorus, and magnesium salts can be made from the analytical data.

  4. Detection of Ganoderic Acid A in Ganoderma lingzhi by an Indirect Competitive Enzyme-Linked Immunosorbent Assay.

    PubMed

    Sakamoto, Seiichi; Kohno, Toshitaka; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2016-05-01

    Ganoderma is a genus of medicinal mushroom traditionally used for treating various diseases. Ganoderic acid A is one of the major bioactive Ganoderma triterpenoids isolated from Ganoderma species. Herein, we produced a highly specific monoclonal antibody against ganoderic acid A (MAb 12 A) and developed an indirect competitive ELISA for the highly sensitive detection of ganoderic acid A in Ganoderma lingzhi, with a limit of detection of 6.10 ng/mL. Several validation analyses support the accuracy and reliability of the developed indirect competitive ELISA for use in the quality control of Ganoderma based on ganoderic acid A content. Furthermore, quantitative analysis of ganoderic acid A in G. lingzhi revealed that the pileus exhibits the highest ganoderic acid A content compared with the stipe and spore of the fruiting body; the best extraction efficiency was found when 50 % ethanol was used, which suggests the use of a strong liquor to completely harness the potential of Ganoderma triterpenoids in daily life. PMID:27093250

  5. In vitro antioxidant assay of medium chain fatty acid rich rice bran oil in comparison to native rice bran oil.

    PubMed

    Sengupta, Avery; Ghosh, Mahua; Bhattacharyya, D K

    2015-08-01

    The study aimed to evaluate the in vitro antioxidant activity of medium chain fatty acid (MCFA) rich-rice bran oils in comparison with native rice bran oil. Different in vitro methods were used to evaluate the free radical scavenging activity, metal chelation activity, reducing acitivity, ABTS radical scavenging activity, thiobarbituric acid (TBA) value and so on at different concentrations of the oils such as 10-100 μg/mL. Inhibition of lipid peroxidation was evaluated measuring thiobarbituric acid responsive substance (TBARS) and conjugated diene formation. All the oils showed potent antioxidant activity at 100 μg/mL concentration. TBARS formation and conjugated diene formation was lower with MCFA rich oils i.e. the inhibition of lipid peroxidation was more in MCFA rich oils than original rice bran oil. Caprylic acid rich rice bran oil showed maximum antioxidant activity in comparison to capric- and lauric acid rich rice bran oils. Overall the MCFA rich rice bran oils showed to be more potent antioxidant than rice bran oil due to their lower unsaturated fatty acid content. PMID:26243941

  6. One-step nucleic acid amplification assay for intraoperative prediction of advanced axillary lymph node metastases in breast cancer patients with sentinel lymph node metastasis

    PubMed Central

    KUBOTA, MICHIYO; KOMOIKE, YOSHIFUMI; HAMADA, MIKA; SHINZAKI, WATARU; AZUMI, TATSUYA; HASHIMOTO, YUKIHIKO; IMOTO, SHIGERU; TAKEYAMA, YOSHIFUMI; OKUNO, KIYOTAKA

    2016-01-01

    The one-step nucleic acid amplification (OSNA) assay is used to semiquantitatively measure the cytokeratin (CK)19 mRNA copy numbers of each sentinel lymph node (SLN) in breast cancer patients. The aim of the present study was to evaluate whether the diagnosis of ≥4 LN metastases is possible using the OSNA assay intraoperatively. Between May, 2010 and December, 2014, a total of 134 patients who underwent axillary lymph node dissection (ALND) of positive SLNs were analyzed. The total tumor load (TTL) was defined as the total CK19 mRNA copies of all positive SLNs. The correlation between TTL and ≥4 LN metastases was evaluated. Of the 134 patients, 31 (23.1%) had ≥4 LN metastases. TTL ≥5.4×104 copies/µl evaluated by receiver operator characteristic curve analysis was examined along with other clinicopathological variables. In the multivariate analysis, only TTL ≥5.4×104 copies/µl was correlated with ≥4 LN metastases (odds ratio = 2.95, 95% confidence interval: 1.17–7.97, P=0.022). Therefore, TTL assessed by the OSNA assay has the potential to be a predictor of ≥4 LN metastases and it may be useful for the selection of patients with positive SLNs in whom ALND may be safely omitted. PMID:26893855

  7. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

    PubMed

    Rutjes, Saskia A; van den Berg, Harold H J L; Lodder, Willemijn J; de Roda Husman, Ana Maria

    2006-08-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment. PMID:16885286

  8. A novel human T-leukemia virus type 1 cell-to-cell transmission assay permits definition of SU glycoprotein amino acids important for infectivity.

    PubMed Central

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Dokhélar, M C

    1997-01-01

    Human T-leukemia virus type 1 (HTLV-1) envelope glycoproteins play a major role in viral transmission, which in the case of this virus occurs almost exclusively via cell-to-cell contact. Until very recently, the lack of an HTLV-1 infectivity assay precluded the determination of the HTLV-1 protein domains required for infectivity. Here, we describe an assay which allows the quantitative evaluation of HTLV-1 cell-to-cell transmission in a single round of infection. Using this assay, we demonstrate that in this system, cell-to-cell transmission is at least 100 times more efficient than transmission with free viral particles. We have examined 46 surface (SU) glycoprotein mutants in order to define the amino acids of the HTLV-1 SU glycoprotein required for full infectivity. We demonstrate that these amino acids are distributed along the entire length of the SU glycoprotein, including the N-terminus and C-terminus regions, which have not been previously defined as being important for HTLV-1 glycoprotein function. For most of the mutated glycoproteins, the capacity to mediate cell-to-cell transmission is correlated with the ability to induce formation of syncytia. This result indicates that the fusion capacity is the main factor responsible for infectivity mediated by the HTLV-1 SU envelope glycoprotein, as is the case for other retroviral glycoproteins. However, other factors must also intervene, since two of the mutated glycoproteins were correctly fusogenic but could not mediate cell-to-cell transmission. Existence of this phenotype shows that capacity for fusion is not sufficient to confer infectivity, even in cell-to-cell transmission, and could suggest that postfusion events involve the SU. PMID:8985345

  9. In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes[S

    PubMed Central

    Ensenauer, Regina; Fingerhut, Ralph; Schriever, Sonja C.; Fink, Barbara; Becker, Marc; Sellerer, Nina C.; Pagel, Philipp; Kirschner, Andreas; Dame, Torsten; Olgemöller, Bernhard; Röschinger, Wulf; Roscher, Adelbert A.

    2012-01-01

    Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO. PMID:22345709

  10. Harmonization of Glutamic Acid Decarboxylase and Islet Antigen-2 Autoantibody Assays for National Institute of Diabetes and Digestive and Kidney Diseases Consortia

    PubMed Central

    Bonifacio, Ezio; Yu, Liping; Williams, Alastair K.; Eisenbarth, George S.; Bingley, Polly J.; Marcovina, Santica M.; Adler, Kerstin; Ziegler, Anette G.; Mueller, Patricia W.; Schatz, Desmond A.; Krischer, Jeffrey P.; Steffes, Michael W.; Akolkar, Beena

    2010-01-01

    Background/Rationale: Autoantibodies to islet antigen-2 (IA-2A) and glutamic acid decarboxylase (GADA) are markers for diagnosis, screening, and measuring outcomes in National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies. A harmonization program was established to increase comparability of results within and among these studies. Methods: Large volumes of six working calibrators were prepared from pooled sera with GADA 4.8–493 World Health Organization (WHO) units/ml and IA-2A 2–235 WHO units/ml. Harmonized assay protocols for IA-2A and GADA using 35S-methionine-labelled in vitro transcribed and translated antigens were developed based on methods in use in three NIDDK laboratories. Antibody thresholds were defined using sera from patients with recent onset type 1 diabetes and healthy controls. To evaluate the impact of the harmonized assay protocol on concordance of IA-2A and GADA results, two laboratories retested stored TEDDY study sera using the harmonized assays. Results: The harmonized assays gave comparable but not identical results in the three laboratories. For IA-2A, using a common threshold of 5 DK units/ml, 549 of 550 control and patient samples were concordantly scored as positive or negative, specificity was greater than 99% with sensitivity 64% in all laboratories. For GADA, using thresholds equivalent to the 97th percentile of 974 control samples in each laboratory, 1051 (97.9%) of 1074 samples were concordant. On the retested TEDDY samples, discordance decreased from 4 to 1.8% for IA-2A (n = 604 samples; P = 0.02) and from 15.4 to 2.7% for GADA (n = 515 samples; P < 0.0001). Conclusion: Harmonization of GADA and IA-2A is feasible using large volume working calibrators and common protocols and is an effective approach to ensure consistency in autoantibody measurements. PMID:20444913

  11. A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture

    PubMed Central

    Zhu, Lingxiang; Shen, Dingxia; Zhou, Qiming; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2015-01-01

    Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates. PMID:25775001

  12. Effects of acid stress on aerobic decomposition of algal and aquatic macrophyte detritus: Direct comparison in a radiocarbon assay

    SciTech Connect

    Schoenberg, S.A.; Benner, R.; Armstrong, A.; Sobecky, P.; Hodson, R.E. )

    1990-01-01

    Radiolabeled phytoplankton and macrophyte lignocelluloses were incubated at pHs 4 and 7 in water from a naturally acidic freshwater wetland (Okefenokee Swamp; ambient pH, 3.8 to 4.2), a freshwater reservoir (L-Lake; pH 6.7 to 7.2), and a marine marsh (Sapelo Island; pH {approximately}7.8). The data suggest that acidity is an important factor in explaining the lower decomposition rates of algae in Okefenokee Swamp water relative to L-Lake or Sapelo Island water. The decomposition of algal substrate was less sensitive to low pH ({approximately}5 to 35% inhibition) than was the decomposition of lignocellulose ({approximately}30 to 70% inhibition). These substrate-dependent differences were greater and more consistent in salt marsh than in L-lake incubations. In both freshwater sites, the extent to which decomposition was suppressed by acidity was greater for green algal substrate than for mixed diatom or blue-green algal (cyanobacteria) substrates. The use of different bases to adjust pH or incubation in a defined saltwater medium had no significant effect on substrate-dependent differences. Although pH differences with lignocellulose were larger in marine incubations, amendment of lake water with marine bacteria or with calcium, known to stabilize exoenzymes in soils, did not magnify the sensitivity of decomposition to acid stress.

  13. Effects Of Haloacetic Acid Mixtures in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    The haloacetic acids (HAAs) are a class of chemicals produced as byproducts of drinking water disinfection. Source water characteristics (such as level of bromide) affects which HAAs are present in drinking water and their concentration. For example, high bromide-source water wil...

  14. Evaluation of bicinchoninic acid as a ligand for copper(I)-catalyzed azide-alkyne bioconjugations.

    PubMed

    Christen, Erik H; Gübeli, Raphael J; Kaufmann, Beate; Merkel, Lars; Schoenmakers, Ronald; Budisa, Nediljko; Fussenegger, Martin; Weber, Wilfried; Wiltschi, Birgit

    2012-09-01

    The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields. PMID:22821135

  15. Detection of teratogenic substances in acidic mine water samples using the frog embryo teratogenesis assay--Xenopus (FETAX).

    PubMed

    Dawson, D A; McCormick, C A; Bantle, J A

    1985-08-01

    The FETAX (Frog Embryo Teratogenesis Assay--Xenopus) whole embryo bioassay has been developed to screen for environmental substances that cause birth defects. We have used this assay to test its effectiveness in working with actual water samples from the field. Tar Creek is contaminated by discharges from abandoned lead and zinc mines. In addition to high concentrations of zinc, iron and other metals, water samples are routinely low in pH and oxygen content. The pH values of three Tar Creek sample sites were below the established tolerance limits of the embryos. Therefore, one group of samples had no pH adjustment while a second group had the pH adjusted to 7.0. Two of the four sites contained agents that reduced embryonic growth and caused high rates of mortality. A third site contained teratogenic substances. We have determined that metal content is responsible, along with the low pH, for the biologic effects. We have identified high concentrations of toxic metals in Tar Creek by water analysis and were able to demonstrate, by removing metals via Chelex 100 ion exchange chromatography, that the observed toxicity and teratogenicity were caused by metal ions. We have concluded that FETAX is an excellent test for complex mixtures. Physicochemical parameters, such as pH, oxygen and metals content, can be altered and the effect of these changes on toxicity and teratogenicity determined using FETAX. The interaction of toxic substances and low pH are important when considering embryo survival and development. PMID:4045096

  16. Boronic acid recognition based-gold nanoparticle-labeling strategy for the assay of sialic acid expression on cancer cell surface by inductively coupled plasma mass spectrometry.

    PubMed

    Zhang, Xing; Chen, Beibei; He, Man; Zhang, Yuan; Peng, Lu; Hu, Bin

    2016-02-01

    Sialic acids are special sugars widely expressed at the termini of glycan chains on the cell surface, and their expression level on the cancer cell surface is much higher than on the normal cell surface. Herein, we reported an inductively coupled plasma mass spectrometry (ICP-MS) based method with elemental tags for the analysis of sialic acids on the cancer cell surface. The method is based on the selective recognition of sialic acids by biotinylated phenylboronic acid (biotin-APBA) at physiological pH and signal enhancement of gold nanoparticles (AuNPs) in ICP-MS when AuNPs were used as elemental tags labeled on biotin-APBA. A specificity test reveals that the proposed method has high specificity towards cancer cells. Taking HepG2 and MCF-7 cells as two model cancer cells, competitive experiments were performed to estimate the expression level of sialic acids on the cancer cell surface, and it was found that the average numbers of sialic acids expressed on the single MCF-7 and HepG2 cell surface were 7.0 × 10(9) and 5.4 × 10(9), respectively. With sialic acid as the biomarker for cancer cells, the method was further used for cell detection. The limits of detection in terms of cell number for HepG2 and MCF-7 cells were 120 and 64, respectively. And the relative standard deviations for nine replicate determinations of ca. 1000 HepG2 and MCF-7 cells were 9.6% and 8.9%, respectively. The linear ranges for HepG2 cells and MCF-7 cells were 300-10 000 and 170-11 000, respectively. The proposed approach is sensitive as well as selective for the analysis of sialic acids on the cancer cell surface, and is potentially applicable for the study of tumor malignancy and metastasis, which is helpful for biological research and clinical diagnostics. PMID:26811850

  17. Development of an Interaction Assay between Single-Stranded Nucleic Acids Trapped with Silica Particles and Fluorescent Compounds

    PubMed Central

    Isoda, T.; Maeda, R.

    2012-01-01

    Biopolymers are easily denatured by heating, a change in pH or chemical substances when they are immobilized on a substrate. To prevent denaturation of biopolymers, we developed a method to trap a polynucleotide on a substrate by hydrogen bonding using silica particles with surfaces modified by aminoalkyl chains ([A-AM silane]/SiO2). [A-AM silane]/SiO2 was synthesized by silane coupling reaction of N-2-(aminoethyl)-3-aminopropyltrimethoxysilane (A-AM silane) with SiO2 particles with a diameter of 5 μm at 100 °C for 20 min. The surface chemical structure of [A-AM silane]/SiO2 was characterized by Fourier transform infrared spectroscopy and molecular orbital calculations. The surface of the silica particles was modified with A-AM silane and primary amine groups were formed. [A-AM silane]/SiO2 was trapped with single-stranded nucleic acids [(Poly-X; X = A (adenine), G (guanine) and C (cytosine)] in PBS solution at 37 °C for 1 h. The single-stranded nucleic acids were trapped on the surface of the [A-AM silane]/SiO2 by hydrogen bonding to form conjugated materials. The resulting complexes were further conjugated by derivatives of acridine orange (AO) as fluorescent labels under the same conditions to form [AO:Poly-X:A-AM silane]/SiO2 complexes. Changes in the fluorescence intensity of these complexes originating from interactions between the single-stranded nucleic acid and aromatic compounds were also evaluated. The change in intensity displayed the order [AO: Poly-G: A-AM silane]/SiO2 > [AO:Poly-A:A-AM silane]/SiO2 >> [AO:Poly-C:A-AM silane]/SiO2. This suggests that the single-stranded nucleic acids conjugated with aminoalkyl chains on the surfaces of SiO2 particles and the change in fluorescence intensity reflected the molecular interaction between AO and the nucleic-acid base in a polynucleotide. PMID:24955635

  18. Development of a Medium-Throughput Targeted LCMS Assay to Detect Endogenous Cellular Levels of Malonyl-CoA to Screen Fatty Acid Synthase Inhibitors.

    PubMed

    Hopcroft, Philip J; Fisher, David I

    2016-02-01

    The fatty acid synthase (FAS) enzyme in mammalian cells is a large multidomain protein responsible for de novo synthesis of fatty acids. The steps catalyzed by FAS involve the condensation of acetyl-CoA and malonyl-CoA moieties in the presence of NADPH until palmitate is formed. Inhibition of FAS causes an accumulation of intracellular malonyl-CoA, as this metabolite is essentially committed to fatty acid synthesis once formed. Detection of intracellular metabolites for screening can be problematic due to a lack of appropriate tools, but here we describe a targeted liquid chromatography-mass spectroscopy (LCMS) method to directly measure endogenous levels of malonyl-CoA to drive a drug development structure-activity relationship (SAR) screening cascade. Our process involves preparation of samples at 96-well scale, normalization postpermeabilization via use of a whole-well imaging platform, and the LCMS detection methodology. The assay is amenable to multiplexing cellular endpoints, has a typical Z' of >0.6, and has high reproducibility of EC50 values. PMID:26586251

  19. Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen

    PubMed Central

    2012-01-01

    Background HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. Method A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. Results The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. Conclusions When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3–4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV. PMID

  20. An instantaneous colorimetric protein assay based on spontaneous formation of a protein corona on gold nanoparticles.

    PubMed

    Ho, Yan Teck; Poinard, Barbara; Yeo, Eugenia Li Ling; Kah, James Chen Yong

    2015-02-21

    Commercial protein assays used ubiquitously in laboratories typically require long incubation times due to the inherently slow protein-reagent reactions. In this study, we report a novel facile technique for the instantaneous measurement of total protein concentration by exploiting the rapid aggregation dynamics of gold nanoparticles (NPs). By adsorbing different amounts of proteins on their surface to form a protein corona, these NPs can be sterically stabilized to different degrees by aggregation, thus exhibiting a spectrum of color change which can be quantitatively characterized by UV-Vis absorption spectroscopy. We evaluated this technique on four model proteins with different structures: bovine serum albumin (BSA), normal mouse immunoglobulin G (IgG), fibrinogen (FBG) and apolipoprotein A-I (Apo-A1) using two approaches, sequential and simultaneous. We obtained an approach-dependent linear concentration range up to 80 μg mL(-1) and 400 μg mL(-1) for sequential and simultaneous approaches, respectively. This linear working range was wider than that of the commercial Bradford assay and comparable to the Micro BCA assay. The simultaneous approach was also able to produce a linear working range of 200 to 1000 μg mL(-1) (R(2) = 0.995) in human urine, while the sequential approach was non-functional in urine. Similar to Micro BCA, the NP-based protein assay was able to elicit a linear response (R(2) > 0.87) for all four proteins with different structures. However, unlike Micro BCA which requires up to 120 min of incubation, we were able to obtain the read-out almost instantaneously without the need for incubation. The NP-based technique using the simultaneous approach can thus be exploited as a novel assay for instantaneous protein quantification to increase the productivity of laboratory processes. PMID:25501998

  1. High Throughput Liquid Chromatography-Tandem Mass Spectrometry Assay for Mercapturic Acids of Acrolein and Crotonaldehyde in Cigarette Smokers’ Urine

    PubMed Central

    Carmella, Steven G.; Chen, Menglan; Zarth, Adam; Hecht, Stephen S.

    2014-01-01

    3-Hydroxypropylmercapturic acid (3-HPMA) and 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) are urinary metabolites of the toxicants acrolein and crotonaldehyde, respectively. Virtually all human urine samples contain these metabolites, resulting from the action of glutathione-S-transferases on acrolein and crotonaldehyde, which are lipid peroxidation products, environmental and dietary contaminants, and constituents of cigarette smoke. We have developed a high throughput liquid chromatography-tandem mass spectrometry method for quantitative analysis of 3-HPMA and HMPMA in large numbers of small urine samples, as would be required in molecular epidemiology and clinical studies relating levels of these metabolites to cancer risk. Solid-phase extraction on mixed mode reverse phase-anion exchange 96-well plates provided sufficient purification for LC-MS/MS analysis, which was performed by auto-injection using a 96-well format, and resulted in clean, readily interpretable chromatograms, with detection limits of 4.5 pmol/mL urine for 3-HPMA and 3.5 pmol/mL urine for HMPMA. Accuracy was 92% for 3-HPMA and 97% for HMPMA while inter-day precision was 9.1% (coefficient of variation) for 3-HPMA and 11.0% for HMPMA. The method was applied to more than 2600 urine samples from smokers; mean levels of 3-HPMA and HMPMA were 4800 ± 5358 (S.D.) pmol/ml and 3302 ± 3341 pmol/ml, respectively. PMID:23934173

  2. The neurotoxic effect of clindamycin - induced gut bacterial imbalance and orally administered propionic acid on DNA damage assessed by the comet assay: protective potency of carnosine and carnitine

    PubMed Central

    2013-01-01

    Background Comet assay is a quick method for assessing DNA damage in individual cells. It allows the detection of single and double DNA strand breaks, which represent the direct effect of some damaging agents. This study uses standard comet quantification models to compare the neurotoxic effect of orally administered propionic acid (PA) to that produced as a metabolite of bacterial overgrowth induced by clindamycin. Additionally, the protective effect of carnosine and carnitine as natural dietary supplements is assessed. Methods Single cell gel electrophoresis (comet assays) were performed on brain cortex and medulla samples after removal from nine groups of hamsters including: a control (untreated) group; PA-intoxicated group; clindamycin treated group; clindamycin-carnosine group and; clindamycin-carnitine group. Results There were significant double strand breaks recorded as tail length, tail moment and % DNA damage in PA and clindamycin-treated groups for the cortex and medulla compared to the control group. Neuroprotective effects of carnosine and carnitine were observed. Receiver Operating Characteristics curve (ROC) analysis showed satisfactory values of sensitivity and specificity of the comet assay parameters. Conclusion Percentage DNA damage, tail length, and tail moment are adequate biomarkers of PA neurotoxicity due to oral administration or as a metabolite of induced enteric bacterial overgrowth. Establishing biomarkers of these two exposures is important for protecting children’s health by documenting the role of the imbalance in gut microbiota in the etiology of autism through the gut-brain axis. These outcomes will help efforts directed at controlling the prevalence of autism, a disorder recently related to PA neurotoxicity. PMID:23587115

  3. Scaling of Nucleic Acid Assays on Microelectrophoresis Array Devices: High-Dynamic Range Multi-gene Readout from less than 10 Transcripts

    PubMed Central

    Ueberfeld, Joern; Ehrlich, Daniel J.

    2009-01-01

    In this paper we describe progress in using the prodigious data-collecting ability of multilane microelectrophoresis instruments to bear on problems in scaled nucleic acid assays. We emphasize compound stacking and Solid-Support loading as means to concentrate <100 pg samples for direct injection. Reaction Mapping is applied to readout of quantitative polymerase chain reaction (qPCR) gene-expression and as a way to practically overcome difficulty in interpreting amplification curves of multiplexed qPCR at 20–50 gene/well complexity. We demonstrate multiplexed readout of gene expression over an abundancy range of 9Log2 units starting with reverse-transcribed samples as small as 5 molecules in each sample. PMID:19544490

  4. Development of an enzyme-linked immunosorbent assay to determine the numbers of chemolithotrophic bacteria at acid-mine-drainage sites. Technical report (Final)

    SciTech Connect

    Blake, R.C.; Revis, N.W.; Holdsworth, G.

    1990-09-01

    Thiobacillus ferrooxidans is a prominent member of a group of chemo-lithotrophic bacteria that bear principal responsibility for the formation of acid mine drainage. A prototype enzyme-linked immunosorbent assay (ELISA) for enumerating and qualifying T. ferrooxidans was assembled and characterized. The immunoassay protocol consisted of sequential incubations of the sample with (i) the primary antibody, (ii) the enzyme-labeled secondary antibody, and (iii) a chromogenic substrate specific for the enzyme lable. The necessary reagents comprised primary polyclonal rabbit antibodies directed against T. ferrooxidans ATCC 23270, alkaline phosphatase-copled goat anti-rabbit polyclonal antibodies, and phenolphrhalein monophosphate. The ELISA developed herein correctly identified whether iron-oxidizing bacteria were present in each of 4 samples supplied and analyzed by an independent laboratory. Sufficient preliminary data was obtained to warrant further research and development activities.

  5. Re-exploration of the Codon Context Effect on Amber Codon-Guided Incorporation of Noncanonical Amino Acids in Escherichia coli by the Blue-White Screening Assay.

    PubMed

    Xu, Huan; Wang, Yan; Lu, Jiaqi; Zhang, Bo; Zhang, Ziwei; Si, Longlong; Wu, Ling; Yao, Tianzhuo; Zhang, Chuanling; Xiao, Sulong; Zhang, Lihe; Xia, Qing; Zhou, Demin

    2016-07-01

    The effect of codon context on amber codon-guided incorporation of noncanonical amino acids (NAAs) has been previously examined by antibiotic selection. Here, we re-explored this effect by screening a library in which three nucleotides upstream and downstream of the amber codon were randomised, and inserted within the lacZ-α gene. Thousands of clones were obtained and distinguished by the depth of blue colour upon exposure to X-gal. Large-scale sequencing revealed remarkable preferences in nucleotides downstream of the amber codon, and moderate preferences for upstream nucleotides. Nucleotide preference was quantified by a dual-luciferase assay, which verified that the optimum context for NAA incorporation, AATTAGACT, was applicable to different proteins. Our work provides a general guide for engineering amber codons into genes of interest in bacteria. PMID:27028123

  6. Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

    PubMed

    Zevgiti, Stella; Zabala, Juliana Gonzalez; Darji, Ayub; Dietrich, Ursula; Panou-Pomonis, Eugenia; Sakarellos-Daitsiotis, Maria

    2012-01-01

    The terminal parts of the influenza hemagglutinin (HA) receptors α2,6- and α2,3-sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -glyco-conjugate bearing two copies of the α2,6-sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6-linkage. SOC(4) -glyco-conjugate bearing two copies of the α2,3-sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well-known α2,3-sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions. PMID:22052803

  7. Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection.

    PubMed

    Zinellu, Angelo; Sotgia, Salvatore; Zinellu, Elisabetta; Chessa, Roberto; Deiana, Luca; Carru, Ciriaco

    2006-03-01

    Guanidinoacetic acid (GAA) measurement has recently become of great interest for the diagnosis of creatine (Cn) metabolism disorders, and research calls for rapid and inexpensive methods for its detection in plasma and urine in order to assess a large number of patients. We propose a new assay for the measurement of GAA by a simple CZE UV-detection without previous sample derivatization. Plasma samples were filtered by Microcon-10 microconcentrators and directly injected into the capillary, while for urine specimens a simple water dilution before injection was needed. A baseline separation was obtained in less than 8 min using a 60.2 cm x 75 microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer pH 2.25 at 15 degrees C. The performance of the developed method was assessed by measuring plasma creatinine and Cn in 32 normal subjects and comparing the data obtained by the new method with those found with the previous CE assay. Our new method seems to be an inexpensive, fast and specific tool to assess a large number of patients both in clinical and in research laboratories. PMID:16605092

  8. Monoclonal antibody production and indirect competitive enzyme-linked immunosorbent assay development of 3-methyl-quinoxaline-2-carboxylic acid based on novel haptens.

    PubMed

    Li, Guopeng; Zhao, Liang; Zhou, Feng; Li, Jiaying; Xing, Yuan; Wang, Tiangang; Zhou, Xilong; Ji, Baoping; Ren, Wanpeng

    2016-10-15

    Two novel immunizing haptens of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) were synthesized and conjugated with cationized bovine serum albumin. Female BALB/c mice were immunized with above conjugates, splenocytes were fused with Sp2/0 cells to produce monoclonal antibody. Compared with previous studies, antibodies raised in this work showed higher sensitivity. Meantime, a novel heterologous coating hapten was also prepared. The indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the optimum condition showed an IC50 of 3.1μg/kg (ppb), and the linear range of 0.46-10.5ppb for MQCA. The limit of detect (LOD) of MQCA in swine muscle, swine liver and chicken was 0.32, 0.54, and 0.28ppb, respectively. The LOD of this assay can satisfy the minimum required performance levels (4ppb) for MQCA. These results indicated that the proposed ELISA, with high sensitivity and specificity, as well as good reproducibility and accuracy, is suitable for determination of MQCA residues in food samples. PMID:27173564

  9. Treatment of cytomegalovirus infection and disease pre- and post-quantitative nucleic acid test standardization: does use of a more sensitive assay lead to longer treatment duration?

    PubMed

    Dioverti, M Veronica; Lahr, Brian; Razonable, Raymund R

    2016-02-01

    Quantitative cytomegalovirus (CMV) nucleic acid testing (NAT) has been standardized using the World Health Organization (WHO) international calibration standard. A new FDA-approved WHO-calibrated assay (CA) was found to be more sensitive than a laboratory-developed test (LDT). We hypothesized that monitoring therapeutic response using a more sensitive assay may lead to longer antiviral therapy in solid organ and hematopoietic stem cell transplant patients with CMV infection. We reviewed transplant patients with CMV disease retrospectively, and divided them into two groups: those diagnosed and managed based on LDT and those managed using WHO-CA. Compared to patients monitored by LDT, the time to reach an undetectable viral load was significantly longer in the group monitored by the WHO-CA. However, a trend toward shorter duration of antiviral treatment was observed (median, 34 vs. 41 d; p = 0.058), with earlier discontinuation of induction antiviral therapy upon reaching undetectable viral load using WHO-CA (11 vs. 18 d; p = 002). We concluded that despite using a more sensitive CMV NAT, the total duration of antiviral treatment was not significantly prolonged in transplant patients with CMV infection and disease. Relapse rates did not differ between the two groups. PMID:26589482

  10. A rapid and sensitive assay of perfluorocarboxylic acids in aqueous matrices by headspace solid phase microextraction-gas chromatography-triple quadrupole mass spectrometry.

    PubMed

    Monteleone, Marcello; Naccarato, Attilio; Sindona, Giovanni; Tagarelli, Antonio

    2012-08-17

    The work aims at developing a rapid and sensitive method for the quantification of perfluorocarboxylic acids in aqueous matrices. The proposed analytical approach is based on the use of solid phase microextraction in headspace mode after a fast derivatization of the carboxylate function by propylchloroformate/propanol mixture. Several fibers were evaluated and the optimization of the parameters affecting the SPME process was carried out using a central composite design. The optimum working conditions in terms of response values were achieved by performing analysis with CAR/PDMS fiber at room temperature, without addition of NaCl, with a sample volume of 6 ml and an extraction time of 10 min. Assay of PFCAs was performed by using a gas chromatography-triple quadrupole mass spectrometry (GC-QqQ MS) system in negative chemical ionization mode with ammonia as reagent gas. An overall evaluation of all analytical parameters shows that the proposed method provides satisfactory results. In particular, the observed accuracies, ranging from 84.4% to 116.8%, and the RSD values in the range 0.4% and 14.5% confirm the effectiveness of the developed protocol in the assay of PFCAs content in aqueous matrices. Moreover, LOD and LOQ values ranging from 0.08 to 6.6 ng l(-1) and from 0.17 to 14.3 ng l(-1), respectively, can be considered very satisfactory. None of the compounds were detected in six samples of river collected in Calabria. PMID:22762954

  11. A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds.

    PubMed

    Lee, Keun Pyo; Piskurewicz, Urszula; Turecková, Veronika; Strnad, Miroslav; Lopez-Molina, Luis

    2010-11-01

    Seed dormancy is an ecologically important adaptive trait in plants whereby germination is repressed even under favorable germination conditions such as imbibition with water. In Arabidopsis and most plant species, dormancy absolutely requires an unidentified seed coat germination-repressive activity and constitutively higher abscisic acid (ABA) levels upon seed imbibition. The mechanisms underlying these processes and their possible relationship are incompletely understood. We developed a "seed coat bedding" assay monitoring the growth of dissected embryos cultured on a layer of seed coats, allowing combinatorial experiments using dormant, nondormant, and various genetically modified seed coat and embryonic materials. This assay, combined with direct ABA measurements, revealed that, upon imbibition, dormant coats, unlike nondormant coats, actively produce and release ABA to repress embryo germination, whatever the embryo origin, i.e., from dormant, nondormant, or never dormant aba seeds, unable to synthesize ABA. The persistent high ABA levels in imbibed dormant seeds requires the permanent expression of the DELLA gene RGL2, where it remains insensitive to gibberellins (GA) unlike in nondormant seeds. These findings present the seed coat as an organ actively controlling germination upon seed imbibition and provide a framework to investigate how environmental factors break seed dormancy. PMID:20956298

  12. Developmental toxicity of mixtures: the water disinfection by-products dichloro-, dibromo- and bromochloro acetic acid in rat embryo culture

    EPA Science Inventory

    The chlorination of drinking water results in production of numerous disinfection by-products (DBPs). One of the important classes of DBPs is the haloacetic acids. We have previously shown that the haloacetic acids (HAs), dichloro (DCA), dibromo (DBA) and bromochloro (BCA) acetic...

  13. A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay.

    PubMed

    Fleming, Jonathan K; Glass, Thomas R; Lackie, Steve J; Wojciak, Jonathan M

    2016-09-01

    Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive signaling lysophospholipids that activate specific G protein-coupled receptors on the cell surface triggering numerous biological events. In circulation, S1P and LPA associate with specific carrier proteins or chaperones; serum albumin binds both S1P and LPA while HDL shuttles S1P via interactions with apoM. We used a series of kinetic exclusion assays in which monoclonal anti-S1P and anti-LPA antibodies competed with carrier protein for the lysophospholipid to measure the equilibrium dissociation constants (Kd) for these carrier proteins binding S1P and the major LPA species. Fatty acid-free (FAF)-BSA binds these lysophospholipids with the following Kd values: LPA(16:0), 68 nM; LPA(18:1), 130 nM; LPA(18:2), 350 nM; LPA(20:4), 2.2 μM; and S1P, 41 μM. FAF human serum albumin binds each lysophospholipid with comparable affinities. By measuring the apoM concentration and expanding the model to include endogenous ligand, we were able to resolve the Kd values for S1P binding apoM in the context of human HDL and LDL particles (21 nM and 2.4 nM, respectively). The novel competitive assay and analysis described herein enables measurement of Kd values of completely unmodified lysophospholipids binding unmodified carrier proteins in solution, and thus provide insights into S1P and LPA storage in the circulation system and may be useful in understanding chaperone-dependent receptor activation and signaling. PMID:27444045

  14. A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters.

    PubMed

    Li, Haiyin; Chang, Jiafu; Hou, Ting; Ge, Lei; Li, Feng

    2016-11-01

    Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a "signal-off" strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring. PMID:27591641

  15. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.

    PubMed

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène; Bastien, Patrick

    2014-11-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10(4) to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥ 6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections. PMID:25187637

  16. Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays

    PubMed Central

    Varlet-Marie, Emmanuelle; Sterkers, Yvon; Brenier-Pinchart, Marie-Pierre; Cassaing, Sophie; Dalle, Frédéric; Delhaes, Laurence; Filisetti, Denis; Pelloux, Hervé; Touafek, Fériel; Yera, Hélène

    2014-01-01

    The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 104 to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections. PMID:25187637

  17. A rapid and sensitive assay for determination of doxycycline using thioglycolic acid-capped cadmium telluride quantum dots.

    PubMed

    Tashkhourian, Javad; Absalan, Ghodratollah; Jafari, Marzieh; Zare, Saber

    2016-01-01

    A rapid, simple and inexpensive spectrofluorimetric sensor for determination of doxycycline based on its interaction with thioglycolic acid-capped cadmium telluride quantum dots (TGA/CdTe QDs) has been developed. Under the optimum experimental conditions, the sensor exhibited a fast response time of <10s. The results revealed that doxycycline could quench the fluorescence of TGA/CdTe QDs via electron transfer from the QDs to doxycycline through a dynamic quenching mechanism. The sensor permitted determination of doxycycline in a concentration range of 1.9×10(-6)-6.1×10(-5)molL(-1) with a detection limit of 1.1×10(-7)molL(-1). The sensor was applied for determination of doxycycline in honey and human serum samples. PMID:26204505

  18. A rapid and sensitive assay for determination of doxycycline using thioglycolic acid-capped cadmium telluride quantum dots

    NASA Astrophysics Data System (ADS)

    Tashkhourian, Javad; Absalan, Ghodratollah; Jafari, Marzieh; Zare, Saber

    2016-01-01

    A rapid, simple and inexpensive spectrofluorimetric sensor for determination of doxycycline based on its interaction with thioglycolic acid-capped cadmium telluride quantum dots (TGA/CdTe QDs) has been developed. Under the optimum experimental conditions, the sensor exhibited a fast response time of <10 s. The results revealed that doxycycline could quench the fluorescence of TGA/CdTe QDs via electron transfer from the QDs to doxycycline through a dynamic quenching mechanism. The sensor permitted determination of doxycycline in a concentration range of 1.9 × 10-6-6.1 × 10-5 mol L-1 with a detection limit of 1.1 × 10-7 mol L-1. The sensor was applied for determination of doxycycline in honey and human serum samples.

  19. Synthesis, DFT and antimicrobial activity assays in vitro for novel cis/trans-but-2-enedioic acid esters

    NASA Astrophysics Data System (ADS)

    Ma, Yan-Long; Zhou, Ru-Jin; Zeng, Xing-Ye; An, Ya-Xiong; Qiu, Song-Shan; Nie, Li-Jun

    2014-04-01

    Six novel cis/trans-but-2-enedioic acid esters had been synthesized to discover the new bioactive molecules that could kill food-related bacteria and fungi. Their structures were analyzed by melting point, LC-MS, 1H NMR and 13C NMR. 4-(Methoxycarbonyl) phenyl ethyl fumarate (6b) was also characterized by single-crystal X-ray diffraction. Their antimicrobial activities were evaluated in vitro by measuring the minimal inhibitory concentration (MIC). Compared with the single monomethyl fumarate and methyl 4-hydroxybenzoate, these compounds had stronger antimicrobial activity against all the eight microorganisms. Among the antibacterial and antifungal compounds, 4-(methoxycarbonyl) phenyl methyl fumarate (6a) showed the best antimicrobial activity. The electronic properties of these compounds were calculated by the density functional theory (DFT) method with 6-31G (d, p) basis set. DFT studies indicated that molecular electrostatic potential (MEP) map, ELUMO, energy gap, electronegativity and electrophilicity index could be helpful to understand the various antimicrobial activities among these compounds. The antimicrobial activity of compound 6a was evaluated in vitro against Salmonellacholeraesuis subsp. choleraesuis, Lactococcus lactis subsp. lactis and Saccharomyces cerevisiae by time-kill, and it was found that compound 6a exhibited significant microbiocidal activity against the three microorganisms.

  20. A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: Heterologous regulation of the same K+ channel

    SciTech Connect

    Kamatchi, G.L.; Ticku, M.K. )

    1991-02-01

    The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.

  1. A fluorescence-coupled assay for gamma aminobutyric acid (GABA) reveals metabolic stress-induced modulation of GABA content in neuroendocrine cancer.

    PubMed

    Ippolito, Joseph E; Piwnica-Worms, David

    2014-01-01

    Pathways involved in the synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) have been implicated in the pathogenesis of high grade neuroendocrine (NE) neoplasms as well as neoplasms from a non-NE lineage. Using The Cancer Genome Atlas, overexpression of the GABA synthetic enzyme, glutamate decarboxylase 1 (GAD1), was found to be associated with decreased disease free-survival in prostate adenocarcinoma and decreased overall survival in clear cell renal cell carcinomas. Furthermore, GAD1 was found to be expressed in castrate-resistant prostate cancer cell lines, but not androgen-responsive cell lines. Using a novel fluorescence-coupled enzymatic microplate assay for GABA mediated through reduction of resazurin in a prostate neuroendocrine carcinoma (PNEC) cell line, acid microenvironment-induced stress increased GABA levels while alkaline microenvironment-induced stress decreased GABA through modulation of GAD1 and glutamine synthetase (GLUL) activities. Moreover, glutamine but not glucose deprivation decreased GABA through modulation of GLUL. Consistent with evidence in prokaryotic and eukaryotic organisms that GABA synthesis mediated through GAD1 may play a crucial role in surviving stress, GABA may be an important mediator of stress survival in neoplasms. These findings identify GABA synthesis and metabolism as a potentially important pathway for regulating cancer cell stress response as well as a potential target for therapeutic strategies. PMID:24551133

  2. Bacteriological analysis of water by potentiometric measurement of lipoic acid reduction: preliminary assays for selective detection of indicator organisms.

    PubMed Central

    Charriere, G; Jouenne, T; Lemeland, J F; Selegny, E; Junter, G A

    1984-01-01

    The practical task of adapting an original potentiometric technique to the bacteriological analysis of water is discussed. Various laboratory strains of organisms belonging to the usual aquatic flora were inoculated one by one in a minimal lactose broth supplied with lipoic (thioctic) acid. The time evolution of the redox potential of the cultures was followed during incubation by combined gold versus reference electrodes. When the incubation temperature was regulated at 36 degrees C, most organisms were able to grow and to reduce the coenzyme, generating changes in the redox potential of the culture. However, very few organisms developed significant reductive activity when the temperature was increased to 41 degrees C and when the broth was provided with sodium deoxycholate. Among the fecal coliform organisms, only Escherichia coli and Klebsiella pneumoniae exhibited early but reproducible potential-time responses. Positive potentiometric responses were also recorded with Acinetobacter calcoaceticus. E. coli showed rapid potentiometric signals as compared with K. pneumoniae. The time required for 100-mV shift of potential to be detected was related to the logarithm of the initial concentration of E. coli or K. pneumoniae in the culture broth. Experiments on natural surface water samples showed the the potentiometric method, associated with the selective incubation conditions, mainly detected E. coli among the bacterial flora of the tested environmental water. The calibration curve relating the time required for a 100-mV shift of potential to be detected to the number of fecal coliforms, as determined by control fecal coliform-selective plate counts, was consistent with the composite standard curve of detection times obtained with six different laboratory strains of E. coli.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6421230

  3. 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

  4. Evaluation of different modifications of acid-fast staining techniques and stool enzyme-linked immunosorbent assay in detecting fecal Cryptosporidium in diarrheic HIV seropositive and seronegative patients

    PubMed Central

    Parghi, Ekta; Dash, Lona; Shastri, Jayanthi

    2014-01-01

    Rational: The role of Cryptosporidium as an agent of human diarrhea has been redefined over the past decade following recognition of the strong association between cases of cryptosporidiosis and immune deficient individuals (such as those with AIDS). Purpose: The purpose of this study is to determine the prevalence of enteric parasites and to compare the diagnostic utility of stool enzyme-linked immunosorbent assay (ELISA) with various modifications of acid-fast (AF) staining in detection of Cryptosporidium in stool samples of diarrheic patients. Materials and Methods: Stool samples from 186 cases comprising of 93 HIV seropositive and 93 seronegative patients were included. These were subjected to routine and microscopic examination as well as various modifications of AF staining for detection of coccidian parasites and ELISA for the detection of Cryptosporidium. Results: The prevalence of enteric parasites was 54.8% and of Cryptosporidium was 17.2% in HIV seropositive patients while it was 29.0% and 5.4%, respectively in seronegative patients. Of the 186 cases, 33 cases (17.7%) were positive for Cryptosporidium by stool ELISA as compared to 21 (11.3%) by modified AF staining (gold standard) showing sensitivity and specificity of 100% and 92.7%, respectively. The maximum cases of Cryptosporidium (21; 11.3%) were detected by AF staining using 3% acid alcohol. Conclusion: ELISA is a simple, useful, and rapid tool for detection of Cryptosporidium in stool, especially for large scale population studies. However, the role of modified AF staining in detection of Cryptosporidium and other coccidian parasites is important. Based on the results of various modifications of AF staining, the present study recommends the use of 3% acid alcohol along with 10% H2SO4. PMID:25250230

  5. Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.

    PubMed

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  6. Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

    PubMed Central

    Ivanov, Delyan P.; Parker, Terry L.; Walker, David A.; Alexander, Cameron; Ashford, Marianne B.; Gellert, Paul R.; Garnett, Martin C.

    2014-01-01

    Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money. PMID:25119185

  7. Production of monoclonal antibody for okadaic acid and its utilization in an ultrasensitive enzyme-linked immunosorbent assay and one-step immunochromatographic strip.

    PubMed

    Liu, Biing-Hui; Hung, Chun-Tse; Lu, Chuan-Chen; Chou, Hong-Non; Yu, Feng-Yih

    2014-02-12

    Okadaic acid (OA) is a common marine biotoxin that accumulates in bivalves and causes diarrhetic shellfish poisoning (DSP). This study generated a monoclonal antibody (mAb) specific to OA from a hybridoma cell line, 6B1A3, which was obtained by fusion of myeloma cells (P3/NS1/1-AG4-1) with spleen cells isolated from a BALB/c mouse immunized with OA-γ-globulin. The 6B1A3 mAb belongs to the immunoglobulin G1 (κ chain) isotype. Both competitive direct and indirect enzyme-linked immunosorbent assays (ELISAs) were established for characterization of the antibody. The concentrations causing 50% inhibition of binding of OA-horseradish peroxidase to the antibody by OA were calculated to be 0.077 ng/mL in the cdELISA. A rapid and sensitive mAb-based gold nanoparticle immunochromatographic strip was also established. This proposed strip has a detection limit of 5 ng/mL for OA and can be finished in 10 min. Extensive analyses of 20 seafood samples with ELISA revealed that 10 were slightly contaminated with OA, with a mean concentration of 0.892 ng/g. Analysis of OA in shellfish samples showed that data acquired by the immunochromatographic strip agreed well with those acquired by the ELISA. The mAb-based ELISA and immunochromatographic strip assay developed in this study have adequate sensitivity and accuracy for rapid screening of OA in shellfish samples. PMID:24446876

  8. ALTERED CRANIOFACIAL GENE EXPRESSION OF NEURULATION STAGED MOUSE EMBRYO CULTURES EXPOSED TO BROMOCHLOROACETIC ACID

    EPA Science Inventory

    An unintended consequence of chemical disinfection of municipal drinking water is the production of chemical disinfection by-products (DBPs). A major class ofDBPs are haloacetic acidsthat may be found at concentrations higher than other DBPs. Bromochloroacetic acid (BCA) is a de...

  9. Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection.

    PubMed

    Yin, Hui-qiong; Jia, Min-xian; Shi, Li-jun; Liu, Jun; Wang, Rui; Lv, Mao-min; Ma, Yu-yuan; Zhao, Xiong; Zhang, Jin-gang

    2014-01-01

    A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10(-2) fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82-6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins. PMID:24164314

  10. Simultaneous determination of rosuvastatin and fenofibric acid in human plasma by LC-MS/MS with electrospray ionization: assay development, validation and application to a clinical study.

    PubMed

    Trivedi, Ravi Kumar; Kallem, Raja Reddy; Mullangi, Ramesh; Srinivas, Nuggehally R

    2005-09-15

    A simple, sensitive and specific LC-MS/MS method for simultaneous determination of rosuvastatin (RST) and fenofibric acid (FFA) was developed and validated with 500 microL human plasma using carbamazepine as an internal standard (IS). The assay procedure involved a simple one-step liquid/liquid extraction of RST and FFA and IS from plasma into ethyl acetate. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto X-Terra MS C-18 column (4.6 mm x 50 mm, 5.0 microm). Separation of RST, FFA and IS was achieved with a mobile phase consisting of 0.05 M formic acid:acetonitrile (45:55, v/v) at a flow rate of 0.40 ml/min. The API-3000LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Positive ion acquisition chromatographic run was used in the present method. Nominal retention times of RST, FFA and IS were 2.35, 4.70 and 2.32 min, respectively. Absolute recovery of RST, FFA and IS was 74, 61 and 69%, respectively. The lower limit of quantification (LLOQ) of RST and FFA was 1.00 ng/ml and 0.50 microg/ml, respectively. Response function was established for the range of concentrations 1.00-50.0 ng/ml and 0.50-20.0 microg/ml for RST and FFA, respectively, with a coefficient of determination (r2) of 0.999 for both the compounds. The inter- and intra-day precision in the measurement of RST quality control (QC) samples 5, 15, 400 and 800 ng/ml, were in the range 8.93-9.37% relative standard deviation (R.S.D.) and 1.74-16.1% R.S.D., respectively. Similarly, the inter- and intra-day precision in the measurement of FFA quality control (QC) samples 0.5, 1.5, 8.0 and 15.0 microg/ml, were in the range 9.78-11.6% relative standard deviation (R.S.D.) and 0.22-17.4% R.S.D., respectively. Accuracy in the measurement of QC samples for RST and FFA were in the range 88.1-108 and 87-115%, respectively, of the nominal

  11. Detection of the marijuana metabolite 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid specimens and its contribution to positive results in screening assays.

    PubMed

    Moore, Christine; Ross, Wayne; Coulter, Cynthia; Adams, Laura; Rana, Sumandeep; Vincent, Michael; Soares, James

    2006-09-01

    The detection of the marijuana metabolite 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid specimens is described, and its contribution to an immunoassay for the detection of cannabinoids is investigated. Oral fluid specimens, screened using an enzyme-linked immunosorbent immunoassay (ELISA), were carried forward to confirmation for both tetrahydrocannabinol (THC) and THC-COOH using gas chromatography-mass spectrometry (GC-MS). One hundred and fifty-three specimens were analyzed, of which 143 screened positive for cannabinoids. Ninety-five (66.4%) of these specimens were positive for both THC and THC-COOH; 14 (9.7%) were positive for THC-COOH only, and 27 (18.8%) were positive for THC only. The GC-MS assay for the detection of THC-COOH in oral fluid was linear to 160 pg/mL with a limit of quantitation of 2 pg/mL. The detection of the marijuana metabolite, THC-COOH, in 76.2% of oral fluid specimens screening positive for cannabinoids is reported. As a potential defense against passive exposure claims, proposed SAMHSA regulations may require the simultaneous collection of a urine sample when oral fluid samples are used. The detection of the metabolite, THC-COOH, is a significant alternative to this approach because its presence in oral fluid minimizes the argument for passive exposure to marijuana in drug testing cases. PMID:16959132

  12. High-throughput LC-MS/MS assay for 6-methoxy-2-naphthylacetic acid, an active metabolite of nabumetone in human plasma and its application to bioequivalence study.

    PubMed

    Patel, Bhavin N; Sharma, Naveen; Sanyal, Mallika; Prasad, Arpana; Shrivastav, Pranav S

    2008-11-01

    A simple, precise and accurate assay for the determination of 6-methoxy-2-naphthylacetic acid (6-MNA), an active metabolite of nabumetone in human plasma, was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte (6-MNA) and propranolol (internal standard, IS) were extracted from 200 microL aliquot of human plasma via solid-phase extraction employing HLB Oasis cartridges and separated on a Discovery HS C18 (50 x 4.6 mm, 5 microm) column. Detection of analyte and IS was done by tandem mass spectrometry with a turbo ion spray interface operating in positive ion and multiple reaction monitoring acquisition mode. The total chromatographic runtime was 3.0 min with retention time for 6-MNA and IS at 1.97 and 1.26 min, respectively. The method was validated over a dynamic linear range of 0.20-60.00 microg/mL for 6-MNA with mean correlation coefficient r > or = 0.9986. The intra-batch and inter-batch precision (%CV) across five validation runs (lower limit of quantiation, low-, medium- and high-quality controls and upper limit of quantitation) was less than 7.5%. The accuracy determined at these levels was within -5.8 to +0.2% in terms of percentage bias. The method was successfully applied for a bioequivalence study of 750 mg nabumetone tablet formulation in 12 healthy Indian male subjects under fasted condition. PMID:18651608

  13. Determination of alachlor and its sulfonic acid metabolite in water by solid-phase extraction and enzyme-linked immunosorbent assay

    USGS Publications Warehouse

    Aga, D.S.; Thurman, E.M.; Pomes, M.L.

    1994-01-01

    Solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) were combined for the trace analysis of the herbicide alachlor and its major soil metabolite, ethanesulfonic acid (ESA). The anti-alachlor antibody cross-reacted with ESA, which produced false-positive detections of alachlor in water samples by immunoassay screens. Alachlor and ESA were isolated from water by SPE on a C18 resin and eluted sequentially with ethyl acetate and methanol. Alachlor is soluble in ethyl acetate while the anionic ESA is not. Thus ESA remained adsorbed on the C18 resin and was eluted later with methanol. The combination of SPE with ELISA effectivety separated and quantified both alachlor and ESA using the same antibody for two ELISA methods. The general method may have applicability for the separation of other herbicides and their ionic metabolites. The SPE-ELISA method has a, detection limit of 0.01 ??g/L for alachlor and 0.05 ??g/L for ESA, with a precision of ?? 10%. Analyses of surface and ground water samples were confirmed by gas chromatography/mass spectrometry and high-performance liquid chromatography with photodiode-array detection. Results showed widespread occurrence of ESA in surface and ground water of the midwestern United States, with concentrations ranging from 10 ??g/L.

  14. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes

    PubMed Central

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  15. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    PubMed

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis. PMID:26305575

  16. Antitubercular specific activity of ibuprofen and the other 2-arylpropanoic acids using the HT-SPOTi whole-cell phenotypic assay

    PubMed Central

    Guzman, Juan D; Evangelopoulos, Dimitrios; Gupta, Antima; Birchall, Kristian; Mwaigwisya, Solomon; Saxty, Barbara; McHugh, Timothy D; Gibbons, Simon; Malkinson, John; Bhakta, Sanjib

    2013-01-01

    Objectives Lead antituberculosis (anti-TB) molecules with novel mechanisms of action are urgently required to fuel the anti-TB drug discovery pipeline. The aim of this study was to validate the use of the high-throughput spot culture growth inhibition (HT-SPOTi) assay for screening libraries of compounds against Mycobacterium tuberculosis and to study the inhibitory effect of ibuprofen (IBP) and the other 2-arylpropanoic acids on the growth inhibition of M tuberculosis and other mycobacterial species. Methods The HT-SPOTi method was validated not only with known drugs but also with a library of 47 confirmed anti-TB active compounds published in the ChEMBL database. Three over-the-counter non-steroidal anti-inflammatory drugs were also included in the screening. The 2-arylpropanoic acids, including IBP, were comprehensively evaluated against phenotypically and physiologically different strains of mycobacteria, and their cytotoxicity was determined against murine RAW264.7 macrophages. Furthermore, a comparative bioinformatic analysis was employed to propose a potential mycobacterial target. Results IBP showed antitubercular properties while carprofen was the most potent among the 2-arylpropanoic class. A 3,5-dinitro-IBP derivative was found to be more potent than IBP but equally selective. Other synthetic derivatives of IBP were less active, and the free carboxylic acid of IBP seems to be essential for its anti-TB activity. IBP, carprofen and the 3,5-dinitro-IBP derivative exhibited activity against multidrug-resistant isolates and stationary phase bacilli. On the basis of the human targets of the 2-arylpropanoic analgesics, the protein initiation factor infB (Rv2839c) of M tuberculosis was proposed as a potential molecular target. Conclusions The HT-SPOTi method can be employed reliably and reproducibly to screen the antimicrobial potency of different compounds. IBP demonstrated specific antitubercular activity, while carprofen was the most selective agent among the

  17. Magnetic levitation as a platform for competitive protein-ligand binding assays.

    PubMed

    Shapiro, Nathan D; Soh, Siowling; Mirica, Katherine A; Whitesides, George M

    2012-07-17

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater

  18. Magnetic Levitation as a Platform for Competitive Protein-Ligand Binding Assays

    PubMed Central

    Shapiro, Nathan D.; Soh, Siowling; Mirica, Katherine A.; Whitesides, George M.

    2012-01-01

    This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (Kd’s within the range of ~ 10 nM to 100 µM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 minutes – 2 hours). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight

  19. Relationship of spermatoscopy, prostatic acid phosphatase activity and prostate-specific antigen (p30) assays with further DNA typing in forensic samples from rape cases.

    PubMed

    Romero-Montoya, Lydia; Martínez-Rodríguez, Hugo; Pérez, Miguel Antonio; Argüello-García, Raúl

    2011-03-20

    In the forensic laboratory the biological analyses for rape investigation commonly include vaginal swabs as sample material combined to biochemical tests including sperm cytology (SC) and detection of acid phosphatase activity (AP) and prostate-specific antigen (PSA, p30) for the conclusive identification of semen components. Most reports comparing these tests relied on analysis of semen samples or donor swabs taken under controlled conditions; however their individual or combined efficacy under real live sampling conditions in different laboratories is largely unknown. We carried out SC, APA and PSA analyses in vaginal swabs collected from casework rapes submitted to Mexican Forensic Laboratories at Texcoco and Toluca. On the basis of positive and negative results from each assay and sample, data were classified into eight categories (I-VIII) and compared with those obtained in the two only similar studies reported in Toronto, Canada and Hong Kong, China. SC and APA assays had the higher overall positivity in Toluca and Texcoco samples respectively and otherwise PSA had a lower but very similar positivity between these two laboratories. When compared to the previous studies some similarities were found, namely similar frequencies (at a ratio of approximately 1 out of 3) of samples being positive or negative by all techniques (Categories I and VI respectively) and a comparable overall positivity of APA and SC but higher than that of PSA. Indeed the combined results of using SC, APA and PSA tests was considered as conclusive for semen detection from approximately 1 out of 3 cases (Category I) to approximately 1 out of 2 cases in a scenario where at least SC is positive, strongly presumptive in 2 out of 3 cases (with at least one test positive) and the remainder 1 out of 3 cases (Category VI) suggested absence of semen. By determining Y-STR polymorphisms (12-loci) in additional samples obtained at Toluca laboratory, complete DNA profiles were determined from all

  20. Li/Ca, B/Ca, and Mg/Ca content in sea urchin spines cultured at different temperatures and pCO2

    NASA Astrophysics Data System (ADS)

    Nguyen, T.; Eagle, R.; Courtney, T.; Ries, J. B.; Brillo, V.; Rollion-Bard, C.; Gabitov, R. I.; Tripati, A. K.

    2012-12-01

    Element/calcium ratios within biogenic calcium carbonate minerals have been used as tools to reconstruct seawater temperature and pH. Most recent studies have focused on examining systematics governing elemental incorporation in coral, foraminifera, and otoliths [1-3, etc.]. In this study we focus on examining Li/Ca, B/Ca, and Mg/Ca ratios in sea urchins cultured at different temperatures and pCO2. We conducted in situ secondary ion mass spectrometry (SIMS) analyses on two different species of sea urchins. A temperate species of sea urchin (Arbacia punctulata) was cultured at variable pCO2 (400, 600, 900, 2850 ppmv) and at a constant temperature (25°C) [4]. We also investigated a tropical species of sea urchins (Echinometra viridis) that was cultured at variable pCO2 (400 and 1000 ppmv) and variable temperature (20°C and 30°C). The highly porous spines were embedded in epoxy and polished with 3 μm diamond suspension. SIMS analyses were performed with an oxygen primary beam and a lateral spatial resolution of about 40 μm. The standard deviation for SIMS spot analysis of Li in the reference synthetic calcite, CAL-HTP, was 3.5 % (1σ). The standard deviation of SIMS spots analyses of coral reference material M93-TB-FC-1 was 9.5 % (1σ). The bulk B content in this reference coral was determined by LA-ICP-MS as 39.3 ppm [6]. The standard deviation for the SIMS spot analysis of Mg in the reference synthetic calcite, UCI, was 1% (1σ). For the temperate species, B/Ca ratios decrease from ~0.39 to 0.29 mmol/mol as pCO2 increase from 400 to 2850 ppmv. This suggests that B/Ca ratios in this species may be a viable proxy for paleo-seawater pH. Other elements such as Li/Ca showed an increase from .047 to .052 mmol/mol as pCO2 increased. However, Mg/Ca did not show any significant trend as pCO2 increased. The tropical species showed a general increase in Li/Ca, B/Ca, Mg/Ca with increasing temperature. When temperature was held constant, there was no significant effect of

  1. Structure-function analysis of the ATP-driven glycolipid efflux pump DevBCA reveals complex organization with TolC/HgdD.

    PubMed

    Staron, Peter; Forchhammer, Karl; Maldener, Iris

    2014-01-31

    In Gram-negative bacteria, trans-envelope efflux pumps have periplasmic membrane fusion proteins (MFPs) as essential components. MFPs act as mediators between outer membrane factors (OMFs) and inner membrane factors (IMFs). In this study, structure-function relations of the ATP-driven glycolipid efflux pump DevBCA-TolC/HgdD from the cyanobacterium Anabaena sp. PCC 7120 were analyzed. The binding of the MFP DevB to the OMF TolC absolutely required the respective tip-regions. The interaction of DevB with the IMF DevAC mainly involved the β-barrel and the lipoyl domain. Efficient binding to DevAC and TolC, substrate recognition and export activity by DevAC were dependent on stable DevB hexamers. PMID:24361095

  2. Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay.

    PubMed

    Bisp, Marianne R; Bor, Mustafa Vakur; Heinsvig, Else-Marie; Kall, Morten A; Nexø, Ebba

    2002-06-01

    Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma. PMID:12018948

  3. Evaluating the role of conserved amino acids in bacterial O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays.

    PubMed

    Musumeci, Matias A; Faridmoayer, Amirreza; Watanabe, Yasuharu; Feldman, Mario F

    2014-01-01

    Bacterial O-Oligosaccharyltransferases (O-OTases) constitute a growing family of enzymes that catalyze the transfer of a glycan from a lipid carrier to protein acceptors. O-OTases are inner membrane proteins that display limited sequence similarity, except for the Wzy_C signature domain also present in a predicted periplasmic loop of the WaaL ligase, the enzyme responsible for transferring the O antigen to the lipid A core. The mechanism of O-OTase-dependent glycosylation is poorly understood. In this work, conserved amino acid residues in the O-OTases were replaced with alanine in PglL, the O-OTase of Neisseria meningitidis. The activity of wild-type PglL and its mutant derivatives were analyzed in vivo in engineered Escherichia coli cells, and in in vitro assays. We identified two additional sites of pilin glycosylated exclusively by PglL in E. coli. Both sites are modified with phosphoglycerol (PG) by different enzymes in Neisseria gonorrhoeae and Neisseria meningitidis. Limited proteolysis experiments revealed a conformational change that is triggered upon interaction of the C-terminal region of PglL with the lipid-linked oligosaccharide (LLO) substrate. These experiments showed that Q178 and Y405 are required for optimal function, whereas H349 is essential for activity and plays a critical role in the interaction with LLO. The equivalent His residue is also essential for WaaL activity, which suggests a common mechanism for both enzymes, and supports the hypothesis that O-glycosylation and lipopolysaccharide (LPS) synthesis are evolutionarily related. These results contribute to the elucidation of the mechanism of O-OTases, which are promising targets for novel antibiotics and present an enormous potential for glycoengineering novel vaccines and therapeutics. PMID:24092836

  4. Development and validation of a quantitative LC-tandem MS assay for hexadeca-4,7,10,13-tetraenoic acid in human and mouse plasma.

    PubMed

    Stigter, Edwin C A; Letsiou, Sophia; vd Broek, Niels J F; Gerrits, Johan; Ishihara, Kenji; Voest, Emile E; Verhoeven-Duif, Nanda M; Brenkman, Arjan B

    2013-04-15

    Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid-liquid extraction, solid phase clean-up and LC-MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1-100 nmol/L (r(2)>0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98-106% and 100-108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at -80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance. PMID:23502461

  5. 96-Well Plate Colorimetric Assay for K(sub i) Determination of (plusmn)-2-Benzylsuccinic Acid, an Inhibitor of Carboxypeptidase A

    ERIC Educational Resources Information Center

    Wentland, Mark P.; Raza, Shaan; Yingtong Gao

    2004-01-01

    An appropriate assay to determine the inhibition potency of carboxypeptidase A (CPA) in 96-well format to illustrate how high throughput screening is used in modern drug discovery to identify bioactive molecules is developed. Efforts in developing a colorimetric 96-well plate assay for determination of the K(sub i) for inhibition of CPA by…

  6. Ability of Two Commercially Available Assays (Abbott RealTime HIV-1 and Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Version 2.0) To Quantify Low HIV-1 RNA Levels (<1,000 Copies/Milliliter): Comparison with Clinical Samples and NIBSC Working Reagent for Nucleic Acid Testing Assays

    PubMed Central

    Marsella, Patrizia; Bloisi, Maria; Forbici, Federica; Angeletti, Claudio; Capobianchi, Maria R.

    2014-01-01

    Concordance between molecular assays may be suboptimal at low HIV-1 viremia levels (<1,000 copies/ml); therefore, it may be difficult to define and compare virologic endpoints for successful and failed therapy. We compared two commercial assays (the Abbott RealTime HIV-1 and the Roche Cobas AmpliPrep/TaqMan HIV-1 version 2.0) for their ability to detect and quantify low viral loads. A comparison was performed using 167 residual clinical samples (with values ranging from “not detected” to 1,000 copies/ml, as measured by the Abbott assay) and the National Institute and Biological Standards and Control (NIBSC) HIV-1 RNA working reagent 1 for nucleic acid amplification techniques (NAT) assays (serially diluted to a range from 1 to 1,000 copies/ml). Quantitative results were compared using Lin's concordance correlation coefficient and a Bland-Altman plot. Concordance with the qualitative results was measured by Cohen's kappa statistic. With clinical samples, the degree of interassay concordance of the qualitative results at a 40-copies/ml HIV-1 RNA threshold was substantial (κ = 0.762); the correlation among the quantified samples was suboptimal (concordance correlation coefficient, 0.728; P < 0.0001); the mean difference of the values between the Roche and Abbott assays was 0.193 log10 copies/ml. Using the HIV-1 RNA working reagent 1 for NAT assays, the results provided by the Roche assay were, on average, 3 times higher than expected, while the Abbott assay showed high accuracy. The Roche assay was highly sensitive, being able to detect a level as low as 3.5 copies/ml HIV-1 RNA with 95% probability. The performance characteristics of each molecular assay should be taken into account when HIV-1 RNA threshold values for “virologic suppression,” “virologic failure,” “persistent low viral loads,” etc., are defined and indicated in the support of clinical decisions. PMID:24671791

  7. Assay of phenolic compounds from four species of ber (Ziziphus mauritiana L.) fruits: comparison of three base hydrolysis procedure for quantification of total phenolic acids.

    PubMed

    Memon, Ayaz Ali; Memon, Najma; Bhanger, Muhammad Iqbal; Luthria, Devanand L

    2013-08-15

    The present study was undertaken to investigate the flavonoid profile in four species of ber (Ziziphus mauritiana Lamk.) fruit. The 12 flavonoids identified were quercetin 3-O-robinobioside, quercetin 3-O-rutinoside, quercetin 3'-O-galactoside, quercetin 3'-O-glucoside, quercetin 3'-O-rhamnoside, quercetin 3'-O-pentosylhexoside, quercetin 3-O-6'malonylglucoside, quercetin 3'-O-malonylglucoside, luteolin 7-O-6'malonylglucoside, luteolin 7-O-malonylglucoside, myricetin 3-O-galactoside, and naringenin tri glycoside. This is the first report on extraction of nine additional flavonoids from the ber fruits. In addition, we also compared the impact of three different base hydrolysis techniques namely ultrasonic assisted base hydrolysis (UABH), microwave assisted base hydrolysis (MWABH), and pressurised liquid assisted base hydrolysis (PLABH) for the quantification of total phenolic acids. Nine phenolic acids, protocatechuic acid, p-hydroxybenzoic acid, ferulic acid, chlorogenic acid, vanillic acid, caffeic acid, vanillin, ortho- and para-coumaric acids, were identified and quantified. The three major phenolic acids identified in all four ber species were p-coumaric acid, vanillin and ferulic acids. Higher amounts (p<0.05) of total phenolic acids in all cultivars were obtained with the PLABH technique as compared to other two procedures (UABH and MWABH). PMID:23561136

  8. Unscheduled deoxyribonucleic acid (DNA) synthesis assays for toxicological studies. May 1977-March 1990 (A Bibliography from the NTIS data base). Report for May 1977-March 1990

    SciTech Connect

    Not Available

    1990-04-01

    This bibliography contains citations concerning the unscheduled DNA synthesis (UDS) assay for toxicological studies. UDS assays provide very sensitive measures of damage to DNA by detecting induction of DNA synthesis in non-S-phase cells. UDS toxicological studies analyzing gamma radiation, drugs, pesticides, nerve gas, jet engine fuels, ultraviolet light, chlorated organic compounds, and aromatic compounds are discussed. UDS studies using both human and animal tissue cultures are described. (Contains 57 citations fully indexed and including a title list.)

  9. High-throughput and sensitive next-generation droplet digital PCR assay for the quantitation of the hepatitis C virus mutation at core amino acid 70.

    PubMed

    Mukaide, Motokazu; Sugiyama, Masaya; Korenaga, Masaaki; Murata, Kazumoto; Kanto, Tatsuya; Masaki, Naohiko; Mizokami, Masashi

    2014-10-01

    The next-generation droplet digital polymerase chain reaction (ddPCR) assay employs an emulsion-based endpoint to quantitate the amount of target DNA and is more robust than real-time PCR when analyzing sequence variations. However, no studies have applied this technique to quantitate mutations in polymorphic viral genomes. To develop this approach, a ddPCR-based assay was designed to quantitate with high-throughput and sensitivity mutations and their frequencies in codon 70 of the hepatitis C virus (HCV) gene that encodes the Core protein. The assay was linear from 2.5 to 10(5) copies per assay, and the limit of detection of mutants in the presence of a 20,000-fold excess of wild type was 0.005%. The results correlated well with those obtained using the COBAS(®) TaqMan(®) HCV Test, which is a real-time PCR assay for the quantitative detection of HCV RNA in human serum (n=87; range, 2.3-7.7log10IU/mL; Pearson's R(2)=0.9120; p<0.0001). The median frequencies of mutations by ddPCR were 0.262% (n=55; range, 0-37.951%) and 99.687% (n=32; range, 52.191-100%) for the wild-type and mutant sequences, respectively, by direct sequencing. The ddPCR assay should be useful for quantitating mutations in other polymorphic viral genomes. PMID:25019167

  10. Assessing the impact of diagenesis on δ11B, δ13C, δ18O, Sr/Ca and B/Ca values in fossil planktic foraminiferal calcite

    NASA Astrophysics Data System (ADS)

    Edgar, Kirsty M.; Anagnostou, Eleni; Pearson, Paul N.; Foster, Gavin L.

    2015-10-01

    The geochemical composition of foraminiferal tests is a valuable archive for the reconstruction of paleo-climatic, -oceanographic and -ecological changes. However, dissolution of biogenic calcite and precipitation of inorganic calcite (overgrowth and recrystallization) at the seafloor and in the sediment column can potentially alter the original geochemical composition of the foraminiferal test, biasing any resulting paleoenvironmental reconstruction. The δ11B of planktic foraminiferal calcite is a promising ocean pH-proxy but the effect of diagenesis is still poorly known. Here we present new δ11B, δ13C, δ18O, Sr/Ca and B/Ca data from multiple species of planktic foraminifera from time-equivalent samples for two low latitude sites: clay-rich Tanzanian Drilling Project (TDP) Site 18 from the Indian Ocean containing well-preserved ('glassy') foraminifera and carbonate-rich Ocean Drilling Program (ODP) Site 865 from the central Pacific Ocean hosting recrystallized ('frosty') foraminifera. Our approach makes the assumption that environmental conditions were initially similar at both sites so most chemical differences are attributable to diagenesis. Planktic foraminiferal δ18O and δ13C records show offsets in both relative and absolute values between the two sites consistent with earlier findings that these isotopic ratios are strongly influenced by diagenetic alteration. Sr/Ca and B/Ca ratios in planktic foraminiferal calcite are also offset between the two sites but there is little change in the relative difference between surface and deep dwelling taxa. In contrast, δ11B values indicate no large differences between well-preserved and recrystallized foraminifera suggesting that despite extensive diagenetic alteration the δ11B of biogenic calcite appears robust, potentially indicative of a lack of free exchange of boron between pore fluids and the recrystallizing CaCO3. Our finding may remove one potential source of uncertainty in δ11B based p

  11. 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. PMID:26608294

  12. A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer.

    PubMed

    Go, Yun Young; Rajapakse, R P V Jayanthe; Kularatne, Senanayake A M; Lee, Pei-Yu Alison; Ku, Keun Bon; Nam, Sangwoo; Chou, Pin-Hsing; Tsai, Yun-Long; Liu, Yu-Lun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas; Balasuriya, Udeni B R

    2016-06-01

    Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3' untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries. PMID:27030492

  13. Targeted epidural patch with n-butyl cyanoacrylate (n-BCA) through a single catheter access site for treatment of a cerebral spinal fluid leak causing spontaneous intracranial hypotension.

    PubMed

    Woolen, Sean; Gemmete, Joseph J; Pandey, Aditya S; Chaudhary, Neeraj

    2015-01-01

    Spontaneous intracranial hypotension (SIH) usually occurs in the setting of a spontaneous cerebral spinal fluid (CSF) leak. We report the first description of a case of SIH caused by a CSF leak which improved after a targeted epidural patch with n-butyl cyanoacrylate (n-BCA) at the right T1-T2 level. An 81-year-old woman presented with an orthostatic headache for 6 days. MRI of the brain with contrast demonstrated low lying cerebellar tonsils, an engorged transverse sinus flow void, bifrontal small subdural fluid collections, and diffuse dural enhancement. CT myelography showed extravasation of intrathecal contrast at the right T1-T2 level. A targeted epidural patch was performed by injection of n-BCA through a catheter at the right T1-T2 level. After treatment, the patient's symptoms immediately improved and she was without a headache at 1-year follow-up. PMID:26038380

  14. Targeted epidural patch with n-butyl cyanoacrylate (n-BCA) through a single catheter access site for treatment of a cerebral spinal fluid leak causing spontaneous intracranial hypotension.

    PubMed

    Woolen, Sean; Gemmete, Joseph J; Pandey, Aditya S; Chaudhary, Neeraj

    2016-07-01

    Spontaneous intracranial hypotension (SIH) usually occurs in the setting of a spontaneous cerebral spinal fluid (CSF) leak. We report the first description of a case of SIH caused by a CSF leak which improved after a targeted epidural patch with n-butyl cyanoacrylate (n-BCA) at the right T1-T2 level. An 81-year-old woman presented with an orthostatic headache for 6 days. MRI of the brain with contrast demonstrated low lying cerebellar tonsils, an engorged transverse sinus flow void, bifrontal small subdural fluid collections, and diffuse dural enhancement. CT myelography showed extravasation of intrathecal contrast at the right T1-T2 level. A targeted epidural patch was performed by injection of n-BCA through a catheter at the right T1-T2 level. After treatment, the patient's symptoms immediately improved and she was without a headache at 1-year follow-up. PMID:26047904

  15. Matrix-assisted laser desorption/ionisation (MALDI) TOF analysis identifies serum angiotensin II concentrations as a strong predictor of all-cause and breast cancer (BCa)-specific mortality following breast surgery.

    PubMed

    Boccardo, Francesco; Rubagotti, Alessandra; Nuzzo, Pier Vitale; Argellati, Francesca; Savarino, Grazia; Romano, Paolo; Damonte, Gianluca; Rocco, Mattia; Profumo, Aldo

    2015-11-15

    MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affected by early BCa. Mortality was analysed based on signal profiling, and appropriate statistics were used. The results indicate that four signals were increased in deceased patients compared with living patients. Three of the four signals were individually associated with all-cause mortality, but only one having mass/charge ratio (m/z) 1,046.49 was associated with BCa-specific mortality and was the only peak to maintain an independent prognostic role after multivariate analysis. Two groups exhibiting different mortality probabilities were identified after clustering patients based on the expression of the four peptides, but m/z 1,046.49 was exclusively expressed in the cluster exhibiting the worst mortality outcome, thus confirming the crucial value of this peptide. The specific role of this peak was confirmed by competing risk analysis. MS findings were validated by ELISA analysis after demonstrating that m/z 1,046.49 structurally corresponded to Angiotensin II (ATII). In fact, mortality results obtained after arbitrarily dividing patients according to an ATII serum value of 255 pg/ml (which corresponds to the 66(th) percentile value) were approximately comparable to those previously demonstrated when the same patients were analysed according to the expression of signal m/z 1,046.49. Similarly, ATII levels were specifically correlated with BCa-related deaths after competing risk analysis. In conclusion, ATII levels were increased in women who exhibited worse mortality outcomes, reinforcing the evidence that this peptide potentially significantly affects the natural history of early BCa. Our findings also confirm that MALDI-TOF MS is an efficient screening tool to identify novel tumour markers and that MS findings can be rapidly validated through less complex techniques, such as ELISA. PMID:25994113

  16. Thermal expansion behaviour in the oxygen deficient perovskites Sr{sub 2}BSbO{sub 5.5} (B=Ca, Sr, Ba). Competing effects of water and oxygen ordering

    SciTech Connect

    Zhou Qingdi; Kennedy, Brendan J.; Avdeev, Maxim

    2011-09-15

    Neutron diffractions studies reveal the presence of oxygen disorder in the oxygen deficient perovskites Sr{sub 2}BSbO{sub 5.5} (B=Ca, Sr, Ba). Synchrotron X-ray studies demonstrate that these oxides have a double perovskite-type structure with the cell size increasing as the size of the B cation increases from 8.2114(2) A for B=Ca to 8.4408(1) A for B=Ba. It is postulated that a combination of local clustering of the anions and vacancies together with water-water and water-host hydrogen bonds plays a role in defining the volume of the encapsulated water clusters and that changes in the local structure upon heating result in anomalous thermal expansion observed in variable temperature diffraction measurements. - Graphical abstract: The oxides Sr{sub 2}BSbO{sub 5.5} (B=Ca, Sr, Ba) have unusual anion disorder. There is a lag in the contraction in the cell size of Sr{sub 2}CaSbO{sub 5.5}nH{sub 2}O established from X-ray diffraction measurements following the loss of water suggesting changes on the local structure are important. Highlights: > The average structures of the defect perovskites Sr{sub 2}MSbO{sub 5.5} established. > Anion and cation disorder quantified by neutron and synchrotron X-ray diffraction. > Anomalous thermal expansion due to local clustering of anions and vacancies observed.

  17. Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures.

    PubMed

    Rödel, Jürgen; Karrasch, Matthias; Edel, Birgit; Stoll, Sylvia; Bohnert, Jürgen; Löffler, Bettina; Saupe, Angela; Pfister, Wolfgang

    2016-03-01

    Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management. PMID:26712265

  18. Specific ionic effect for simple and rapid colorimetric sensing assays of amino acids using gold nanoparticles modified with task-specific ionic liquid.

    PubMed

    Wu, Datong; Cai, Pengfei; Tao, Zhihao; Pan, Yuanjiang

    2016-01-01

    In this study, a novel task-specific ionic liquid functionalized gold nanoparticle (TSIL-GNP) was successfully prepared and applied in the recognition of amino acids. Particularly, the surface of GNP was modified with the ionic liquid containing carbamido and ester group via thiol, which was characterized by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The stability of this material in aqueous solution improves apparently and can remain unchanged for more than three months. The effect of pH was also discussed in this study. Attractive ionic interaction would effectively weaken intensity of the covalent coupling between the metal ion and the functional groups of amino acids. Thus, TSIL-GNP was successfully applied to recognizing serine, aspartic acid, lysine, arginine, and histidine in the presence of Cu(2+) through distinctive color changes. Suspension would be generated once a spot of cysteine was added into the GNPs solution. Results indicated that it had a good linear relationship between extinction coefficients and concentration of amino acids in a wide range of 10(-3)-10(-6) M. Moreover, the proposed strategy was successfully used to analyze the histidine in urinary samples. In brief, TSIL-GNP is a suitable substrate for discrimination of five amino acids in a rapid and simple way without sophisticated instruments. PMID:26703268

  19. Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

    PubMed Central

    Debussche, L; Couder, M; Thibaut, D; Cameron, B; Crouzet, J; Blanche, F

    1992-01-01

    Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin. Images PMID:1429466

  20. Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.

    PubMed

    Takasawa, Hironao; Takashima, Rie; Narumi, Kazunori; Kawasako, Kazufumi; Hattori, Akiko; Kawabata, Masayoshi; Hamada, Shuichi

    2015-07-01

    As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone. PMID:26212305

  1. Effects Of Haloacetic Acids and their major metabolites in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    EPA Science Inventory

    The haloacetic acids (HAAs) are a class of chemicals produced by disinfection of drinking water. Many of the HAAs are developmental toxicants when administered to rodents producing a variety of developmental effects. We have previously shown that the HAAs can produce direct effec...

  2. Assay of phenolic compounds from four species of Ber (Ziziphus mauritiana L.) Fruits: Comparision of three base hydrolysis procedure for quantification of total phenolic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present study was undertaken to investigate the flavonoids profile in four species of ber (Ziziphus mauritiana Lamk) fruit and to compare various techniques for the analysis of total phenolic acids. The 12 flavonoids identified were quercetin 3-O-robinobioside, quercetin 3-O-rutinoside, querceti...

  3. Complexation induced fluorescence and acid-base properties of dapoxyl dye with γ-cyclodextrin: a drug-binding application using displacement assays.

    PubMed

    Pal, Kaushik; Mallick, Suman; Koner, Apurba L

    2015-06-28

    Host-guest complexation of dapoxyl sodium sulphonate (DSS), an intramolecular charge transfer dye with water-soluble and non-toxic macrocycle γ-cyclodextrin (γ-CD), has been investigated in a wide pH range. Steady-state absorption, fluorescence and time-resolved fluorescence measurements confirm the positioning of DSS into the hydrophobic cavity of γ-CD. A large fluorescence enhancement ca. 30 times, due to 1 : 2 complex formation and host-assisted guest-protonation have been utilised for developing a method for the utilisation of CD based drug-delivery applications. A simple fluorescence-displacement based approach is implemented at physiological pH for the assessment of binding strength of pharmaceutically useful small drug molecules (ibuprofen, paracetamol, methyl salicylate, salicylic acid, aspirin, and piroxicam) and six important antibiotic drugs (resazurin, thiamphenicol, chloramphenicol, ampicillin, kanamycin, and sorbic acid) with γ-CD. PMID:26028009

  4. Oxidative potential of ambient water-soluble PM2.5 in the southeastern United States: contrasts in sources and health associations between ascorbic acid (AA) and dithiothreitol (DTT) assays

    NASA Astrophysics Data System (ADS)

    Fang, Ting; Verma, Vishal; Bates, Josephine T.; Abrams, Joseph; Klein, Mitchel; Strickland, Matthew J.; Sarnat, Stefanie E.; Chang, Howard H.; Mulholland, James A.; Tolbert, Paige E.; Russell, Armistead G.; Weber, Rodney J.

    2016-03-01

    The ability of certain components of particulate matter to induce oxidative stress through the generation of reactive oxygen species (ROS) in vivo may be one mechanism accounting for observed linkages between ambient aerosols and adverse health outcomes. A variety of assays have been used to measure this so-called aerosol oxidative potential. We developed a semi-automated system to quantify oxidative potential of filter aqueous extracts utilizing the dithiothreitol (DTT) assay and report here the development of a similar semi-automated system for the ascorbic acid (AA) assay. Approximately 500 PM2.5 filter samples collected in contrasting locations in the southeastern US were analyzed for a host of aerosol species, along with AA and DTT activities. We present a detailed contrast in findings from these two assays. Water-soluble AA activity was higher in summer and fall than in winter, with highest levels near heavily trafficked highways, whereas DTT activity was higher in winter compared to summer and fall and more spatially homogeneous. AA activity was nearly exclusively correlated with water-soluble Cu (r = 0.70-0.94 at most sites), whereas DTT activity was correlated with organic and metal species. Source apportionment models, positive matrix factorization (PMF) and a chemical mass balance method with ensemble-averaged source impact profiles (CMB-E), suggest a strong contribution from traffic emissions and secondary processes (e.g., organic aerosol oxidation or metals mobilization by secondary acids) to both AA and DTT activities in urban Atlanta. In contrast, biomass burning was a large source for DTT activity, but insignificant for AA. AA activity was not correlated with PM2.5 mass, while DTT activity co-varied strongly with mass (r = 0.49-0.86 across sites and seasons). Various linear models were developed to estimate AA and DTT activities for the central Atlanta Jefferson Street site, based on the CMB-E sources. The models were then used to estimate daily

  5. Oxidative potential of ambient water-soluble PM2.5 measured by Dithiothreitol (DTT) and Ascorbic Acid (AA) assays in the southeastern United States: contrasts in sources and health associations

    NASA Astrophysics Data System (ADS)

    Fang, T.; Verma, V.; Bates, J. T.; Abrams, J.; Klein, M.; Strickland, M. J.; Sarnat, S. E.; Chang, H. H.; Mulholland, J. A.; Tolbert, P. E.; Russell, A. G.; Weber, R. J.

    2015-11-01

    The ability of certain components of particulate matter to induce oxidative stress through catalytic generation of reactive oxygen species (ROS) in vivo may be one mechanism accounting for observed linkages between ambient aerosols and adverse health outcomes. A variety of assays have been used to measure this so-called aerosol oxidative potential. We developed a semi-automated system to quantify oxidative potential of filter aqueous extracts utilizing the dithiothreitol (DTT) assay and have recently developed a similar semi-automated system using the ascorbic acid (AA) assay. Approximately 500 PM2.5 filter samples collected in contrasting locations in the southeastern US were analyzed using both assays. We found that water-soluble DTT activity on a per air volume basis was more spatially uniform than water-soluble AA activity. DTT activity was higher in winter than in summer/fall, whereas AA activity was higher in summer/fall compared to winter, with highest levels near highly trafficked highways. DTT activity was correlated with organic and metal species, whereas AA activity was correlated with water-soluble metals (especially water-soluble Cu, r=0.70-0.91 at most sites). Source apportionment models, Positive Matrix Factorization (PMF) and a Chemical Mass Balance Method with ensemble-averaged source impact profiles (CMB-E), suggest a strong contribution from secondary processes (e.g., organic aerosol oxidation or metal mobilization by formation of an aqueous particle with secondary acids) and traffic emissions to both DTT and AA activities in urban Atlanta. Biomass burning was a large source for DTT activity, but insignificant for AA. DTT activity was well correlated with PM2.5 mass (r=0.49-0.86 across sites/seasons), while AA activity did not co-vary strongly with mass. A linear model was developed to estimate DTT and AA activities for the central Atlanta Jefferson Street site, based on the CMB-E sources that are statistically significant with positive

  6. Effects of processing and of storage on the stability of pantothenic acid in sea buckthorn products (Hippophaë rhamnoides L. ssp. rhamnoides) assessed by stable isotope dilution assay.

    PubMed

    Gutzeit, Derek; Klaubert, Bernd; Rychlik, Michael; Winterhalter, Peter; Jerz, Gerold

    2007-05-16

    A stable isotope dilution assay for quantification of pantothenic acid in sea buckthorn berries, juice, and concentrate using a four-fold labeled isotopologue of vitamin B5 as the internal standard was adopted using reversed phase liquid chromatography-mass spectrometry with electrospray ionization. Because of a rapid sample clean up procedure without the necessity of external calibration, this methodology permits the accurate analysis of a high number of samples within a short time. Sea buckthorn juice was stored at 25 and 40 degrees C for up to 7 days to determine the effects of storage temperature on the stability of pantothenic acid. Analysis of kinetic data suggested that the degradation follows a first-order model. The results of the experiments showed that storage of sea buckthorn juice for 7 days at ambient temperature (25 degrees C) already resulted in a significant degradation of pantothenic acid of about 18%. The processing effects of juice production and subsequent concentration revealed a decrease of about 6-7% in the juice and of 23% in the juice concentrate. PMID:17447792

  7. ΔCO3 from benthic U/Ca in Caribbean core VM28-122: comparison with benthic B/Ca, δ11B, and δ13C

    NASA Astrophysics Data System (ADS)

    Russell, A. D.; Yu, J.; Schmidt, M. W.; Spero, H. J.

    2012-12-01

    Here we present a new U/Ca record from C. wuellerstorfi in the Caribbean Sea (VM28-122), and compare trends in bottom-water ΔCO3 (CO3bw - ΔCO3sat) over the last 30 kyr, calculated from U/Ca, B/Ca, and δ11B. All three proxies produce very similar ΔCO3 records during the Holocene, reflecting saturated but variable conditions. They diverge during the Bolling-Allerod (BA) and during MIS 2 and late MIS 3. During the BA, U/Ca peaks at 18 nmol/mol, suggesting a brief period of undersaturation (ΔCO3] = -20 μmol/kg). During MIS 2 and 3, U/Ca-ΔCO3 is lowest (25-35 μmol/kg); B/Ca- ΔCO3 is highest (60 μmol/kg)and δ11B-ΔCO3 lies between them. It is not clear why ΔCO3 based on the three proxies should agree so well during the Holocene but so poorly during the glacial period; we present two possible explanations. First, the different sensitivities of B/Ca, δ11B and U/Ca to ΔCO3 may be caused by differences between U and B in their incorporation at different calcification rates. This would suggest a non-linear relationship between U/Ca and ΔCO3, with a higher sensitivity at high ΔCO3. The existing core-top U/Ca-ΔCO3 calibration covers a lower range of ΔCO3 (Raitzsch et al. 2011) than that suggested by B/Ca data, so it does not establish the form of the relationship at higher ΔCO3. Secondly, U/Ca may be influenced by cycles in sediment oxidation-reduction conditions. The high δ13C of glacial C. wuellerstorfi (1.2‰) indicates that upper NAIW entering the Caribbean basin was replete with oxygen. If U/Ca is controlled primarily by ΔCO3, benthic calcite should reflect the low U/Ca expected for oxic, high ΔCO3 bottom waters. The U/Ca peak during the BA occurs in both G. ruber and C. wuellerstorfi, within a period of dramatically increased sedimentation rate. These higher sedimentation rates may have buried solids deeper below the O2 = 0 horizon, reducing and remobilizing both Fe-Mn oxides and reduced or adsorbed U. This may have elevated the concentration of

  8. A high-performance liquid chromatography assay with a triazole-bonded column for evaluation of d-amino acid oxidase activity.

    PubMed

    Iwasaki, Megumi; Kashiwaguma, Yoshiyuki; Nagashima, Chihiro; Izumi, Mao; Uekusa, Ayano; Iwasa, Sumiko; Onozato, Mayu; Ichiba, Hideaki; Fukushima, Takeshi

    2016-03-01

    Elution profiles of kynurenic acid (KYNA) and 7-chlorokynurenic acid (Cl-KYNA) were examined by high-performance liquid chromatography (HPLC) using a triazole-bonded stationary phase column (Cosmosil® HILIC) under isocratic elution of a mobile phase consisting of CH3 CN-aqueous 10 mm ammonium formate between pH 3.0 and 6.0. The capacity factors of KYNA and Cl-KYNA varied with both the CH3 CN content and the pH of the mobile phase. The elution order of KYNA and Cl-KYNA was reversed between the CH3 CN- and H2 O-rich mobile phases, suggesting that hydrophilic interactions and anion-exchange interactions caused retention of KYNA and Cl-KYNA in the CH3 CN- and H2 O-rich mobile phases, respectively. The present HPLC method using a triazole-bonded column and fluorescence detection (excitation 250 nm, emission 398 nm) was applied to monitor in vitro production of KYNA from d-kynurenine (d-KYN) by d-amino acid oxidase (DAO) using Cl-KYNA as an internal standard. A single KYNA peak was clearly observed after enzymatic reaction of d-KYN with DAO. Production of KYNA from d-KYN was suppressed by the addition of commercial DAO inhibitors. The present HPLC method can be used to evaluate DAO activity and DAO inhibitory effects in candidate drugs for the treatment of schizophrenia. PMID:26174062

  9. A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

    ERIC Educational Resources Information Center

    Harding, Ethelynda E.; Kimsey, R. Scott

    1998-01-01

    Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

  10. Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

    PubMed

    Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

    2016-06-01

    Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990224

  11. The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay.

    PubMed

    Zheng, Joanna J; Shields, Eric E; Snow, Kimberly J; Nelson, David M; Olah, Timothy V; Reily, Michael D; Robertson, Donald G; Shipkova, Petia A; Stryker, Steven A; Xin, Baomin; Drexler, Dieter M

    2016-06-15

    The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time. PMID:27033006

  12. Multiwell Assay for the Analysis of Sugar Gut Permeability Markers: Discrimination of Sugar Alcohols with a Fluorescent Probe Array Based on Boronic Acid Appended Viologens.

    PubMed

    Resendez, Angel; Panescu, Priera; Zuniga, Ruth; Banda, Isaac; Joseph, Jorly; Webb, Dominic-Luc; Singaram, Bakthan

    2016-05-17

    With the aim of discerning between different sugar and sugar alcohols of biomedical relevance, such as gut permeability, arrays of 2-component probes were assembled with up to six boronic acid-appended viologens (BBVs): 4,4'-o-BBV, 3,3'-o-BBV, 3,4'-o-BBV, 4,4'-o,m-BBV, 4,7'-o-PBBV, and pBoB, each coupled to the fluorophore 8-hydroxypyrene, 1,3,6-trisulfonic acid trisodium salt (HPTS). These probes were screened for their ability to discriminate between lactulose, l-rhamnose, 3-O-methyl-d-glucose, and xylose. Binding studies of sugar alcohols mannitol, sorbitol, erythritol, adonitol, arabitol, galactitol, and xylitol revealed that diols containing threo-1,2-diol units have higher affinity for BBVs relative diols containing erythro-1,2 units. Those containing both threo-1,2- and 1,3-syn diol motifs showed high affinity for boronic acid binding. Fluorescence from the arrays were examined by principle component analysis (PCA) and linear discriminant analysis (LDA). Arrays with only three BBVs sufficed to discriminate between sugars (e.g., lactulose) and sugar alcohols (e.g., mannitol), establishing a differential probe. Compared with 4,4'-o-BBV, 2-fold reductions in lower limits of detection (LOD) and quantification (LOQ) were achieved for lactulose with 4,7-o-PBBV (LOD 41 μM, LOQ 72 μM). Using a combination of 4,4'-o-BBV, 4,7-o-PBBV, and pBoB, LDA statistically segregated lactulose/mannitol (L/M) ratios from 0.1 to 0.5, consistent with values encountered in small intestinal permeability tests. Another triad containing 3,3'-o-BBV, 4,4'-o-BBV, and 4,7-o-PBBV also discerned similar L/M ratios. This proof-of-concept demonstrates the potential for BBV arrays as an attractive alternate to HPLC to analyze mixtures of sugars and sugar alcohols in biomedical applications and sheds light on structural motifs that make this possible. PMID:27116118

  13. Cholinesterase assay for monitoring the kinetics of the JD6. 5 organophosphorus acid anhydrase in detoxification of diisopropylfluorophosphate. Final report, January-June 1988

    SciTech Connect

    Yeh, H.R.; Cheng, T.C.; DeFrank, J.J.

    1992-06-01

    In the present studies, cholinesterase was used for monitoring the enzymatic activities of the JD6.5 organophosphorus acid anhydrase. The kinetic data indicated that: (1) the first order of kinetic constants (k) and Vmax values of the enzymatic reactions increased as the concentrations of the enzyme increased; (2) while the half-life (tl/2) of diisopropylfluorophosphate (DFP) hydrolysis decreased as the enzyme concentrations increased; (3) the minimum time required for hydrolysis of 9mM of DFP was 3 min at the concentrations of the enzyme present; Km values of DFP were found to be in range of 5mM; and (4) both MnCl2 and NaCl were found to be required for the optimal activity of the enzyme.

  14. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    PubMed

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  15. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR

    PubMed Central

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage. PMID:27313572

  16. Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost, and versatile nucleic acid extraction assay for downstream analysis.

    PubMed

    Shin, Yong; Lim, Swee Yin; Lee, Tae Yoon; Park, Mi Kyoung

    2015-01-01

    Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis. PMID:26370251

  17. Dimethyl adipimidate/Thin film Sample processing (DTS); A simple, low-cost, and versatile nucleic acid extraction assay for downstream analysis

    PubMed Central

    Shin, Yong; Lim, Swee Yin; Lee, Tae Yoon; Park, Mi Kyoung

    2015-01-01

    Sample processing, especially that involving nucleic acid extraction, is a prerequisite step for the isolation of high quantities of relatively pure DNA for downstream analyses in many life science and biomedical engineering studies. However, existing methods still have major problems, including labor-intensive time-consuming methods and high costs, as well as requirements for a centrifuge and the complex fabrication of filters and membranes. Here, we first report a versatile Dimethyl adipimidate/Thin film based Sample processing (DTS) procedure without the limitations of existing methods. This procedure is useful for the extraction of DNA from a variety of sources, including 6 eukaryotic cells, 6 bacteria cells, and 2 body fluids in a single step. Specifically, the DTS procedure does not require a centrifuge and has improved time efficiency (30 min), affordability, and sensitivity in downstream analysis. We validated the DTS procedure for the extraction of DNA from human body fluids, as well as confirmed that the quality and quantity of the extracted DNA were sufficient to allow robust detection of genetic and epigenetic biomarkers in downstream analysis. PMID:26370251

  18. Statistical methods for assays with limits of detection: Serum bile acid as a differentiator between patients with normal colons, adenomas, and colorectal cancer

    PubMed Central

    LaFleur, Bonnie; Lee, Wooin; Billhiemer, Dean; Lockhart, Craig; Liu, Junmei; Merchant, Nipun

    2011-01-01

    In analytic chemistry a detection limit (DL) is the lowest measurable amount of an analyte that can be distinguished from a blank; many biomedical measurement technologies exhibit this property. From a statistical perspective, these data present inferential challenges because instead of precise measures, one only has information that the value is somewhere between 0 and the DL (below detection limit, BDL). Substitution of BDL values, with 0 or the DL can lead to biased parameter estimates and a loss of statistical power. Statistical methods that make adjustments when dealing with these types of data, often called left-censored data, are available in many commercial statistical packages. Despite this availability, the use of these methods is still not widespread in biomedical literature. We have reviewed the statistical approaches of dealing with BDL values, and used simulations to examine the performance of the commonly used substitution methods and the most widely available statistical methods. We have illustrated these methods using a study undertaken at the Vanderbilt-Ingram Cancer Center, to examine the serum bile acid levels in patients with colorectal cancer and adenoma. We have found that the modern methods for BDL values identify disease-related differences that are often missed, with statistically naive approaches. PMID:21712958

  19. Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

    PubMed

    Peano, C; Lesignoli, F; Gulli, M; Corradini, R; Samson, M C; Marchelli, R; Marmiroli, N

    2005-09-15

    In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found. PMID:16055074

  20. Discovery of the unique self-assembly behavior of terminal suckers-contained dsDNA onto GNP and novel "light-up" colorimetric assay of nucleic acids.

    PubMed

    Qiu, Liping; Shen, Zhifa; Wu, Zai-Sheng; Shen, Guo-Li; Yu, Ruqin

    2015-02-15

    Noble metal nanoparticles are currently of great interest because of their unique optical properties and potential applications in disease diagnostics and cancer treatment. In the present work, a discovery was reported that dsDNA with terminal thiols at its two ends could lie easily flat onto the gold nanoparticle (GNP) surface rather than cross linked different GNPs, indicating an unique self-assembly behavior of newly-designed molecules on GNPs. This could intensively stabilize gold nanoparticles against aggregation even at a high salt concentration. On the basis of this discovery, a novel light-up colorimetric sensing strategy was developed for the detection of p53 gene by combining with the cyclical nucleic acid strand-displacement polymerization (CNDP). For the described colorimetric system, GNPs require no any surface functionalization, and target recognition reaction and CNDP amplification could be conducted under the optimized conditions to achieve a high efficiency. The high detection sensitivity and desirable selectivity are achieved, and the potential practical application was demonstrated. Besides, this sensing system can function in a wide range of salts, making it a suitable platform to cooperate with many biological processes. PMID:25240129

  1. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. PMID:25749303

  2. Chiral recognition and enantiomer assays of N-(3,5-dinitrobenzoyl)amino acid derivatives using electrospray ionization-mass spectrometry

    NASA Astrophysics Data System (ADS)

    Zu, Chengli; Woolfolk, Jonathan A.; Koscho, Michael E.

    2009-12-01

    A pair of pseudoenantiomers, anilide derivatives of N-pivaloylproline were prepared and used as chiral selectors for enantiomer discrimination of amides or esters of N-(3,5-dinitrobenzoyl)amino acids in single-stage electrospray ionization/mass spectrometric experiments. Addition of a chiral analyte to a solution of the two pseudoenantiomeric chiral selectors affords selector-analyte complexes in the electrospray ionization mass spectrum where the ratio of these complexes is dependent on the enantiomeric composition of the analyte. The relationship between the ratio of the selector-analyte complexes in the electrospray ionization mass spectrum and the enantiomeric composition of the analyte can be used to relate the extent of the measured enantioselectivity and for quantitative enantiomeric composition determinations. Effects of the added cationic ions (H+, Li+, Na+ and K+) and instrument conditions on the selector-analyte ion intensity and the enantioselectivity ([alpha]MS) were investigated. The percent ratio of the sum of the selector-analyte ion counts and the total ion counts decreases accordingly with the increase of the desolvation temperature for H+, Na+ and K+. The ratio for Li+ kept almost constant. The best [alpha]MS was observed at a desolvation temperature of 200 °C with the added H+. The cone voltage has little effects on the [alpha]MS values though the intensities of selector-analyte complexes are decreased at higher cone voltages. The observed MS enantioselectivities are comparable to the HPLC enantioselectivities and the sense of chiral recognition by MS is consistent with what is observed chromatographically. Quantitative enantiomeric composition determinations for five different samples of N-(3,5-dinitrobenzoyl)leucinyl butylamide at four different concentrations were performed. The average % difference between the HPLC and MS enantiomer determinations is 6.8% and 3.7% for the calibration lines constructed at a concentration of the analyte of

  3. Assay of intermediates in bile acid biosynthesis using isotope dilution--mass spectrometry: hepatic levels in the normal state and in cerebrotendinous xanthomatosis

    SciTech Connect

    Bjoerkhem, I.; Oftebro, H.; Skrede, S.; Pedersen, J.I.

    1981-02-01

    The synthesis of 2H4-labeled 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one is described. A mixture of these compounds, together with 2H3-labeled 5-cholestene-3 beta, 7 alpha-diol, was added to extracts of different subcellular fractions of liver. After purification by high performance liquid chromatography and conversion into trimethylsilyl ethers, the amounts of different endogenous unlabeled steroids were determined by selected ion monitoring. In normal liver, the concentration of 5-cholestene-3 beta, 7 alpha-diol was higher than the concentration of the other steroids. The concentration of the different steroids was highest in the microsomal fraction of the liver homogenate. In a liver sample from a patient with cerebrotendinous xanthomatosis (CTX), the amounts of the 12 alpha-hydroxylated steroids were considerably higher than in the normal liver. The levels of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol were similar or only slightly higher than in the liver of the control patients. The concentration of 5-cholestene-3 beta, 7 alpha-diol was very high in the mitochondrial fraction of the CTX-liver. The findings are in accordance with the previous demonstration that the basic metabolic defect in CTX is a lack of the mitochondrial 26-hydroxylase. The results are further compatible with the contention that 7 alpha,26-dihydroxy-4-cholesten-3-one is an important intermediate in the normal bile acid biosynthesis.

  4. Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells.

    PubMed

    Maremanda, Nihal G; Roy, Kislay; Kanwar, Rupinder K; Shyamsundar, Vidyarani; Ramshankar, Vijayalakshmi; Krishnamurthy, Arvind; Krishnakumar, Subramanian; Kanwar, Jagat R

    2015-09-01

    The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10-100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning. PMID:26487896

  5. Bacterial mutagenicity assays: test methods.

    PubMed

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  6. Development of a new microtiter plate format for clinically relevant assays.

    PubMed

    Piletska, Elena V; Piletsky, Stanislav S; Whitcombe, Michael J; Chianella, Iva; Piletsky, Sergey A

    2012-02-21

    A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care. PMID:22264028

  7. The pro-apoptotic role of autophagy in breast cancer

    PubMed Central

    Suman, S; Das, T P; Reddy, R; Nyakeriga, A M; Luevano, J E; Konwar, D; Pahari, P; Damodaran, C

    2014-01-01

    Background: Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines. Methods: Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis. Results: LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells. Conclusions: Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa. PMID:24945999

  8. Methods and devices for protein assays

    DOEpatents

    Chhabra, Swapnil; Cintron, Jose M.; Shediac, Renee

    2009-11-03

    Methods and devices for protein assays based on Edman degradation in microfluidic channels are disclosed herein. As disclosed, the cleaved amino acid residues may be immobilized in an array format and identified by detectable labels, such as antibodies, which specifically bind given amino acid residues. Alternatively, the antibodies are immobilized in an array format and the cleaved amino acids are labeled identified by being bound by the antibodies in the array.

  9. Urine protein concentration estimation for biomarker discovery.

    PubMed

    Mistry, Hiten D; Bramham, Kate; Weston, Andrew J; Ward, Malcolm A; Thompson, Andrew J; Chappell, Lucy C

    2013-10-01

    Recent advances have been made in the study of urinary proteomics as a diagnostic tool for renal disease and pre-eclampsia which requires accurate measurement of urinary protein. We compared different protein assays (Bicinchoninic acid (BCA), Lowry and Bradford) against the 'gold standard' amino-acid assay in urine from 43 women (8 non-pregnant, 34 pregnant, including 8 with pre-eclampsia). BCA assay was superior to both Lowry and Bradford assays (Bland Altman bias: 0.08) compared to amino-acid assay, which performed particularly poorly at higher protein concentrations. These data highlight the need to use amino-acid or BCA assays for unprocessed urine protein estimation. PMID:26103798

  10. [Interference of ethylene glycol on lactate assays].

    PubMed

    Graïne, H; Toumi, K; Roullier, V; Capeau, J; Lefèvre, G

    2007-01-01

    Ethylene glycol is broken down to three main organic acids: glycolic acid, glyoxylic acid and oxalic acid which cause severe metabolic acidosis. Effect of these three acids on lactate assays was evaluated in five blood gas analysers and two clinical chemistry analysers. For all systems, no influence of oxalic acid on lactate results could be demonstrated. No interference of glycolic acid could be observed on lactate assay performed with Rapid Lab 1265 (R: 104,9 +/- 12,1%), Vitros 950 (R: 105,7 +/- 5,3 %) and Architect ci8200 (R: 104,9 +/- 4,7%), but on the contrary, CCX 4, OMNI S, ABL 725 and 825 demonstrated a concentration-dependent interference. No interference of glyoxylic acid could be observed with Vitros 950, but a positive interference could be observed with ABL 725 and 825, OMNI S, CCX4 and Architect ci8200 A linear relationship between apparent lactate concentration found with ABL 725 and 825, OMNI S, CCX 4, and glyoxylic acid could be observed (0,94 < r < 0,99), a weaker interference being observed with Rapid Lab 1265 and Architect ci 8200. Our results demonstrated that in case of ethylene glycol poisoning, cautious interpretation of lactate assay should be done, since wrong results of lactacidemia could lead to misdiagnostic and delay patient treatment. PMID:17627925

  11. Radiometric assays for glycerol, glucose, and glycogen.

    PubMed

    Bradley, D C; Kaslow, H R

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays. PMID:2817333

  12. Radiometric assays for glycerol, glucose, and glycogen

    SciTech Connect

    Bradley, D.C.; Kaslow, H.R. )

    1989-07-01

    We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with (32P)ATP and glycerokinase, residual (32P)ATP is hydrolyzed by heating in acid, and free (32P)phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.

  13. Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.

    PubMed

    Hsieh, Ming-Lung; Tseng, Min-Jen; Tseng, Ming-Chung; Chu, Yen-Ho

    2006-07-01

    As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced. PMID:16701071

  14. Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay.

    PubMed

    Kluk, Michael J; Abo, Ryan P; Brown, Ronald D; Kuo, Frank C; Dal Cin, Paola; Pozdnyakova, Olga; Morgan, Elizabeth A; Lindeman, Neal I; DeAngelo, Daniel J; Aster, Jon C

    2015-10-01

    We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy. PMID:27148563

  15. Myeloid neoplasm demonstrating a STAT5B-RARA rearrangement and genetic alterations associated with all-trans retinoic acid resistance identified by a custom next-generation sequencing assay

    PubMed Central

    Kluk, Michael J.; Abo, Ryan P.; Brown, Ronald D.; Kuo, Frank C.; Dal Cin, Paola; Pozdnyakova, Olga; Morgan, Elizabeth A.; Lindeman, Neal I.; DeAngelo, Daniel J.; Aster, Jon C.

    2015-01-01

    We describe the case of a patient presenting with several weeks of symptoms related to pancytopenia associated with a maturation arrest at the late promyelocyte/early myelocyte stage of granulocyte differentiation. A diagnosis of acute promyelocytic leukemia was considered, but the morphologic features were atypical for this entity and conventional tests for the presence of a PML-RARA fusion gene were negative. Additional analysis using a custom next-generation sequencing assay revealed a rearrangement producing a STAT5B-RARA fusion gene, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and supplementary cytogenetic studies, allowing the diagnosis of a morphologically atypical form of acute promyelocytic leukemia to be made. Analysis of the sequencing data permitted characterization of both chromosomal breakpoints and revealed two additional alterations, a small deletion in RARA exon 9 and a RARA R276W substitution, that have been linked to resistance to all-trans retinoic acid. This case highlights how next-generation sequencing can augment currently standard testing to establish diagnoses in difficult cases, and in doing so help guide selection of therapy. PMID:27148563

  16. Potential probiotic lactic acid bacteria Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019) do not degrade gastric mucin in vitro.

    PubMed

    Zhou, J S; Gopal, P K; Gill, H S

    2001-01-22

    The mucus layer (mucin) coating the surface of the gastrointestinal tract (GIT) plays an important role in the mucosal barrier system. Any damage or disturbance of this mucin layer will compromise the host's mucosal defence function. In the present study, the ability of three potential probiotic lactic acid bacteria (LAB) strains (Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, Bifidobacterium lactis HN019) to degrade mucin in vitro was evaluated, in order to assess their potential pathogenicity and local toxicity. The LAB strains were incubated in medium containing hog gastric mucin (HGM, 0.3%) at 37 degrees C for 48 h, following which any decrease in carbohydrate and protein concentration in the ethanol-precipitated portion of the culture medium was determined, using phenol-sulphuric acid and bicinchonic acid (BCA) protein assays, respectively. The change in molecular weight of mucin glycoproteins, following incubation with the test strains, was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In order to expose any ability of the test strains to degrade mucin visually and more directly, the test strains were also cultured on agarose containing 0.3% HGM and incubated anaerobically for 72 h at 37 degrees C. No significant change in the carbohydrate or protein concentration in mucin substrates was found following incubation with the test strains. No mucin fragments were derived from the mucin suspension incubated with test strains, and no mucinolysis zone was identified on agarose. These results demonstrate that the potential probiotic LAB strains tested here were unable to degrade gastrointestinal mucin in vitro, which suggests that these novel probiotic candidates are likely to be non-invasive and non-toxic at the mucosal interface. PMID:11205957

  17. Absolute nuclear material assay

    DOEpatents

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

    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.

  18. 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.

  19. Hemoglobin assay for validation and quality control of medical device reprocessing.

    PubMed

    Frey, Justin; Guan, Allan; Li, Zhenyu; Turtil, Steven; Phillips, K Scott

    2015-09-01

    Hemoglobin (Hb) is an important analyte in medicine, forensics, and research. One area of crucial need for real-world Hb quantitation is the validation and quality control (QC) of reprocessed medical device cleaning. Here, we show how a microplate reader and colorimetric blood test strips can be used to quantitate nanogram (ng) quantities of Hb in a 1-min assay. The assay had a linear range of 0-50 ng (0-370 ng on a log scale) for Hb, with a limit of detection (LOD) of 3.3 ng, which was ∼500-fold more sensitive than the micro-BCA reagent (LOD = 1.6 μg) and on the same order of magnitude as detection of labeled Hb with fluorescence (LOD = 1.9 ng). For validation of medical device cleaning, the assay was specific for Hb in the presence of artificial test soil and was unaffected by interferences from common cleaning reagents at 10 ppm. Lubricant and sodium dodecyl sulfate did not significantly affect the assay at 10 ppm but affected the assay at 1 % g/g. The method showed 100 % recovery of hemoglobin in extracted soils, with extraction from silicone having the greatest variability in recovery, while Teflon and stainless steel had <10 % RSD. The assay makes it possible for medical device companies and health-care providers to obtain crucially needed information on the cleanliness of reused devices. PMID:26173785

  20. Immunochromatographic assay on thread.

    PubMed

    Zhou, Gina; Mao, Xun; Juncker, David

    2012-09-18

    Lateral-flow immunochromatographic assays are low-cost, simple-to-use, rapid tests for point-of-care screening of infectious diseases, drugs of abuse, and pregnancy. However, lateral flow assays are generally not quantitative, give a yes/no answer, and lack multiplexing. Threads have recently been proposed as a support for transporting and mixing liquids in lateral-flow immunochromatographic assays, but their use for quantitative high-sensitivity immunoassays has yet to be demonstrated. Here, we introduce the immunochromatographic assay on thread (ICAT) in a cartridge format that is suitable for multiplexing. The ICAT is a sandwich assay performed on a cotton thread knotted to a nylon fiber bundle, both of which are precoated with recognition antibodies against one target analyte. Upon sample application, the assay results become visible to the eye within a few minutes and are quantified using a flatbed scanner. Assay conditions were optimized, the binding curves for C-reactive protein (CRP) in buffer and diluted serum were established and a limit of detection of 377 pM was obtained. The possibility of multiplexing was demonstrated using three knotted threads coated with antibodies against CRP, osteopontin, and leptin proteins. The performance of the ICAT was compared with that of the paper-based and conventional assays. The results suggest that thread is a suitable support for making low-cost, sensitive, simple-to-use, and multiplexed diagnostic tests. PMID:22889381

  1. THE INDUCTION OF ABERRANT CRYPT FOCI (ACF) IN MALE AND FEMALE F344/N RATS BY BROMOCHLOROACETIC ACID (BCA) ADMINISTERED IN THE DRINKING WATER

    EPA Science Inventory

    The Induction of Aberrant Crypt Foci (ACF) in Male and Female F344/N Rats by Bromochloroacetic Acid (BCA) Administered in the Drinking Water.

    M.H. George1, D. Delker1, D.R. Geter1, C.Herbert2, J. Roycroft3, R. Melnick3, D.W.
    Rosenberg4, and A.B. DeAngelo1. 1USEPA, Resea...

  2. Rapid mercury assays

    SciTech Connect

    Szurdoki, S.; Kido, H.; Hammock, B.D.

    1996-10-01

    We have developed rapid assays with the potential of detecting mercury in environmental samples. our methods combine the simple ELISA-format with the selective, high affinity complexation of mercuric ions by sulfur-containing ligands. The first assay is based on a sandwich chelate formed by a protein-bound ligand immobilized on the wells of a microliter plate, mercuric ion of the analyzed sample, and another ligand conjugated to a reporter enzyme. The second assay involves competition between mercuric ions and an organomercury-conjugate to bind to a chelating conjugate. Several sulfur containing chelators (e.g., dithiocarbamates) and organomercurials linked to macromolecular carriers have been investigated in these assay formats. The assays detect mercuric ions in ppb/high ppt concentrations with high selectivity.

  3. Miniaturization of hydrolase assays in thermocyclers.

    PubMed

    Lucena, Severino A; Moraes, Caroline S; Costa, Samara G; de Souza, Wanderley; Azambuja, Patrícia; Garcia, Eloi S; Genta, Fernando A

    2013-03-01

    We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein. PMID:23123426

  4. 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.

  5. Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems.

    PubMed

    Glyk, Anna; Heinisch, Sandra L; Scheper, Thomas; Beutel, Sascha

    2015-05-15

    In the current study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and bicinchoninic acid (BCA) assays. Each phase-forming component above 1 and 5 wt% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing poly(ethylene glycol) (PEG)/salt concentration and PEG molecular weight. Therefore, a convenient dilution of both components (up to 1 and 5 wt%) before protein quantification is recommended in both assays, respectively, where the BCA assay is favored in comparison with the Bradford assay. PMID:25684109

  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. 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

  8. 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

  9. 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.

  10. 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. PMID:27154596

  11. Co2+-dependent gene expression in Streptococcus pneumoniae: opposite effect of Mn2+ and Co2+ on the expression of the virulence genes psaBCA, pcpA, and prtA

    PubMed Central

    Manzoor, Irfan; Shafeeq, Sulman; Kloosterman, Tomas G.; Kuipers, Oscar P.

    2015-01-01

    Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+) play significant roles in transcriptional gene regulation, physiology, and virulence of Streptococcus pneumoniae. So far, the effect of the important transition metal ion cobalt (Co2+) on gene expression of S. pneumoniae has not yet been explored. Here, we study the impact of Co2+ stress on the transcriptome of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+-transport operon (cbi operon), the expression of which we show here to be induced by a high Co2+ concentration. Furthermore, we found that Co2+, as has been shown previously for Zn2+, can cause derepression of the genes of the PsaR virulence regulon, encoding the Mn2+-uptake system PsaBCA, the choline binding protein PcpA and the cell-wall associated serine protease PrtA. Interestingly, although Mn2+ represses expression of the PsaR regulon and Co2+ leads to derepression, both metal ions stimulate interaction of PsaR with its target promoters. These data will be discussed in the light of previous studies on similar metal-responsive transcriptional regulators. PMID:26257722

  12. Practical assay for nitrite and nitrosothiol as an alternative to the Griess assay or the 2,3-diaminonaphthalene assay.

    PubMed

    Shen, Yanming; Zhang, Quanjuan; Qian, Xuhong; Yang, Youjun

    2015-01-20

    Nitrite is a heavily assayed substrate in the fields of food safety, water quality control, disease diagnosis, and forensic investigation and more recently in basic biological studies on nitric oxide physiology and pathology. The colorimetric Griess assay and the fluorimetric 2,3-diaminonaphthalene (DAN) assay are the current gold standards for nitrite quantification. They are not without limitations, yet have amazingly survived 156 and 44 years, respectively, due to the lack of a practical alternative. Both assays exhibit slow detection kinetics due to inactivation of nucleophiles under strongly acidic media, require an extensive incubation time for reaction to go completion, and hence offer a limited detection throughput. By converting an intermolecular reaction of the Griess assay intramolecularly, we designed a novel probe (NT555) for nitrite detection, which displays superior detection kinetics and sensitivity. NT555 was constructed following our "covalent-assembly" probe design principle. Upon detection, it affords a gigantic bathochromic shift of the absorption spectrum and a sensitive turn-on fluorescence signal from a zero background, both of which are typical of an "assembly" type probe. Overall, NT555 has addressed various difficulties associated with the Griess and the DAN assays and represents an attractive alternative for practical applications. PMID:25519711

  13. 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.

  14. Against vaccine assay secrecy

    PubMed Central

    Herder, Matthew; Hatchette, Todd F; Halperin, Scott A; Langley, Joanne M

    2015-01-01

    Increasing the transparency of the evidence base behind health interventions such as pharmaceuticals, biologics, and medical devices, has become a major point of critique, conflict, and policy focus in recent years. Yet the lack of publicly available information regarding the immunogenicity assays upon which many important, widely used vaccines are based has received no attention to date. In this paper we draw attention to this critical public health problem by reporting on our efforts to secure vaccine assay information in respect of 10 vaccines through Canada's access to information law. We argue, under Canadian law, that the public health interest in having access to the methods for these laboratory procedures should override claims by vaccine manufacturers and regulators that this information is proprietary; and, we call upon several actors to take steps to ensure greater transparency with respect to vaccine assays, including regulators, private firms, researchers, research institutions, research funders, and journal editors. PMID:25826194

  15. 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.

  16. Immobilization of carbonic anhydrase on spherical SBA-15 for hydration and sequestration of CO2.

    PubMed

    Vinoba, Mari; Bhagiyalakshmi, Margandan; Jeong, Soon Kwan; Yoon, Yeo Ii; Nam, Sung Chan

    2012-02-01

    Bovine carbonic anhydrase (BCA) was immobilized on spherical SBA-15 through various approaches, including covalent attachment (BCA-CA), adsorption (BCA-ADS), and cross-linked enzyme aggregation (BCA-CLEA). The spherical SBA-15 was characterized by XRD, BET, and FE-SEM analysis. (29)Si CP-MAS NMR was used to confirm the 3-aminopropyltriethoxysilane grafting (an intermediate step in the immobilization technique), and the immobilization of BCA was confirmed by FT-IR spectrum. The catalytic activities for hydration of CO(2) were calculated for free and immobilized BCA with and without buffer. The K(cat) values for free BCA, BCA-CLEA, BCA-CA and BCA-ADS were 0.79, 0.78, 0.58 and 0.36 s(-1), respectively, indicating that BCA-CLEA showed a comparatively higher hydration of CO(2) than BCA-CA and BCA-ADS, which was nearly the same as free BCA. The amount of CaCO(3) precipitated over free BCA, BCA-CLEA, BCA-CA and BCA-ADS were 140, 138, 135 and 130 mg, respectively. Performance studies, including assays on reusability, thermal stability and storage stability, were also carried out for BCA-CLEA. The results confirmed that BCA-CLEA is reusable, thermally stable and, withstands storage, and is thus a suitable candidate for use in hydration and sequestration of CO(2). PMID:22024402

  17. Lateral flow strip assay

    SciTech Connect

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    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.

  18. Development of a new Laser Photofragmentation/Fluorescent Assay by Gas Expansion (LP/FAGE) technique for the quantification of tropospheric nitrous acid (HONO) at low parts-per-trillion mixing ratios

    NASA Astrophysics Data System (ADS)

    Mielke, L. H.; Lew, M.; Bottorff, B.; Berke, A.; Raff, J. D.; Stevens, P. S.; Dusanter, S.

    2013-12-01

    Determining the full oxidative capacity of the atmosphere is vital to understanding the production of secondary pollutants such as ozone and secondary organic aerosols and for regulating the lifetime of pollutants leading to climate change. The hydroxyl radical is the primary oxidant of volatile organic compounds (VOCs) in the troposphere. Nocturnal nitrous acid (HONO) is an important radical reservoir species and releases OH upon photolysis the next morning. In addition, recent studies have indicated higher than expected mixing ratios of HONO in the daytime. As daytime HONO mixing ratios usually maximize at only a couple hundred part-per-trillion, it is important to have a technique that is accurate, sensitive, and precise. Here we outline an instrumental technique called Laser Photofragmention/Fluorescent Assay by Gas Expansion (LP/FAGE). Ambient air is drawn through an inlet composed of a 1' diameter metal disk with a 0.025' cylindrically bored hole where it undergoes expansion into a cell held at ~3 torr. Fiber coupled laser emission (YILF: 355 nm, 2.2 W) induces photofragmentation of HONO to OH and NO whereby the OH is quantified by the FAGE technique using a fiber coupled 308 nm (6 mW) laser emission. The 355 nm and 308 nm emission are single pass, collinear, and separated only by the time delay of the pulses. To differentiate ambient OH from HONO-generated-OH, a shutter is used to block the 355 nm laser emission for a given period of time. Fluorescence from OH vs. fluorescence from interfering species can be differentiated by scanning on and off a specific rovibronic feature in the OH absorbance spectra. In this presentation we outline the instrumental technique, including its calibration in which effluent from an HCl permeation device is humidified and passed over a bed of sodium nitrate coated glass beads. The calibrator output is varied from 1 to several tens of parts-per-billions (ppb) and is detected using a chemiluminescence NOx analyzer. The

  19. 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.

  20. 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.

  1. Macroautophagic cargo sequestration assays.

    PubMed

    Seglen, Per O; Luhr, Morten; Mills, Ian G; Sætre, Frank; Szalai, Paula; Engedal, Nikolai

    2015-03-01

    Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy. PMID:25576638

  2. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  3. A Luciferase Reporter Gene Assay to Measure Ebola Virus Viral Protein 35-Associated Inhibition of Double-Stranded RNA-Stimulated, Retinoic Acid-Inducible Gene 1-Mediated Induction of Interferon β.

    PubMed

    Cannas, Valeria; Daino, Gian Luca; Corona, Angela; Esposito, Francesca; Tramontano, Enzo

    2015-10-01

    During Ebola virus (EBOV) infection, the type I interferon α/β (IFN-α/β) innate immune response is suppressed by EBOV viral protein 35 (VP35), a validated drug target. Identification of EBOV VP35 inhibitors requires a cellular system able to assess the VP35-based inhibitory functions of viral double-stranded RNA (dsRNA) IFN-β induction. We established a miniaturized luciferase gene reporter assay in A549 cells that measures IFN-β induction by viral dsRNA and is dose-dependently inhibited by VP35 expression. When compared to influenza A virus NS1 protein, EBOV VP35 showed improved inhibition of viral dsRNA-based IFN-β induction. This assay can be used to screen for EBOV VP35 inhibitors. PMID:25926684

  4. 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.

  5. Radioreceptor assay for oxyphenonium.

    PubMed

    Ensing, K; de Zeeuw, R A

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. PMID:6428927

  6. Robust quantitative scratch assay

    PubMed Central

    Vargas, Andrea; Angeli, Marc; Pastrello, Chiara; McQuaid, Rosanne; Li, Han; Jurisicova, Andrea; Jurisica, Igor

    2016-01-01

    The wound healing assay (or scratch assay) is a technique frequently used to quantify the dependence of cell motility—a central process in tissue repair and evolution of disease—subject to various treatments conditions. However processing the resulting data is a laborious task due its high throughput and variability across images. This Robust Quantitative Scratch Assay algorithm introduced statistical outputs where migration rates are estimated, cellular behaviour is distinguished and outliers are identified among groups of unique experimental conditions. Furthermore, the RQSA decreased measurement errors and increased accuracy in the wound boundary at comparable processing times compared to previously developed method (TScratch). Availability and implementation: The RQSA is freely available at: http://ophid.utoronto.ca/RQSA/RQSA_Scripts.zip. The image sets used for training and validation and results are available at: (http://ophid.utoronto.ca/RQSA/trainingSet.zip, http://ophid.utoronto.ca/RQSA/validationSet.zip, http://ophid.utoronto.ca/RQSA/ValidationSetResults.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975.zip, http://ophid.utoronto.ca/RQSA/ValidationSet_H1975Results.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip, http://ophid.utoronto.ca/RQSA/RobustnessSet.zip). Supplementary Material is provided for detailed description of the development of the RQSA. Contact: juris@ai.utoronto.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26722119

  7. Evaluation of lot-to-lot repeatability and effect of assay media choice in the recombinant Factor C assay.

    PubMed

    McKenzie, Jennifer Helen; Alwis, K Udeni; Sordillo, Joanne E; Kalluri, Kesava Srinivas; Milton, Donald Kirby

    2011-06-01

    Measurement of environmental endotoxin exposures is complicated by variability encountered using current biological assay methods arising in part from lot-to-lot variability of the Limulus-amebocyte lysate (LAL) reagents. Therefore, we investigated the lot-to-lot repeatability of commercially available recombinant Factor C (rFC) kits as an alternative to LAL. Specifically, we compared endotoxin estimates obtained from rFC assay of twenty indoor dust samples, using four different extraction and assay media, to endotoxin estimates previously obtained by Limulus amebocyte lysate (LAL) assay and amounts of 3-hydroxy fatty acids (3-OHFA) in lipopolysaccharide (LPS) using gas-chromatography mass spectroscopy (GC-MS). We found that lot-to-lot variability of the rFC assay kits does not significantly alter endotoxin estimates in house dust samples when performed using three of the four assay media tested and that choice of assay media significantly altered endotoxin estimates obtained by rFC assay of house dust samples. Our findings demonstrate lot-to-lot reproducibility of rFC assay of environmental samples and suggest that use of rFC assay performed with Tris buffer or water as the extraction and assay medium for measurement of endotoxin in dust samples may be a suitable choice for developing a standardized methodology. PMID:21552635

  8. High-throughput assay for optimising microbial biological control agent production and delivery

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialization. A myriad of variables associated with BCA cultivation, formulation, drying, storage, and reconstitution processes complicates agent quality maximization. An efficie...

  9. Comparison of Luminescence ADP Production Assay and Radiometric Scintillation Proximity Assay for Cdc7 Kinase

    PubMed Central

    Takagi, Toshimitsu; Shum, David; Parisi, Monika; Santos, Ruth E.; Radu, Constantin; Calder, Paul; Rizvi, Zahra; Frattini, Mark G.; Djaballah, Hakim

    2013-01-01

    Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [33P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [33P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay. PMID:21564015

  10. Macrophage Inflammatory Assay

    PubMed Central

    Ylostalo, Joni H.

    2016-01-01

    Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’. In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16–18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).

  11. C. elegans chemotaxis assay.

    PubMed

    Margie, Olivia; Palmer, Chris; Chin-Sang, Ian

    2013-01-01

    Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior. The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress (1-10). The method described here aims to address these issues by modifying the assay developed by Bargmann et al.(1). A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired. PMID:23644543

  12. 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+.

  13. 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+.

  14. 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.

  15. 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 ...

  16. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  17. HIV-1 Fusion Assay

    PubMed Central

    Cavrois, Marielle; Neidleman, Jason; Greene, Warner C.

    2016-01-01

    The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

  18. 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. PMID:26498795

  19. Molecular inversion probe assay.

    PubMed

    Absalan, Farnaz; Ronaghi, Mostafa

    2007-01-01

    We have described molecular inversion probe technologies for large-scale genetic analyses. This technique provides a comprehensive and powerful tool for the analysis of genetic variation and enables affordable, large-scale studies that will help uncover the genetic basis of complex disease and explain the individual variation in response to therapeutics. Major applications of the molecular inversion probes (MIP) technologies include targeted genotyping from focused regions to whole-genome studies, and allele quantification of genomic rearrangements. The MIP technology (used in the HapMap project) provides an efficient, scalable, and affordable way to score polymorphisms in case/control populations for genetic studies. The MIP technology provides the highest commercially available multiplexing levels and assay conversion rates for targeted genotyping. This enables more informative, genome-wide studies with either the functional (direct detection) approach or the indirect detection approach. PMID:18025701

  20. Peroxynitrite scavenging by different antioxidants. Part I: convenient assay.

    PubMed

    Balavoine, G G; Geletii, Y V

    1999-01-01

    A convenient "tube" assay to quantify relative antioxidant activities in aqueous solutions has been developed. Peroxynitrite was employed as a biologically relevant source of radicals with Pyrogallol Red as a detecting molecule. A variety of compounds have been examined, namely polyphenols, uric acid, glutathione, and ascorbic acid. Competition kinetics were observed for the majority of examined compounds, except thymol and ascorbic acid. Pyrogallol Red was fully protected by ascorbic acid against the bleaching by peroxynitrite until its total consumption. The deviation from competition kinetics in the case of thymol was due to the formation of radicals from thymol and their subsequent reaction with Pyrogallol Red. Quercetin was the most efficient scavenger of free radicals. The measurements of relative antioxidant activities using Pyrogallol Red and other detecting molecules, such as gallocyanine and carminic acid, were in fair agreement. The assay was successfully used for a screening of antioxidant activity of plant extracts of unknown composition. PMID:10355895

  1. A specific endpoint assay for ubiquitin.

    PubMed Central

    Rose, I A; Warms, J V

    1987-01-01

    Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. The method for measuring Ub makes use of the reaction of iodoacetamide-treated Ub-activating enzyme (E): [3H]ATP + Ub + E----E X [3H]AMP-Ub + PPi and PPi----2Pi (in the presence of pyrophosphatase). The Ub is then measured by determining the acid-insoluble radioactivity. The reaction is accompanied by a slow enzyme-catalyzed hydrolysis of the complex to AMP plus Ub. The presence of ubiquitin-activating enzyme in excess of Ub by approximately equal to 0.1 microM assures that the steady state will be close to the endpoint for total Ub. A preparation of the activating enzyme from human erythrocytes that does not depend on affinity chromatography is described. Several applications of the assay are presented. PMID:3031643

  2. Evaluation of Chikungunya Diagnostic Assays: Differences in Sensitivity of Serology Assays in Two Independent Outbreaks

    PubMed Central

    Yap, Grace; Pok, Kwoon-Yong; Lai, Yee-Ling; Hapuarachchi, Hapuarachchige-Chanditha; Chow, Angela; Leo, Yee-Sin; Tan, Li-Kiang; Ng, Lee-Ching

    2010-01-01

    Background The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested. Methods and Findings For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green. Conclusion Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different

  3. A more sensitive and specific radioenzymatic assay for catecholamines

    SciTech Connect

    Kennedy, B.; Ziegler, M.G. )

    1990-01-01

    This modification of the catechol-O-methyltransferase (COMT) based radioenzymatic assay for norepinephrine (NE) and epinephrine (E) improves sensitivity, selectivity and eliminates many inhibitors of COMT. Prior to assay, samples are extracted into heptane with diphenylborate, then into dilute acetic acid. This extraction procedure has an efficiency of 78% for NE but less than 2% for S-adenosylmethionine (SAM). The extraction procedure also excludes calcium and other COMT inhibitors present in urine, plasma and every tissue tested. This eliminates the requirement for individual standardization of tissue and urine samples. Sensitivity of the assay for NE and E is 10 and 6 pg/ml respectively in 1 ml of plasma. The intraassay coefficients of variation for NE and E are 4 and 13% and the interassay coefficients of variation for NE and E are 10 and 16% in a human plasma sample containing low catecholamine levels. The assay permits quantitation of plasma E levels that were undetectable in prior assays.

  4. Comparison of antioxidant activities of different parts from snow chrysanthemum (Coreopsis tinctoria Nutt.) and identification of their natural antioxidants using high performance liquid chromatography coupled with diode array detection and mass spectrometry and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt-based assay.

    PubMed

    Chen, L X; Hu, D J; Lam, S C; Ge, L; Wu, D; Zhao, J; Long, Z R; Yang, W J; Fan, B; Li, S P

    2016-01-01

    Snow chrysanthemum (Coreopsis tinctoria Nutt.), a world-widely well-known flower tea material, has attracted more and more attention because of its beneficial health effects such as antioxidant activity and special flavor. In this study, a high performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) and 2,2'-azinobis(3-ethylbenzthiazoline-sulfonic acid)diammonium salt (ABTS) based assay was employed for comparison and identification of antioxidants in different samples of snow chrysanthemum. The results showed that snow chrysanthemum flowers possessed the highest while stems presented the lowest antioxidant capacities. Fourteen detected peaks with antioxidant activity were temporarily identified as 3,4',5,6,7-pentahydroxyflavanone-O-hexoside, chlorogenic acid, 2R-3',4',8-trihydroxyflavanone-7-O-glucoside, flavanomarein, flavanocorepsin, flavanokanin, quercetagitin-7-O-glucoside, 3',5,5',7-tetrahydroxyflavanone-O-hexoside, marein, maritimein, 1,3-dicaffeoylquinic acid, coreopsin, okanin and acetyl-marein by comparing their UV spectra, retention times and MS data with standards or literature data. Antioxidants existed in snow chrysanthemum are quite different from those reported in Chrysanthemum morifolium, a well-known traditional beverage in China, which indicated that snow chrysanthemum may be a promising herbal tea material with obvious antioxidant activity. PMID:26521095

  5. Toxicity of adipic acid.

    PubMed

    Kennedy, Gerald L

    2002-05-01

    Adipic acid has very low acute toxicity in rats with an LD50 > 5000 mg/kg. Adipic acid produced mild to no skin irritation on intact guinea pig skin as a 50% concentration in propylene glycol; it was not a skin sensitizer. Adipic acid caused mild conjunctival irritation in washed rabbit eyes; in unwashed rabbit eyes, there was mild conjunctival irritation, minimal iritis, but no corneal effects. Adipic acid dust may irritate the mucous membranes of the lungs and nose. In a 2-year feeding study, rats fed adipic acid at concentrations up to 5% in the diet exhibited only weight loss. Adipic acid is not genetically active in a wide variety of assay systems. Adipic acid caused no developmental toxicity in mice, rats, rabbits, or hamsters when administered orally. Adipic acid is partially metabolized in humans; the balance is eliminated unchanged in the urine. Adipic acid is slightly to moderately toxic to fish, daphnia, and algae in acute tests. PMID:12024802

  6. Non-separation assay for glycohemoglobin.

    PubMed

    Blincko, S; Edwards, R

    1998-06-01

    The determination of glycohemoglobin [HbA1c, HbA1, or total glycohemoglobin (GHb)] has become an established procedure in the management of diabetes mellitus. Here, we describe the development of a simple, fluorescence, non-separation assay for the percentage of GHb (%GHb). The fluorescence of an eosin-boronic acid derivative when it was mixed with hemolysates of unwashed erythrocytes was quenched in proportion to the percentage of glycohemoglobin. Measurement of the fluorescence intensity gave an estimate of GHb in the sample, and measurement of light absorbance gave an estimate of total hemoglobin. A combination of the two measurements gave the assay response. Comparison with HPLC (Menarini-Arkray HA-8140 fully automated analyzer) for the percentage of HbA1 (%HbA1) gave %GHb(NETRIA) = 1.1(SD +/-0.03)%HbA1 +0.6(SD +/-0.3), S(y/x) = 0.821, r = 0.972, n = 80; comparison for HbA1c gave %GHb(NETRIA) = 1.3(SD +/-0.04)%HbA1c + 1.8(SD +/-0.3), S(y/x) = 0.813, r = 0.973, n = 80. Precision, estimated as the percentage of the CV of the %GHb assay results, was <2% (intraassay, range 5-22% GHb) and <4.2% (interassay, range 4-16% GHb). Dilution of a high-percentage GHb sample lysate showed that the assay was linear, and addition of glucose (60 mmol/L), bilirubin (250 micromol/L), and triglycerides (14 mmol/L) to low, medium, and high %GHb samples showed no clinical interference in assay results. PMID:9625057

  7. An assay for adjuvanticity

    PubMed Central

    Dresser, D. W.

    1968-01-01

    Adult mice injected with an adequate amount of a non-immunogenic antigen progress to a specific state of immunological paralysis, unless a substance with `extrinsic' adjuvanticity is injected before the induction of paralysis is completed. Consequently incipiently paralysed mice can be used to assay substances for adjuvanticity. Conventional adjuvants such as Freund's adjuvant and pertussis possess adjuvanticity; other substances with varying degrees of adjuvanticity are listed in the tables. It has been shown that the adjuvanticity effect of an injection of pertussis lasts for only a few days, although the effect of such an injection of pertussis on phagocytosis of carbon particles does not reach a maximum until 2 weeks after the injection. The dose-effectiveness of alum precipitated (highly phagocytosable) bovine γ-globulin was greatly increased by the intraperitoneal injection of pertussis. The evidence is considered to be incompatible with increased phagocytosis being either an essential factor in the role of pertussis as a conventional adjuvant, or in the adjuvanticity effect of pertussis. PMID:4179956

  8. Membrane Flotation Assay

    PubMed Central

    Vogt, Dorothee A; Ott, Melanie

    2016-01-01

    Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10–20–30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.

  9. Chicken glucagon: sequence and potency in receptor assay.

    PubMed

    Huang, J; Eng, J; Yalow, R S

    1987-11-01

    Glucagon is a 29 amino acid peptide that is generally highly conserved. Among mammalian glucagons the only one that has been shown to differ significantly is that of the guinea pig which differs from the others in 5 of the 9 COOH-terminus amino acids. The amino acid content and partial sequencing of chicken glucagon had been reported earlier. This report describes the purification and complete amino acid sequencing of chicken glucagon and demonstrates that it differs from the usual mammalian glucagon by the replacement of asparagine at position 28 with serine. Chicken glucagon is indistinguishable from porcine glucagon in the rat liver receptor assay system. PMID:2828209

  10. Biocontrol agents-mediated suppression of oxalic acid induced cell death during Sclerotinia sclerotiorum-pea interaction.

    PubMed

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Sarma, Birinchi Kumar; Singh, Harikesh Bahadur

    2015-05-01

    Oxalic acid (OA) is an important pathogenic factor during early Sclerotinia sclerotiorum-host interaction and might work by reducing hydrogen peroxide production (H2 O2 ). In the present investigation, oxalic acid-induced cell death in pea was studied. Pea plants treated with biocontrol agents (BCAs) viz., Pseudomonas aeruginosa PJHU15, Bacillus subtilis BHHU100, and Trichoderma harzianum TNHU27 either singly and/or in consortium acted on S. sclerotiorum indirectly by enabling plants to inhibit the OA-mediated suppression of oxidative burst via induction of H2 O2 . Our results showed that BCA treated plants upon treatment with culture filtrate of the pathogen, conferred the resistance via. significantly decreasing relative cell death of pea against S. sclerotiorum compared to control plants without BCA treatment but treated with the culture filtrate of the pathogen. The results obtained from the present study indicate that the microbes especially in consortia play significant role in protection against S. sclerotiorum by modulating oxidative burst and partially enhancing tolerance by increasing the H2 O2 generation, which is otherwise suppressed by OA produced by the pathogen. PMID:24920251

  11. New lipase assay using Pomegranate oil coating in microtiter plates.

    PubMed

    Ülker, Serdar; Placidi, Camille; Point, Vanessa; Gadenne, Benoît; Serveau-Avesque, Carole; Canaan, Stéphane; Carrière, Frédéric; Cavalier, Jean-François

    2016-01-01

    Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step. PMID:26343557

  12. On-column labeling technique and chiral ligand-exchange CE with zinc(II)-L-arginine complex as a chiral selector for assay of dansylated D,L-amino acids.

    PubMed

    Qi, Li; Yang, Gengliang

    2009-08-01

    A novel on-column labeling method of amino acid (AA) enantiomers by using dansyl chloride (Dns-Cl) has been explored combined with chiral ligand-exchange CE (CLE-CE) technique and UV detection. Efficient labeling was achieved by sequential injection of buffer, Dns-Cl, AA enantiomers, Dns-Cl and buffer at 0.2 psi for 10.0, 3.0, 24.0, 3.0, and 10.0 s, respectively. After injection, the sandwich sections were allowed to react at room temperature for 35.0 min. With this procedure, successful on-column labeling and CLE-CE separation of 17 pairs AA enantiomers have been achieved with a buffer of 100.0 mM boric acid, 5.0 mM ammonium acetate, 3.0 mM ZnSO4 and 6.0 mM L-Arg at pH 8.4, giving nine pairs fully enantioresolved with resolution in between 2.0 and 5.1. CLE-CE of some individual and mixed pairs was also demonstrated, much the same as using pre-column labeling. As validated by both artificially prepared solutions and serum samples, this new method was shown to be applicable to the quantitative analysis, with a linear range between 14.0 muM and 3.7 mM, correlation coefficient above 0.99 and recovery in between 90.4% and 111.7%. It was also demonstrated that the migration time-temperature based curve allows for temperature determination in CE by using this new method. PMID:19655328

  13. Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders.

    PubMed

    Lu, Hao; Kopcho, Lisa; Ghosh, Kaushik; Witmer, Mark; Parker, Michael; Gupta, Sumit; Paul, Marilyn; Krishnamurthy, Prasad; Laksmaiah, Basanth; Xie, Dianlin; Tredup, Jeffrey; Zhang, Litao; Abell, Lynn M

    2016-05-15

    Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies. PMID:26874021

  14. Characterization of Leptospiral Chemoreceptors Using a Microscopic Agar Drop Assay.

    PubMed

    Affroze, Samia; Islam, Md Shafiqul; Takabe, Kyosuke; Kudo, Seishi; Nakamura, Shuichi

    2016-08-01

    Bacterial chemotaxis is induced by sensing chemical stimuli via chemoreceptors embedded in the cytoplasmic membrane, enabling the cells to migrate toward nutrients or away from toxins. The chemoreceptors of Escherichia coli and Salmonella spp. have been well studied and are functionally classified on the basis of detectable substrates. The spirochete Leptospira possesses more than ten chemoreceptors and shows attractive or repellent responses against some sugars, amino acids, and fatty acids. However, the roles of these chemoreceptors have not been investigated. In this study, we conducted a chemotaxis assay called microscopic agar drop assay in combination with competition experiments, determining whether two kinds of attractants are recognized by the same type of chemoreceptor in the saprophytic Leptospira strain, Leptospira biflexa. Analyzing the competition effect observed between several pairs of chemicals, we found that L. biflexa senses sugars via chemoreceptors different from those that sense amino acids and fatty acids. PMID:27109059

  15. Transformation Assay for Identification of Psychrotrophic Achromobacters

    PubMed Central

    Juni, Elliot; Heym, Gloria A.

    1980-01-01

    The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototrophy. Genetically interacting bacteria included strains that lacked one or more of the characteristics typical for most achromobacters. An oxidase-negative mutant of one of these strains reacted positively in the transformation assay, unlike other oxidase-negative bacteria. Achromobacters were derived from fish, poultry, irradiated foods, seawater, and other sources. One strain previously classified as Micrococcus cryophilus has been shown to be related to the achromobacters. Two achromobacters had an optimum growth temperature of 35°C and behaved as typical mesophiles. The moraxellae and Acinetobacter were shown to be unrelated to the achromobacters by using the transformation assay. The ready demonstration of genetic relatedness provides a new basis for taxonomic grouping of the psychrotrophic achromobacters. Images PMID:16345673

  16. Development of a specific real-time PCR assay targeting the poly-γ-glutamic acid synthesis gene, pgsB, for the quantification of Bacillus amyloliquefaciens in solid-state fermentation.

    PubMed

    Yong, Xiaoyu; Zhang, Ruifu; Zhang, Nan; Chen, Yilu; Huang, Xinqi; Zhao, Jun; Shen, Qirong

    2013-02-01

    A TaqMan real-time PCR procedure was developed for specific detection and quantification of strains belonging to Bacillus amyloliquefaciens group. The primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the poly-γ-glutamic acid synthesis gene (pgsB) of B. amyloliquefaciens. The detection limit was approximately between 10(2)-10(3) cells/mL. A linear correlation between the log10 input pMD-pgsB plasmid DNA copies and the threshold cycle values were observed with a magnitude of linearity in the range of 9.415×10(3)-10(7) copies/mL for the standard curve, which exhibited a slope of -3.35 and an R2 value of 99.8%. Results of validation of this method with artificially contaminated and natural solid-state fermentation samples showed that it was suitable for specific and sensitive detection and quantification for the target strains in solid-state fermentation samples. This could be more useful to understand the fermentation starting strain and the final microbiological properties of fermentation products. PMID:23266849

  17. 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

  18. Assay of vtg, ERs and PPARs as endpoint for the rapid in vitro screening of the harmful effect of Di-(2-ethylhexyl)-phthalate (DEHP) and phthalic acid (PA) in zebrafish primary hepatocyte cultures.

    PubMed

    Maradonna, Francesca; Evangelisti, Matteo; Gioacchini, Giorgia; Migliarini, Beatrice; Olivotto, Ike; Carnevali, Oliana

    2013-02-01

    In the last years the concern about the negative effects of phthalates on reproduction significantly increased. Considering that, at date data available dealing with the adverse outcome of Di-(2-ethylhexyl)-phthalate (DEHP) on the reproduction of several species are still contrasting, in this study, the effects induced by DEHP (0.05, 0.1, 1, 10 and 100 nM) and its active metabolite, phthalic acid (PA) (0.01, 0.1, 1 and 10 μM), were analyzed in zebrafish, Danio rerio, primary hepatocyte cultures, using target molecules involved in fish reproduction (vitellogenin--vtg and estrogen receptors--ERα, β1 and β2) and metabolism (peroxisome proliferators activated receptors--PPAR α, β, γ). The use of in vitro culture, in fact, has the potential to significantly reduce the number of animals sacrificed for research allowing a precise control of the physical and chemical parameters that is often not possible in vivo. Moreover, since many toxicological studies revealed a sex specific response to toxicants, male and female primary hepatocyte cultures were set up to elucidate the possible gender specific effects of two common environmental phthalates. The increase of vtg levels observed in the culture media of male or female hepatocytes strongly evidenced the phthalates E2-like action. Moreover, the data obtained suggested that the observed different ERs isoforms modulation is otherwise associated with the vtg increase, depending on fish gender. Regarding PPARs, a similar trend of expression was found in both males and females. In conclusion, this study enforces the role of vtg as biomarker for evaluate the presence of environmental doses of DEHP and PA. Considering the similar gender modulation observed for vtg and PPARs, these molecules could be used for the rapid screening of the presence of DEHP and PA. Noteworthy the gender specific modulation observed for ERs opens a debate on the estrogenic mechanism of action of DEHP and PA and their role on vtg induction. PMID

  19. 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. PMID:25027375

  20. The assay of diphtheria toxin

    PubMed Central

    Gerwing, Julia; Long, D. A.; Mussett, Marjorie V.

    1957-01-01

    A precise assay of diphtheria toxin is described, based on the linear relationship between the diameter of the skin reaction to, and logarithm of the dose of, toxin. It eliminates the need for preliminary titrations, is economical, provides information about the slope of the log-dose response lines and, therefore, of the validity of the assay, and yields limits of error of potency from the internal evidence of the assay. A study has been made of the effects of avidity, combining power, toxicity and buffering on the assay of diphtheria toxins against the International Standards for both Diphtheria Antitoxin and Schick-Test Toxin. All the toxins assayed against the standard toxin, whatever their other properties might be, gave log-dose response lines of similar slope provided that they were diluted in buffered physiological saline. The assays were therefore valid. These experiments were repeated concurrently in non-immune and in actively immunized guinea-pigs, and comparable figures for potency obtained in both groups. The result was not significantly affected by the avidity or combining power of the toxin. However, non-avid toxins gave low values in Schick units when assayed, by the Römer & Sames technique, in terms of the International Standard for Diphtheria Antitoxin. The problem of the ultimate standard and the implications of these findings are discussed. PMID:13511133

  1. Transwell(®) invasion assays.

    PubMed

    Marshall, John

    2011-01-01

    The need to identify inhibitors of cancer invasion has driven the development of quantitative in vitro invasion assays. The most common assays used are based on the original Boyden assay system. Today commercially available plastic inserts for multi-well plates, which possess a cell-permeable membrane, as typified by Transwell(®) Permeable Supports, permit accurate repeatable invasion assays. When placed in the well of a multi-well tissue culture plate these inserts create a two-chamber system separated by the cell-permeable membrane. To create an invasion assay the pores in the membrane are blocked with a gel composed of extracellular matrix that is meant to mimic the typical matrices that tumour cells encounter during the invasion process in vivo. By placing the cells on one side of the gel and a chemoattractant on the other side of the gel, invasion is determined by counting those cells that have traversed the cell-permeable membrane having invaded towards the higher concentration of chemoattractant. In this chapter, in addition to protocols for performing Transwell invasion assays, there is consideration of the limitations of current assay designs with regard to available matrices and the absence of tumour microenvironment cells. PMID:21748672

  2. MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)

    EPA Science Inventory

    An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

  3. Molecular amplification assays for the detection of flaviviruses.

    PubMed

    Lanciotti, Robert S

    2003-01-01

    Over the past 10 years, a number of molecular amplification assays have been developed for the detection of flaviviruses. Most of these assays utilize the reverse transcriptase-polymerase chain reaction (RT-PCR) as the amplification format with detection by either agarose gel electrophoresis and ethidium bromide staining or hybridization with molecular probes. Recently, a modification of the standard RT-PCR using fluorescent-labeled oligonucleotide probes for detection (TaqMan) has been described. As a result, several assays for detecting flaviviruses have been developed using this approach. In addition, another amplification format, nucleic acid sequence based amplification (NASBA), has been developed and utilized for the detection of several flaviviruses. The various assay formats will be described and their utility discussed. PMID:14714430

  4. Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments.

    PubMed

    Afolayan, Olamide T; Webb, Cathy C; Cannon, Jennifer L

    2016-03-01

    Human noroviruses (NoVs) are a major source of foodborne illnesses worldwide. Since human NoVs cannot be cultured in vitro, methods that discriminate infectious from non-infectious NoVs are needed. The purpose of this study was to evaluate binding of NoV genotypes GI.1 and GII.4 to histo-blood group antigens expressed in porcine gastric mucin (PGM) as a surrogate for detecting infectious virus following thermal (99 °C/5 min), 70% ethanol or 0.5% levulinic acid (LV) plus 0.01 or 0.1% sodium dodecyl sulfate (SDS) sanitizer treatments and to determine the limit of detection of GI.1 and GII.4 binding to PGM. Treated and control virus samples were applied to 96-well plates coated with 1 µg/ml PGM followed by RNase A (5 ng/µl) treatment for degradation of exposed RNA. Average log genome copies per ml (gc/ml) reductions and relative differences (RD) in quantification cycle (Cq) values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) and 1.73/5.50 and 1.56/6.58 (no RNase A) for GI.1 and GII.4, respectively. Treatment of NoVs with 70% EtOH resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and average RD in Cq value, respectively. LV (0.5%) combined with 0.1 % SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5% LV/0.01 % SDS. Virus recovery after PGM binding was variable with GII.4 > GI.1. PGM binding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction, however, results vary depending on virus strain and inactivation method. PMID:26514820

  5. Development of an inhibitive enzyme assay for copper.

    PubMed

    Shukor, M Y; Bakar, N A; Othman, A R; Yunus, I; Shamaan, N A; Syed, M A

    2009-01-01

    In this work the development of an inhibitive assay for copper using the molybdenum-reducing enzyme assay is presented. The enzyme is assayed using 12-molybdophosphoric acid at pH 5.0 as an electron acceptor substrate and NADH as the electron donor substrate. The enzyme converts the yellowish solution into a deep blue solution. The assay is based on the ability of copper to inhibit the molybdenum-reducing enzyme from the molybdate-reducing Serratia sp. Strain DRY5. Other heavy metals tested did not inhibit the enzyme at 10 mg l(-1). The best model with high regression coefficient to measure copper inhibition is one-phase binding. The calculated IC50 (concentration causing 50% inhibition) is 0.099 mg l(-1) and the regression coefficient is 0.98. The comparative LC50, EC50 and IC50 data for copper in different toxicity tests show that the IC50 value for copper in this study is lower than those for immobilized urease, bromelain, Rainbow trout, R. meliloti, Baker's Yeast dehydrogenase activity Spirillum volutans, P. fluorescens, Aeromonas hydrophilia and synthetic activated sludge assays. However the IC50 value is higher than those for Ulva pertusa and papain assays, but within the reported range for Daphnia magna and Microtox assays. PMID:20112861

  6. A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

    PubMed Central

    Baumeister, Mark A.; Zhang, Nan; Beas, Hilda; Brooks, Jesse R.; Canchola, Jesse A.; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R.

    2012-01-01

    Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements. PMID:22479381

  7. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  8. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  9. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium salt may be safely used in accordance...: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum 98 percent (dry basis). Sodium salt...

  10. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  11. 21 CFR 172.130 - Dehydroacetic acid.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Dehydroacetic acid. 172.130 Section 172.130 Food... Food Preservatives § 172.130 Dehydroacetic acid. The food additive dehydroacetic acid and/or its sodium... meets the following specifications: Dehydroacetic acid: Melting point, 109 °C-111 °C; assay, minimum...

  12. Methods to assay Drosophila behavior.

    PubMed

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

    2012-01-01

    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

  13. 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).

  14. Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

    SciTech Connect

    Ratcliffe, W.A.; Corrie, J.E.; Dalziel, A.H.; Macpherson, J.S.

    1982-06-01

    Researchers compared two direct radioimmunoassays for progesterone in 50 microL of unextracted serum or plasma with assays involving extraction of serum. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11 alpha hemisuccinyl conjugate and the radioligand /sup 125/I-labeled progesterone 11 alpha-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r greater than 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. Researchers conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.

  15. High mannose-binding lectin with preference for the cluster of alpha1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses.

    PubMed

    Sato, Yuichiro; Hirayama, Makoto; Morimoto, Kinjiro; Yamamoto, Naoki; Okuyama, Satomi; Hori, Kanji

    2011-06-01

    The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1-2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1-2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1-2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 10(8) M(-1)) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent. PMID:21460211

  16. High Mannose-binding Lectin with Preference for the Cluster of α1–2-Mannose from the Green Alga Boodlea coacta Is a Potent Entry Inhibitor of HIV-1 and Influenza Viruses*

    PubMed Central

    Sato, Yuichiro; Hirayama, Makoto; Morimoto, Kinjiro; Yamamoto, Naoki; Okuyama, Satomi; Hori, Kanji

    2011-01-01

    The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1–2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1–2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1–2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 108 m−1) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent. PMID:21460211

  17. Validation of real-time PCR assays for bioforensic detection of model plant pathogens.

    PubMed

    James, Mindy; Blagden, Trenna; Moncrief, Ian; Burans, James P; Schneider, Katherine; Fletcher, Jacqueline

    2014-03-01

    The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real-time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence. PMID:24261870

  18. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    PubMed

    Boortz, Kayla A; Syring, Kristen E; Pound, Lynley D; Wang, Yingda; Oeser, James K; O'Brien, Richard M

    2016-01-01

    Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans. PMID:27611587

  19. Gel mobility shift assays for RNA binding viral RNAi suppressors.

    PubMed

    Csorba, Tibor; Burgyán, József

    2011-01-01

    The host-virus interaction is a continuous coevolutionary race involving both host defence strategies and virus escape mechanisms. RNA silencing is one of the main processes employed by eukaryotic organisms to fight viruses. However, viruses encode suppressor proteins to counteract this antiviral mechanism. Virtually all plant viruses encode at least one suppressor. In spite of being highly diverse at the protein level, a large group of these proteins inhibit RNA silencing very similarly, by sequestration of double-stranded RNA or small-interfering RNA molecules, the central players of the pathway. The RNA binding capacity of virus suppressor proteins can be studied by the electrophoretic mobility shift assay method. Also known as gel retardation assay, gel mobility assay, gel shift assay or band shift assay, EMSA is an in vitro technique used to characterize protein:DNA or protein:RNA interactions. The method had been developed based on the observation that protein: nucleic acid complexes migrate slower through a non-denaturing polyacrylamide gel than the free nucleic acid fragments. Here, we provide a detailed protocol for the analysis of crucifer-infecting Tobacco mosaic tobamovirus (cr-TMV) silencing suppressor protein p122 RNA binding capacity. PMID:21431690

  20. 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. PMID:24123550

  1. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  2. Assay of Endocannabinoid Oxidation by Cyclooxygenase-2.

    PubMed

    Kudalkar, Shalley N; Kingsley, Philip J; Marnett, Lawrence J

    2016-01-01

    The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays. PMID:27245906

  3. Assay of DAGLα/β Activity.

    PubMed

    Bisogno, Tiziana

    2016-01-01

    The endocannabinoid 2-arachidonoylglycerol (2-AG) exerts its physiological action by binding to and functionally activating type-1 (CB1) and type-2 (CB2) cannabinoid receptors. It is thought to be produced through the action of sn-1 selective diacylglycerol lipase (DAGL) that catalyzes 2-AG biosynthesis from sn-2-arachidonate-containing diacylglycerols. Since 2-AG biosynthetic enzymes have been identified only recently, little information on methodological approaches for measuring DAGL activity is as yet available. Here, a highly sensitive radiometric assay to measure DAGL activity by using 1-oleoyl[1-(14)C]-2-arachidonoylglycerol as the substrate is reported. All the steps needed to perform lipid extraction, fractionation by thin-layer chromatography (TLC), and quantification of radiolabeled [(14)C]-oleic acid via scintillation counting are described in detail. PMID:27245901

  4. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  5. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  6. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  7. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  8. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  9. 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.

  10. 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.

  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. 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.

  13. Assays for B lymphocyte function.

    PubMed

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  14. 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... Laboratory assays. Where the results of laboratory assays of drug components, including assays by State...

  15. 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...

  16. 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...

  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...

  18. 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...

  19. HALOACID INDUCED ALTERATIONS IN FERTILITY AND THE SPERM BIOMARKER SP22 IN THE RAT ARE ADDITIVE: VALIDATION OF AN ELISA

    EPA Science Inventory

    Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm an...

  20. More insights into a human adipose tissue GPAT activity assay

    PubMed Central

    Morgan-Bathke, Maria; Chen, Liang; Oberschneider, Elisabeth; Harteneck, Debra; Jensen, Michael D

    2016-01-01

    ABSTRACT Adipose tissue fatty acid storage varies according to sex, adipose tissue depot and degree of fat gain. However, the mechanism(s) for these variations is not completely understood. We recently published findings based on the glycerol 3-phosphate acyltransferase (GPAT) enzyme activity assay we optimized for use with human adipose tissue. These findings include a decrease in total GPAT and GPAT1 as a function of adipocyte size in both omental and subcutaneous adipose tissue and a strong, positive correlations between ACS, GPAT, and DGAT activities for both sexes and depots and between these storage factors and palmitate storage rates into TAG. The aim of this commentary is to expand upon the data from our recent publication. We describe here additional details on the optimization of the GPAT enzyme activity assay, a correlation between DGAT and percentage palmitate in the diacylglycerol fraction, and sex differences in fatty acid storage factors and storage rates into TAG at high palmitate concentrations. PMID:27144101

  1. Capillary Electrophoresis-Based Assay of Phosphofructokinase-1

    PubMed Central

    Malina, Andrew; Bryant, Sherrisse K.; Chang, Simon H.; Waldrop, Grover L.; Gilman, S. Douglass

    2013-01-01

    An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by UV absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by addition of Mg2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors. PMID:24444856

  2. An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience

    PubMed Central

    Kaja, Simon; Payne, Andrew J.; Singh, Tulsi; Ghuman, Jasleen K.; Sieck, Erin G.; Koulen, Peter

    2015-01-01

    Background Quantification of lactate dehydrogenase (LDH) release is a widely accepted assay for the quantitative determination of cell viability and late-stage apoptosis. Major disadvantages of commercially available LDH assay kits include proprietary formulations, limited options for optimization and high cost, all resulting in limited reproducibility in research applications. New Method Here, we describe a novel, custom LDH assay suitable in the context of plate reader-based screening of drug candidates for glioprotection, but with wide applicability to other cell types and experimental paradigms. Results We developed a novel and highly reproducible LDH release assay that is more cost-effective than commercially available assays with comparable performance. The assay was validated by assessing 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid antioxidant protection against tert-butylhydroperoxide-induced oxidative stress in C6 astroglioma cells. Assay performance was validated by direct comparison and compatible with other methods of measuring cellular viability, namely 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate assays. Comparison with Existing Method(s) There was no statistically significant difference between results obtained with the novel custom assay and a commercially available assay CytoTox96® (Promega, Madison, WI). Conclusions The novel custom LDH release assay allows the reproducible quantification of cell viability and is highly cost-effective when compared to commercially available assays (approximately 25 times cheaper). In addition and in contrast to commercially available assays, the identification and detailed description of all assay components and procedures provide greater control over experimental conditions and design. We provide a detailed standard operating procedure permitting our novel assay to be readily adapted depending on experimental requirements

  3. World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA

    PubMed Central

    Baylis, Sally A.; Hanschmann, Kay-Martin; Montag-Lessing, Thomas; Chudy, Michael; Kreß, Julia; Ulrych, Ursula; Czurda, Stefan; Rosengarten, Renate

    2015-01-01

    Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the “1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection” (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing. PMID:26070671

  4. 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.

  5. 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.

  6. 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. PMID:26608293

  7. 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.

  8. 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.

  9. A homogeneous biochemiluminescent assay for detection of influenza

    NASA Astrophysics Data System (ADS)

    Hui, Kwok Min; Li, Xiao Jing; Pan, Lu; Li, X. J.

    2015-05-01

    Current methods of rapid detection of influenza are based on detection of the nucleic acids or antigens of influenza viruses. Since influenza viruses constantly mutate leading to appearance of new strains or variants of viruses, these detection methods are susceptible to genetic changes in influenza viruses. Type A and B influenza viruses contain neuraminidase, an essential enzyme for virus replication which enables progeny influenza viruses leave the host cells to infect new cells. Here we describe an assay method, the homogeneous biochemiluminescent assay (HBA), for rapid detection of influenza by detecting viral neuraminidase activity. The assay mimics the light production process of a firefly: a viral neuraminidase specific substrate containing a luciferin moiety is cleaved in the presence of influenza virus to release luciferin, which becomes a substrate to firefly luciferase in a light production system. All reagents can be formulated in a single reaction mix so that the assay involves only one manual step, i.e., sample addition. Presence of Type A or B influenza virus in the sample leads to production of strong, stable and easily detectable light signal, which lasts for hours. Thus, this influenza virus assay is suitable for use in point-of-care settings.

  10. Development of a new colorimetric assay for lipoxygenase activity.

    PubMed

    Lu, Weiqiang; Zhao, Xue; Xu, Zhongyu; Dong, Ningning; Zou, Shien; Shen, Xu; Huang, Jin

    2013-10-15

    Lipoxygenases (LOXs) are a family of non-heme iron-containing dioxygenases that catalyze the hydroperoxidation of lipids, containing a cis,cis-1,4-pentadiene structure. A rapid and reliable colorimetric assay for determination of the activity of three human functional lipoxygenase isoforms (5-lipoxygenase, platelet 12-lipoxygenase, and 15-lipoxygenase-1) is developed in this article. In the new assay, LOX-derived lipid hydroperoxides oxidize the ferrous ion (Fe²⁺) to the ferric ion (Fe³⁺), the latter of which binds with thiocyanate (SCN⁻) to generate a red ferrithiocyanate (FTC) complex. The absorbance of the FTC complex can be easily measured at 480 nm. Because 5-LOX can be stimulated by many cofactors, the effects of its cofactors (Ca²⁺, ATP, dithiothreitol, glutathione, L-α-phosphatidylcholine, and ethylenediaminetetraacetic acid) on the color development of the FTC complex are also determined. The assay is adaptive for purified LOXs and cell lysates containing active LOXs. We use the new colorimetric assay in a 96-well format to evaluate several well-known LOX inhibitors, the IC₅₀ values of which are in good agreement with previously reported data. The reliability and reproducibility of the assay make it useful for in vitro screening for inhibitors of LOXs and, therefore, should accelerate drug discovery for clinical application. PMID:23811155

  11. Homogeneous assay for whole blood folate using photon upconversion.

    PubMed

    Arppe, Riikka; Mattsson, Leena; Korpi, Krista; Blom, Sami; Wang, Qi; Riuttamäki, Terhi; Soukka, Tero

    2015-02-01

    Red blood cell folate is measured for folate deficiency diagnosis, because it reflects the long-term folate level in tissues, whereas serum folate only represents the dietary intake. Direct homogeneous assay from whole blood would be ideal but conventional fluorescence techniques in blood suffer from high background and strong absorption of light at ultraviolet and visible wavelengths. In this study, a new photon upconversion-based homogeneous assay for whole blood folate is introduced based on resonance energy transfer from upconverting nanophosphor donor coated with folate binding protein to a near-infrared fluorescent acceptor dye conjugated to folate analogue. The sensitized acceptor emission is measured at 740 nm upon 980 nm excitation. Thus, optically transparent wavelengths are utilized for both donor excitation and sensitized acceptor emission to minimize the sample absorption, and anti-Stokes detection completely eliminates the Stokes-shifted autofluorescence. The IC50 value of the assay was 6.0 nM and the limit of detection (LOD) was 1 nM. The measurable concentration range was 2 orders of magnitude between 1.0-100 nM, corresponding to 40-4000 nM folate in the whole blood sample. Recoveries of added folic acid were 112%-114%. A good correlation was found when compared to a competitive heterogeneous assay based on the DELFIA-technology. The introduced assay provides a simple and fast method for whole blood folate measurement. PMID:25548870

  12. Parallel Force Assay for Protein-Protein Interactions

    PubMed Central

    Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

    2014-01-01

    Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay. PMID:25546146

  13. Comparison of two methods for assaying reducing sugars in the determination of carbohydrase activities.

    PubMed

    Gusakov, Alexander V; Kondratyeva, Elena G; Sinitsyn, Arkady P

    2011-01-01

    The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of carbohydrase activities against different polysaccharides. Using twelve commercial enzyme preparations, the comparison of the NS and DNS assays in determination of cellulase, β-glucanase, xylanase, and β-mannanase activities was carried out. When cellulase activities against CMC were measured, the DNS assay gave activity values, which were typically 40-50% higher than those obtained with the NS assay. In the analysis of the xylanase, β-mannanase, and β-glucanase activities, the overestimations by the DNS assay were much more pronounced (the observed differences in the activities were 3- to 13-fold). Reasons for preferential use of the NS assay for measuring activities of carbohydrases other than cellulases are discussed. PMID:21647284

  14. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India

    PubMed Central

    2016-01-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  15. Automated Triplex (HBV, HCV and HIV) NAT Assay Systems for Blood Screening in India.

    PubMed

    Rajput, Manoj Kumar

    2016-02-01

    This review is confined to triplex nucleic acid testing (NAT) assays to be used on fully automated platform. Around the world, these assays are being used at various transfusion medicine centres or blood banks to screen blood units for HBV, HCV and HIV. These assay systems can screen up to 1000 blood units for HBV, HCV and HIV simultaneously in a day. This area has been dominated by mainly two manufacturers: M/s Gen-Probe-Novartis and M/s Roche Molecular Systems. The triplex NAT assay systems of both manufacturers are licensed by United States Food and Drug Administration. There is not much awareness about the technology and procedures used in these assays. The main objective of this review is to create awareness about the technology and procedure of these assays. PMID:27042485

  16. SLP assay: a rapid assay for detection of red blood cell antibodies in the serology of pregnancies.

    PubMed

    Giannitsis, D J; Hofstätter, I; Wild, J; Häcker-Shahin, B

    1992-01-01

    A rapid manual test for the detection of red cell antibodies called SLP assay has been developed and compared with the sensitivity of the antiglobulin assay. Acid-soluble proteins (SLP) from human leukocytes cause aggregation of human red blood cells. SLP represents a group of proteins consisting of 5 fractions of different positively charged macromolecules, which are able to reduce the negative charge of the red cells. Reduction of the negative charge results in a nonspecific hemagglutination of different strengths, depending on the SLP fractions used. This hemagglutination can be reversed by neutralizing the SLP with heparin. In the case of blood group-antibody-mediated aggregation the hemagglutination is nonreversible despite neutralization with heparin and remains stable for several hours. Because of the high sensitivity of the SLP assay all blood group antibodies from the IgM type as well as from the IgG type are detectable even in low concentrations. The sensitivity of the SLP assay is comparable to the antiglobulin assay. PMID:1284754

  17. Predictive assays in radiation therapy

    SciTech Connect

    West, C.M.L.

    1994-12-31

    There are reports of promising correlations between patient response to radiotherapy and laboratory measurements of tumor radiosensitivity, fibroblast radiosensitivity, tumor proliferation, and tumor oxygenation status. These all need to be substantiated in large clinical studies. The development of rapid, reliable assays, in particular for determining intrinsic radiosensitivity, would greatly facilitate this work. If the results illustrated in the figures in the chapter can be combined and shown to be feasible on a routine clinical basis, then radiobiologists would be able to provide radiotherapists with a useful aid for the individualization of patient treatment. 162 refs., 6 figs., 6 tabs.

  18. Automated cytopathic effect (CPE) assays.

    PubMed

    McAleer, W J; Miller, W J; Hurni, W M; Machlowitz, R A; Hilleman, M R

    1983-07-01

    An automated CPE procedure has been developed that increases the precision and ease of performing titrations of measles, mumps and rubella viruses in vaccine materials. By this procedure, additions of cell suspensions and reagents and the dilution of samples are performed automatically by a modified Dynatiter instrument, using 96-well microtitre plates. Cell monolayers are stained with carbolfuchsin dye to eliminate the need for microscopic examination. Finally, the trays are read in an optical scanner and the end points calculated automatically by a programmable calculator. The increased accuracy and precision attained by performing greater numbers of replicate assays at reasonable cost will be of particular value to vaccine manufacturers. PMID:6885830

  19. New enzymatic assay for serum urea nitrogen using urea amidolyase.

    PubMed

    Kimura, Shigeki; Iyama, Shigeru; Yamaguchi, Yoshihisa; Kanakura, Yuzuru

    2003-01-01

    We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L [(values by reference method - that of present method) +/- SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52-56, 2003. PMID:12640627

  20. Novel applications of locked nucleic acids.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion High Fidelity DNA polymerase and 9( degrees )Nm(TM) DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN(m) polymerase proved to be the best for amplification employing the LNA-A nucleotide. PMID:18029570

  1. Indirect conductimetric assay of antibacterial activities.

    PubMed

    Sawai, J; Doi, R; Maekawa, Y; Yoshikawa, T; Kojima, H

    2002-11-01

    The applicability of indirect conductimetric assays for evaluation of antibacterial activity was examined. The minimal inhibitory concentration (MIC) obtained by the indirect method was consistent with that by the direct conductimetric assay and the turbidity method. The indirect assay allows use of growth media, which cannot be used in the direct conductimetric assay, making it possible to evaluate the antibacterial activity of insoluble or slightly soluble materials with high turbidity, such as antibacterial ceramic powders. PMID:12407467

  2. The chemistry behind antioxidant capacity assays.

    PubMed

    Huang, Dejian; Ou, Boxin; Prior, Ronald L

    2005-03-23

    This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays. PMID:15769103

  3. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  4. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    PubMed

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  5. Serum indices: managing assay interference.

    PubMed

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  6. 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.

  7. 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.

  8. The validity of androgen assays

    PubMed Central

    Carruthers, Malcolm; Trinick, Tom R.; Wheeler, Michael J.

    2007-01-01

    Problems in the measurement of androgens and in interpreting results have been reviewed and classified as follows: Preanalytical factors The exact sampling conditions in relation to circadian and seasonal variations, diet, alcohol, physical activity and posture. Physiological and medical factors Androgen levels vary according to the patient's general health, stress, sexual activity and smoking habits. Analytical variables Sample preservation and storage variables are often unknown. The different androgen assays used have widely differing accuracy and precision and are subject to large inter-laboratory variation, which especially in women and children can render the results of routinely available direct immunoassays meaningless. Interpretation of results Laboratory reference ranges vary widely, largely independent of methodology, and fail to take into account the log-normal distribution of androgen values, causing errors in clinical diagnosis and treatment. Other unknowns are antagonists such as SHBG, estrogens, catecholamines, cortisol, and anti-androgens. As well as age, androgen receptor polymorphisms play a major role in regulating androgen levels and resistance to their action. Conclusions Though laboratory assays can support a diagnosis of androgen deficiency in men, they should not be used to exclude it. It is suggested that there needs to be greater reliance on the history and clinical features, together with careful evaluation of the symptomatology, and where necessary a therapeutic trial of androgen treatment given. PMID:17701661

  9. In vitro Tumorsphere Formation Assays

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.

  10. 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.

  11. Proteasome Assay in Cell Lysates

    PubMed Central

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  12. 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.

  13. 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.

  14. 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.

  15. Amino acids

    MedlinePlus

    Amino acids are organic compounds that combine to form proteins . Amino acids and proteins are the building blocks of life. When proteins are digested or broken down, amino acids are left. The human body uses amino acids ...

  16. Photon upconversion in homogeneous fluorescence-based bioanalytical assays.

    PubMed

    Soukka, Tero; Rantanen, Terhi; Kuningas, Katri

    2008-01-01

    Upconverting phosphors (UCPs) are very attractive reporters for fluorescence resonance energy transfer (FRET)-based bioanalytical assays. The large anti-Stokes shift and capability to convert near-infrared to visible light via sequential absorption of multiple photons enable complete elimination of autofluorescence, which commonly impairs the performance of fluorescence-based assays. UCPs are ideal donors for FRET, because their very narrow-banded emission allows measurement of the sensitized acceptor emission, in principle, without any crosstalk from the donor emission at a wavelength just tens of nanometers from the emission peak of the donor. In addition, acceptor dyes emitting at visible wavelengths are essentially not excited by near-infrared, which further emphasizes the unique potential of upconversion FRET (UC-FRET). These characteristics result in favorable assay performance using detection instrumentation based on epifluorometer configuration and laser diode excitation. Although UC-FRET is a recently emerged technology, it has already been applied in both immunoassays and nucleic acid hybridization assays. The technology is also compatible with optically difficult biological samples, such as whole blood. Significant advances in assay performance are expected using upconverting lanthanide-doped nanocrystals, which are currently under extensive research. UC-FRET, similarly to other fluorescence techniques based on resonance energy transfer, is strongly distance dependent and may have limited applicability, for example in sandwich-type assays for large biomolecules, such as viruses. In this article, we summarize the essentials of UC-FRET, describe its current applications, and outline the expectations for its future potential. PMID:18596348

  17. Development of a homogeneous binding assay for histamine receptors.

    PubMed

    Crane, Kathy; Shih, Daw-Tsun

    2004-12-01

    Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts. PMID:15519569

  18. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

    2015-01-01

    Background Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. Methodology/Principal Findings Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. Conclusions/Significance During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. PMID:26075598

  19. beta. -Sulfopyruvate: chemical and enzymatic syntheses and enzymatic assay

    SciTech Connect

    Weinstein, C.L.; Griffith, O.W.

    1986-01-01

    BETA-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. ..beta..-(/sup 35/S)Sulfopyruvate is prepared by transamination between (/sup 35/)cysteinesulfonate (cysteate) and ..cap alpha..-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis the crude reaction product is conveniently purified by chromatography on Dowex 1; ..beta..-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. ..beta..-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, ..beta..-sulfopyruvate is reduced with an apparent K/sub m/ of 6.3 mM and a V/sub max/ equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of ..beta..-sulfopyruvate.

  20. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a n...

  1. Development of a thyroperoxidase inhibition assay for high-throughput screening.

    PubMed

    Paul, Katie B; Hedge, Joan M; Rotroff, Daniel M; Hornung, Michael W; Crofton, Kevin M; Simmons, Steven O

    2014-03-17

    High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach. PMID:24383450

  2. 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

  3. Fluorometric enzymatic assay of l-arginine.

    PubMed

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of l-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6μМ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples. PMID:27450117

  4. Strategies for Assaying Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Hafner Česen, Maruša; Turk, Boris

    2016-01-01

    Late endosomal organelles have an acidic pH and contain hydrolytic enzymes to degrade cargo delivered either from the extracellular environment by endocytosis or from within the cell itself by autophagy. In the event of lysosomal membrane permeabilization (LMP), the contents of late endosomes and lysosomes can be released into the cytosol and then initiate apoptosis. Compounds that can trigger LMP are therefore candidates for the induction of apoptosis, in particular in anticancer therapy. Alternatively, drug-delivery systems, such as nanoparticles, can have side effects that can include LMP, which has toxic consequences for the cells. To determine when, to what extent, and with what consequences LMP occurs is therefore of paramount importance for the evaluation of new potentially LMP-inducing compounds. In this introduction, we provide an overview of some basic assays for assessing LMP, such as staining with lysosomotropic dyes and measurement of cysteine cathepsin activity, and discuss additional strategies for the detection of the release of endogenous lysosomal molecules or preloaded exogenous tracers into the cytosol. PMID:27250949

  5. Liquid chromatographic assay for dicloxacillin in plasma.

    PubMed

    Alderete, Oscar; González-Esquivel, Dinora F; Del Rivero, L Misael; Castro Torres, Nelly

    2004-06-15

    A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans. PMID:15135112

  6. A mercury saturation assay for measuring metallothionein in fish

    SciTech Connect

    Dutton, M.D. . Dept. of Zoology); Stephenson, M. . Environmental Science Branch); Klaverkamp, J.F. )

    1993-07-01

    An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution of enzymatic and nonenzymatic thiols, including rabbit liver MT-2, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of [sup 203]Hg in 10% trichloroacetic acid. Excess Hg was bound to an removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with [sup 203]Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.

  7. Hydroponic Growth and the Nondestructive Assay for Dinitrogen Fixation 1

    PubMed Central

    Imsande, John; Ralston, Edward J.

    1981-01-01

    Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pKa 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day. PMID:16662112

  8. Northwest Australia's Saladin crude assayed

    SciTech Connect

    Rhodes, A.K.

    1993-10-18

    High-quality Saladin crude oil from offshore Western Australia has been assayed. The 48.2[degree] API, 0.02 wt % sulfur crude's characteristics--determined in 1990--are presented here for the first time. The estimated 30--40 million bbl field, south of Barrow Island, is produced from two platforms in 58 ft of water in block TP 3. Production began in late 1989 from three platforms with three wells each and from two wells drilled directionally from Thevenard Island. The paper lists data on the following properties: API gravity, density, sulfur content, pour point, flash point, viscosity, salinity, heat of combustion, ash content, asphaltene content, wax content, and metal content for the whole crude and various fractions.

  9. 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

  10. 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. PMID:24978020

  11. 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.

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

    PubMed

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

    2016-01-01

    The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively. PMID:26188195

  13. β-Glucuronidase-coupled assays of glucuronoyl esterases.

    PubMed

    Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

    2016-10-01

    Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. PMID:27452816

  14. Radiometric-microbiologic assay fo vitamin B-6: analysis of plasma samples

    SciTech Connect

    Guilarte, T.R.; McIntyre, P.A.

    1981-11-01

    A radiometric microbiologic assay for the analysis of vitamin B-6 in plasma was developed. The method is based on the measurement of 14CO2 generated from the metabolism of DL-l-14C-valine (L-l-14C-valine) by Kloeckera brevis. The assay is specific for the biologically active forms of the vitamin, that is, pyridoxine, pyridoxal and pyridoxamine, and their respective phosphorylated forms. The biologically inert vitamin B-6 metabolite (4-pyridoxic acid) did not generate a response at concentrations tested. The radiometric technique was shown to be sensitive to the 1 nanogram level. Reproducibility and recovery studies gave good results. Fifteen plasma samples were assayed using the radiometric and turbidimetric techniques. The correlation coefficient was r . 0.98. Turbid material or precipitated debris did not interfere with the radiometric microbiologic assay, thus allowing for simplification of assay procedure.

  15. Comparative study of xylanase kinetics using dinitrosalicylic, arsenomolybdate, and ion chromatographic assays.

    PubMed

    Jeffries, T W; Yang, V W; Davis, M W

    1998-01-01

    Xylanases are commonly assayed by the dinitrosalicylic acid (DNS) or the arsenomolybdate (ARS) method. However, specific activities are many times higher with DNS than with ARS. This is because the DNS assay is more reactive and the ARS assay is less reactive with xylooligosaccharides than with xylose. Xylose is often used as a standard, even though oligosaccharides are prevalent, so the DNS method overestimates and the ARS method underestimates specific activity. Ion chromatography, with pulsed amperometric detection, separates and measures all products and intermediates, but quantitation on a molar basis is difficult, because few xylooligosaccharide response factors are known. This report directly compares these three assay methods for the assay of xylanase activities. PMID:18575995

  16. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters

    PubMed Central

    Dold, S. -M.; Zimmermann, S.; Hamacher, K.; Schmitz, K.; Rudat, J.

    2016-01-01

    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods—namely, the classical Z’-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  17. Statistical Evaluation of HTS Assays for Enzymatic Hydrolysis of β-Keto Esters.

    PubMed

    Buß, O; Jager, S; Dold, S-M; Zimmermann, S; Hamacher, K; Schmitz, K; Rudat, J

    2016-01-01

    β-keto esters are used as precursors for the synthesis of β-amino acids, which are building blocks for some classes of pharmaceuticals. Here we describe the comparison of screening procedures for hydrolases to be used for the hydrolysis of β-keto esters, the first step in the preparation of β-amino acids. Two of the tested high throughput screening (HTS) assays depend on coupled enzymatic reactions which detect the alcohol released during ester hydrolysis by luminescence or absorption. The third assay detects the pH shift due to acid formation using an indicator dye. To choose the most efficient approach for screening, we assessed these assays with different statistical methods-namely, the classical Z'-factor, standardized mean difference (SSMD), the Kolmogorov-Smirnov-test, and t-statistics. This revealed that all three assays are suitable for HTS, the pH assay performing best. Based on our data we discuss the explanatory power of different statistical measures. Finally, we successfully employed the pH assay to identify a very fast hydrolase in an enzyme-substrate screening. PMID:26730596

  18. A supplemented soft agar chemotaxis assay demonstrates the Helicobacter pylori chemotactic response to zinc and nickel

    PubMed Central

    Sanders, Lisa; Andermann, Tessa M.

    2013-01-01

    Directed motility, or chemotaxis, is required for Helicobacter pylori to establish infection in the stomach, although the full repertoire of this bacterium’s chemotactic responses is not yet known. Here we report that H. pylori responds to zinc as an attractant and nickel as a repellent. To reach this conclusion, we employed both a temporal chemotaxis assay based on bacterial reversals and a supplemented soft agar spatial assay. We refined the temporal assay using a previously described chemorepellent, acid, and found that H. pylori requires rich media with serum to maintain optimal swimming motility. Surprisingly, we found that some strains respond to acid as an attractant, and that the TlpC chemoreceptor correlated with whether acid was sensed as an attractant or repellent. Using this same assay, we detected weak repellent responses to nickel and copper, and a varied response to zinc. We thus developed an alternative spatial chemotactic assay called the supplemented soft agar assay, which utilizes soft agar medium supplemented with the test compound. With Escherichia coli, the attractant serine slowed overall bacterial migration, while the repellent nickel increased the speed of overall migration. In H. pylori we detected slowed migration with doubled tryptone media, as well as zinc, consistent with an attractant response. In contrast, nickel increased migration, consistent with repulsion. PMID:23139399

  19. Expert system for transuranic waste assay

    SciTech Connect

    Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

    1989-01-01

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

  20. Sodium borohydride/chloranil-based assay for quantifying total flavonoids.

    PubMed

    He, Xiangjiu; Liu, Dong; Liu, Rui Hai

    2008-10-22

    A novel sodium borohydride/chloranil-based (SBC) assay for quantifying total flavonoids, including flavones, flavonols, flavonones, flavononols, isoflavonoids, flavanols, and anthocyanins, has been developed. Flavonoids with a 4-carbonyl group were reduced to flavanols using sodium borohydride catalyzed with aluminum chloride. Then the flavan-4-ols were oxidized to anthocyanins by chloranil in an acetic acid solution. The anthocyanins were reacted with vanillin in concentrated hydrochloric acid and then quantified spectrophotometrically at 490 nm. A representative of each common flavonoid class including flavones (baicalein), flavonols (quercetin), flavonones (hesperetin), flavononols (silibinin), isoflavonoids (biochanin A), and flavanols (catechin) showed excellent linear dose-responses in the general range of 0.1-10.0 mM. For most flavonoids, the detection limit was about 0.1 mM in this assay. The recoveries of quercetin from spiked samples of apples and red peppers were 96.5 +/- 1.4% (CV = 1.4%, n = 4) and 99.0 +/- 4.2% (CV = 4.2%, n = 4), respectively. The recovery of catechin from spiked samples of cranberry extracts was 97.9 +/- 2.0% (CV = 2.0%, n = 4). The total flavonoids of selected common fruits and vegetables were measured using this assay. Among the samples tested, blueberry had the highest total flavonoid content (689.5 +/- 10.7 mg of catechin equiv per 100 g of sample), followed by cranberry, apple, broccoli, and red pepper. This novel SBC total flavonoid assay can be widely used to measure the total flavonoid content of fruits, vegetables, whole grains, herbal products, dietary supplements, and nutraceutical products. PMID:18798633