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Sample records for cytotoxicity assay simultaneously

  1. Application of the direct beta counter Matrix 96 for cytotoxic assays: simultaneous processing and reading of 96 wells using a 51Cr-retention assay.

    Science.gov (United States)

    Hillman, G G; Roessler, N; Fulbright, R S; Pontes, J E; Haas, G P

    1993-06-01

    To assess the cytotoxic activity of immune cells, we have developed a 51Cr-retention assay in which the radioactivity retained by 51Cr-labeled target cells, following coincubation with cytotoxic cells, is monitored using the automated Matrix 96 beta counter. The Matrix 96 is designed for simultaneously counting 96 samples isolated from a 96-well microplate. It uses 96 uniform and independent detectors operating on the principle of avalanche gas ionization in the Geiger-Muller mode. Samples must be dry because the detectors are of the open-window type. Therefore, samples from the 96 wells of the microplate are simultaneously harvested onto a filter using the MicroMate 196, a 96-well cell harvester, dried and quantified in the Matrix 96. Usually the 51Cr isotope is measured by the detection of gamma radiation in gamma counters. The Matrix 96, however, monitors Auger electrons, which are also emitted by 51Cr. We have shown that the retention assay can be used to monitor the cytotoxic activity of activated lymphocytes including lymphokine-activated killer cells and tumor-infiltrating lymphocytes against various tumor cell lines. This assay is most suitable for experiments in which low E/T ratios are sufficient to detect highly cytotoxic cells, such as clone screening in cloning assays or in limiting-dilution analysis assays. These assays involve processing and reading large numbers of microplates. In this case, the retention assay monitored in the Matrix 96 will improve the work flow and decrease the amount of radioactive waste.

  2. Cell Proliferation and Cytotoxicity Assays.

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    Adan, Aysun; Kiraz, Yağmur; Baran, Yusuf

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

  3. A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products

    Science.gov (United States)

    Carballo, José Luis; Hernández-Inda, Zaira L; Pérez, Pilar; García-Grávalos, María D

    2002-01-01

    Background The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity. Material and Methods The bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity. Results Two sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%. Conclusions Our results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological

  4. Sulforhodamine B colorimetric assay for cytotoxicity screening.

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    Vichai, Vanicha; Kirtikara, Kanyawim

    2006-01-01

    The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening.

  5. Preliminary in vitro cytotoxic assay of human liver carcinoma cells ...

    African Journals Online (AJOL)

    From the preliminary in vitro cytotoxic assay study, triorganotin(IV) complexes (2 and 5) were found to exhibit better activity as compared to diorganotin(IV) complexes (1, 3 and 4) but lower activity as compared to the reference drug. In addition, within the diorganotin(IV) complexes, monomeric type (3) exhibited a slightly ...

  6. Lactate dehydrogenase assay for assessment of polycation cytotoxicity

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Andersen, Helene; Moghimi, Seyed Moien

    2013-01-01

    cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early...

  7. Influence of radiolysis products of water in cytotoxicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Camillo, Maria A.P.; Luly, Juliana H.; Silva, Jose A. Alves da; Magalhaes, Vanessa D.; Higa, Olga Z. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Biotecnologia]. E-mail: mcamillo@ipen.br

    2007-07-01

    The ionizing radiation has shown to be an useful tool in the improvement of immunogens such as toxins; venoms; protozoa among others. A recent study has demonstrated that hydroxyl radical is the main radiolysis product of water involved in the protein irradiation. It promotes important structural modifications in the toxins meanwhile the immunogenic properties are preserved. Tests of biological activity are essential to determine the detoxification of the samples and the safety of the inoculum. The in vitro cytotoxicity assay, determining the cellular viability, was chosen for this analysis. This test has good reproducibility; it can be carried out with little amount of sample and has been useful for biological evaluation of biomaterials. Herein are presented the results gotten with gyroxin (a serine protease, glycoprotein with 26 kDa) isolated from the rattlesnake Crotalus durissus terrificus venom. The toxin was irradiated with 2 kGy of gamma ray of cobalt-60 in saline solution. The evaluation of the biological activity was carried out with different cell lines, using MTS dye or {sup 3}H-thymidine incorporation as indicators of cells viability. The best correlation between the cellular viability assay and lethality assay in vivo was obtained with endothelial cells (HUVECs) and {sup 3}H-thymidine incorporation. The MTS evaluation presented a false-positive result of cytotoxicity, probably due to the reaction with free radicals formed by radiolysis of water during the irradiation process. (author)

  8. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  9. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    Multiplex polymerase chain reaction (PCR) assay for simultaneous detection of shiga-like toxin ( stx 1 and stx 2), intimin (eae) and invasive plasmid antigen H ( ipaH ) genes in diarrheagenic Escherichia coli.

  10. Assessment of bacterial biodetoxification of herbicide atrazine using Aliivibrio fischeri cytotoxicity assay with prolonged contact time.

    Science.gov (United States)

    Háhn, Judit; Szoboszlay, Sándor; Tóth, Gergő; Kriszt, Balázs

    2017-07-01

    In our study, we determined and compared the atrazine-biodetoxification ability of 41 bacterial strains and 21 consortia created of those with over 50% degradation rate in pure cultures. Biodegradation capacity was measured with GC-MS. Detoxification was assessed based on the cytotoxic effect of end-products to Aliivibrio fischeri in chronic bioluminescence inhibition assay with 25 h contact time. Chronic A. fischeri assay adapted to a microplate, which is suitable for examine numerous residues simultaneously, also appeared to be significantly more sensitive to atrazine compared to the standard acute (30 min) test. Due to its sensitivity, the chronic assay could be a valuable tool to provide a more comprehensive view of the ecological risks of atrazine and other chemicals. Thirteen strains were able to degrade more than 50% of 50 ppm atrazine. Four of these belong to Rhodococcus aetherivorans, R. qingshengii, Serratia fonticola and Olivibacter oleidegradans which species' atrazine degrading ability has never been reported before. Four consortia degrading ability was more effective than that of the creating individual strains; moreover, their residues did not show cytotoxic effects to A. fischeri. However, in several cases, the degradation products of sole strains and consortia resulted in significant bioluminescence inhibition. Thus high biodegradation (>90%) does not certainly mean the reduction or cessation of toxicity highlighting the importance of the evaluation of biological effects of degradation residues to improve the efficiency and abate the ecological risks of bioremediation techniques.

  11. Cytotoxicity of eight cigarette smoke condensates in three test systems: comparisons between assays and condensates.

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    Richter, Patricia A; Li, Albert P; Polzin, Gregory; Roy, Shambhu K

    2010-12-01

    Cytotoxic properties of tobacco smoke are associated with chronic tobacco-related diseases. The cytotoxicity of tobacco smoke can be tested with short-term predictive assays. In this study, we compare eight mainstream cigarette smoke condensates (CSCs) from commercial and experimental cigarettes in three different cytotoxicity assays with unique and overlapping endpoints. The CSCs demonstrated cytotoxicity in all assays. In the multiple cytotoxicity endpoint (MCE) assay with TK-6 cells, the cigarette varieties that had the highest EC50s for reduced cell growth also showed a positive dose-response relationship for necrotic cells. In the IdMOC multiple cell-type co-culture (MCTCC) system, all CSCs reduced the viability of the cells. Low concentrations of some CSCs had a stimulatory effect in lung microvascular endothelial cells and small airway epithelial cells. In the neutral dye assay (NDA), except for a 100% flue-cured tobacco CSC, there was little consistency between CSCs producing morphological evidence of moderate or greater toxicity and the CSCs with the lowest EC50s in the MCE or MCTCC assays. Overall, cigarettes made with flue-cured tobacco were the most cytotoxic across the assays. When results were expressed on a per-mg of nicotine basis, lower tar cigarettes were the most cytotoxic in primary human respiratory cells. Published by Elsevier Inc.

  12. The low molecular weight DNA diffusion assay as an indicator of cytotoxicity for the in vitro comet assay.

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    Speit, Günter; Vesely, Alexandra; Schütz, Petra; Linsenmeyer, Regina; Bausinger, Julia

    2014-07-01

    The low molecular weight DNA diffusion assay (LMW assay) has been recommended as a measure for cytotoxicity for the in vivo comet assay. To better understand the relationship between effects in the LMW assay, DNA migration in the comet assay and effects in established cytotoxicity tests, we performed in vitro experiments with cultured human cell lines (TK6, A549) and comparatively investigated five test substances (methyl methanesulfonate, (±)-benzo[a]pyrene diol epoxide, sodium dodecyl sulphate, menthol and sodium arsenite). We measured DNA migration (tail intensity) in the comet assay and the frequency of 'hedgehogs' (cells with almost all DNA in the tail), DNA diffusion in the LMW assay, cell viability (trypan blue and fluorescein diacetate/ethidium bromide staining) and inhibition of proliferation (relative cell counts). Our in vitro experiments indicate that effects in the LMW assay occur independently from DNA effects in the comet assay and are not related to the occurrence of hedgehogs. Results from the LMW assay are in good agreement with results from viability assays and seem to allow discriminating genotoxic from non-genotoxic substances when appropriate preparation times are considered. Measurements of cytotoxicity by these methods only at an early preparation time after exposure to genotoxic substances may lead to erroneous results. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Accurate non-invasive image-based cytotoxicity assays for cultured cells

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    Brouwer Jaap

    2010-06-01

    Full Text Available Abstract Background The CloneSelect™ Imager system is an image-based visualisation system for cell growth assessment. Traditionally cell proliferation is measured with the colorimetric MTT assay. Results Here we show that both the CloneSelect Imager and the MTT approach result in comparable EC50 values when assaying the cytotoxicity of cisplatin and oxaliplatin on various cell lines. However, the image-based technique was found non-invasive, considerably quicker and more accurate than the MTT assay. Conclusions This new image-based technique has the potential to replace the cumbersome MTT assay when fast, unbiased and high-throughput cytotoxicity assays are requested.

  14. Clonogenic assays measure leukemia stem cell killing not detectable by chromium release and flow cytometric cytotoxicity assays.

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    Williams, Brent A; Wang, Xing-Hua; Keating, Armand

    2010-11-01

    NK-92, a permanent natural killer (NK) cell line, shows cytotoxicity against a variety of tumors and has been tested in a phase I trial. We tested the toxicity of NK-92 and chemotherapy drugs against the stem cell capacity of the acute leukemia cell line, KG1. While the chromium-release assay is the most common method for assessing cytotoxicity of immune effectors, and flow cytometry is increasingly used, the relationship of either assay to clonogenic readouts remains unknown. KG1 was assessed for stem cell frequency by serial dilution, single-cell sorting and colony growth in methylcellulose. KG1 was sorted into CD34(+) CD38(+) and CD34(+) CD38⁻ populations and recultured in liquid medium or methylcellulose to determine the proliferative capacity of each fraction. Cytotoxicity of NK-92, daunorubicin and cytarabine against KG1 was measured using the chromium-release assay, flow cytometry and clonogenic assays. The culture-initiating cell frequency of whole KG1 was between 1 in 100 to 1000 by serial dilution and single-cell sorting. Although a rare (1-3%) CD34(+) CD38⁻ population could be demonstrated in KG1, both fractions had equivalent proliferative capacity. The cumulative flow cytotoxicity assay was more sensitive than the chromium-release assay in detecting target cell killing. At a 10:1 ratio NK-92 eliminated the clonogenic capacity of KG1, which was not predicted by the chromium-release assay. Clonogenic assays provide a more sensitive means of assessing the effect of a cytotoxic agent against putative cancer stem cells within cell lines, provided that they grow well in liquid culture medium or methylcellulose.

  15. The application of sulforhodamine B as a colorimetric endpoint in a cytotoxicity assay.

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    Fricker, S P

    1994-08-01

    Sulforhodamine B (SRB), an aminoxanthene dye, has been used as an assay for total cell protein, initially developed as an endpoint assay for in vitro screening of antitumour agents. In this paper it was investigated as a possible endpoint for a cytotoxicity assay using CHO cells. It is a robust assay with a stable colorimetric endpoint, capable of semi-automation using microtitre equipment. At optimum concentrations (0.05-0.1% SRB) the assay is linear with respect to cell number over a range of 5 x 10(3) to 10(5) cells. In comparative studies with the neutral red assay the SRB assay was more sensitive, and in cytotoxicity assays with test compounds gave comparable dose-response curves. The cytotoxicity of five divalent metal chlorides was assessed using the SRB assay. The order of toxicity was Cd > Hg > Zn > Mn > Mg, that is similar to the expected in vivo ranking. 16 compounds with reported oral LD(50) (rat) ranging from 25,800 mg/kg (glucose) to 1 mg/kg (mercuric chloride) were tested in the assay. The relative toxicities of the compounds in the in vitro SRB assay were similar to the relative in vivo toxicities. The exceptions could be explained by the chemistry of the compounds and could be attributed to pharmacokinetic properties or mechanism of action. This assay can therefore be used to rank chemically similar compounds but is unsuitable as a precise predictor of in vivo toxicity.

  16. Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.

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    Karimi, Mobin A; Lee, Eric; Bachmann, Michael H; Salicioni, Ana Maria; Behrens, Edward M; Kambayashi, Taku; Baldwin, Cynthia L

    2014-01-01

    The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

  17. Simultaneous assay of pigments, carbohydrates, proteins and lipids in microalgae.

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    Chen, Yimin; Vaidyanathan, Seetharaman

    2013-05-07

    Biochemical compositional analysis of microbial biomass is a useful tool that can provide insight into the behaviour of an organism and its adaptational response to changes in its environment. To some extent, it reflects the physiological and metabolic status of the organism. Conventional methods to estimate biochemical composition often employ different sample pretreatment strategies and analytical steps for analysing each major component, such as total proteins, carbohydrates, and lipids, making it labour-, time- and sample-intensive. Such analyses when carried out individually can also result in uncertainties of estimates as different pre-treatment or extraction conditions are employed for each of the component estimations and these are not necessarily standardised for the organism, resulting in observations that are not easy to compare within the experimental set-up or between laboratories. We recently reported a method to estimate total lipids in microalgae (Chen, Vaidyanathan, Anal. Chim. Acta, 724, 67-72). Here, we propose a unified method for the simultaneous estimation of the principal biological components, proteins, carbohydrates, lipids, chlorophyll and carotenoids, in a single microalgae culture sample that incorporates the earlier published lipid assay. The proposed methodology adopts an alternative strategy for pigment assay that has a high sensitivity. The unified assay is shown to conserve sample (by 79%), time (67%), chemicals (34%) and energy (58%) when compared to the corresponding assay for each component, carried out individually on different samples. The method can also be applied to other microorganisms, especially those with recalcitrant cell walls. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango ...

    African Journals Online (AJOL)

    The three samples contained negligible amounts of anthocyanins (<0.67 ng/g) compared with the other constituents (1.4 to 2.2 mg/g), largely of gallotannins and flavonoids. However, PBMK had the least flavonoid content, averaging 43%. Due to relative sensitivity of assay techniques to phenolic composition of sample ...

  19. Functional NK cell cytotoxicity assays against virus infected cells.

    Science.gov (United States)

    Aicheler, Rebecca J; Stanton, Richard J

    2013-01-01

    Natural Killer (NK) cells are crucial to the control of many viral infections. They are able to kill infected cells directly through the secretion of cytotoxic granules or through binding to death receptors on target cells. They also secrete cytokines and chemokines and, through interactions with dendritic cells, can shape adaptive immunity. The activity of NK cells can be controlled by a balance of activating and inhibitory signals conveyed through ligands on target cells binding to receptors on the NK cell. As a result viruses have devised mechanisms to modulate the expression of NK ligands on target cells, interfering with NK cell recognition and prolonging the life of infected cells. An understanding of how viruses modulate the NK response can lead to an understanding both of NK cell function, and of virus pathogenesis. Measuring the ability of NK cells to kill target cells infected with different viruses, or expressing different viral proteins, is an invaluable technique to identify the proteins and mechanisms by which viruses modulate the NK response. Here we describe two methods to measure this; one method measures sodium dichromate (51)Cr that is released from target cells as they are killed, and the other uses 7-amino-actinomycin D (7-AAD) to measure apoptosis and death of target cells following incubation with NK cells.

  20. [Simultaneous screening method for Bordetella species by conventional PCR assay].

    Science.gov (United States)

    Akiyama, Yumi; Saito, Etsuko; Enomoto, Miki; Tsuji, Hidetaka; Chikahira, Masatsugu; Yoshida, Masashi

    2013-11-01

    A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.

  1. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    proliferation index. (CFSE/PI) and flow ... Key words: Flow cytometry, CFSE, cytokine-induced killer cell, proliferation, cytotoxicity. .... PI-treated target cells, not incubated with effectors, were used to set marker for dead cells (M1).

  2. Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices.

    Directory of Open Access Journals (Sweden)

    Diana Pauly

    Full Text Available BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed. Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex

  3. Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices.

    Science.gov (United States)

    Pauly, Diana; Worbs, Sylvia; Kirchner, Sebastian; Shatohina, Olena; Dorner, Martin B; Dorner, Brigitte G

    2012-01-01

    In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.

  4. Optimization of Compound Plate Preparation to Address Precipitation Issue in Mammalian A549 Cytotoxicity Assay.

    Science.gov (United States)

    Raghavendra Achar, Vijayashree Gauribidanur; Barde, Shubhada Pramod; Mallya, Meenakshy Venkatesh; Awasthy, Disha; Narayan, Chandan

    2016-06-01

    This study illustrates the optimization of low-volume dispensing on a liquid handling system (LHS) to overcome the precipitation of compounds in the mammalian cytotoxicity assay with low dimethyl sulfoxide (DMSO) tolerance. All compounds at AstraZeneca Bangalore are tested in the mammalian cytotoxicity assay. In order to maintain the DMSO levels, serially diluted plates were prepared in DMSO/water. It was observed that some of the compounds precipitated. The IC50 data for such compounds were therefore erratic. To circumvent the problem of compound precipitation, the LHS was optimized to dispense low volumes (<1 µL). The plates were serially diluted using neat DMSO. Since the dilution was done using neat DMSO, there were no issues with precipitation. The serially diluted sample (0.5 µL) from the plate was stamped onto the assay plate to give the desired DMSO concentration. No significant differences in IC50 data were observed for 1 µL dispenses made from DMSO/water and 0.5 µL dispenses from neat DMSO for the samples with no precipitation issues. These data therefore gave us the confidence to switch over to 0.5 µL dispenses for the cytotoxicity assay to address the precipitation issue. However, precipitation of samples in the assay buffer is beyond the scope of this discussion. © 2015 Society for Laboratory Automation and Screening.

  5. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Science.gov (United States)

    Tomitaka, Asahi; Hirukawa, Atsuo; Yamada, Tsutomu; Morishita, Shin; Takemura, Yasushi

    2009-05-01

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe 3O 4 (20-30 nm), ZnFe 2O 4 (15-30 nm) and NiFe 2O 4 (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe 3O 4 sample was found to be biocompatible on HeLa cells. While ZnFe 2O 4 and NiFe 2O 4 were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 μg/ml nanoparticles.

  6. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail: d07gd158@ynu.ac.jp; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)

    2009-05-15

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  7. Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity

    DEFF Research Database (Denmark)

    Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik

    2013-01-01

    and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well......-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine...... exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously...

  8. Epitope Prediction Assays Combined with Validation Assays Strongly Narrows down Putative Cytotoxic T Lymphocyte Epitopes.

    Science.gov (United States)

    Ip, Peng Peng; Nijman, Hans W; Daemen, Toos

    2015-03-24

    Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

  9. In vitro combined cytotoxic effects of pesticide cocktails simultaneously found in the French diet.

    Science.gov (United States)

    Takakura, Natsuko; Sanders, Pascal; Fessard, Valérie; Le Hégarat, Ludovic

    2013-02-01

    Although human populations may be constantly exposed to complex pesticide mixtures through their diet, the human health risk of pesticide exposure is currently assessed on the basis of toxicity data on individual compounds. To investigate the combined toxic effects of pesticide cocktails previously identified in the French diet, we first studied the cytotoxicity induced by seven cocktails composed of two to six pesticides on human hepatic (HepG2) and colon (Caco-2) cell lines using the MTT and neutral red uptake assays. Secondly, we challenged to assess the combined effects of the two most cytotoxic cocktails by comparing the measured effects of the mixtures with the predictions based on additive effects on two concepts-independent action (IA) and concentration addition (CA). For the cocktail composed of dichlorodiphenyltrichloroethane (DDT) and dieldrin, the cytotoxicity of the equimolar cocktail proved greater than the additive effect estimated by the two concepts. Furthermore, apoptosis induction was higher in equimolar cocktail than predicted by summing the effects of DDT and dieldrin. Thus, some supra-additive toxicity was found in the DDT-dieldrin cocktail. Nevertheless, if IA and CA models could reveal combined effects of pesticide cocktails, an accurate evaluation remains challenging. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Design of a multiplex PCR assay for the simultaneous detection and confirmation of Neisseria gonorrhoeae.

    LENUS (Irish Health Repository)

    O'Callaghan, Isabelle

    2010-05-01

    To improve the detection of Neisseria gonorrhoeae by designing a multiplex PCR assay using two N gonorrhoeae-specific genes as targets, thereby providing detection and confirmation of a positive result simultaneously.

  11. Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L. Using In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Claudia R. da Silva

    2010-01-01

    This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains. Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest and Plasmid-DNA treatment, and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties. Papain exhibited negative results for all tests. This agent presented an activity protecting cells against H2O2-induced mutagenesis.

  12. The agar diffusion scratch assay - A novel method to assess the bioactive and cytotoxic potential of new materials and compounds

    Science.gov (United States)

    Pusnik, Mascha; Imeri, Minire; Deppierraz, Grégoire; Bruinink, Arie; Zinn, Manfred

    2016-01-01

    A profound in vitro evaluation not only of the cytotoxic but also of bioactive potential of a given compound or material is crucial for predicting potential effects in the in vivo situation. However, most of the current methods have weaknesses in either the quantitative or qualitative assessment of cytotoxicity and/or bioactivity of the test compound. Here we describe a novel assay combining the ISO 10993-5 agar diffusion test and the scratch also termed wound healing assay. In contrast to these original tests this assay is able to detect and distinguish between cytotoxic, cell migration modifying and cytotoxic plus cell migration modifying compounds, and this at higher sensitivity and in a quantitative way. PMID:26861591

  13. Validation of a Multiparametric, High-Content-Screening Assay for Predictive/Investigative Cytotoxicity: Evidence from Technology Transfer Studies and Literature Review.

    Science.gov (United States)

    O'Brien, Peter James; Edvardsson, Anna

    2017-03-20

    A multiparametric, live-cell, high-content-screening (HCS) cytotoxicity assay was first demonstrated in 2006 ( Arch. Toxicol. 2006 , 80 , 580 - 604 ) to be highly concordant with human hepatotoxicity, including idiosyncratic hepatotoxicities and other target organ toxicities in contrast to historical assays. The success of the assay was attributed to its simultaneous measurement of multiple appropriate "cytobiomarkers": use of human cells with xenometabolic competence for toxicities mediated by metabolites, 72 h exposure to enable expression of slower-acting toxicants, exposure to a wide-range of concentrations from 30- to 100-fold the efficacious concentration, and normalizing the in vitro cytotoxic concentration to an estimate of the in vivo concentration of exposure. An overwhelming volume of evidence has accumulated over the last 10 years to support this approach as necessary in predictive toxicology. Equivalent assays have now been successfully applied in ∼50 studies across a wide variety of toxicants, toxicities, cell types, and disciplines. Review herein of the wider literature on cytotoxicity since the first assay was reported 100 years ago supports the selection of key cytobiomarkers along a final common pathway of cell injury, including cell proliferation, mitochondrial activity, apoptosis, lysosomal mass, oxidative stress, and cell membrane permeability. HCS studies without inclusion of such key cytobiomarkers or without testing to sufficiently high concentration have not been as successful. Furthermore, a subset of the original toxicants has been reanalyzed herein using the original HCS assay and has confirmed their high sensitivities and specificities across locations, HCS technologies, staff, laboratories, and time. A protocol is demonstrated for operational validation of the assay within laboratories to demonstrate proficiency and quality management.

  14. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2017-12-21

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  15. A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

    NARCIS (Netherlands)

    R. Alghafri (Rashed); W. Goodwin (Will); A. Ralf (Arwin); M.H. Kayser (Manfred); S. Hadi (Sibte)

    2015-01-01

    textabstractAbstract A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other

  16. Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

    Directory of Open Access Journals (Sweden)

    Marilanda Ferreira Bellini

    2008-01-01

    Full Text Available Functional food investigations have demonstrated the presence of substances that could be beneficial to human health when consumed. However, the toxic effects of some substances contained in foods have been determined. Reported medicinal and nutritive properties have led to the extensive commercialization of the basidiomycete fungi Agaricus blazei Murrill (sensu Heinemann, also known as Agaricus brasiliensis Wasser et al., Agaricus subrufescens Peck or the Brazilian medical mushroom (BMM. Different methanolic extract fractions (ME of this mushroom were submitted to the cytokinesis-block micronucleus (CBMN clastogenic assay and the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT assay for gene mutation, both using Chinese hamster ovary cells clone K1 (CHO-K1. The results suggest that all the fractions tested possess cytotoxic and mutagenic potential but no clastogenic effects. Further information is needed on the biochemical components of the A. blazei methanol fractions to identify any substances with cytotoxic and/or mutagenicity potential. These findings indicate that A. blazei methanolic extract should not be used due to their genotoxicity and care should be taken in the use of A. blazei by the general population until further biochemical characterization of this fungi is completed.

  17. Efficient fixation procedure of human leukemia cells in sulforhodamine B cytotoxicity assay.

    Science.gov (United States)

    Kim, H M; Han, S B; Kim, M S; Kang, J S; Oh, G T; Hong, D H

    1996-11-01

    The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.

  18. Cytotoxicity assay of Typhonium flagelliforme Lodd against breast and cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Endang Purwaningsih

    2015-12-01

    Ethanolic extract of Typhonium flagelliforme Lodd has cytotoxic effects against HeLa cells and MCF-7 cells. The cytotoxic effects against MCF-7 cells are greater than the cytotoxic effects against HeLa cells.

  19. Cytotoxicity of solubilization vehicles for Trichomonas gallinae and Tritrichomonas foetus measured by the resazurin microtiter assay.

    Science.gov (United States)

    Duarte, Mariana; Giordani, Raquel Brandt; Carli, Geraldo Attilio De; Zuanazzi, José Angelo; Macedo, Alexandre José; Tasca, Tiana

    2009-12-03

    The parasites Tritrichomonas foetus and Trichomonas gallinae present veterinary and economic importance since they cause bovine and avian trichomonosis, respectively. The absence of a specific treatment and the necessity of effective and safe drugs against these etiologic agents have stimulated the search for new antiprotozoal drugs with high activity, low toxicity to the animal, and low cost. Screening of potential antiprotozoal molecules is currently a common practice and different kinds of organic solvents and surfactant vehicles are used, since many bioactive compounds have low water solubility. Thus, it is important to determine the toxicity and to provide the minimal inhibitory concentration (MIC) values of the most common solubilization vehicles used in biological activity in vitro evaluation: ethanol, methanol, isopropanol, acetone, DMSO, tween 20 and tween 80. The assays were conducted employing the resazurin microtiter assay, which demonstrated a rapid, safe, and quantitative method for the in vitro determination of T. foetus and T. gallinae trophozoites viability. In summary, all solvents and surfactants, except ethanol, could be used in cytotoxicity assays against T. foetus, and acetone, tween 20 and tween 80 are the preferential vehicles for in vitro analysis of potential bioactive compounds against T. gallinae, though these must be used with caution.

  20. New approach in multipurpose optical diagnostics: fluorescence based assay for simultaneous determination of physicochemical parameters

    OpenAIRE

    Moczko, Ewa

    2009-01-01

    The development of sensors assays for comprehensive characterisation of biological samples and effective minimal-invasive diagnostics is highly prioritised. Last decade this research area has been actively developing due to possibility of simultaneous, real- time, in vivo detection and monitoring of diverse physicochemical parameters and analytes. The new approach which has been introduced in this thesis was to develop and examine an optical diagnostic assay consisting of a ...

  1. Cytotoxic Effect of Iron Oxide Nanoparticles on Mouse Embryonic Stem Cells by MTT Assay

    Directory of Open Access Journals (Sweden)

    Homa Mohseni Kouchesfehani

    2016-07-01

    Full Text Available Background: Despite the wide range of applications, there is a serious lack of information on the impact of the nanoparticles on human health and the environment. The present study was done to determine the range of dangerous concentrations of iron oxide nanoparticle and their effects on mouse embryonic stem cells. Methods: Iron oxide nanoparticles with less than 20 nanometers diameter were encapsulated by a PEG-phospholipid. The suspension of iron oxide nanoparticles was prepared using the culture media and cell viability was determined by MTT assay. Results: MTT assay was used to examine the cytotoxicity of iron oxide nanoparticle s. Royan B1 cells were treated with medium containing different concentrations (10, 20, 30, 40, 50, and 60µg/ml of the iron oxide nanoparticle. Cell viability was determined at 12 and 24 hours after treatment which showed significant decreases when concentration and time period increased. Conclusion: The main mechanism of nanoparticles action is still unknown, but in vivo and in vitro studies in different environments suggest that they are capable of producing reactive oxygen species (ROS. Therefore, they may have an effect on the concentration of intracellular calcium, activation of transcription factors, and changes in cytokine. The results of this study show that the higher concentration and duration of treatment of cells with iron oxide nanoparticles increase the rate of cell death.

  2. The comet assay, DNA damage, DNA repair and cytotoxicity: hedgehogs are not always dead.

    Science.gov (United States)

    Lorenzo, Yolanda; Costa, Solange; Collins, Andrew R; Azqueta, Amaya

    2013-07-01

    DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.

  3. A high-performance, non-radioactive potency assay for measuring cytotoxicity: A full substitute of the chromium-release assay targeting the regulatory-compliance objective.

    Science.gov (United States)

    Rossignol, Alexis; Bonnaudet, Véronique; Clémenceau, Béatrice; Vié, Henri; Bretaudeau, Laurent

    2017-04-01

    Standardized and biologically relevant potency assays are required by the regulatory authorities for the characterization and quality control of therapeutic antibodies. As critical mechanisms of action (MoA) of antibodies, the antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) must be characterized by appropriate potency assays. The current reference method for measuring cytotoxicity is the 51Cr-release method. However, radioactivity handling is difficult to implement in an industrial context because of environmental and operator protection constraints. Alternative non-radioactive methods suffer from poor validation performances and surrogate assays that measure FcγR-dependent functions do not comply with the regulatory requirement of biological relevance. Starting from these observations, we developed a non-radioactive luminescent method that is specific for target cell cytolysis. In adherent and non-adherent target cell models, the ADCC (using standardized effector cells) or CDC activities of rituximab, trastuzumab and adalimumab were compared in parallel using the 51Cr or luminescent methods. We demonstrated that the latter method is highly sensitive, with validation performances similar or better than the 51Cr method. This method also detected apoptosis following induction by a chemical agent or exposure to ultraviolet light. Moreover, it is more accurate, precise and specific than the concurrent non-radioactive calcein- and TR-FRET-based methods. The method is easy to use, versatile, standardized, biologically relevant and cost effective for measuring cytotoxicity. It is an ideal candidate for developing regulatory-compliant cytotoxicity assays for the characterization of the ADCC, CDC or apoptosis activities from the early stages of development to lot release.

  4. Cytotoxicity of artemisinin, a dimer of dihydroartemisinin, artemisitene and eupatoriopicrin as evaluated by the MTT and clonogenic assay

    NARCIS (Netherlands)

    Beekman, AC; Woerdenbag, HJ; Kampinga, HH; Konings, AWT

    Artemisinin and its derivatives possess an endoperoxide bridge, which is thought to lead to the production of free-radical species, The cytotoxicity of some of these agents to a murine Ehrlich ascites (EN19) and a human HeLa S3 cancer cell line was determined using the MTT and the clonogenic assay,

  5. Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure.

    Science.gov (United States)

    Zhu, Xuena; Hondroulis, Evangelia; Liu, Wenjun; Li, Chen-Zhong

    2013-05-27

    The increased utilization of nanomaterials could affect human health and the environment due to increased exposure. Several mechanisms regarding the negative effects of nanomaterials have been proposed, one of the most discussed being oxidative stress. Many studies have shown that some metal oxide nanoparticles can enhance reactive oxygen species generation, inducing oxidative stress, DNA damage, and unregulated cell signaling, and eventually leading to changes in cell motility, apoptosis, and even carcinogenesis. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of oxidative DNA damage, and has therefore been widely used as a biomarker for oxidative stress and carcinogenesis. Ther are two major objectives to this study. Firstly, the development of a novel lateral flow immunoassay (LFIA) is presented to measure the concentration of 8-OHdG in cells and thus reveal the nanotoxicity on the genomic level. The feasibility of this new method is validated by comparison with two other established methods: Alamar Blue assay and a recently developed electrical impedance sensing (EIS) system on the level of cell proliferation/viability. Secondly, the toxicological effects of three metallic nanoparticles (CuO, CdO, and TiO2 ) are investigated and compared using these three methods with completely different mechanisms. The results show that there is a high variation among different nanoparticles concerning their ability to cause toxic effects. CuO nanoparticles are the most potent regarding cytotoxicity and DNA damage. CdO shows a fallen cell viability as well as DNA damage, however, to a lesser extent than CuO nanoparticles. TiO2 particles only cause very limited cytotoxicity, and there is no obvious increase in 8-OHdG levels. In conclusion, LFIA as well as the EIS system are useful methods for quantitative or qualitative nanotoxicity assessments with high sensitivity, specificity, speed of performance, and simplicity. Copyright © 2013 WILEY-VCH Verlag Gmb

  6. Lateral flow assay for simultaneous detection of cellular- and humoral immune responses.

    Science.gov (United States)

    Corstjens, Paul L A M; de Dood, Claudia J; van der Ploeg-van Schip, Jolien J; Wiesmeijer, Karien C; Riuttamäki, Terhi; van Meijgaarden, Krista E; Spencer, John S; Tanke, Hans J; Ottenhoff, Tom H M; Geluk, Annemieke

    2011-10-01

    The development of a cytokine detection assay suitable for detection of multiple biomarkers for improved diagnosis of mycobacterial diseases. A lateral flow (LF) assay to detect IL-10 was developed utilizing the up-converting phosphor (UCP) reporter-technology. The assay was evaluated using blood samples of leprosy patients. Multiplex applications were explored targeting: 1) IL-10 and IFN-γ in assay buffer; 2) IL-10 and anti-phenolic glycolipid (PGL-I) antibodies in serum from leprosy patients. Detection of IL-10 below the targeted level of 100pg/mL in serum was shown. Comparison with ELISA showed a quantitative correlation with R(2) value of 0.92. Multiplexing of cytokines and simultaneous detection of cytokine and antibody was demonstrated. The UCP-LF IL-10 assay is a user-friendly, rapid alternative for IL-10 ELISAs, suitable for multiplex detection of different cytokines and can be merged with antibody-detection assays to simultaneously detect cellular- and humoral immunity. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. A multiparametric assay to compare the cytotoxicity of soy milk with different storage media.

    Science.gov (United States)

    Silva, Emmanuel J N L; Rollemberg, Carolina B; de Souza Coutinho-Filho, Tauby; Zaia, Alexandre A

    2013-08-01

    The aim of this study was to evaluate the cytotoxicity of soy milk compared with several other storage media [coconut water, Hank's Balanced Salt Solution (HBSS) and whole milk], assessed through a multiparametric analysis employing 3T3 cells. Plates containing confluent 3T3 fibroblasts were exposed to the various media for 24 h, at 37°C with 5% CO₂, and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (XTT), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). Results from each test were compared by two-way analysis of variance (ANOVA). Statistical analysis showed that whole milk, HBSS and soy milk were the most effective media in maintaining cell viability at all tested times (P soy milk in maintaining the viability of 3T3 fibroblasts is similar to that of HBSS and milk, as shown by three different cell viability tests. © 2012 John Wiley & Sons A/S.

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

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Zor, Kinga; Canepa, Silvia

    2015-01-01

    between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell–substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity......We investigated the combined effect of the initial cell density (12 500, 35 000, 75 000, and 100 000 cells cm−2) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing timedependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation...

  9. Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays.

    Science.gov (United States)

    Guo, Xiaoqing; Li, Yan; Yan, Jian; Ingle, Taylor; Jones, Margie Yvonne; Mei, Nan; Boudreau, Mary D; Cunningham, Candice K; Abbas, Mazhar; Paredes, Angel M; Zhou, Tong; Moore, Martha M; Howard, Paul C; Chen, Tao

    2016-11-01

    The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

  10. Simultaneous assay for amatoxins and phallotoxins in Amanita phalloides Fr. by high-performance liquid chromatography.

    Science.gov (United States)

    Enjalbert, F; Gallion, C; Jehl, F; Monteil, H; Faulstich, H

    1992-05-15

    A reversed-phase high-performance liquid chromatographic method is described that allows the simultaneous determination of up to eight amatoxins and phallotoxins. The method identifies both neutral toxins (alpha- and gamma-amanitin, phalloidin, phallisin and phalloin) and acidic toxins (beta-amanitin, phallacidin and phallisacin). Toxins were separated, identified and determined by gradient elution with 0.02 M aqueous ammonium acetate-acetonitrile and simultaneous monitoring of the absorbances at 214 and 295 nm. The assay was successfully applied to the analysis of the toxins in a crude extract of Amanita phalloides. The limit of detection for each toxin was 10 ng/ml of extraction medium. The assay was further validated by analysing the toxin content in Galerina marginata, a species containing only amatoxins. This relatively simple method should be suitable for the detection of amatoxins and phallotoxins in almost any species of mushrooms.

  11. Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses.

    Science.gov (United States)

    Wagner, Bettina; Freer, Heather

    2009-02-15

    The detection and quantification of equine cytokines has been hampered by the lack of antibodies for many years. With the development of antibody pairs for equine cytokines during the past years, the quantification of these essential regulators of the immune response became possible. After being successfully tested by enzyme-linked immunosorbent assays (ELISA), three of these anti-cytokine reagents were used here to establish the first cytokine multiplex assay for equine IL-4, IL-10 and IFN-alpha. A fluorescent bead-based system was used as matrix for this assay that allows the simultaneous detection of the cytokines in a single sample by a Luminex analyzer. Equine recombinant cytokine/IgG fusion proteins were validated as standards for quantification of the individual cytokines. The analytical sensitivities of the multiplex assay were found to be 40 pg/ml for IL-4 and 15 pg/ml for IL-10 and IFN-alpha. The sensitivity of cytokine detection by the multiplex assay was increased by 13- to 150-fold compared to the corresponding ELISA. The specificity of the multiplex assay was validated using cell culture supernatants from equine peripheral blood mononuclear cells (PBMC) stimulated with different mitogens or infected with equine herpesvirus type 1 (EHV-1). As predicted, supernatants from PBMC stimulated with different mitogens contained IL-4 and IL-10, but no IFN-alpha. EHV-1 infection of PBMC resulted in a dose-dependent secretion of IFN-alpha. Low concentrations of IL-10 were also measured. IL-4 was not detectable in these samples. The resulting detection pattern found for the multiplex analysis and assays performed with individual standard cytokines indicated that individual bead assays did not interfere or cross-react during simultaneous detection of equine IL-4, IL-10 and IFN-alpha. The equine cytokine multiplex assay is a valuable and cost-effective tool for quantification of IL-4, IL-10 and IFN-alpha and can be used for manifold immunological applications. In

  12. Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom.

    Science.gov (United States)

    Liu, Bin-Sin; Wu, Wen-Guey; Lin, Min-Han; Li, Chi-Han; Jiang, Bo-Rong; Wu, Suh-Chin; Leng, Chih-Hsiang; Sung, Wang-Chou

    2017-12-25

    Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA) simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV) and Vietnamese snake antivenom-Naja (SAV-Naja) exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX) and cytotoxicity of cardiotoxin/cytotoxin (CTX), respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

  13. Identification of Immunoreactive Peptides of Toxins to Simultaneously Assess the Neutralization Potency of Antivenoms against Neurotoxicity and Cytotoxicity of Naja atra Venom

    Directory of Open Access Journals (Sweden)

    Bing-Sin Liu

    2017-12-01

    Full Text Available Assessing the neutralization capability of nonlethal but medically relevant toxins in venom has been a challenging task. Nowadays, neutralization efficacy is evaluated based simply on the survival rates of animals injected with antivenom together with a predefined dose of venom, which can determine potency against neurotoxicity but not validate the capability to neutralize cytotoxin-induced complications. In this study, a high correlation with in-vivo and in-vitro neutralization assays was established using the immunoreactive peptides identified from short-chain neurotoxin and cytotoxin A3. These peptides contain conserved residues associated with toxin activities and a competition assay indicated that these peptides could specifically block the antibody binding to toxin and affect the neutralization potency of antivenom. Moreover, the titers of peptide-specific antibody in antivenoms or mouse antisera were determined by enzyme-linked immunosorbent assay (ELISA simultaneously, and the results indicated that Taiwanese bivalent antivenom (BAV and Vietnamese snake antivenom-Naja (SAV-Naja exhibited superior neutralization potency against the lethal effect of short-chain neurotoxin (sNTX and cytotoxicity of cardiotoxin/cytotoxin (CTX, respectively. Thus, the reported peptide ELISA shows not only its potential for antivenom prequalification use, but also its capability of justifying the cross-neutralization potency of antivenoms against Naja atra venom toxicity.

  14. [Cytotoxicity of DMSO for MRC5, Chang liver and CV1 cells evaluated in vitro by LK, MTT and NR assays].

    Science.gov (United States)

    Siennicka, Joanna; Gut, Włodzimierz; Zuk, Anna; Litwińska, Bogumiła

    2003-01-01

    Evaluation of chemicals cytotoxicity plays fundamental role in many in vitro investigations. The way of assessment of cytotoxicity depend on aim of study, characteristic of used cells and mode of action of investigated chemicals. The principal aspect of these investigations is validation of used method. In this paper validation of three different cytotoxicity assays is presented: total cell number measurement (LK), microplate assay measured mitochondrial dehydrogenase activity (MTT) and colorimetric assay measured ability of live cell to uptake neutral red (NR). The investigation was performed on different cells (MRC5, CV1 i Chang Liver) with DMSO as reference agent.

  15. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    Science.gov (United States)

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

  16. Comparison of the TOX A/B test to a cell culture cytotoxicity assay for the detection of Clostridium difficile in stools.

    Science.gov (United States)

    Aldeen, W E; Bingham, M; Aiderzada, A; Kucera, J; Jense, S; Carroll, K C

    2000-04-01

    The TOX A/B Test (Techlab, Blacksburg, VA, USA) was compared to cell culture cytotoxicity assay on 1109 consecutive diarrheal stool samples collected from patients with the presumptive diagnosis of Clostridium difficile disease. The TOX A/B Test is an enzyme immunoassay in a microtiter format that detects both toxins A and B. The procedure used for this study takes approximately 1.5 h to perform. Cell culture cytotoxicity was performed by using a fibroblast cell line in a microtiter format read at 4 h, 24 h, and 48 h. One hundred ninety-four of the 1109 samples were positive by the "gold standard" cytotoxicity assay, whereas 189 were positive by EIA. There was a 98.5% agreement between the two assays. When compared to the cytotoxicity assay, the EIA had an initial sensitivity of 94.3% and a specificity of 99.3%. However, after resolution of six discrepants using another ELISA for toxin A detection the sensitivity, specificity, positive and negative predictive values for the TOX A/B test are as follows: 94.5%; 100%; 100%; 98.8%. The corresponding values for the cytotoxicity assay are: 97%; 100%; 100%; and 99.3%. This test seems to have excellent sensitivity and specificity as compared to an in-house cell culture cytotoxicity assay. It is sensitive enough to use as a stand-alone test for the detection of C. difficile toxin in laboratories that do not have cell culture cytotoxicity testing capability.

  17. Phase-dependent preference of thermosensation and chemosensation during simultaneous presentation assay in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Shingai Ryuzo

    2008-11-01

    Full Text Available Abstract Background Multi-sensory integration is necessary for organisms to discriminate different environmental stimuli and thus determine behavior. Caenorhabditis elegans has 12 pairs of amphid sensory neurons, which are involved in generating behaviors such as thermotaxis toward cultivation temperature, and chemotaxis toward chemical stimuli. This arrangement of known sensory neurons and measurable behavioral output makes C. elegans suitable for addressing questions of multi-sensory integration in the nervous system. Previous studies have suggested that C. elegans can process different chemoattractants simultaneously. However, little is known about how these organisms can integrate information from stimuli of different modality, such as thermal and chemical stimuli. Results We studied the behavior of a population of C. elegans during simultaneous presentation of thermal and chemical stimuli. First, we examined thermotaxis within the radial temperature gradient produced by a feedback-controlled thermoregulator. Separately, we examined chemotaxis toward sodium chloride or isoamyl alcohol. Then, assays for simultaneous presentations of 15°C (colder temperature than 20°C room temperature and chemoattractant were performed with 15°C-cultivated wild-type worms. Unlike the sum of behavioral indices for each separate behavior, simultaneous presentation resulted in a biased migration to cold regions in the first 10 min of the assay, and sodium chloride-regions in the last 40 min. However, when sodium chloride was replaced with isoamyl alcohol in the simultaneous presentation, the behavioral index was very similar to the sum of separate single presentation indices. We then recorded tracks of single worms and analyzed their behavior. For behavior toward sodium chloride, frequencies of forward and backward movements in simultaneous presentation were significantly different from those in single presentation. Also, migration toward 15°C in simultaneous

  18. A duplex PCR assay for simultaneous detection and differentiation of Capripoxvirus and Orf virus.

    Science.gov (United States)

    Zheng, Min; Liu, Qi; Jin, Ningyi; Guo, Jiangang; Huang, Xia; Li, Huaming; Zhu, Wei; Xiong, Yi

    2007-08-01

    A duplex polymerase chain reaction (PCR) was developed and optimized for simultaneous detection and differentiation of Capripoxvirus (CPV) (including goat pox virus and sheep pox virus) and orf virus (ORFV). Two sets of specific oligonucleotide primers were designed and used for CPV and ORFV, respectively. The duplex PCR DNA products, which consisted of fragments of 413 bp for A29L gene of CPV, and 708 bp for P55 gene of ORFV, were visualized by gel electrophoresis. The developed assay was found to be highly specific and sensitive with a detection limit of 1 plaque forming unit (pfu) for both CPV and CPV.

  19. Comparison of five different in vitro assays for assessment of sodium metavanadate cytotoxicity in Chinese hamster ovary cells (CHO-K1 line).

    Science.gov (United States)

    Zwolak, Iwona

    2015-08-01

    This investigation was undertaken to compare five different in vitro cytotoxicity assays for their power in revealing vanadium-mediated toxicity in Chinese hamster ovary (CHO)-K1 cells. The cells were exposed to sodium metavanadate (NaVO(3)) in the range of 10-1000 µM for 24 h and thereafter the cytotoxic effects of NaVO(3) were measured by colorimetric in vitro assays: the neutral red (NR) test, the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay, the resazurin assay, the sulforhodamine B (SR-B) assay, and by microscopic assessment of cell viability using the trypan blue (TB) staining method. Among the assays used, the NR test was the most sensitive, since it revealed metavanadate cytotoxicity at the lowest NaVO(3) dose (=50 µM). Also, NaVO(3) cytotoxicity expressed as inhibitory concentration (IC) showed the lowest values for the NR test. Three other tests XTT, resazurin, and SR-B assays showed intermediate sensitivity revealing the cytotoxicity of NaVO(3) at 100 µM. The corresponding IC10 and IC50 values calculated for the XTT, resazurin, and SR-B tests were similar. The TB staining method was the least sensitive, since it recorded metavanadate cytotoxicity at the highest NaVO(3) concentration tested (=600 µM). Based on the cytotoxicity end points measured with the above assays, it can be concluded that lysosomal/Golgi apparatus damage (measured by NR assay) may be the primary effect of NaVO(3) on CHO-K1 cells. The disintegration of mitochondria (assessed with the XTT and resazurin assays) probably follows lysosomal impairment. Plasma membrane permeability (staining with TB) occurs at a late stage of NaVO(3)-induced cytotoxicity on CHO-K1 cells. The results obtained in this research work show that the NR test can be recommended as a very sensitive assay for the assessment of NaVO(3) cytotoxicity in the CHO-K1 cell culture model. Considering the convenience of assay performance along with adequate sensitivity

  20. Genotoxicity and cytotoxicity of dental materials in human lymphocytes as assessed by the single cell microgel electrophoresis (comet) assay.

    Science.gov (United States)

    Kleinsasser, Norbert H; Wallner, Barbara C; Harréus, Ulrich A; Kleinjung, Tobias; Folwaczny, Matthias; Hickel, Reinhard; Kehe, Kai; Reichl, Franz-Xaver

    2004-03-01

    Resin monomers may be released from restorative dental materials and can diffuse into the tooth pulp or the gingiva, and can reach the saliva and the circulating blood. Whereas the cytotoxic potential of some components has been clearly documented, possible genotoxicity in human target cells demands further investigation. The Comet assay was used to quantify DNA single strand breaks, alkali labile and incomplete excision repair sites in lymphocytes of 10 volunteers. The xenobiotics investigated were 2-hydroxyethylmethacrylate (HEMA), triethyleneglycoldimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A-glycidyl methacrylate (Bis-GMA) with N-methyl-N'-nitro-N-nitrosoguanidine and dimethyl sulfoxide as controls. DNA migration was quantified using the tail moment according to Olive (OTM) and DNA migration was considered to be elevated at OTM levels above 2. Cytotoxicity was monitored using trypan blue. In the negative controls, OTM ranged between 1.0 and 1.2. With HEMA concentrations above 10(-6)M, TEGDMA 10(-3)M, Bis-GMA 10(-4)M, and UDMA above 10(-6)M relevant enhancements of DNA migration (OTM>2) were achieved. At higher concentrations of up to 2.5x10(-2) induced DNA migration was expressed by OTM of 3.3 for HEMA, 4.5 for TEGDMA, 7.4 for Bis-GMA, and 2.8 for UDMA. Relevant cytotoxic effects were also seen but vitality levels were at a critical range of 71% for Bis-GMA and 73% for TEGDMA, only. In higher concentration levels, all tested substances induced significant but minor enhancement of DNA migration in the Comet assay as a possible sign for limited genotoxic effects. However, with the highest levels of DNA migration being combined with elevated cytotoxic effects, a low in vivo genotoxic strain appears to be posed by the resin components.

  1. Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

    Science.gov (United States)

    Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

    2015-04-01

    Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC50 values in WST-1 assays. The IC50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles.

  2. Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

    Science.gov (United States)

    Xu, Yan; Hadjiargyrou, M; Rafailovich, Miriam; Mironava, Tatsiana

    2017-07-11

    Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). Herein, we report that titanium dioxide (TiO2) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

  3. Ultrasensitive Detection of RNA and DNA Viruses Simultaneously Using Duplex UNDP-PCR Assay

    Science.gov (United States)

    Wang, Zengguo; Zhang, Xiujuan; Zhao, Xiaomin; Du, Qian; Chang, Lingling; Tong, Dewen

    2015-01-01

    Mixed infection of multiple viruses is common in modern intensive pig rearing. However, there are no methods available to detect DNA and RNA viruses in the same reaction system in preclinical level. In this study, we aimed to develop a duplex ultrasensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) that was able to simultaneously detect DNA and RNA viruses in the same reaction system. PCV2 and TGEV are selected as representatives of the two different types of viruses. PCV2 DNA and TGEV RNA were simultaneously released from the serum sample by boiling with lysis buffer, then magnetic beads and gold nanoparticles coated with single and/or duplex specific probes for TGEV and PCV2 were added to form a sandwich-like complex with nucleic acids released from viruses. After magnetic separation, DNA barcodes specific for PCV2 and TGEV were eluted using DTT and characterized by specific PCR assay for specific DNA barcodes subsequently. The duplex UNDP-PCR showed similar sensitivity as that of single UNDP-PCR and was able to detect 20 copies each of PCV2 and TGEV in the serum, showing approximately 250-fold more sensitivity than conventional duplex PCR/RT-PCR assays. No cross-reaction was observed with other viruses. The positive detection rate of single MMPs- and duplex MMPs-based duplex UNDP-PCR was identical, with 29.6% for PCV2, 9.3% for TGEV and 3.7% for PCV2 and TGEV mixed infection. This duplex UNDP-PCR assay could detect TGEV (RNA virus) and PCV2 (DNA virus) from large-scale serum samples simultaneously without the need for DNA/RNA extraction, purification and reverse transcription of RNA, and showed a significantly increased positive detection rate for PCV2 (29%) and TGEV (11.7%) preclinical infection than conventional duplex PCR/RT-PCR. Therefore, the established duplex UNDP-PCR is a rapid and economical detection method, exhibiting high sensitivity, specificity and reproducibility. PMID:26544710

  4. Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers

    Energy Technology Data Exchange (ETDEWEB)

    Boswell, G.S.; Dimitrijevich, S.D.; Gracy, R.W. (Univ. of North Texas/Texas College of Osteopathic Medicine, Fort Worth (USA))

    1989-10-01

    A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developed an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ((3H)thymidine, (14C)leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.

  5. A three-dimensional organotypic assay to measure target cell killing by cytotoxic T lymphocytes.

    NARCIS (Netherlands)

    Weigelin, B.; Friedl, P.H.A.

    2010-01-01

    Cytotoxic T lymphocytes (CTL) mediate antigen- and cell-cell contact dependent killing of target cells, such as cancer cells and virus-infected cells. In vivo, this process requires the active migration of CTL towards and away from target cells. We here describe an organotypic 3D collagen matrix

  6. The agar diffusion scratch assay--A novel method to assess the bioactive and cytotoxic potential of new materials and compounds.

    Science.gov (United States)

    Pusnik, Mascha; Imeri, Minire; Deppierraz, Grégoire; Bruinink, Arie; Zinn, Manfred

    2016-02-10

    A profound in vitro evaluation not only of the cytotoxic but also of bioactive potential of a given compound or material is crucial for predicting potential effects in the in vivo situation. However, most of the current methods have weaknesses in either the quantitative or qualitative assessment of cytotoxicity and/or bioactivity of the test compound. Here we describe a novel assay combining the ISO 10993-5 agar diffusion test and the scratch also termed wound healing assay. In contrast to these original tests this assay is able to detect and distinguish between cytotoxic, cell migration modifying and cytotoxic plus cell migration modifying compounds, and this at higher sensitivity and in a quantitative way.

  7. Direct contact cytotoxicity assays for filter-collected, carbonaceous (soot) nanoparticulate material and observations of lung cell response

    Science.gov (United States)

    Soto, K. F.; Garza, K. M.; Shi, Y.; Murr, L. E.

    A simple, direct contact, cytotoxicity (in vitro) assay has been developed where particulate matter (PM) collected on glass fiber filters was exposed to human epithelial (lung) cells. Carbonaceous (soot) PM included tire, wood, diesel, candle, and variously combusted natural gas PM from a kitchen stove range. Black carbon PM and a commercial multiwall carbon nanotube aggregate PM was also examined in vitro as surrogate materials, and all experimental PM was characterized by field emission scanning electron microscopy and transmission electron microscopy. Assay results for 48 h cultures showed toxicity for all carbonaceous PM with various natural gas PM being the most toxic; this was comparable to the toxicity induced by the surrogate PM. Light microscopy examination of affected epithelial cells confirmed the semi-quantitative results. Comparison of polycyclic aromatic hydrocarbon (PAH) content and concentration for the carbonaceous PM showed no PAH correlation with relative cell viability (cell death) after 48 h.

  8. Cytotoxicity and Antiproliferative Activity Assay of Clove Mistletoe (Dendrophthoe pentandra (L. Miq. Leaves Extracts

    Directory of Open Access Journals (Sweden)

    Vida Elsyana

    2016-01-01

    Full Text Available Clove mistletoe (Dendrophthoe pentandra (L. Miq. is a semiparasitic plant that belongs to Loranthaceae family. Clove mistletoe was traditionally used for cancer treatment in Indonesia. In the present study, we examined cytotoxicity of clove mistletoe leaves extracts against brine shrimps and conducted their antiproliferative activity on K562 (human chronic myelogenous leukemia and MCM-B2 (canine benign mixed mammary cancer cell lines in vitro. The tested samples were water extract, ethanol extract, ethanol fraction, ethyl acetate fraction, and n-hexane fraction. Cytotoxicity was screened using Brine Shrimp Lethality Test (BSLT. Antiproliferative activity was conducted using Trypan Blue Dye Method and cells were counted using haemocytometer. The results showed that n-hexane fraction exhibited significant cytotoxicity with LC50 value of 55.31 μg/mL. The n-hexane fraction was then considered for further examination. The n-hexane fraction of clove mistletoe could inhibit growth of K562 and MCM-B2 cancer cell lines in vitro. The inhibition activity of clove mistletoe n-hexane fraction at concentration of 125 μg/mL on K562 cancer cell lines was 38.69%, while on MCM-B2 it was 41.5%. Therefore, it was suggested that clove mistletoe had potential natural anticancer activity.

  9. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  10. Reversed-phase high-performance Liquid Chromatography-ultraviolet Photodiode Array Detector Validated Simultaneous Quantification of six Bioactive Phenolic Acids in Roscoea purpurea Tubers and their In vitro Cytotoxic Potential against Various Cell Lines

    Science.gov (United States)

    Srivastava, Sharad; Misra, Ankita; Kumar, Dharmesh; Srivastava, Amit; Sood, Anil; Rawat, AKS

    2015-01-01

    Background: Roscoea purpurea or Roscoea procera Wall. (Zingiberaceae) is traditionally used for nutrition and in the treatment of various ailments. Objective: Simultaneous reversed-phase high-performance liquid chromatography-ultraviolet (RP-HPLC) photodiode array detector identification of phenolic acids (PA's) was carried out in whole extract of tuber and their cytotoxic potential was estimated along with radical scavenging action. Bioactivity guided fractionation was also done to check the response potential against the same assay. Materials and Methods: Identification and method validation was performed on RP-HPLC column and in vitro assays were used for bioactivity. Results: Protocatechuic acid, syringic acid, ferulic acid, rutin, apigenin, and kaempferol were quantified as 0.774%, 0.064%, 0.265%, 1.125%, 0.128%, and 0.528%, respectively. Validated method for simultaneous determination of PA's was found to be accurate, reproducible, and linearity was observed between peak area response and concentration. Recovery of identified PA's was within the acceptable limit of 97.40–104.05%. Significant pharmacological response was observed in whole extract against in vitro cytotoxic assay, that is, Sulforhodamine B assay, however, fractionation results in decreased action potential. Similar pattern of results were observed in the antioxidant assay, as total phenolic content and total flavonoid content were highest in whole extract and decreases with fractionation. Radical scavenging activity was prominent in chloroform fraction, exhibiting IC50 at 0.25 mg/mL. Conclusion: Study, thus, reveals that R. purpurea exhibit significant efficacy in cytotoxic activity with the potentiality of scavenging free radicals due the presence of PA's as reported through RP-HPLC. SUMMARY Proto-catechuic acid, syringic acid, ferulic acid, rutin, apigenin and kaempferol were quantified as 0.774, 0.064, 0.265, 1.125, 0.128 and 0.528 %Preliminary cytotoxic activity revealed that whole

  11. Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

    Directory of Open Access Journals (Sweden)

    Göbbert Christian

    2011-02-01

    Full Text Available Abstract Background Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. Results Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. Conclusion In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints.

  12. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

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    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

  13. A Multichannel Calorimetric Simultaneous Assay Platform Using a Microampere Constant-Current Looped Enthalpy Sensor Array

    Directory of Open Access Journals (Sweden)

    Hsien-Chin Wei

    2017-02-01

    Full Text Available Calorimetric biochemical measurements offer various advantages such as low waste, low cost, low sample consumption, short operating time, and labor-savings. Multichannel calorimeters can enhance the possibility of performing higher-throughput biochemical measurements. An enthalpy sensor (ES array is a key device in multichannel calorimeters. Most ES arrays use Wheatstone bridge amplifiers to condition the sensor signals, but such an approach is only suitable for null detection and low resistance sensors. To overcome these limitations, we have developed a multichannel calorimetric simultaneous assay (MCSA platform. An adjustable microampere constant-current (AMCC source was designed for exciting the ES array using a microampere current loop measurement circuit topology. The MCSA platform comprises a measurement unit, which contains a multichannel calorimeter and an automatic simultaneous injector, and a signal processing unit, which contains multiple ES signal conditioners and a data processor. This study focused on the construction of the MCSA platform; in particular, construction of the measurement circuit and calorimeter array in a single block. The performance of the platform, including current stability, temperature sensitivity and heat sensitivity, was evaluated. The sensor response time and calorimeter constants were given. The capability of the platform to detect relative enzyme activity was also demonstrated. The experimental results show that the proposed MCSA is a flexible and powerful biochemical measurement device with higher throughput than existing alternatives.

  14. Cytotoxic assay of endophytic fungus 1.2.11 secondary metabolites from Brucea javanica (L Merr towards cancer cell in vitro

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    Pratiwi Sudarmono

    2006-09-01

    Full Text Available Cytotoxic assay of secondary metabolite endophytic fungus 1.2.11 from Brucea javanica (L Merr has been carried out. Brucea javanica fruit collected from Cianjur was used in this experiment. Cytotoxic assay was done on Raji, NS-1, HeLa and Vero cells. The observation was done for 24 hours and also for 48 hours. IC50 was calculated using the Rich and Muench theory. To observe the working mechanism of cytotoxic process, DNA staining with etidium bromide and acridine orange was conducted. The cytotoxic assay of endophytic fungi 1.2.11 showed an IC50 of 58.35 μg/ml, 88.39 μg/ml on Raji cell,; 162.09 μg/ml, 66.24 μg/ml on NS cell; 361.21 μg/ml, 219.97 μg/ml on HeLa cell; and lastly 1075.18 μg/ml, 656.82 μg/ml on Vero cell after 24 and 48 hour incubation respectively. The results of this study showed that secondary metabolite of endophytic fungus 1.2.11 has selective cytotoxic effect towards cancer cell and also showed that it might cause apoptosis in NS-1cell. (Med J Indones 2006; 15:137-44 Keywords: Brucea javanica (L. Merr, endophytic microbe, Cytotoxic assay, endophytic isolate 1.2.11, apoptosis

  15. A gold immunochromatographic assay for the rapid and simultaneous detection of fifteen β-lactams

    Science.gov (United States)

    Chen, Yanni; Wang, Yongwei; Liu, Liqiang; Wu, Xiaoling; Xu, Liguang; Kuang, Hua; Li, Aike; Xu, Chuanlai

    2015-10-01

    A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively. Since goat anti-mouse IgG does not combine with receptors, negative serum from mice labelled with gold nanoparticles (GNP) was mixed with GNP-labelled receptors. Results were obtained within 20 min using a paper-based sensor. The utility of the assay was confirmed by the analysis of milk samples. The limits of detection (LOD) for amoxicillin, ampicillin, penicillin G, penicillin V, cloxacillin, dicloxacillin, nafcillin, oxacillin, cefaclor, ceftezole, cefotaxime, ceftiofur, cefoperazone, cefathiamidine, and cefepime were 0.25, 0.5, 0.5, 0.5, 1, 5, 5, 10, 25, 10, 100, 10, 5, 5, and 2 ng mL-1, respectively, which satisfies the maximum residue limits (MRL) set by the European Union (EU). In conclusion, our newly developed GICA-based anti-β-lactam receptor assay provides a rapid and effective method for one-site detection of multiple β-lactams in milk samples.A novel gold immunochromatographic assay (GICA) based on anti-β-lactam receptors was innovatively developed that successfully allowed rapid and simultaneous detection of fifteen β-lactams in milk samples in 5-10 minutes. By replacing the antibodies used in traditional GICA with anti-β-lactam receptors, the difficulty in producing broad specific antibodies against β-lactams was overcome. Conjugates of ampicillin with BSA and goat anti-mouse immunoglobulin (IgG) were immobilized onto the test and control lines on the nitrocellulose membrane, respectively

  16. Honey is cytotoxic towards prostate cancer cells but interacts with the MTT reagent: Considerations for the choice of cell viability assay.

    Science.gov (United States)

    Abel, Sean D A; Baird, Sarah K

    2018-02-15

    Honey is a complex biological substance, consisting mainly of sugars, phenolic compounds and enzymes. Using five quick and accessible assays for measuring honey's cytotoxicity in vitro, we found honey is cytotoxic towards prostate cancer cells PC3 and DU145. However, the level of cell death varied with assay. The MTT assay was confounded by the reduction of the MTT reagent by honey's reducing sugars and phenolic compounds, and the lactate dehydrogenase assay was invalidated by honey oxidising the enzyme cofactor NADH. The sulforhodamine B assay gave valid results, but measures only protein content, providing no information about cell death in the remaining cells. The trypan blue assay and a microscope-based propidium iodide/Hoechst staining assay assess only late stage membrane permeability. However, the propidium iodide/Hoechst assay gives morphological information about cell death mechanism. A combination of the sulforhodamine B and propidium iodide/Hoechst assays would provide the most accurate quantification of honey cytotoxicity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    Science.gov (United States)

    Chandler, Kelly J; Barrier, Marianne; Jeffay, Susan; Nichols, Harriette P; Kleinstreuer, Nicole C; Singh, Amar V; Reif, David M; Sipes, Nisha S; Judson, Richard S; Dix, David J; Kavlock, Robert; Hunter, Edward S; Knudsen, Thomas B

    2011-01-01

    The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

  18. Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.

    Directory of Open Access Journals (Sweden)

    Kelly J Chandler

    Full Text Available The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7 and cytotoxicity (DRAQ5™/Sapphire700™ were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀ values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500 revealed significant associations for a subset of chemicals (26 that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.

  19. In vitro cytotoxicity of four calcium silicate-based endodontic cements on human monocytes, a colorimetric MTT assay

    Directory of Open Access Journals (Sweden)

    Sedigheh Khedmat

    2014-08-01

    Full Text Available Objectives This study was performed to evaluate the cytotoxicity of four calcium silicate-based endodontic cements at different storage times after mixing. Materials and Methods Capillary tubes were filled with Biodentine (Septodont, Calcium Enriched Mixture (CEM cement, BioniqueDent, Tech Biosealer Endo (Tech Biosealer and ProRoot MTA (Dentsply Tulsa Dental. Empty tubes and tubes containing Dycal were used as negative and positive control groups respectively. Filled capillary tubes were kept in 0.2 mL microtubes and incubated at 37℃. Each material was divided into 3 groups for testing at intervals of 24 hr, 7 day and 28 day after mixing. Human monocytes were isolated from peripheral blood mononuclear cells and cocultered with 24 hr, 7 day and 28 day samples of different materials for 24 and 48 hr. Cell viability was evaluated using an MTT assay. Results In all groups, the viability of monocytes significantly improved with increasing storage time regardless of the incubation time (p < 0.001. After 24 hr of incubation, there was no significant difference between the materials regarding monocyte viability. However, at 48 hr of incubation, ProRoot MTA and Biodentine were less cytotoxic than CEM cement and Biosealer (p < 0.01. Conclusions Biodentine and ProRoot MTA had similar biocompatibility. Mixing ProRoot MTA with PBS in place of distilled water had no effect on its biocompatibility. Biosealer and CEM cement after 48 hr of incubation were significantly more cytotoxic to on monocyte cells compared to ProRoot MTA and Biodentine.

  20. Development and validation of a nonaplex assay for the simultaneous quantitation of antibodies to nine Streptococcus pneumoniae serotypes.

    Science.gov (United States)

    Lal, Gouri; Balmer, Paul; Stanford, Elaine; Martin, Sarah; Warrington, Rosalind; Borrow, Ray

    2005-01-01

    Serum IgG levels specific for Streptococcus pneumoniae are currently quantified using the ELISA. However, this method has significant limitations in that a separate test is required for each serotype-specific antibody. Here, we describe a rapid and simple method for the simultaneous quantitation of IgG to nine pneumococcal serotypes (1, 4, 5, 6B, 9V, 14, 18C, 19F, 23F). Pneumococcal polysaccharides were covalently attached to fluorescent microspheres and these beads were serologically assessed using 89-SF standard serum. The multiplex assay was linear over a 24-fold serum dilution range and comparison of monoplex and nonaplex assays revealed no evidence of bead interference. The nonaplex assay was shown to be specific with <30% heterologous inhibition occurring and assay sensitivity was high with the LOD ranging between 32.3 and 109.7 pg/ml. The assay was shown to have low inter- and intra-assay variability and conjugated microspheres were shown to remain stable over 12 months. When samples were assayed there was a good correlation of the nonaplex assay with the ELISA. Finally, four bead sets coated with meningococcal polysaccharides (serogroups A, C, Y and W135) were added into the nonaplex assay, with no effect on the pneumococcal or meningococcal IgG levels generated. The pneumococcal nonaplex assay will prove to be a valuable tool to aid in the evaluation of pneumococcal capsular polysaccharide-based vaccines.

  1. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    Science.gov (United States)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  2. A simple protocol for using a LDH-based cytotoxicity assay to assess the effects of death and growth inhibition at the same time.

    Science.gov (United States)

    Smith, Shilo M; Wunder, Michael B; Norris, David A; Shellman, Yiqun G

    2011-01-01

    Analyzing the effects on cell growth inhibition and/or cell death has been an important component of biological research. The MTS assay and LDH-based cytotoxicity assays are two of the most commonly used methods for this purpose. However, data here showed that MTS cell proliferation assay could not distinguish the effects of cell death or cell growth inhibition. In addition, the original LDH-based cytotoxicity protocol grossly underestimated the proportion of dead cells in conditions with growth inhibition. To overcome the limitation, we present here a simple modified LDH-based cytotoxicity protocol by adding additional condition-specific controls. This modified protocol thus can provide more accurate measurement of killing effects in addition to the measurement of overall effects, especially in conditions with growth inhibition. In summary, we present here a simple, modified cytotoxicity assay, which can determine the overall effects, percentage of cell killing and growth inhibition in one 96-well based assay. This is a viable option for primary screening for many laboratories, and could be adapted for high throughput screening.

  3. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Viral and Bacterial Pathogens of Infectious Diarrhea

    Directory of Open Access Journals (Sweden)

    Ji Wang

    2014-01-01

    Full Text Available Diarrhea caused by viral and bacterial infections is a major health problem in developing countries. The purpose of this study is to develop a two-tube multiplex PCR assay using automatic electrophoresis for simultaneous detection of 13 diarrhea-causative viruses or bacteria, with an intended application in provincial Centers for Diseases Control and Prevention, China. The assay was designed to detect rotavirus A, norovirus genogroups GI and GII, human astrovirus, enteric adenoviruses, and human bocavirus (tube 1, and Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Campylobacter jejuni, Shigella, Yersinia, and Vibrio cholera (tube 2. The analytical specificity was examined with positive controls for each pathogen. The analytical sensitivity was evaluated by performing the assay on serial tenfold dilutions of in vitro transcribed RNA, recombinant plasmids, or bacterial culture. A total of 122 stool samples were tested by this two-tube assay and the results were compared with those obtained from reference methods. The two-tube assay achieved a sensitivity of 20–200 copies for a single virus and 102-103 CFU/mL for bacteria. The clinical performance demonstrated that the two-tube assay had comparable sensitivity and specificity to those of reference methods. In conclusion, the two-tube assay is a rapid, cost-effective, sensitive, specific, and high throughput method for the simultaneous detection of enteric bacteria and virus.

  4. The cytotoxic activity of cisplatin, carboplatin and teniposide alone and combined determined on four human small cell lung cancer cell lines by the clonogenic assay

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Christensen, I J

    1988-01-01

    Using the clonogenic assay to compare the cytotoxic activity of cisplatin and carboplatin on four human small cell lung cancer cell lines, cisplatin was shown to be equally or more potent than carboplatin at equitoxic doses with 1 h incubation. Increased potency of carboplatin was revealed when...

  5. Die Sulforhodamien B Seltellingsmetode in vergelyking met drie algemene seltellingsmetodes vir sitotoksisiteitstoetse/The Sulforhodamine B Assay in comparison with three commonly used cytotoxicity assays

    National Research Council Canada - National Science Library

    A van Tonder; A Joubert; A D Cromarty

    2014-01-01

      The sulforhodamine B (SRB) assay was compared to the MTT assay, the neutral red uptake assay and the resazurin reduction assay with regards to sensitivity, interference with glycolysis inhibitors, reproducibility and cost-effectiveness...

  6. The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism

    Directory of Open Access Journals (Sweden)

    Hoskins Clare

    2012-04-01

    Full Text Available Abstract Background Iron oxide magnetic nanoparticles (MNP's have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface coated with poly(ethylenimine (PEI and poly(ethylene glycol (PEG to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM, was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS production and lipid peroxidation (LPO, LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines. Results Successful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (−16.9 mV which increased to 40 nm (+55.6 mV upon coating with PEI and subsequently 50 nm (+31.2 mV with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling. Conclusions

  7. A simple method to measure cell viability in proliferation and cytotoxicity assays.

    Science.gov (United States)

    Borra, Ricardo Carneiro; Lotufo, Mônica Andrade; Gagioti, Sonia Maria; Barros, Fabiana de Mesquita; Andrade, Priscila Maria

    2009-01-01

    Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  8. Ferrocenes as potential chemotherapeutic drugs: Synthesis, cytotoxic activity, reactive oxygen species production and micronucleus assay

    Science.gov (United States)

    Pérez, Wanda I.; Soto, Yarelys; Ortíz, Carmen; Matta, Jaime; Meléndez, Enrique

    2014-01-01

    Three new ferrocene complexes were synthesized with 4-(1H-pyrrol-1-yl)phenol group appended to one of the Cp ring. These are: 1,1′-4-(1H-pyrrol-1-yl)phenyl ferrocenedicarboxylate, (“Fc-(CO2-Ph-4-Py)2”), 1,4-(1H-pyrrol-1-yl)phenyl, 1′-carboxyl ferrocenecarboxylate (“Fc-(CO2-Ph-4-Py)CO2H”) and 4-(1H-pyrrol-1-yl)phenyl ferroceneacetylate (“Fc-CH2CO2-Ph-4-Py”). The new species were characterized by standard analytical methods. Cyclic voltammetry experiments showed that Fc-CH2CO2-Ph-4-Py has redox potential very similar to the Fc/Fc+ redox couple whereas Fc-(CO2-Ph-4-Py)2 and Fc-(CO2-Ph-4-Py)CO2H have redox potentials of over 400 mV higher than Fc/Fc+ redox couple. The in vitro studies on Fc-(CO2-Ph-4-Py)2 and Fc-(CO2-Ph-4-Py)CO2H revealed that these two compounds have moderate anti-proliferative activity on MCF-7 breast cancer cell line. In contrast Fc-CH2CO2-Ph-4-Py which displayed low anti-proliferative activity. In the HT-29 colon cancer cell line, the new species showed low anti-proliferaive activity. Cytokinesis-block micronucleus assay (CBMN) was performed on these ferrocenes and it was determined they induce micronucleus formation on binucleated cells and moderate genotoxic effects on the MCF-7 breast cancer cell line. There is a correlation between the IC50 values of the ferrocenes and the amount of micronucleus formation activity on binucleated cells and the reactive oxygen species (ROS) production on MCF-7 cell line. PMID:25555734

  9. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra

    2009-09-01

    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  10. Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

    Science.gov (United States)

    Noya, Oscar; Losada, Sandra; Toledo, Marilyan; Gauna, Adriana; Lorenzo, María Angelita; Bermúdez, Henry; de Noya, Belkisyolé Alarcón

    2015-01-01

    This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

  11. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

    Science.gov (United States)

    van Geffen, Mark; Loof, Arnoud; Lap, Paul; Boezeman, Jan; Laros-van Gorkom, Britta A P; Brons, Paul; Verbruggen, Bert; van Kraaij, Marian; van Heerde, Waander L

    2011-11-01

    Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.

  12. Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin.

    Science.gov (United States)

    Kitpipit, Thitika; Thongjued, Kantima; Penchart, Kitichaya; Ouithavon, Kanita; Chotigeat, Wilaiwan

    2017-03-01

    Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate (N=140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space

    Data.gov (United States)

    U.S. Environmental Protection Agency — Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here,...

  14. Annatto Tocotrienol Induces a Cytotoxic Effect on Human Prostate Cancer PC3 Cells via the Simultaneous Inhibition of Src and Stat3.

    Science.gov (United States)

    Sugahara, Ryosuke; Sato, Ayami; Uchida, Asuka; Shiozawa, Shinya; Sato, Chiaki; Virgona, Nantiga; Yano, Tomohiro

    2015-01-01

    Prostate cancer is one of the most frequently occurring cancers and often acquires the potential of androgen-independent growth as a malignant phenotype. Androgen-independent prostate cancer has severe chemoresistance towards conventional chemotherapeutic agents, so a new treatment approach is required for curing such prostate cancer. In this context, the present study was undertaken to check if annatto tocotrienol (main component δ-tocotrienol) could suppress cell growth in human prostate cancer (PC3, androgen-independent type) cells via the inhibition of Src and Stat3. The tocotrienol showed cytotoxic effects on PC3 cells in a dose-dependent manner, and the effect depended on G1 arrest in the cell cycle and subsequent induction of apoptosis. In a cytotoxic dose, the tocotrienol suppressed cellular growth via the simultaneous inhibition of Src and Stat3. Similarly, the treatment combination of both Src and Stat3 inhibitors induced cytotoxic effects in PC3 cells in an additive manner compared to each by itself. With respect to cell cycle regulation and the induction of apoptosis, the combination treatment showed a similar effect to that of the tocotrienol treatment. These results suggest that annatto tocotrienol effectively induces cytotoxicity in androgen-independent prostate cancer cells via the suppression of Src and Stat3.

  15. Development of real-time PCR assay for simultaneous detection and genotyping of cystic echinococcosis in humans and livestock

    Directory of Open Access Journals (Sweden)

    Mohamed Elamin Ahmed

    2017-11-01

    Full Text Available Objective: To develop and evaluate a single-tube one-step real-time quantitative PCR (qPCR assay for simultaneous diagnosis and genotyping of cystic echinococcosis (CE in humans and livestock in the Sudan, and to compare it with conventional PCR assay. Methods: Hydatid cysts were obtained from slaughtered animals and from humans after surgical interventions. DNA from the hydatid cysts and associated germ layers was extracted using a commercially available kit. The mitochondrial NADH dehydrogenase subunit 1 (NAD1 was used as a target for PCR amplifications. qPCR and conventional nested PCR assays were compared in this study. Results: The qPCR assay amplified the NAD 1 gene of hydatid cysts on melting temperature generated at 80 °C. Ten-folds serial dilutions of DNA with known dilution of 1 × 106 to 1 × 101 (1 ng–1 fg resulted in detection of as little as 1 fg of DNA with an R2 value equivalent to 0.997. Similar sensitivities were encountered from both qPCR and the conventional nested PCR. The two assays did not amplify DNAs from Fasciola gigantica, Taenia saginata, Schistosoma bovis and DNA-free samples (negative controls. The PCR amplified products were purified for subsequent sequencing. The sequence data were analysed to insure the specificity of the amplified PCR products and to identify the genotype(s of hydatid cysts. All cysts were identified as E. canadensis genotype 6 (G6. Conclusions: The developed qPCR should be used as a rapid and reliable assay for diagnosis and genotyping of CE. The assay is highly recommended for the epidemiological surveillance in humans and livestock in endemic countries.

  16. A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.

    NARCIS (Netherlands)

    Geffen, M. van; Loof, A.H.; Lap, P.; Boezeman, J.B.M.; Laros-van Gorkom, B.A.P.; Brons, P.; Verbruggen, B.; Kraaij, M.G.J. van; Heerde, W.L. van

    2011-01-01

    Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and

  17. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    Science.gov (United States)

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.

    Science.gov (United States)

    Shin, Yoojin; Han, Sewoon; Jeon, Jessie S; Yamamoto, Kyoko; Zervantonakis, Ioannis K; Sudo, Ryo; Kamm, Roger D; Chung, Seok

    2012-06-07

    This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

  19. Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Rosemary S Turingan

    Full Text Available BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental

  20. Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins.

    Energy Technology Data Exchange (ETDEWEB)

    Jenko, Kathryn; Zhang, Yanfeng; Kostenko, Yulia; Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Marks, James D.; Varnum, Susan M.

    2014-10-21

    Plant and microbial toxins are considered bioterrorism threat agents because of their extreme toxicity and/or ease of availability. Additionally, some of these toxins are increasingly responsible for accidental food poisonings. The current study utilized an ELISA-based protein antibody microarray for the multiplexed detection of ten biothreat toxins, botulinum neurotoxins (BoNT) A, B, C, D, E, F, ricin, shiga toxins 1 and 2 (Stx), and staphylococcus enterotoxin B (SEB), in buffer and complex biological matrices. The multiplexed assay displayed a sensitivity of 1.3 pg/mL (BoNT/A, BoNT/B, SEB, Stx-1 and Stx-2), 3.3 pg/mL (BoNT/C, BoNT/E, BoNT/F) and 8.2 pg/mL (BoNT/D, ricin). All assays demonstrated high accuracy (75-120 percent recovery) and reproducibility (most coefficients of variation < 20%). Quantification curves for the ten toxins were also evaluated in clinical samples (serum, plasma, nasal fluid, saliva, stool, and urine) and environmental samples (apple juice, milk and baby food) with overall minimal matrix effects. The multiplex assays were highly specific, with little crossreactivity observed between the selected toxin antibodies. The results demonstrate a multiplex microarray that improves current immunoassay sensitivity for biological warfare agents in buffer, clinical, and environmental samples.

  1. Thaw-and-use target cells pre-labeled with calcein AM for antibody-dependent cell-mediated cytotoxicity assays.

    Science.gov (United States)

    Chung, Shan; Nguyen, Van; Lin, Yuwen Linda; Kamen, Lynn; Song, An

    2017-08-01

    In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assays are routinely performed to support the research and development of therapeutic antibodies. In ADCC assays, target cells bound by the antibodies are lysed by activated effector cells following interactions between the Fc region of the bound antibody and Fcγ receptors on effector cells. Target cell lysis is typically measured by quantification of released endogenous enzymes, e.g., lactate dehydrogenase, or measurement of released exogenous labels, e.g., (51)Cr, europium or calcein. ADCC assays based on the detection of exogenous labels released from lysed target cells generally show higher sensitivity and require shorter incubation times. However, target cells are usually labeled immediately prior to assay, which inadvertently introduces additional assay variations due to differences in target cell conditions and labeling/handling processes. In this report, we describe the use of thaw-and-use pre-labeled target cells for ADCC assays. Thaw-and-use target cells in our experiments were pre-labeled with the fluorescent dye calcein AM, cryopreserved in single-use aliquots and used directly in assays after thawing. Upon thaw, the pre-labeled cells displayed viability and label retention comparable to freshly labeled cells, responded to ADCC mediated by both peripheral blood mononuclear cells and engineered natural killer cells, performed stably for at least 3 years and provided favorable precision and accuracy to ADCC assays. Implementation of thaw-and-use pre-labeled target cells in ADCC assays can help to alleviate both cell culture and dye labeling derived variability, increase the flexibility of assay scheduling and improve assay consistency and robustness. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A validated assay for the simultaneous quantification of six tyrosine kinase inhibitors and two active metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

    NARCIS (Netherlands)

    Erp, N. van; Wit, D. de; Guchelaar, H.J.; Gelderblom, H.; Hessing, T.J.; Hartigh, J. den

    2013-01-01

    A sensitive, sophisticated and practical bioanalytical assay for the simultaneous determination of six tyrosine kinase inhibitors (imatinib, sunitinib, nilotinib, dasatinib, pazopanib, regorafenib) and two active metabolites (N-desmethyl imatinib and N-desethyl sunitinib) was developed and

  3. A versatile size-coded flow cytometric bead assay for simultaneous detection of multiple microRNAs coupled with a two-step cascading signal amplification.

    Science.gov (United States)

    Qiu, Liying; Zhang, Yuecheng; Liu, Chenghui; Li, Zhengping

    2017-03-07

    A versatile flow cytometric bead assay is developed for the sensitive and simultaneous detection of multiple microRNAs that combines an elegant enzyme-mediated cascading signal amplification on magnetic beads with a novel bead size-encoding mechanism.

  4. In vitro comparative cytotoxic effect of Nimbolide: A limonoid from Azadirachta indica (Neem tree) on cancer cell lines and normal cell lines through MTT assay.

    Science.gov (United States)

    Kashif, Muhammad; Hwang, Yawon; Hong, Gyeongmi; Kim, Gonhyung

    2017-05-01

    The present study was conducted to find the cytotoxicity in vitro of nimbolide, limonoids derivative of flowers and leaves from Azadirachta indica (neem tree) on the selected cell lines of cancer (Du-145, PC-3, A-549) and normal fibroblast cell lines (NIH3T3, CCD-18Co) using MTT assay. The cells were seeded in 96 multi-well tissue plate using different concentrations of nimbolide for 24hrs and 48hrs. The percentage of viability of cell lines was calculated by optical density obtained by micro plate reader and cytotoxic effect in term of IC50 value was determined by using linear regression analysis. The percentages of viability of cells treated with different concentrations of nimbolide were significantly lower (P0.05) between treated and the non-treated cells was observed. Nimbolide exerted time and dose dependent cytotoxic effect on the cancer lines and mild effect on the normal cell lines. It was further confirmed through PKH 26. Results of the present study suggested nimbolide as a potent chemotherapeutic and chemopreventive agent as it exerted a more cytotoxic effect on cancer cell lines as compared with the normal cell lines. Nimbolide may be a new hope as an anticancer drug in future.

  5. Development of a GeXP-multiplex PCR assay for the simultaneous detection and differentiation of six cattle viruses.

    Directory of Open Access Journals (Sweden)

    Qing Fan

    Full Text Available Foot-and-mouth disease virus (FMDV, Bluetongue virus (BTV, Vesicular stomatitis Virus (VSV, Bovine viral diarrheal (BVDV, Bovine rotavirus (BRV, and Bovine herpesvirus 1 (IBRV are common cattle infectious viruses that cause a great economic loss every year in many parts of the world. A rapid and high-throughput GenomeLab Gene Expression Profiler (GeXP analyzer-based multiplex PCR assay was developed for the simultaneous detection and differentiation of these six cattle viruses. Six pairs of chimeric primers consisting of both the gene-specific primer and a universal primer were designed and used for amplification. Then capillary electrophoresis was used to separate the fluorescent labeled PCR products according to the amplicons size. The specificity of GeXP-multiplex PCR assay was examined with samples of the single template and mixed template of six viruses. The sensitivity was evaluated using the GeXP-multiplex PCR assay on serial 10-fold dilutions of ssRNAs obtained via in vitro transcription. To further evaluate the reliability, 305 clinical samples were tested by the GeXP-multiplex PCR assay. The results showed that the corresponding virus specific fragments of genes were amplified. The detection limit of the GeXP-multiplex PCR assay was 100 copies/μL in a mixed sample of ssRNAs containing target genes of six different cattle viruses, whereas the detection limit for the Gexp-mono PCR assay for a single target gene was 10 copies/μL. In detection of viruses in 305 clinical samples, the results of GeXP were consistent with simplex real-time PCR. Analysis of positive samples by sequencing demonstrated that the GeXP-multiplex PCR assay had no false positive samples of nonspecific amplification. In conclusion, this GeXP-multiplex PCR assay is a high throughput, specific, sensitive, rapid and simple method for the detection and differentiation of six cattle viruses. It is an effective tool that can be applied for the rapid differential diagnosis

  6. Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I; Fernández, José Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

    2017-01-01

    A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

  7. A novel HRM assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance

    Science.gov (United States)

    Gelaye, Esayas; Mach, Lukas; Kolodziejek, Jolanta; Grabherr, Reingard; Loitsch, Angelika; Achenbach, Jenna E.; Nowotny, Norbert; Diallo, Adama; Lamien, Charles Euloge

    2017-01-01

    Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus]. The assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans. PMID:28216667

  8. Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay.

    Science.gov (United States)

    Pauly, Diana; Kirchner, Sebastian; Stoermann, Britta; Schreiber, Tanja; Kaulfuss, Stefan; Schade, Rüdiger; Zbinden, Reto; Avondet, Marc-André; Dorner, Martin B; Dorner, Brigitte G

    2009-10-01

    Proteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 microL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 microL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.

  9. Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers.

    Science.gov (United States)

    Kuwayama, Masaru; Shigemoto, Naoki; Oohara, Sachiko; Tanizawa, Yukie; Yamada, Hiroko; Takeda, Yoshihiro; Matsuo, Takeshi; Fukuda, Shinji

    2011-07-01

    We have developed simultaneous detection of eight genes associated with the five categories of diarrheagenic Escherichia coli by the multiplex PCR assay with Alexa Fluor-labeled primers. This assay can easily distinguish eight genes based on the size and color of amplified products without gel staining. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. A new assay for the simultaneous identification and differentiation of Klebsiella oxytoca strains.

    Science.gov (United States)

    Stojowska-Swędrzyńska, Karolina; Krawczyk, Beata

    2016-12-01

    Klebsiella oxytoca is the second most frequently identified species of Klebsiella isolated from hospitalized patients. Klebsiella spp. is difficult to identify using conventional methods and is often misclassified in clinical microbiology laboratories. K. oxytoca is responsible for an increasing number of multi-resistant infections in hospitals because of insufficient detection and identification. In this study, we propose a new simple method called pehX-LM PCR/XbaI, which simultaneously indicates K. oxytoca species and genotype by the fingerprint pattern. The pehX-LM PCR/XbaI is a combination of the following two methods: species-specific amplification of pehX gene and non-specific amplification of short restriction fragments by the LM PCR method. The specificity and the discrimination power of the pehX-LM PCR/XbaI method were determined by typing 209 K. oxytoca strains (included 9 reference strains), 28 K. pneumoniae, and other 25 strains belonging to the Enterobacteriaceae. The typing results were confirmed by the PCR melting profile method. Unlike the known fingerprinting methods, the pehX-LM PCR/XbaI leads to a clear pattern (approx. 3-5 bands) with a sufficient, relatively high discriminatory power. As a result, the time and cost of a single analysis are lower. The method can be used both in clinical and environmental research.

  11. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J

    1981-01-01

    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunolog...

  12. cis-Restricted 3-aminopyrazole analogues of combretastatins: synthesis from plant polyalkoxybenzenes and biological evaluation in the cytotoxicity and phenotypic sea urchin embryo assays.

    Science.gov (United States)

    Tsyganov, Dmitry V; Konyushkin, Leonid D; Karmanova, Irina B; Firgang, Sergei I; Strelenko, Yuri A; Semenova, Marina N; Kiselyov, Alex S; Semenov, Victor V

    2013-08-23

    We have synthesized a series of novel cis-restricted 4,5-polyalkoxydiaryl-3-aminopyrazole analogues of combretastatins via short synthetic sequences using building blocks isolated from dill and parsley seed extracts. The resulting compounds were tested in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM concentration, exhibiting microtubule-destabilizing properties. Compounds 12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multi-drug-resistant cancer cells.

  13. A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces.

    Science.gov (United States)

    Evangelopoulos, A; Spanakos, G; Patsoula, E; Vakalis, N; Legakis, N

    2000-04-01

    The detection of and differentiation between Entamoeba histolytica and Entamoeba dispar are of great importance, both for diagnosis and for epidemiological studies. Most PCR-based methods for the discrimination of these two species employ complex procedures for DNA extraction and require different protocols for E. histolytica and E. dispar, leading to relatively high expenditure, labour costs and turnaround times. A simple, rapid, cost-effective and yet sensitive and specific multiplex PCR technique has now been developed for the simultaneous detection and differentiation of E. histolytica and E. dispar in faecal samples. The detection limit is 200 trophozoites of E. dispar or 1000 trophozoites of E. histolytica/g stool sample. The sensitivity of the assay remains practically unchanged, even in the presence of 20,000 trophozoites of the other species/g stool sample. Thus, this technique may also easily reveal mixed infections, without the danger of misdiagnosis caused by one strain displacing the other in culture.

  14. Simultaneous high-performance liquid chromatography assay of acetylsalicylic acid and salicylic acid in film-coated aspirin tablets.

    Science.gov (United States)

    Fogel, J; Epstein, P; Chen, P

    1984-12-28

    A reversed-phase high-performance liquid chromatography (HPLC) method has been developed for the simultaneous assay of acetylsalicylic acid (I) and salicylic acid (II) in film-coated aspirin tablets. As little as 0.1% II (relative to I) can be quantitatively determined. Using a 5-microns octadecylsilane column with water-acetonitrile-phosphoric acid (76:24:0.5) as the mobile phase enabled the chromatographic separation to be completed in 4 min. Due to the slow rate of decomposition of I to II in the extraction solvent, acetonitrile-methanol-phosphoric acid (92:8:0.5), the analysis of many samples was routinely performed by means of automated HPLC equipment. Other compounds (non-aspirin salicylates, caffeine and acetaminophen) were also separated by the chromatographic system.

  15. Xanthium strumarium L. Extracts Produce DNA Damage Mediated by Cytotoxicity in In Vitro Assays but Does Not Induce Micronucleus in Mice

    Directory of Open Access Journals (Sweden)

    Janet Piloto Ferrer

    2014-01-01

    Full Text Available Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats. In CHO cells, high concentrations (25–100 μg/mL revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses. The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations.

  16. Xanthium strumarium L. Extracts Produce DNA Damage Mediated by Cytotoxicity in In Vitro Assays but Does Not Induce Micronucleus in Mice

    Science.gov (United States)

    Piloto Ferrer, Janet; Cozzi, Renata; Cornetta, Tommaso; Stano, Pasquale; Fiore, Mario; Degrassi, Francesca; De Salvia, Rosella; Remigio, Antonia; Francisco, Marbelis; Quiñones, Olga; Valdivia, Dayana; González, Maria L.; Pérez, Carlos; Sánchez-Lamar, Angel

    2014-01-01

    Xanthium strumarium L. is a member of the Asteraceae commonly used in Cuba, mainly as diuretic. Some toxic properties of this plant have also been reported and, to date, very little is known about its genotoxic properties. The present work aims was to evaluate the potential cytotoxic and genotoxic risk of whole extract from Xanthium strumarium L. whole extract of aerial parts. No positive response was observed in a battery of four Salmonella typhimurium strains, when exposed to concentrations up to 5 mg/plate, with and without mammalian metabolic activation (liver microsomal S9 fraction from Wistar rats). In CHO cells, high concentrations (25–100 μg/mL) revealed significant reduction in cell viability. Results from sister chromatid exchanges, chromosome aberrations, and comet assay showed that X. strumarium extract is genotoxic at the highest concentration used, when clear cytotoxic effects were also observed. On the contrary, no increase in micronuclei frequency in bone marrow cells was observed when the extract was orally administered to mice (100, 500, and 2000 mg/Kg doses). The data presented here constitute the most complete study on the genotoxic potential of X. strumarium L. and show that the extract can induce in vitro DNA damage at cytotoxic concentrations. PMID:25025061

  17. Simultaneous screening of multiple mutations by invader assay improves molecular diagnosis of hereditary hearing loss: a multicenter study.

    Directory of Open Access Journals (Sweden)

    Shin-ichi Usami

    Full Text Available Although etiological studies have shown genetic disorders to be a common cause of congenital/early-onset sensorineural hearing loss, there have been no detailed multicenter studies based on genetic testing. In the present report, 264 Japanese patients with bilateral sensorineural hearing loss from 33 ENT departments nationwide participated. For these patients, we first applied the Invader assay for screening 47 known mutations of 13 known deafness genes, followed by direct sequencing as necessary. A total of 78 (29.5% subjects had at least one deafness gene mutation. Mutations were more frequently found in the patients with congenital or early-onset hearing loss, i.e., in those with an awareness age of 0-6 years, mutations were significantly higher (41.8% than in patients with an older age of awareness (16.0%. Among the 13 genes, mutations in GJB2 and SLC26A4 were mainly found in congenital or early-onset patients, in contrast with mitochondrial mutations (12S rRNA m.1555A>G, tRNA(Leu(UUR m.3243A>G, which were predominantly found in older-onset patients. The present method of simultaneous screening of multiple deafness mutations by Invader assay followed by direct sequencing will enable us to detect deafness mutations in an efficient and practical manner for clinical use.

  18. Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX(®) test.

    Science.gov (United States)

    Kouvelis, Vassili N; Wang, Chengshu; Skrobek, Anke; Pappas, Katherine M; Typas, Milton A; Butt, Tariq M

    2011-05-18

    The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites. 2011 Elsevier B.V. All rights reserved.

  19. Development of a highly sensitive cytotoxicity assay for the detection of immune responses against tumor associated antigens

    OpenAIRE

    Stanke, Jonas

    2012-01-01

    Cytotoxic CD8+ T cells (CTL) recognize specific intracellular peptides of processed antigens in the context of certain major histocompatibility complexes class I (MHC class I) or human leukocyte antigens class I (HLA-A, B, C) and can lyse these cells. Therefore CTL effectors are essential to eliminate such virus-infected cells or tumor cells and their presence provides information about a current event or the immune potential. The functional characterization of usually very...

  20. Cytotoxic evaluation of medicinal smoke \\"Anbar Nasara\\" against cancer cells (Hela & KB and a normal cell line (L929, using MTT assay

    Directory of Open Access Journals (Sweden)

    hojat Sadeghi-aliabadi

    2013-09-01

    Full Text Available Background: Medicinal smoke has been in natural health products by many nations all over the worlds. Dung's smoke called "Anbar Nasara" is one of the most used medicinal smokes in Iranian traditional medicine. Anber Nasara has been administered as inhalation in the treatment of viral or bacterial infections as well as for tumors and cysts in vaginal diseases in women. Its cytotoxic effects have been proposed in these studies. Materials and Methods: Dung's of female donkey have been collected from Shahreza, Isfahan province in summer 2011. The samples were burned in a specially designed apparatus and its smoke was collected in n-hexane solvent over ice-cold water. Solvent was evaporated in the air and dried residue was dissolved in DMSO and diluted in RPMI So that the final concentrations of 0.4, 0.3, 0.25, 0.2, 0.125, 0.1, 0.0625, 0.0312 mg/ml were obtained for MTT assay as a cytotoxic evaluation method against two cancer cells (KB and Hela and a normal one (L929. After 48h incubation percent cell survival and IC50 values were obtained using ELISA plate reader at 540nm. Results: Results showed that dung's smoke were cytotoxic in tested concentrations against cancer cell lines in a dose dependent manner. The IC50 was calculated ≥ 0.2 mg/ml. Conclusion: Dung's smoke consists of too many constituents and its cytotoxic effect may belong to one or complexes of constituents. More researches are under way to clarify these aspects of the study.

  1. The histone deacetylase inhibitor SAHA simultaneously reactivates HIV-1 from latency and up-regulates NKG2D ligands sensitizing for natural killer cell cytotoxicity.

    Science.gov (United States)

    Desimio, Maria Giovanna; Giuliani, Erica; Doria, Margherita

    2017-10-01

    In pilot HIV-1 eradication studies, patients' immune responses were ineffective at killing viral reservoirs reactivated through latency reversing agents (LRAs) like suberoylanilide hydroxamic acid (SAHA). We hypothesized that T cells harboring reactivated HIV-1 express MIC and ULBP ligands for the activating NKG2D receptor of natural killer (NK) cells. Here, we demonstrated that MICA/B and ULBP2 are induced by SAHA on primary T cells harboring reactivated virus. Using latently HIV-1-infected J-Lat 6.3/8.4/9.2 and J1.1 cell lines, we showed that SAHA reverts latency and, simultaneously, up-regulates MICA/B and ULBP2 acting at the transcriptional level and through ATR activation, thus sensitizing T cells with reactivated virus to NKG2D-mediated killing by NK cells. Moreover, IL-2 and IL-15 potently boosted NKG2D expression and cytotoxicity of NK cells against SAHA-reactivated p24(+) target cells. Therefore, immunotherapy with cytokines enhancing NKG2D-mediated NK-cell cytotoxicity combined with administration of LRAs up-modulating NKG2D ligands, represents a promising approach towards HIV-1 eradication. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Development and Validation of a Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Three Papaya Viruses

    Directory of Open Access Journals (Sweden)

    Decai Tuo

    2014-10-01

    Full Text Available Papaya ringspot virus (PRSV, Papaya leaf distortion mosaic virus (PLDMV, and Papaya mosaic virus (PapMV produce similar symptoms in papaya. Each threatens commercial production of papaya on Hainan Island, China. In this study, a multiplex reverse transcription PCR assay was developed to detect simultaneously these three viruses by screening combinations of mixed primer pairs and optimizing the multiplex RT-PCR reaction conditions. A mixture of three specific primer pairs was used to amplify three distinct fragments of 613 bp from the P3 gene of PRSV, 355 bp from the CP gene of PLDMV, and 205 bp from the CP gene of PapMV, demonstrating the assay’s specificity. The sensitivity of the multiplex RT-PCR was evaluated by showing plasmids containing each of the viral target genes with 1.44 × 103, 1.79 × 103, and 1.91 × 102 copies for the three viruses could be detected successfully. The multiplex RT-PCR was applied successfully for detection of three viruses from 341 field samples collected from 18 counties of Hainan Island, China. Rates of single infections were 186/341 (54.5%, 93/341 (27.3%, and 3/341 (0.9%, for PRSV, PLDMV, and PapMV, respectively; 59/341 (17.3% of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting multiple viruses in papaya and can be used for routine molecular diagnosis and epidemiological studies in papaya.

  3. Evaluation of an Immunochromatographic Assay for the Rapid and Simultaneous Detection of Rotavirus and Adenovirus in Stool Samples

    Science.gov (United States)

    Kim, Jayoung; Kim, Han-Sung; Kim, Jae-Seok; Song, Wonkeun; Lee, Kyu Man; Lee, Sunhwa; Park, Kyoung Un; Lee, Woochang; Hong, Young Jun

    2014-01-01

    Background We evaluated the analytical and clinical performances of the SD BIOLINE Rota/Adeno Rapid kit (SD Rota/Adeno Rapid; Standard Diagnostics, Inc., Korea), an immunochromatographic assay (ICA), for the simultaneous detection of rotaviruses and adenoviruses in human stool samples. Methods We tested 400 clinical stool samples from patients with acute gastroenteritis and compared the ICA results with the results obtained by using ELISA, enzyme-linked fluorescent assays (ELFA), PCR, and multiplex reverse transcription-PCR (mRT-PCR). To assess the analytical performance of the SD BIOLINE Rota/Adeno Rapid kit, we determined its detection limit, reproducibility, cross-reactivity, and analytical reactivity for adenovirus subtypes, and performed interference studies. Results The overall agreement rates among the tested methods were 91.5% for rotavirus and 85.5% for adenovirus. On the basis of mRT-PCR, the overall agreement, positive agreement, and negative agreement rates of the ICA were 95.6%, 100%, and 94.9% for rotavirus, and 94.0%, 71.4%, and 94.8% for adenovirus, respectively. Using the ICA, we detected all the subtypes of adenovirus tested, but the analytical reactivities for adenovirus subtypes were different between the 4 adenovirus detection methods. The high reproducibility was confirmed, and no cross-reactivity or interference was detected. Conclusions The SD BIOLINE Rota/Adeno Rapid kit showed acceptable analytical and clinical performances. However, interpretation of adenovirus positive/negative result should be cautious because of different detectability for adenovirus subtypes among adenovirus detection methods. PMID:24790909

  4. Cytotoxic and genotoxic effects of resin monomers in human salivary gland tissue and lymphocytes as assessed by the single cell microgel electrophoresis (Comet) assay.

    Science.gov (United States)

    Kleinsasser, Norbert H; Schmid, Katharina; Sassen, Andrea W; Harréus, Ulrich A; Staudenmaier, Rainer; Folwaczny, Matthias; Glas, Jürgen; Reichl, Franz-Xaver

    2006-03-01

    Malignant tumors of the three major pairs and the numerous minor salivary glands in humans are rare, and little is known about their various etiologies. Considering the fact that resin monomers from dental restorative materials are released into the saliva and diffuse into the tooth pulp or gingiva, mucosa, and salivary glands, this may potentially contribute to tumorigenesis. Resin monomers may also be reabsorbed and reach the circulating blood as well. Whereas the cytotoxic potential of some components has been clearly documented, data on genotoxicity in human target cells require further investigation. In the present study, genotoxic and cytotoxic effects of three common methacrylates are investigated in human samples of salivary glands and peripheral lymphocytes. The Comet assay was used to quantify DNA single strand breaks, alkali labile and incomplete excision repair sites in salivary gland probes and lymphocytes of 10 volunteers. The xenobiotics investigated were triethyleneglycoldimethacrylate (TEGDMA), urethanedimethacrylate (UDMA), and 2-hydroxyethylmethacrylate (HEMA), with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) as controls. DNA migration was analyzed using the tail moment according to Olive (OTM). Cytotoxicity was monitored using trypan blue staining. With TEGDMA concentrations at 10(-5)m (10(-3)m), UDMA at 10(-7)m (10(-7)m), and HEMA at 10(-3)m (10(-5)m) significant enhancements of DNA migration were achieved in tissue cells (lymphocytes) as compared to the negative controls. At higher concentrations of up to 2.5x10(-2)m, induced DNA migration was expressed by OTM at 10.7 for TEGDMA in tissue cells (8.7 in lymphocytes), 10.5 for UDMA (6.4), and 9.7 for HEMA (6.1). The viability of the cell systems was not affected as concerns the threshold level for the assay of 75% viable cells except for the highest concentration tested for TEGDMA and UDMA in tissue cells. At higher concentration levels, all tested substances

  5. Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

    Science.gov (United States)

    Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

    2014-11-01

    The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. © 2014 FEBS.

  6. A rapid, accurate and robust particle-based assay for the simultaneous screening of plasma samples for the presence of five different anti-cytokine autoantibodies

    DEFF Research Database (Denmark)

    Guldager, Daniel Kring Rasmussen; von Stemann, Jakob Hjorth; Larsen, Rune

    2015-01-01

    PURPOSE: To establish and validate a rapid, cost-effective and accurate screening assay for the simultaneous testing of human naturally occurring anti-cytokine autoantibodies (c-aAb) targeting interleukin-1α (IL-1α), interleukin-6 (IL-6), interleukin-10 (IL-10), granulocyte-macrophage colony...

  7. Safety evaluation of Chlorella sorokiniana strain CK-22 based on an in vitro cytotoxicity assay and a 13-week subchronic toxicity trial in rats.

    Science.gov (United States)

    Himuro, Sayaka; Ueno, Sugi; Noguchi, Naoto; Uchikawa, Takuya; Kanno, Toshihiro; Yasutake, Akira

    2017-08-01

    The genus Chlorella contains unicellular green algae that have been used as food supplements. The purpose of this work was to evaluate the safety of the Chlorella sorokiniana strain CK-22 using powdered preparation (CK-22P) both by in vitro and in vivo assays. These included an experiment for cytotoxicity using Chinese hamster lung fibroblasts (V79 cells) and a 13-week repeated-dose oral toxicity trial using Wistar rats. The cytotoxicity was evaluated by MTT assay of a hot water extract (Hw-Ex) and 80% ethanol extract (Et-Ex) of CK-22P, and no effect on cell viability was observed. The 50% viability inhibitory effect (IC50) value for Hw-Ex and Et-Ex were estimated as greater than 73 and 17 μg/ml, respectively. In the subchronic toxicity test, pelleted rodent diet containing 0%, 2.5%, 5% or 10% CK-22P was given to Wistar rats (ten animals/sex/groups) for 13 weeks. During the experimental period, no CK-22P treatment-induced differences in general condition, body weight gain, food and water consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, histopathology, or animal death were observed. The no-observed-adverse-effect levels (NOAEL) were estimated to be 5.94 g/kg body-weight/day for males and 6.41 g/kg body-weight/day for females. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Simultaneous HPLC assay for pretomanid (PA-824), moxifloxacin and pyrazinamide in an inhaler formulation for drug-resistant tuberculosis.

    Science.gov (United States)

    Momin, Mohammad A M; Thien, Sim J; Krittaphol, Woravimol; Das, Shyamal C

    2017-02-20

    A simple and sensitive reversed phase HPLC method has been developed for the simultaneous quantitation of pretomanid (PA-824), moxifloxacin and pyrazinamide in a combination spray-dried powder formulation for inhalation, without any use of an internal standard. Good resolution of the analytes was achieved on a Luna C18 (2), 150×4.6mm, 5μm, 100Å column using gradient elution with a mobile phase containing methanol and triethylamine phosphate buffer (pH 2.5) at a flow rate of 1.0mL/min in a total run time of 25min. Pyrazinamide, moxifloxacin and pretomanid (PA-824) were detected at wavelengths (retention times) of 269nm (3.80min), 296nm (7.94min) and 330nm (17.46min), respectively. The assay was linear for all analytes in the concentration range 2.5-100μg/mL (correlation coefficients >0.999) with LODs and LLOQs (μg/mL) of pretomanid (PA-824) 0.51 and 1.56, moxifloxacin 0.06 and 0.19 and pyrazinamide 0.35 and 1.06, respectively. Recoveries of the three drugs were 99.6-106.8% with intra- and inter-day precisions (as relative standard deviation) of <7%. The method was successfully applied to an evaluation of content uniformity and freedom from interference by l-leucine of a spray-dried combination powder for inhalation. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Verification of the ProPneumo-1 assay for the simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in clinical respiratory specimens.

    Science.gov (United States)

    Higgins, Rachel R; Lombos, Ernesto; Tang, Patrick; Rohoman, Karl; Maki, Anne; Brown, Shirley; Jamieson, Frances; Drews, Steven J

    2009-03-10

    Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples. The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay. A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea). Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool. The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.

  10. Verification of the ProPneumo-1 assay for the simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in clinical respiratory specimens

    Directory of Open Access Journals (Sweden)

    Higgins Rachel R

    2009-03-01

    Full Text Available Abstract Background Mycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples. Objective The aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay. Methods A total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands. We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea. Results Following discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool. Conclusion The ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.

  11. Differential toxicity of Disperse Red 1 and Disperse Red 13 in the Ames test, HepG2 cytotoxicity assay, and Daphnia acute toxicity test.

    Science.gov (United States)

    Ferraz, E R A; Umbuzeiro, G A; de-Almeida, G; Caloto-Oliveira, A; Chequer, F M D; Zanoni, M V B; Dorta, D J; Oliveira, D P

    2011-10-01

    Azo dyes are of environmental concern due to their degradation products, widespread use, and low-removal rate during conventional treatment. Their toxic properties are related to the nature and position of the substituents with respect to the aromatic rings and amino nitrogen atom. The dyes Disperse Red 1 and Disperse Red 13 were tested for Salmonella mutagenicity, cell viability by annexin V, and propidium iodide in HepG2 and by aquatic toxicity assays using daphnids. Both dyes tested positive in the Salmonella assay, and the suggestion was made that these compounds induce mainly frame-shift mutations and that the enzymes nitroreductase and O-acetyltransferase play an important role in the observed effect. In addition, it was shown that the presence of the chlorine substituent in Disperse Red 13 decreased the mutagenicity about 14 times when compared with Disperse Red 1, which shows the same structure as Disperse Red 13, but without the chlorine substituent. The presence of this substituent did not cause cytotoxicity in HepG2 cells, but toxicity to the water flea Daphnia similis increased in the presence of the chlorine substituent. These data suggest that the insertion of a chlorine substituent could be an alternative in the design of dyes with low-mutagenic potency, although the ecotoxicity should be carefully evaluated. Copyright © 2010 Wiley Periodicals, Inc.

  12. Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples.

    Science.gov (United States)

    Xirogianni, Athanasia; Tzanakaki, Georgina; Karagianni, Eleni; Markoulatos, Panayiotis; Kourea-Kremastinou, Jenny

    2009-02-01

    This study describes the development and evaluation of a multiplex single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. used as target species-specific or genus-specific genes. The assay enables the detection of 5 to 50 pg of bacterial DNA. The sensitivity of the assay was evaluated as 100% for P. aeruginosa, S. aureus, and Streptococcus spp., and 94.3% for H. influenzae; the specificity was 100% for all 4 microorganisms (positive predictive value, 100%; negative predictive value, 98.2%). The assay permits rapid and accurate detection of these 4 microorganisms in a wide range of clinical samples such as whole blood, cerebrospinal, ear, pleural and ophthalmic fluids, as well as bronchoalveolar lavage and bronchial secretions.

  13. Simultaneous detection and differentiation of three Potyviridae viruses in sweet potato by a multiplex TaqMan real time RT-PCR assay.

    Science.gov (United States)

    Lan, Pingxiu; Li, Fan; Abad, Jorge; Pu, Lingling; Li, Ruhui

    2018-02-01

    A multiplex TaqMan real time RT-PCR was developed for detection and differentiation of Sweet potato virus G, Sweet potato latent virus and Sweet potato mild mottle virus in one tube. Amplification and detection of a fluorogenic cytochrome oxidase gene was included as an internal control. The assay was compared with a multiplex RT-PCR developed in the initial study for the detection and differentiation of the three viruses and host 18S rRNA. Primers and/or probes of the two assays were designed from conserved regions of each virus. The two assays were optimized for primers/probes and primer concentrations and thermal cycling conditions. Sensitivity and specificity of the assays were compared each other and with other assay. Both assays were evaluated by 74 field samples original from five different provinces of China. showed that the TaqMan real time RT-PCR offered rapid, sensitive, effective and reliable for the simultaneous detection and differentiation of the three viruses in sweet potato plants. The assay will be useful to quarantine and certification programs and virus surveys when large numbers of samples are tested. Copyright © 2017. Published by Elsevier B.V.

  14. The development of a GeXP-based multiplex reverse transcription-PCR assay for simultaneous detection of sixteen human respiratory virus types/subtypes

    Directory of Open Access Journals (Sweden)

    Li Jin

    2012-08-01

    Full Text Available Abstract Background Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. Methods A GeXP-based multiplex RT-PCR assay (GeXP assay was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay. Results The GeXP assay achieved a sensitivity of 20–200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51% of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours. Conclusions In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

  15. A validated high-performance thin-layer chromatography method for the identification and simultaneous quantification of six markers from Platanus orientalis and their cytotoxic profiles against skin cancer cell lines.

    Science.gov (United States)

    Khan, Imran; Sangwan, Payare L; Dar, Alamgir A; Rafiq, Rather A; Farrukh, Mufti R; Dhar, Jagdish K; Tasduq, Sheikh A; Koul, Surrinder

    2013-08-01

    Betulinic acid (1), betulinic acid-3-acetate (2), 3-acetylbetulinaldehyde (3), oleanolic acid-3-acetate (4), 3-β-hydroxy-28,19-β-olenolide (5), and β-sitosterol (6) were isolated from Platanus orientalis and a high-performance thin-layer chromatography method was developed for their simultaneous quantification. The markers were first derivatized on the chromatogram with ceric ammonium sulfate and then high-performance thin-layer chromatography densitometry was carried out. Chromatographic separation of these markers was carried out on silica gel 60 plates using a ternary solvent system n-hexane/toluene/acetone (6:3.5:1 v/v/v) as a mobile phase. For marker 1, a deuterium (D2) lamp and wavelength of 420 nm was used. A tungsten (W) lamp was used for markers 2 and 3 at 550 nm and for 4-6 at 500 nm. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.9919). The precision evaluated by an intra- and interday study showed RSDs < 2.51% and accuracy validation recovery between 95.54 and 99.33% with RSDs < 1.55%. The successful application of the validated method showed 1 as the most abundant component (4.63%) and 5 (0.017%) the least. The markers displayed a significant cytotoxic effect against human keratinocyte, mouse melanoma, and human skin epithelial carcinoma cancer cells by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    Science.gov (United States)

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

  17. Developmental validation of a real-time PCR assay for the simultaneous quantification of total human and male DNA.

    Science.gov (United States)

    Krenke, Benjamin E; Nassif, Nadine; Sprecher, Cynthia J; Knox, Curtis; Schwandt, Melissa; Storts, Douglas R

    2008-12-01

    Multiplex human short tandem repeat analysis demands reliable DNA quantification to consistently produce interpretable genotypes. The Plexor HY System is a multiplex quantitative PCR assay to quantify total human and male DNA. We performed developmental validation of the Plexor HY System to demonstrate the performance capabilities and limitations of the assay for forensic applications. Validation studies examined: (a) human specificity, (b) sensitivity, (c) quantification of degraded DNA, (d) impact of inhibitors, (e) male/female mixture and Y-assay male specificity, (f) reproducibility and concordance and (g) population studies.

  18. Use of in vitro assays to assess the potential antiproliferative and cytotoxic effects of saffron (Crocus sativus L.) in human lung cancer cell line

    Science.gov (United States)

    Samarghandian, Saeed; Boskabady, Mohammad Hossein; Davoodi, Saideh

    2010-01-01

    Background: Saffron is harvested from the dried, dark red stigmas of Crocus sativus flowers. It is used as a spice for flavoring and coloring food as a perfume. It is often used for treating several diseases. We investigated the potential of the ethanolic extract of saffron to induce antiproliferative and cytotoxic effects in cultured carcinomic human alveolar basal epithelial cells in comparison with non-malignant (L929) cells. Materials and Methods: Both cells were cultured in Dulbecco’s modified Eagle’s medium and treated with the ethanolic extract of saffron at various concentrations for two consecutive days. Our study resulted in sequences of events marked by apoptosis, such as loss of cell viability, morphology changes that were evaluated by MTT assay and invert-microscope, respectively. Results: The results showed that the ethanolic extract of saffron decreased cell viability in malignant cells as a concentration and time-dependent manner. The IC 50 values against the lung cancer cell line were determined as 1500 and 565 μg/ml after 24 and 48 h, respectively. However, the extract at different concentrations could not significantly decrease the cell viability in L929 cells. Morphology of MCF7 cells treated with the ethanolic extract confirmed the MTT results. Conclusion: We also showed that even higher concentrations of saffron is safe for L929, but the extract exerts pro-apoptotic effects in a lung cancer-derived cell line and could be considered as a potential chemotherapeutic agent in lung cancer. PMID:21120034

  19. Synthesis, binding assays, cytotoxic activity and docking studies of benzimidazole and benzothiophene derivatives with selective affinity for the CB2 cannabinoid receptor.

    Science.gov (United States)

    Romero-Parra, Javier; Mella-Raipán, Jaime; Palmieri, Vittoria; Allarà, Marco; Torres, Maria Jose; Pessoa-Mahana, Hernán; Iturriaga-Vásquez, Patricio; Escobar, Rossy; Faúndez, Mario; Di Marzo, Vincenzo; Pessoa-Mahana, C David

    2016-11-29

    Herein we report the design, synthesis, bioinformatic and biological studies of benzimidazole and benzothiophene derivatives as new cannabinoid receptor ligands. To test the hypothesis that the lack of a hydrogen bond interaction between benzimidazole and benzothiophene derivatives with Lys192 reduces their affinity for CB1 receptors (as we previously reported) and leads to CB2 selectivity, most of the tested compounds do not exhibit hydrogen bond acceptors. All compounds displayed mostly CB2 selectivity, although this was more pronounced in the benzimidazoles derivatives. Furthermore, docking assays revealed a ∏-cation interaction with Lys109 which could play a key role for the CB2 selectivity index. The series displayed low toxicity on five different cell lines. Derivative 8f presented the best binding profile (Ki = 0.08 μM), high selectivity index (KiCB1/KiCB2) and a low citoxicity. Interestingly, in cell viability experiments, using HL-60 cells (expressing exclusively CB2 receptors), all synthesised compounds were shown to be cytotoxic, suggesting that a CB2 agonist response may be involved. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Multiplex Assay for Simultaneous Detection of Mycoplasma genitalium and Macrolide Resistance Using PlexZyme and PlexPrime Technology.

    Directory of Open Access Journals (Sweden)

    Sepehr N Tabrizi

    Full Text Available Mycoplasma genitalium is a cause of non-gonoccocal urethritis (NGU in men and cervicitis and pelvic inflammatory disease in women. Recent international data also indicated that the first line treatment, 1 gram stat azithromycin therapy, for M. genitalium is becoming less effective, with the corresponding emergence of macrolide resistant strains. Increasing failure rates of azithromycin for M. genitalium has significant implications for the presumptive treatment of NGU and international clinical treatment guidelines. Assays able to predict macrolide resistance along with detection of M. genitalium will be useful to enable appropriate selection of antimicrobials to which the organism is susceptible and facilitate high levels of rapid cure. One such assay recently developed is the MG 23S assay, which employs novel PlexZyme™ and PlexPrime™ technology. It is a multiplex assay for detection of M. genitalium and 5 mutations associated with macrolide resistance. The assay was evaluated in 400 samples from 254 (186 males and 68 females consecutively infected participants, undergoing tests of cure. Using the MG 23S assay, 83% (331/440 of samples were positive, with 56% of positives carrying a macrolide resistance mutation. Comparison of the MG 23S assay to a reference qPCR method for M. genitalium detection and high resolution melt analysis (HRMA and sequencing for detection of macrolide resistance mutations, resulted in a sensitivity and specificity for M. genitalium detection and for macrolide resistance of 99.1/98.5% and 97.4/100%, respectively. The MG 23S assay provides a considerable advantage in clinical settings through combined diagnosis and detection of macrolide resistance.

  1. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

    Directory of Open Access Journals (Sweden)

    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  2. A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria.

    Science.gov (United States)

    Zhang, Chen; Niu, Peihua; Hong, Yanying; Wang, Ji; Zhang, Jingyun; Ma, Xuejun

    2015-11-01

    We aim to develop a multiplex real-time PCR assay to detect the most common pathogens causing community outbreaks of diarrhea. Four reaction systems of fluorescence dye-based real-time PCR assay were performed to amplify genes of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio parahaemolyticus, Salmonella spp., Escherichia coli, and Shigella spp. PCR products of each pathogen were identified by characteristic peaks in melting curves. The assay was able to achieve detection limit of 50 copies/reaction for each individual virus target, and 140-500CFU/mL for each individual bacterium target. A total of 122 clinical specimens from hospitalized children with acute diarrhea were used to evaluate the assay. The clinical sensitivity was very similar to that of reference methods. Norovirus genogroup II revealed the highest detectable rate (45/122, 36.9%). Coinfection was found in 28 out of 122 (23%) clinical specimens. This assay proved to be a cost-effective, sensitive and reliable method for simultaneous detection of enteric viruses and bacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients

    Directory of Open Access Journals (Sweden)

    Elena L. Enache

    2015-09-01

    Full Text Available Chronic hepatitis C (CHC is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3 gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724–1 accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism. This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

  4. A duplex real-time PCR assay based on TaqMan technology for simultaneous detection and differentiation of canine adenovirus types 1 and 2.

    Science.gov (United States)

    Dowgier, Giulia; Mari, Viviana; Losurdo, Michele; Larocca, Vittorio; Colaianni, Maria Loredana; Cirone, Francesco; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio; Decaro, Nicola

    2016-08-01

    Canine adenoviruses are a major cause of disease in dogs, coyotes, red foxes and wolves, as well as in other carnivores and marine mammals. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB), respectively. In this study, a duplex real-time PCR assay for simultaneous detection and characterisation of CAdV-1 and CAdV-2 was developed by using a single primer pair and virus-specific probes. The assay was validated testing standard DNAs produced on purpose and clinical samples of various matrices known to be positive for CAdV-1, CAdV-2 or both viruses. Precise calculation of DNA loads in samples containing a wide range of viral amounts was allowed by generating a standard curve for absolute quantification. The assay was proven to be highly specific, since no cross-reactions with the different CAdV type was observed, and sensitive, being able to detect less than 10 copies of CAdV-1/CAdV-2 DNA. The low intra-assay and interassay coefficient of variations demonstrated a high repeatability, thus confirming the potential use of this assay for quantitative detection of CAdV-1 and CAdV-2 for rapid diagnosis and epidemiological investigations. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Simultaneously typing nine serotypes of enteroviruses associated with hand, foot, and mouth disease by a GeXP analyzer-based multiplex reverse transcription-PCR assay.

    Science.gov (United States)

    Hu, Xiumei; Zhang, Yong; Zhou, Xiaomian; Xu, Banglao; Yang, Mengjie; Wang, Miao; Zhang, Chen; Li, Jin; Bai, Ruyin; Xu, Wenbo; Ma, Xuejun

    2012-02-01

    Hand, foot, and mouth disease (HFMD) is a contagious enteroviral disease occurring primarily in young children and caused by enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and other serotypes of coxsackievirus and echovirus. In this study, a GeXP analyzer-based multiplex reverse transcription (RT)-PCR assay (GeXP assay) consisting of chimeric primer-based PCR amplification with fluorescent labeling and capillary electrophoresis separation was developed to simultaneously identify nine serotypes of enteroviruses associated with HFMD in China, including EV71, CVA16, CVA4, -5, -9, and -10, and CVB1, -3, and -5. The RNAs extracted from cell cultures of viral isolates and synthetic RNAs via in vitro transcription were used to analyze the specificity and sensitivity of the assay. The GeXP assay detected as little as 0.03 tissue culture infective dose (TCID(50)) of EV71 and CVA16, 10 copies of panenterovirus, EV71, CVA16, CVB1, and CVB5, and 100 copies of 10 (including panenterovirus) premixed RNA templates. A total of 180 stool specimens collected from HFMD patients and persons suspected of having HFMD were used to evaluate the clinical performance of this assay. In comparison with the results of conventional methods, the sensitivities of the GeXP assay for detection of panenterovirus, EV71, and CVA16 were 98.79% (163/165), 91.67% (44/48), and 91.67% (33/36), respectively, and the specificities were 80.00% (12/15), 98.48% (130/132), and 100% (144/144), respectively. The concordance of typing seven other serotypes of enteroviruses with the results of conventional methods was 92.59% (25/27). In conclusion, the GeXP assay is a rapid, cost-effective, and high-throughput method for typing nine serotypes of HFMD-associated enteroviruses.

  6. Use of a new single multiplex PCR-based assay for direct simultaneous characterization of six Neisseria meningitidis serogroups.

    Science.gov (United States)

    Fraisier, Christophe; Stor, Richard; Tenebray, Bernard; Sanson, Yannick; Nicolas, Pierre

    2009-08-01

    We developed a new Neisseria meningitidis multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping N. meningitidis from cerebrospinal fluid that could be useful in Africa.

  7. Use of a New Single Multiplex PCR-Based Assay for Direct Simultaneous Characterization of Six Neisseria meningitidis Serogroups▿

    Science.gov (United States)

    Fraisier, Christophe; Stor, Richard; Tenebray, Bernard; Sanson, Yannick; Nicolas, Pierre

    2009-01-01

    We developed a new Neisseria meningitidis multiplex PCR to determine six serogroups, including X-specific primers, and to allow direct W135/Y discrimination. This assay offers a simple and low-cost method for serogrouping N. meningitidis from cerebrospinal fluid that could be useful in Africa. PMID:19553584

  8. Multiplex PCR assay for identification of six different Staphylococcus spp. and simultaneous detection of methicillin and mupirocin resistance.

    Science.gov (United States)

    Campos-Peña, E; Martín-Nuñez, E; Pulido-Reyes, G; Martín-Padrón, J; Caro-Carrillo, E; Donate-Correa, J; Lorenzo-Castrillejo, I; Alcoba-Flórez, J; Machín, F; Méndez-Alvarez, S

    2014-07-01

    We describe a new, efficient, sensitive, and fast single-tube multiple-PCR protocol for the identification of the most clinically significant Staphylococcus spp. and the simultaneous detection of the methicillin and mupirocin resistance loci. The protocol identifies at the species level isolates belonging to S. aureus, S. epidermidis, S. haemolyticus, S. hominis, S. lugdunensis, and S. saprophyticus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Assessment of cytotoxicity of carbon nanoparticles using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay.

    Science.gov (United States)

    Schrand, Amanda M; Lin, Jonathan B; Hussain, Saber M

    2012-01-01

    Ever since the discovery of carbon nanotubes, there has been an increasing interest in technologies that rely upon these incredibly small particles for their unique properties. However, assessment of their biological consequences has been riddled with assay limitations. Here, we describe application of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay to study cytotoxicity of various carbon-based nanomaterials on cells and discuss some pitfalls of this method.

  10. Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

    Science.gov (United States)

    2010-02-01

    respiratory syncytial virus, rubella virus, and poxvirus [variola]) and bacteria (Bacillus [anthracis], Bordetella, Chlamydophila and Chlamydia ...those diagnosed with pneumonia (confirmed by chest x-ray). A group of 298 specimens were analyzed using both the RPM- Flu assay and a validated...adenoviruses of subgroups B1, B2 and E; Mycoplasma pneumoniae ; results not shown). The benchmark RT-PCR type A influenza virus test used to assess clinical

  11. [Development of a GeXP based multiplex RT-PCR assay for simultaneous differentiation of nine human hand food mouth disease pathogens].

    Science.gov (United States)

    Hu, Xiu-Mei; Zhang, Yong; Xu, Bang-Lao; Yang, Meng-Jie; Wang, Miao; Zhang, Chen; Li, Jin; Bai, Ru-Yin; Zhou, Xiao-Mian; Xu, Wen-Bo; Ma, Xue-Jun

    2011-07-01

    A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.

  12. Development and validation of a duplex real-time PCR assay for the simultaneous detection of three mustard species (Sinapis alba, Brassica nigra and Brassica juncea) in food.

    Science.gov (United States)

    Palle-Reisch, Monika; Cichna-Markl, Margit; Hochegger, Rupert

    2014-06-15

    The paper presents a duplex real-time PCR assay for the simultaneous detection of three potentially allergenic mustard species commonly used in food: white mustard (Sinapis alba), black mustard (Brassica nigra) and brown mustard (Brassica juncea). White mustard is detected in the "green" and black/brown mustard in the "yellow" channel. The duplex real-time PCR assay does not show cross-reactivity with other Brassicaceae species including broccoli, cauliflower, radish and rapeseed. Low cross-reactivities (difference in the Ct value ⩾ 11.91 compared with the positive control) were obtained with cumin, fenugreek, ginger, rye and turmeric. When applying 500 ng DNA per PCR tube, the duplex real-time PCR assay allowed the detection of white, black and brown mustard in brewed model sausages down to a concentration of 5mg/kg in 10 out of 10 replicates. The duplex real-time PCR assay was applied to verify correct labelling of commercial foodstuffs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Development and validation of a triplex real-time PCR assay for the simultaneous detection of three mustard species and three celery varieties in food.

    Science.gov (United States)

    Palle-Reisch, Monika; Hochegger, Rupert; Cichna-Markl, Margit

    2015-10-01

    The paper presents a triplex real-time PCR assay allowing the simultaneous detection of three mustard species (white, black and brown mustard) and three celery varieties (celery roots, celery stalks and leaf celery) in foodstuffs. The triplex assay does not show cross-reactivity with other Brassicaceae. Low cross-reactivities were observed with fenugreek, cumin, ginger, caraway, turmeric, lovage and rye, the ΔCt values were, however, ⩾ 12 compared to positive controls. The triplex assay allows the detection of traces of DNA of the allergenic components in spite of an excess of the other DNA templates. Analysis of extracts from model sausages containing defined concentrations of mustard and celery showed that the triplex assay is applicable to both raw and processed foods. It was found to allow the detection of 1 ppm black/brown mustard and 50 ppm white mustard and celery in raw and brewed sausages with a probability ⩾ 95%. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Saitou, Yasumasa; Kobayashi, Yuki; Hirano, Shinji; Mochizuki, Nobuyuki; Itou, Takuya; Ito, Fumio H; Sakai, Takeo

    2010-09-01

    At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field. Copyright 2010 Elsevier B.V. All rights reserved.

  15. Development and application of a triplex real-time PCR assay for the simultaneous detection of avian influenza virus subtype H5, H7 and H9.

    Science.gov (United States)

    Liu, Junli; Yao, Lu; Zhai, Feifei; Chen, Yuqing; Lei, Jing; Bi, Zhenwei; Hu, Jianhua; Xiao, Qian; Song, Suquan; Yan, Liping; Zhou, Jiyong

    2017-11-10

    Avian influenza virus (AIV), especially subtypes H5, H7 and H9, has contributed to enormous economic losses and poses a potential pandemic threat to global human public health. Early screening of suspected cases is key to controlling the spread of AIVs. In this study, an accurate, rapid, and triplex real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of AIV subtypes H5, H7 and H9. The sensitivity of the real-time PCR was at least 100 times higher than that of the conventional PCR, with a detection limit of 50 copies and an EID50 of 1 (50% egg infections dose) for the H5, H7, and H9 subtypes. The lack of cross-reaction with other avian respiratory viruses suggested that the real-time PCR assay was highly specific. The reproducibility of the assay was confirmed using plasmids containing targets genes. Furthermore, 362 clinical field samples were evaluated. Subtypes H5, H7 and H9 were detected in 102 (28.18%) samples by real-time PCR and in 35 (9.67%) samples by conventional virus isolation. These results indicate that the triplex real-time PCR assay has good sensitivity, specificity and reproducibility and that it might be useful for laboratory surveillance and rapid diagnosis of the H5, H7 and H9 subtypes of influenza A viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

    Directory of Open Access Journals (Sweden)

    Soukka Tero

    2010-11-01

    Full Text Available Abstract A highly specific and novel dual-label time-resolved immunofluorometric assay was developed exploiting the unique emission wavelengths of the intrinsically fluorescent terbium (Tb3+ and europium (Eu3+ tracers for the simultaneous detection of human immunodeficiency virus 1 (HIV-1 and hepatitis B virus (HBV infections, respectively. HIV-1 infection was detected using a double antigen sandwich format wherein anti-HIV-1 antibodies were captured using an in vivo biotinylated version of a chimeric HIV-1 antigen and revealed using the same antigen labeled with Tb3+ chelate. Hepatitis B surface antigen (HBsAg, which served as the marker of HBV infection, was detected in a double antibody sandwich using two monoclonal antibodies (mAbs, one chemically biotinylated to capture, and the other labeled with Eu3+ nanoparticles, to reveal. The performance of the assay was evaluated using a collection (n = 60 of in-house and commercially available human sera panels. This evaluation showed the dual-label assay to possess high degrees of specificity and sensitivity, comparable to those of commercially available, single analyte-specific kits for the detection of HBsAg antigen and anti-HIV antibodies. This work demonstrates the feasibility of developing a potentially time- and resource-saving multiplex assay for screening serum samples for multiple infections in a blood bank setting.

  17. Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens.

    Science.gov (United States)

    Cheng, Fang; Wu, Jiajie; Zhang, Jin; Pan, Aihu; Quan, Sheng; Zhang, Dabing; Kim, HaeYeong; Li, Xiang; Zhou, Shan; Yang, Litao

    2016-05-15

    Food allergies cause health risks to susceptible consumers and regulations on labeling of food allergen contents have been implemented in many countries and regions. To achieve timely and accurate food allergen labeling, the development of fast and effective allergen detection methods is very important. Herein, a decaplex polymerase chain reaction (PCR) assay combined with capillary electrophoresis was developed to detect simultaneously 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean and pecan. The absolute limit of detection (LODa) of this system is between 2 and 20 copies of haploid genome, and the relative LOD (LODr) is as low as 0.005% (w/w) in simulated food mixtures. The developed assay was subsequently applied to 20 commercial food products and verified the allergen ingredients stated on the labels. Furthermore, results using this decaplex PCR assay was successfully replicated in three other laboratories, demonstrating the repeatability and applicability of this assay in routine analysis of the 10 food allergens. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    Science.gov (United States)

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum.

    Science.gov (United States)

    Wagner, B; Freer, H; Rollins, A; Erb, H N

    2011-04-15

    established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes.

    Science.gov (United States)

    Okolie, Charles Emeka; Wooldridge, Karl G; Turner, David P J; Cockayne, Alan; James, Richard

    2015-08-05

    Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x10(3) CFU/mL for the 16S rRNA marker and 1.0x10(4) CFU/mL for six other markers and completes cycling in less than one hour. The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow

  1. Rapid quantitative PCR assays for the simultaneous detection of herpes simplex virus, varicella zoster virus, cytomegalovirus, Epstein-Barr virus, and human herpesvirus 6 DNA in blood and other clinical specimens

    NARCIS (Netherlands)

    Engelmann, I.; Petzold, D. R.; Kosinska, A.; Hepkema, B. G.; Schulz, T. F.; Heim, A.

    Rapid diagnosis of human herpesvirus primary infections or reactivations is facilitated by quantitative PCRs. Quantitative PCR assays with a standard thermal cycling profile permitting simultaneous detection of herpes simplex virus (HSV), varicella zoster virus (VZV), cytomegalovirus (CMV),

  2. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... identification of VSV....

  3. A multiplex PCR assay for the rapid and sensitive detection of methicillin-resistant Staphylococcus aureus and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci.

    Science.gov (United States)

    Xu, Benjin; Liu, Ling; Liu, Li; Li, Xinping; Li, Xiaofang; Wang, Xin

    2012-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern, which had been detected in food and food production animals. Conventional testing for detection of MRSA takes 3 to 5 d to yield complete information of the organism and its antibiotic sensitivity pattern. So, a rapid method is needed to diagnose and treat the MRSA infections. The present study focused on the development of a multiplex PCR assay for the rapid and sensitive detection of MRSA. The assay simultaneously detected 4 genes, namely, 16S rRNA of the Staphylococcus genus, femA of S. aureus, mecA that encodes methicillin resistance, and one internal control. It was rapid and yielded results within 4 h. The analytical sensitivity and specificity of the multiplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the multiplex PCR assay at the DNA level was 10 ng DNA. The analytical specificity was evaluated with 10 reference staphylococci strains and was 100%. The diagnostic evaluation of MRSA was carried out using 360 foodborne staphylococci isolates, and showed 99.1% of specificity, 96.4% of sensitivity, 97.5% of positive predictive value, and 97.3% of negative predictive value compared to the conventional method. The inclusion of an internal control in the multiplex PCR assay is important to exclude false-negative cases. This test can be used as an effective diagnostic and surveillance tool to investigate the spread and emergence of MRSA. © 2012 Institute of Food Technologists®

  4. Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis.

    Science.gov (United States)

    Ishida, Natsumi; Sakurada, Makoto; Kusunoki, Hiroshi; Ueno, Yasuhiro

    2017-11-26

    We developed a simple and rapid method for animal species identification in the forensic science field based on mitochondrial DNA using two multiplex real-time PCRs and analysis of the resultant SYBR Green I melting curves. This method was designed to identify nine domestic animals simultaneously (dog, cat, rabbit, cattle, pig, chicken, goat, sheep and horse) and four wild animals (deer, raccoon-dog, monkey and bear) by comparing the different melting temperatures of the amplicons produced from samples originating from each species. For this analysis, we targeted various mitochondrial genes, including those encoding cytochrome b (cytb), NADH dehydrogenase 5 (ND5), cytochrome c oxidase 3 (COX3), tRNA-ND5, and tRNA-ATP synthase 8 (ATP8). For practical applications, this study presents a validation of this assay including its specificity, sensitivity and robustness. The limits of detection in the multiplex reactions were 10 pg for eight of the nine animals, excluding horse (1 pg for horse). The method was able to correctly identify the animal species from artificial forensic samples including blood stains, saliva, hair and bone, and samples digested in artificial gastric fluid, and for 17 forensic casework samples. The data from the multiplex real-time PCR assays are obtainable only 30 min after DNA extraction of the samples, making the assays useful for screening samples containing DNA from unknown animal origin in the forensic field. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

    Science.gov (United States)

    McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

    2017-06-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

  6. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

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    Cengiz Ikten

    Full Text Available Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.. Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  7. Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

    Science.gov (United States)

    Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

    2016-01-01

    Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

  8. UHPLC-PDA Assay for Simultaneous Determination of Vitamin D3 and Menaquinone-7 in Pharmaceutical Solid Dosage Formulation

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    Muhammad Jehangir

    2017-01-01

    Full Text Available A newly developed method based on ultrahigh performance liquid chromatography (UHPLC was optimized for the simultaneous determination of vitamin D3 and menaquinone-7 (MK-7 in tablet formulation in the present study. UHPLC separation of vitamin D3 and MK-7 was performed with ACE Excel 2 C18-PFP column (2 μm, 2.1 × 100 mm at 0.6 mL min−1 flow rate, whereas the mobile phase consisted of methanol/water (19:1, v/v, phase A and isopropyl alcohol (99.9%, phase B containing 0.5% triethylamine. Isocratic separation of both the analytes was performed at 40°C by pumping the mobile phases A and B in the ratio of 50:50 (v/v, pH, 6.0. Both analytes were detected at a wavelength of 265 nm and the injection volume was 1.0 μL. The overall runtime per sample was 4.5 min with retention time of 1.26 and 3.64 min for vitamin D3 and MK-7, respectively. The calibration curve was linear from 5.0 to 100 μg mL−1 for vitamin D3 and MK-7 with a coefficient of determination (R2 ≥ 0.9981, while repeatability and reproducibility (expressed as relative standard deviation were lower than 1.46 and 2.21%, respectively. The proposed HPLC method was demonstrated to be simple and rapid for the determination of vitamin D3 and MK-7 in tablets.

  9. A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting.

    Science.gov (United States)

    Wedajo, Wassihun; Schön, Thomas; Bedru, Ahmed; Kiros, Teklu; Hailu, Elena; Mebrahtu, Tesfamariam; Yamuah, Lawrence; Ängeby, Kristian; Werngren, Jim; Onyebujoh, Philip; Dagne, Kifle; Aseffa, Abraham

    2014-08-10

    Early detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas. Baseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks. The 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for

  10. Screening of Indian medicinal plants for cytotoxic activity by Brine Shrimp Lethality (BSL assay and evaluation of their total phenolic content

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    Mahesh Biradi

    2014-01-01

    Full Text Available Objective: Plant-derived cytotoxic constituents and polyphenolic compounds have played an important role in the development of clinically useful anticancer agents. In this context, we have selected six Indian medicinal plants based on the literature claims and an attempt was made to evaluate the cytotoxic potential and total phenolic content (TPC of their methanol extracts and fractions. Materials and Methods: Six plants have been selected for the study, namely, Artemisia absinthium Linn. (Asteraceae, Oroxylum indicum (Linn. Vent. (Bignoniaceae, Heliotropium indicum Linn. (Boraginaceae, Amorphophallus sylvaticus (Roxb. Kunth. (Araceae, Mimosa pudica Linn. (Mimosaceae, and Premna serratifolia Linn. (Verbenaceae. Authenticated plant materials were subjected to extraction with methanol by cold maceration and hot percolation methods. The extracts were fractionated into four fractions (F1, F2, F3, and F4. Preliminary phytochemical investigation was carried out for all extracts and fractions. All extracts and their fractions were subjected to cytotoxicity screening by brine shrimp lethality (BSL bioassay. The plants with significant cytotoxicity were evaluated for TPC by using Folin-Ciocalteu reagent. Results: F1, F2, and F3 fractions of A. absinthium and P. serratifolia and F1 fraction of M. pudica have shown significant cytotoxicity (lethal concentration (LC 50 < 100 ppm compared with other fractions. F1, F2, and F3 fractions of A. absinthium show the LC 50 values 32.52, 14.27, and 24.02, respectively; F1, F2, and F3 of P. serratifolia show LC 50 values 7.61, 4.01, and 10.91 and same for F1 fraction of M. pudica was 34.82 μg/ml, respectively. TPC was found to be significantly higher (39.11 mg gallic acid equivalent (GAE/g in P. serratifolia compared with other two plants. Conclusion: The cytotoxicity screening system confirmed the proposed anticancer plants used by traditional healers and literature claims.

  11. The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

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    Hae-Ryun Kwak

    2014-12-01

    Full Text Available Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV and sweet potato virus C (SPVC were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1, Sweet potato virus G (SPVG, Sweet potato leaf curl virus (SPLCV, Sweet potato virus 2 ( SPV2, Sweet potato chlorotic fleck virus (SPCFV, and Sweet potato latent virus (SPLV were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1 in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  12. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    Science.gov (United States)

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  13. A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

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    Sanchita Das

    Full Text Available CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR. The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively. The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus.

  14. Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle.

    Science.gov (United States)

    Karanikola, Sofia N; Krücken, Jürgen; Ramünke, Sabrina; de Waal, Theo; Höglund, Johan; Charlier, Johannes; Weber, Corinna; Müller, Elisabeth; Kowalczyk, Slawomir J; Kaba, Jaroslaw; von Samson-Himmelstjerna, Georg; Demeler, Janina

    2015-06-19

    A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100% for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. A versatile bead-based assay using fluorescence detection (x

  15. [In vitro cytotoxicity test of medical ultrasonic couplant].

    Science.gov (United States)

    Gao, Jingxian; Wang, Shasha; Jin, Meng; Yan, Xiaoli

    2013-05-01

    The cytotoxicity of medical ultrasonic couplant was tested by MTT assay and agar overlay test. By MTT assay, when the inoculum density was high, the cytotoxicity level was low, or vice versa. The cytotoxicity grade tested by agar overlay was not accord to MTT assay's too. MTT assay is suitable to test the cytotoxicity of medical ultrasonic couplant because it is quantitative and more sensitive, however, the experimental condition and the preparative method of extraction should be determined.

  16. Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

    Science.gov (United States)

    Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

    2016-04-01

    A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

    Science.gov (United States)

    Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

    1996-02-15

    Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

  18. A one-step duplex rRT-PCR assay for the simultaneous detection of grass carp reovirus genotypes I and II.

    Science.gov (United States)

    Zeng, Weiwei; Wang, Yingying; Liang, Hongru; Liu, Cun; Song, Xinjian; Shi, Chunbin; Wu, Shuqin; Wang, Qing

    2014-12-15

    Hemorrhagic disease of grass carp, caused by grass carp reovirus (GCRV), leads to severe economic losses in the grass carp farming industry in China. GCRV has been divided into three genotypes based on genome sequence. Genotypes I and II (GCRV-1 and GCRV-II, respectively) are the dominant genotypes and co-infections of GCRV-I and GCRV-II are common in grass carp aquaculture. A one-step duplex real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay was developed for simultaneous detection of GCRV-I and GCRV-II. The PCR assay is suitable for early diagnosis of grass carp hemorrhagic disease and for epidemiological surveillance. The detection limit of the assay is 10 copies for both GCRV-I and GCRV-II, which is as high as single-target rRT-PCR and higher than conventional RT-PCR. No cross reactivity with other GCRV subtypes or other viruses was observed. One hundred and twelve samples from grass carp suspected of hemorrhagic disease were collected from South and Central China. Eleven samples were positive for GCRV-I by RT-PCR alone, and fourteen samples were positive by single-target and duplex rRT-PCR. Forty two samples were positive for GCRV-II by RT-PCR alone and forty seven samples were positive by single-target and duplex rRT-PCR. Mixed infections were found in eight samples when analyzed by RT-PCR alone and in ten samples analyzed by single-target and duplex rRT-PCR. The duplex rRT-PCR system provides a sensitive and specific method to detect and differentiate between GCRV-I and GCRV-II in a single sample. This rRT-PCR assay could be a useful tool for the routine diagnosis of these two viruses and for epidemiology studies in grass carp aquaculture. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

    Science.gov (United States)

    Foo, Phiaw Chong; Chan, Yean Yean; See Too, Wei Cun; Tan, Zi Ning; Wong, Weng Kin; Lalitha, Pattabhiraman; Lim, Boon Huat

    2012-09-01

    Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

  20. A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

    Science.gov (United States)

    Hu, Qin; Zhu, Dekang; Ma, Guangpeng; Cheng, Anchun; Wang, Mingshu; Chen, Shun; Jia, Renyong; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Chen, Xiaoyue

    2016-10-01

    Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains. Copyright © 2016. Published by Elsevier B.V.

  1. Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

    Science.gov (United States)

    Piret, Jean-Pascal; Bondarenko, Olesja M; Boyles, Matthew S P; Himly, Martin; Ribeiro, Ana R; Benetti, Federico; Smal, Caroline; Lima, Braulio; Potthoff, Annegret; Simion, Monica; Dumortier, Elise; Leite, Paulo Emilio C; Balottin, Luciene Bottentuit; Granjeiro, José Mauro; Ivask, Angela; Kahru, Anne; Radauer-Preiml, Isabella; Tischler, Ulrike; Duschl, Albert; Saout, Christelle; Anguissola, Sergio; Haase, Andrea; Jacobs, An; Nelissen, Inge; Misra, Superb K; Toussaint, Olivier

    2017-06-01

    The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-β and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-β and TNF-α measurements.

  2. Genetically modified semisynthetic bioluminescent photoprotein variants: simultaneous dual-analyte assay in a single well employing time resolution of decay kinetics.

    Science.gov (United States)

    Rowe, Laura; Combs, Kelly; Deo, Sapna; Ensor, C; Daunert, Sylvia; Qu, Xiaoge

    2008-11-15

    Progress in the miniaturization and automation of complex analytical processes depends largely on increasing the sensitivity, diversity, and robustness of current labels. Because of their ubiquity and ease of use, fluorescent, enzymatic, and bioluminescent labels are often employed in such miniaturized and multiplexed formats, with each type of label having its own unique advantages and drawbacks. The ultrasensitive detection limits of bioluminescent reporters are especially advantageous when dealing with very small sample volumes and biological fluids. However, bioluminescent reporters currently do not have the multiplexing capability that fluorescent labels do. In an effort to address this limitation, we have developed a method of discriminating two semisynthetic aequorin variants from one another using time resolution. In this work we paired two aequorin conjugates with different coelenterazine analogues and then resolved the two signals from one another using the difference in decay kinetics and half-life times. Utilizing this time-resolution, we then developed a simultaneous, dual-analyte, single well assay for 6-keto-prostaglandin-FI-alpha and angiotensin II, two important cardiovascular molecules.

  3. Utilizing three monoclonal antibodies in the development of an immunochromatographic assay for simultaneous detection of sulfamethazine, sulfadiazine, and sulfaquinoxaline residues in egg and chicken muscle.

    Science.gov (United States)

    Guo, Yancheng; Ngom, Babacar; Le, Tao; Jin, Xiue; Wang, Liping; Shi, Deshi; Wang, Xiliang; Bi, Dingren

    2010-09-15

    A rapid and sensitive immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneous detection of sulfamethazine (SM(2)), sulfadiazine (SDZ), and sulfaquinoxaline (SQX) in chicken breast muscle and egg samples. For this purpose, three monoclonal antibodies raised against those three sulfonamides were conjugated to colloidal gold particles and applied to the conjugate pads of the test strip. The competitors of the sulfonamides (SM(2)/SDZ/SQX-bovine serum albumin conjugates) were immobilized onto a nitrocellulose membrane at three detection zones to form T(1), T(2), and T(3), respectively. With this method, the cutoff values for the three test lines were achieved at 80 μg/kg, which is lower than the maximum residue levels (MRLs) established for sulfonamides. The recoveries in negative samples spiked at concentrations of 10, 50, and 100 μg/kg ranged from 75% to 82% for egg samples and from 78% to 81% for chicken samples. The method was compared with the HPLC method by testing 180 eggs and chicken breast samples from local markets, and an agreement rate of 99.7% was obtained between the two methods.

  4. Development of multiplex PCR assay for simultaneous detection of Salmonella genus, Salmonella subspecies I, Salm. Enteritidis, Salm. Heidelberg and Salm. Typhimurium.

    Science.gov (United States)

    Park, S H; Ricke, S C

    2015-01-01

    The aim of this research was to develop multiplex PCR assay that could simultaneously detect Salmonella genus, Salmonella subsp. I, Salm. Enteritidis, Heidelberg and Typhimurium because these Salmonella serovars are the most common isolates associated with poultry products. Five primers were utilized to establish multiplex PCR and applied to Salmonella isolates from chickens and farm environments. These isolates were identified as Salmonella subsp. I and 16 of 66 isolates were classified as Salm. Enteritidis, while Heidelberg or Typhimurium was not detected. We also spiked three Salmonella strains on chicken breast meat to evaluate the specificity and sensitivity of multiplex PCR as well as qPCR to optimize quantification of Salmonella in these samples. The optimized multiplex PCR and qPCR could detect approx. 2·2 CFU of Salmonella per gram after 18 h enrichment. The multiplex PCR and qPCR would provide rapid and consistent results. Also, these techniques would be useful for the detection and quantification of Salmonella in contaminated poultry, foods and environmental samples. The strategy for the rapid detection of Salmonella serovars in poultry is needed to further reduce the incidence of salmonellosis in humans. The optimized multiplex PCR will be useful to detect prevalent Salmonella serovars in poultry products. © 2014 The Society for Applied Microbiology.

  5. Data supporting the rat brain sample preparation and validation assays for simultaneous determination of 8 neurotransmitters and their metabolites using liquid chromatography–tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Aneta Wojnicz

    2016-06-01

    Full Text Available The data presented in this article supports the rat brain sample preparation procedure previous to its injection into the liquid chromatography–tandem mass spectrometry (LC–MS/MS system to monitor levels of adrenaline, noradrenaline, glutamic acid, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine, 5-hydroxyindole acetic acid, and 3-methoxy-4-hydroxyphenylglycol. In addition, we describe the method validation assays (such as calibration curve, lower limit of quantification, precision and accuracy intra- and inter-day, selectivity, extraction recovery and matrix effect, stability, and carry-over effect according to the United States Food and Drug Administration and European Medicine Agency to measure in one step different neurotransmitters and their metabolites. The data supplied in this article is related to the research study entitled: “Simultaneous determination of 8 neurotransmitters and their metabolite levels in rat brain using liquid chromatography in tandem with mass spectrometry: application to the murine Nrf2 model of depression” (Wojnicz et al. 2016 [1].

  6. Forced degradation study of racecadotril: Effect of co-solvent, characterization of degradation products by UHPLC-Q-TOF-MS/MS, NMR and cytotoxicity assay.

    Science.gov (United States)

    Chiguru, Vishnuvardhan; Lingesh, Allakonda; R, Srinivas; N, Satheeshkumar

    2016-09-05

    Racecadotril, an enkephalinase inhibitor, was subjected to hydrolysis (acidic and alkaline), oxidation, photolysis and thermal stress, as per ICH specified conditions. The drug showed extensive degradation under acidic, basic hydrolysis and oxidative stress conditions whereas, it was stable under other stress conditions. A total of seven degradation products (DPs) were observed. The chromatographic separation was optimized on Acquity HSS Cyano (100×2.1mm, 1.8μ) column using 0.1% formic acid and acetonitrile as mobile phase in gradient mode. Six DPs were characterised by LC-MS/MS and DP1 by GC-MS. The major DPs (DP 2 and DP 5) were isolated and characterised by NMR. This is a typical case of degradation where co solvent methanol reacts with racecadotril leading to the formation of pseudo DPs, DP 6 and DP 5. Interestingly the MS/MS spectra of protonated drug, DP 4 and DP 7 showed product ions which were formed due to intramolecular benzyl migrations. In vitro cytotoxic activity studies on isolated DP 2 and DP 5 revealed that the former has no cytotoxic nature, whereas the latter has potential pulmonary and hepatic toxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. The antioxidant properties, cytotoxicity and monoamine oxidase ...

    African Journals Online (AJOL)

    The antioxidant activities were assessed using the thiobarbituric acid-reactive substances (TBARS) assay and the nitroblue tetrazolium (NBT) assay. The cytotoxicity assays were performed according to the microculture MTT method. From the MTT assay, it was determined that at a concentration of 10 mg/ml of crude extract, ...

  8. Mathematical models of cytotoxic effects in endpoint tumor cell line assays: critical assessment of the application of a single parametric value as a standard criterion to quantify the dose-response effects and new unexplored proposal formats.

    Science.gov (United States)

    Calhelha, Ricardo C; Martínez, Mireia A; Prieto, M A; Ferreira, Isabel C F R

    2017-10-23

    The development of convenient tools for describing and quantifying the effects of standard and novel therapeutic agents is essential for the research community, to perform more precise evaluations. Although mathematical models and quantification criteria have been exchanged in the last decade between different fields of study, there are relevant methodologies that lack proper mathematical descriptions and standard criteria to quantify their responses. Therefore, part of the relevant information that can be drawn from the experimental results obtained and the quantification of its statistical reliability are lost. Despite its relevance, there is not a standard form for the in vitro endpoint tumor cell lines' assays (TCLA) that enables the evaluation of the cytotoxic dose-response effects of anti-tumor drugs. The analysis of all the specific problems associated with the diverse nature of the available TCLA used is unfeasible. However, since most TCLA share the main objectives and similar operative requirements, we have chosen the sulforhodamine B (SRB) colorimetric assay for cytotoxicity screening of tumor cell lines as an experimental case study. In this work, the common biological and practical non-linear dose-response mathematical models are tested against experimental data and, following several statistical analyses, the model based on the Weibull distribution was confirmed as the convenient approximation to test the cytotoxic effectiveness of anti-tumor compounds. Then, the advantages and disadvantages of all the different parametric criteria derived from the model, which enable the quantification of the dose-response drug-effects, are extensively discussed. Therefore, model and standard criteria for easily performing the comparisons between different compounds are established. The advantages include a simple application, provision of parametric estimations that characterize the response as standard criteria, economization of experimental effort and enabling

  9. A battery of assays as an integrated approach to evaluate fungal and mycotoxin inhibition properties and cytotoxic/genotoxic side-effects for the prioritization in the screening of thiosemicarbazone derivatives.

    Science.gov (United States)

    Zani, Claudia; Bisceglie, Franco; Restivo, Francesco Maria; Feretti, Donatella; Pioli, Marianna; Degola, Francesca; Montalbano, Serena; Galati, Serena; Pelosi, Giorgio; Viola, Gaia V C; Carcelli, Mauro; Rogolino, Dominga; Ceretti, Elisabetta; Buschini, Annamaria

    2017-07-01

    Aflatoxins represent a serious problem for a food economy based on cereal cultivations used to fodder animal and for human nutrition. The aims of our work are two-fold: first, to perform an evaluation of the activity of newly synthesized thiosemicarbazone compounds as antifungal and anti-mycotoxin agents and, second, to conduct studies on the toxic and genotoxic hazard potentials with a battery of tests with different endpoints. In this paper we report an initial study on two molecules: S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazone and its metal complex, bis(S-4-isopropenylcyclohexen-1-carbaldehydethiosemicarbazonato)nickel (II). The outcome of the assays on fungi growth and aflatoxin production inhibition show that both molecules possess good antifungal activities, without inducing mutagenic effects on bacteria. From the assays to ascertain that the compounds have no adverse effects on human cells, we have found that they are cytotoxic and, in the case of the nickel compound, they also present genotoxic effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

    Science.gov (United States)

    Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

    2014-07-01

    A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

  11. Two zinc-aminoclays' in-vitro cytotoxicity assessment in HeLa cells and in-vivo embryotoxicity assay in zebrafish.

    Science.gov (United States)

    Chun, Hang-Suk; Park, Duckshin; Eun Lim, Song; Jeong, Kwang-Hun; Park, Ji-Seon; Park, Han-Jin; Kang, Shinyoung; Kang, Kyoung Suk; Park, Hyun Gyu; An, Ha-Rim; Huh, Yun Suk; Lee, Young-Chul

    2017-03-01

    Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49µg/mL and 18.12/15.19/46.10µg/mL of IC50 values for 24 and 48h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36µg/mL and 18.37/17.09µg/mL for 24 and 48h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn(2+) ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50-100µg/mL (corresponding to 37.88-75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs' potential applications in the environmental and biomedical fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Multiplex PCR assay for the simultaneous detection of C. perfringens, P. aeruginosa and K. pneumoniae

    Directory of Open Access Journals (Sweden)

    Pradeepkiran Jangampalli Adi

    2015-11-01

    Results and conclusions: Through this approach, the above pathogens were detected simultaneously with high specificity in pure cultures and from the blood and urine samples. The results were correlated with normal diagnostic process, and proved to be more sensitive and specific diagnostic technique in the simultaneous detection of C. perfringens, P. aeruginosa and K. pneumoniae.

  13. Simultaneous total antioxidant capacity assay of lipophilic and hydrophilic antioxidants in the same acetone-water solution containing 2% methyl-beta-cyclodextrin using the cupric reducing antioxidant capacity (CUPRAC) method.

    Science.gov (United States)

    Ozyürek, Mustafa; Bektaşoğlu, Burcu; Güçlü, Kubilay; Güngör, Nilay; Apak, Reşat

    2008-12-07

    Antioxidants are health beneficial compounds that can protect cells from the damage caused by unstable molecules known as reactive oxygen species (ROS). This work reports the capacity assay of both lipophilic and hydrophilic antioxidants simultaneously, by making use of their 'host-guest' complexes with methyl-beta-cyclodextrin (M-beta-CD), a cyclic oligosaccharide, in acetonated aqueous medium using the cupric reducing antioxidant capacity (CUPRAC) method. Thus the order of antioxidant potency of various compounds irrespective of their lipophilicity could be established in the same solvent medium. M-beta-CD was introduced as the water solubility enhancer for lipophilic antioxidants. Two percent M-beta-CD (w/v) in an acetone-H(2)O (9:1, v/v) mixture was found to sufficiently solubilize beta-carotene, lycopene, vitamin E, vitamin C, synthetic antioxidants and other phenolic antioxidants. This assay was validated through linearity, additivity, precision, and recovery. The validation results demonstrate that the CUPRAC assay is reliable and robust. In acetonated aqueous solution of M-beta-CD, only CUPRAC and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays were capable of measuring carotenoids together with hydrophilic antioxidants. The CUPRAC antioxidant capacities of a wide range of polyphenolics and flavonoids were experimentally reported in this work as trolox equivalent antioxidant capacity (TEAC) in the CUPRAC assay, and compared to those found by reference methods, ABTS/horseradish peroxidase (HRP)-H(2)O(2) and ferric reducing antioxidant power (FRAP) assays.

  14. Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method

    Directory of Open Access Journals (Sweden)

    Wan Zhixiang

    2005-07-01

    Full Text Available Abstract Background Viral hepatitis due to hepatitis B virus and hepatitis C virus are major public health problems all over the world. Traditional detection methods including polymerase chain reaction (PCR-based assays and enzyme-linked immunosorbent assays (ELISA are expensive and time-consuming. In our assay, a protein chip assay using Nano-gold Immunological Amplification and Silver Staining (NIASS method was applied to detect HBV and HCV antibodies rapidly and simultaneously. Methods Chemically modified glass slides were used as solid supports (named chip, on which several antigens, including HBsAg, HBeAg, HBcAg and HCVAg (a mixture of NS3, NS5 and core antigens were immobilized respectively. Colloidal nano-gold labelled staphylococcal protein A (SPA was used as an indicator and immunogold silver staining enhancement technique was applied to amplify the detection signals, producing black image on array spots, which were visible with naked eyes. To determine the detection limit of the protein chip assay, a set of model arrays in which human IgG was spotted were structured and the model arrays were incubated with different concentrations of anti-IgG. A total of 305 serum samples previously characterized with commercial ELISA were divided into 4 groups and tested in this assay. Results We prepared mono-dispersed, spherical nano-gold particles with an average diameter of 15 ± 2 nm. Colloidal nano-gold-SPA particles observed by TEM were well-distributed, maintaining uniform and stable. The optimum silver enhancement time ranged from 8 to 12 minutes. In our assay, the protein chips could detect serum antibodies against HBsAg, HBeAg, HBcAg and HCVAg with the absence of the cross reaction. In the model arrays, the anti-IgG as low as 3 ng/ml could be detected. The data for comparing the protein chip assay with ELISA indicated that no distinct difference (P > 0.05 existed between the results determined by our assay and ELISA respectively. Conclusion

  15. Rapid Assay for Simultaneous Detection and Differentiation of Immunoglobulin G Antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2

    OpenAIRE

    Vallari, Ana S.; Hickman, Robert K.; Hackett, John R.; Brennan, Catherine A.; Varitek, Vincent A.; Devare, Sushil G.

    1998-01-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western b...

  16. Performance evaluation of a rapid molecular diagnostic, MultiCode based, sample-to-answer assay for the simultaneous detection of Influenza A, B and respiratory syncytial viruses

    NARCIS (Netherlands)

    J. Voermans (Jolanda); S. Seven-Deniz; P.L.A. Fraaij (Pieter); A.A. Eijck (Annemiek); M.P.G. Koopmans D.V.M. (Marion); S.D. Pas (Suzan)

    2016-01-01

    textabstractBackground Clinical signs and symptoms of different airway pathogens are generally indistinguishable, making laboratory tests essential for clinical decisions regarding isolation and antiviral therapy. Immunochromatographic tests (ICT) and direct immunofluorescence assays (DFA) have

  17. Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

    Science.gov (United States)

    Vallari, A S; Hickman, R K; Hackett, J R; Brennan, C A; Varitek, V A; Devare, S G

    1998-12-01

    A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected

  18. Click one pot synthesis, spectral analyses, crystal structures, DFT studies and brine shrimp cytotoxicity assay of two newly synthesized 1,4,5-trisubstituted 1,2,3-triazoles

    Science.gov (United States)

    Ahmed, Muhammad Naeem; Yasin, Khawaja Ansar; Ayub, Khurshid; Mahmood, Tariq; Tahir, M. Nawaz; Khan, Bilal Ahmad; Hafeez, Muhammad; Ahmed, Madiha; ul-Haq, Ihsan

    2016-02-01

    Methyl-2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (1) and ethyl-2-(1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)-2-oxoacetate (2) were synthesized by one pot three component strategy, and characterized by FT-IR, NMR (1H and 13C) spectroscopy and TOF-MS spectrometry. Finally, the structures were unequivocally confirmed by single crystal X-ray diffraction analyses. Both compounds, 1 and 2 exist in monoclinic crystal packing having space group P21/n and P21/c, respectively. Crystal structures investigations revealed that the molecular structures of the title compounds are stabilized by weak intermolecular hydrogen bonding interactions to form dimers. Density functional theory (DFT) calculations were performed not only to compare with the experimental spectroscopic results but also to probe structural properties. The molecular electrostatic potential (MEP) mapped over the entire stabilized geometries of the molecules delivered information about the electrophilic and nucleophilic sites. Furthermore, frontier molecular orbital analysis gave the idea about stability and reactivity of compounds. Both compounds were also screened for brine shrimp cytotoxicity assay.

  19. In vitro cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity to the transmissible venereal tumor of the dog.

    Science.gov (United States)

    Cohen, D

    1980-02-01

    Cell-mediated immunity to the transmissible venereal tumor (TVT) of the dog was studied by use of a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes (PBL) of dogs in which the TVT had regressed were shown to be cytotoxic to the tumor cells in contrast to PBL of normal dogs and animals during progressive tumor growth, which were not cytotoxic. In addition, sera of dogs in which the TVT had regressed could be demonstrated to mediate antibody-dependent cellular cytotoxicity (ADCC) with normal dog lymphocytes or cytotoxic PBL as effector cells. With cytotoxic lymphocytes, the ADCC effect could be observed in addition to the direct cytotoxic effect.

  20. Structure-cytotoxicity relationships for dietary flavonoids

    DEFF Research Database (Denmark)

    Breinholt, V.; Dragsted, L.O.

    1998-01-01

    The cytotoxicity of a large series of dietary flavonoids was tested in a non-tumorigenic mouse and two human cancer cell lines, using the neutral red dye exclusion assay. All compounds tested exhibited a concentration-dependent cytotoxic action in the employed cell lines. The relative cytotoxicity...... of the flavonoids, however, Tvas found to vary greatly among the different cell Lines. With a few exceptions, the investigated flavonoids were more cytotoxic to the human cancer cell lines, than the mouse cell line. The differences in cytotoxicity were accounted for in part by differences in cellular uptake...... and metabolic capacity among the different cell types. In 3T3 cells fairly consistent structure-cytotoxicity relationships were found. The most cytotoxic structures tested in 3T3 cells were flavonoids with adjacent 3',4' hydroxy groups on the B-ring, such as luteolin, quercetin, myricetin, fisetin, eriodictyol...

  1. Development of a one-tube multiplex reverse transcriptase-polymerase chain reaction assay for the simultaneous amplification of HIV type 1 group M gag and env heteroduplex mobility assay fragments

    OpenAIRE

    Cham, F.; Heyndrickx, L; Janssens, W; Vereecken, K.; Houwer, K.; Coppens, S.; Van Der Auwera, G.; Whittle, H; van der Groen, G

    2000-01-01

    The emergence of intersubtype recombinant HIV-1 isolates has made it imperative to analyze different regions of HIV-1 genomes. For this purpose a one-tube multiplex RT-PCR, coamplifying first-round amplicons that allow amplification of gag and env heteroduplex mobility assay (HMA) fragments from different HIV-1 group M isolates, was developed, starting with plasma samples. The multiplex RT-PCR assay is sensitive: 115 of 136 (84.5%) samples were positive for both gag and env, positive amplific...

  2. Simultaneously Detection of 50 Mutations at 20 Sites in the BRAF and RAS Genes by Multiplexed Single-Nucleotide Primer Extension Assay Using Fine-Needle Aspirates of Thyroid Nodules.

    Science.gov (United States)

    Stence, Aaron A; Gailey, Michael P; Robinson, Robert A; Jensen, Chris S; Ma, Deqin

    2015-12-01

    Fine-needle aspiration (FNA) is commonly used for primary evaluation of thyroid nodules. Twenty to 30 percent of thyroid nodules remain indeterminate after FNA evaluation. Studies show the BRAF p.V600E to be highly specific for papillary thyroid carcinoma (PTC), while RAS mutations carry up to 88 percent positive predictive value for malignancy. We developed a two-tube multiplexed PCR assay followed by single-nucleotide primer extension assay for simultaneous detection of 50 mutations in the BRAF (p.V600E, p.K601E/Q) and RAS genes (KRAS and NRAS codons 12, 13, 19, 61 and HRAS 61) using FNA smears of thyroid nodules. Forty-two FNAs and 27 paired formalin-fixed, paraffin-embedded (FFPE) tissues were tested. All BRAF p.V600E-positive FNA smears (five) carried a final diagnosis of PTC on resection. RAS mutations were found in benign as well as malignant lesions. Ninety-two percent concordance was observed between FNA and FFPE tissues. In conclusion, our assay is sensitive and reliable for simultaneous detection of multiple BRAF/RAS mutations in FNA smears of thyroid nodules.

  3. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    Science.gov (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  4. Multiplex PCR assays for simultaneous detection of six major serotypes and two virulence-associated phenotypes of Streptococcus suis in tonsillar specimens from pigs

    NARCIS (Netherlands)

    Wisselink, H.J.; Joosten, J.J.; Smith, H.E.

    2002-01-01

    Multiplex PCR assays for the detection and identification of various Streptococcus suis strains in tonsillar specimens from pigs were developed and evaluated. In two separate reactions, five distinct DNA targets were amplified. Three targets, based on the S. suis capsular polysaccharide (cps) genes

  5. A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

    Science.gov (United States)

    Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

    2014-06-01

    Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Evaluation of a new generation synthetic peptide combination assay for detection of antibodies to HIV-1, HIV-2, HTLV-I, and HTLV-II simultaneously.

    Science.gov (United States)

    Zhang, X; Constantine, N T; Bansal, J; Callahan, J D; Marsiglia, V C

    1992-09-01

    A new generation combination test (Detect-Plus, IAF BioChem, Montreal, Canada) based on synthetic peptides for HIV-1, HIV-2, HTLV-I, and HTLV-II was compared with three routine commercial screening assays and confirmatory assays to determine its sensitivity and specificity and to evaluate it as a substitute screening method. Samples from 356 sexually transmitted disease (STD) patients were tested by the four screening tests. All initially reactive samples were retested in duplicate by the corresponding EIA and repeatedly reactive samples were confirmed by Western blots for HIV-1, HIV-2, and HTLV-I/II. The confirmed positives detected by each screening assay were HIV-1 (23/356, 6.46%), HIV-2 (11/356, 3.09%), and HTLV-I/II (5/356, 1.4%). The new generation Detect-Plus test produced only two results (2/356, 0.56%) that were presumed to be false-positives in comparison to the screening tests, but the OD/CO values were just slightly high (1.5 and 1.9). There were no false-negative results, indicating that the sensitivity of the new combination test was excellent (100%). Compared with routine retroviral EIA assays, the test is easy to perform--the total time requirement is only 2 hr and there is no need for incubation equipment. The OD/CO values were very high when samples were positive, making even visual interpretation possible. We conclude that this new combination assay is an excellent screening method for detection of antibodies to the human retroviruses, and may be particularly useful for screening blood for transfusion and in epidemiological investigations.

  7. Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria

    Directory of Open Access Journals (Sweden)

    Hye-young Wang

    2017-08-01

    Conclusions: The entire QMAP system assay takes about 3 h to complete, while results from the culture-based conventional method can take up to 48–72 h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples.

  8. A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

    Directory of Open Access Journals (Sweden)

    Shiyin Zhang

    Full Text Available The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD, which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71 and coxsackie A16 (CA16 are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

  9. Cytotoxicity of Sambucus ebulus on cancer cell lines and protective ...

    African Journals Online (AJOL)

    Cytotoxicity of Sambucus ebulus on cancer cell lines and protective effects of vitamins C and E against its cytotoxicity on normal cell lines. ... Cytotoxicity of SEE on cancer (HepG2 and CT26) and normal (CHO and rat fibroblast) cell lines was evaluated by MTT assay. IC50 of SEE on ... African Journal of Biotechnology Vol.

  10. Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria.

    Science.gov (United States)

    Wang, Hye-Young; Uh, Young; Kim, Seoyong; Shim, Tae-Sun; Lee, Hyeyoung

    2017-08-01

    The differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. A total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. The sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15-100%, ptuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529-534, 524-529, and 514-520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). The entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48-72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

    Science.gov (United States)

    Hao, Min; Zhang, Pingping; Li, Baisheng; Liu, Xiao; Zhao, Yong; Tan, Hailing; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Qiu, Haiyan; Wang, Duochun; Diao, Baowei; Jing, Huaiqi; Yang, Ruifu; Kan, Biao; Zhou, Lei

    2017-01-01

    Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

  12. Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

    Science.gov (United States)

    Cross, Kristen E; Mercante, Jeffrey W; Benitez, Alvaro J; Brown, Ellen W; Diaz, Maureen H; Winchell, Jonas M

    2016-07-01

    Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease. Published by Elsevier Inc.

  13. Development and validation of a 3-Plex RT-qPCR assay for the simultaneous detection and quantitation of the three PML-RARa fusion transcripts in acute promyelocytic leukemia.

    Directory of Open Access Journals (Sweden)

    Zhanguo Chen

    Full Text Available Rapid diagnosis of acute promyelocytic leukemia (APL with promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa contributes to a highly effective therapy with all-trans retinoic acid (ATRA. Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR is a valuable tool to diagnose APL with PML-RARa. However, a single RT-qPCR analysis, which is laborious and costly, has to be performed in three reactions to determine whether one of the three PML-RARa transcripts is present and to quantify the involved transcript. This paper describes a novel TaqMan MGB probe-based 3-plex RT-qPCR assay in a single reaction to detect simultaneously the three PML-RARa transcripts. Specific primers and probe were designed, and the results were further normalized to the Abelson gene. The detection results for the serially diluted plasmid indicate that the analytical sensitivity was 10 copies per reaction for PML-RARa bcr1, bcr2, and bcr3. A relatively high sensitivity of 10-4 was achieved with this assay when analyzing the bcr1 transcripts obtained from the NB4 cell line. The reproducibility was satisfactory because the coefficients of variation of cycle threshold values were less than 3% for both inter- and intra-assays. After testing 319 newly diagnosed patients with leukemia (including 61 APL cases, the results of the 3-plex RT-qPCR assay completely agreed with the traditional methods used for the detection of PML-RARa. The quantitative results of the 3-plex RT-qPCR were highly correlated with the single RT-qPCR and showed similar assay sensitivity for 60 PML-RARa positive APL samples at diagnosis and 199 samples from 57 patients during follow-up. Interestingly, one PML-RARa bcr2 case at diagnosis with breakpoint at 1579, which was not detected by the single RT-q-PCR, was detected by the 3-plex RT-qPCR assay. The 3-plex RT-qPCR assay is a specific, sensitive, stable, and cost-effective method that can be used for the rapid diagnosis and

  14. Optimization of the diffusive gradients in thin films (DGT) method for simultaneous assay of potassium and plant-available phosphorus in soils.

    Science.gov (United States)

    Zhang, Yulin; Mason, Sean; McNeill, Ann; McLaughlin, Michael J

    2013-09-15

    Potassium (K) and phosphorus (P) are two important macronutrients for crops, and are usually applied to soils as granular fertilizer before seeding. Therefore, accurate soil tests prior to planting to predict crop response to fertilizers are important in optimizing crop yields. Traditional methods used for testing both available K and P in soils, which are based on chemical extraction procedures, are to be soil-type dependent, and the predictive relationships across a broad range of soils are generally poor. The diffusive gradients in thin films (DGT) technique, based on diffusion theory, is extensively used to measure the diffusive supply of trace elements, metals and some nutrients in soils and water. When DGT is used to assess plant-available P in soils, a good relationship is found between crop response to P fertilizer and concentrations of P in soil measured by DGT, and therefore the DGT method provides a more precise recommendation of P fertilizer requirements. Adaptation of the DGT method to measure plant-available K in soils has already been attempted [1], but limitations were reported due to the non-uniform size of the resin gel, decreased K binding rate of the gel at long deployment times and a limited ability to measure a wide range of K concentrations. To eliminate these problems, a new resin gel has been developed by combining Amberlite and ferrihydrite. This mixed Amberlite and ferrihydrite (MAF) gel has improved properties in terms of handling and even distribution of Amberlite in the gel. The elution efficiencies of the MAF gel for K and P were 90% and 96%, respectively. The diffusion coefficient of K through the diffusive gel was 1.30 × 10(-5)cm(2)s(-1) at 22 ± 1°C and was stable through time. Since ferrihydrite is already used in DGT P testing, the ability of the MAF gel to assess available P simultaneously was also assessed. The MAF gel performed the same as the traditional ferrihydrite gel for available P assessment in a wide variety of

  15. Evaluation of cytotoxicity of aqueous extract of Graviola leaves on squamous cell carcinoma cell-25 cell lines by 3-(4,5-dimethylthiazol-2-Yl -2,5-diphenyltetrazolium bromide assay and determination of percentage of cell inhibition at G2M phase of cell cycle by flow cytometry: An in vitro study

    Directory of Open Access Journals (Sweden)

    Visveswaraiah Paranjyothi Magadi

    2015-01-01

    Full Text Available Introduction: Malignancies constitute a wide variety of disorders having high mortality and morbidity rates. Current protocols for management include surgical intervention, chemotherapy, and radiation which possess numerous adverse effects. Many phytochemicals are available with anticancer properties similar to anticancer drugs. Major benefit of these compounds is apparent lack of toxicity to normal tissues. Graviola (botanical name: Annona Muricata contain bioactive compound “annonaceous acetogenins” known for anticancer activity on cancer cell lines. Aims: To determine cytotoxicity of Graviola and percentage cell inhibition at G2M phase of cell cycle. Settings and Design: The cytotoxicity of aqueous extract of Graviola leaves on squamous cell carcinoma (SCC-25 cell lines at various concentrations evaluated using 3-(4,5-dimethylthiazol-2-Yl-2,5-diphenyltetrazolium bromide (MTT assay. The percentage of SCC-25 cell inhibition at G2M phase of cell cycle determined using flow cytometry. Methods: Graviola Leaves, American Type Culture Collection SCC-25 cell lines were procured from Skanda Laboratories, Bengaluru. The cytotoxicity of aqueous extract of Graviola on SCC-25 cells at various concentrations evaluated using MTT assay. The percentage of SCC-25 cell inhibition at G2M phase of cell cycle determined using flow cytometry. Statistical Analysis: Statistical analysis was done using one-way ANOVA. Results: MTT assay showed statistically significant (P < 0.001 dose-dependent inhibition of SCC-25 cell lines by Graviola with IC50 value of 12.42 μg/ml. Flow cytometry revealed that Graviola at 25 and 50 g/ml arrested 53.39% and 52.09% cells in G2M phase of cell cycle respectively, which was statistically significant. Conclusion: Graviola showed significant cytotoxic activity and percentage of cell inhibition at G2M phase cell cycle against SCC-25 cell lines.

  16. The multicenter study of a new assay for simultaneous detection of multiple anti-aminoacyl-tRNA synthetases in myositis and interstitial pneumonia.

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    Ran Nakashima

    Full Text Available OBJECTIVE: Autoantibodies to aminoacyl-tRNA synthetases (ARSs are useful in the diagnosis of idiopathic inflammatory myopathy (IIM with interstitial pneumonia (IP. We developed an enzyme-linked immunosorbent assay (ELISA system using a mixture of recombinant ARS antigens and tested its utility in a multicenter study. METHODS: We prepared six recombinant ARSs: GST-Jo-1, His-PL-12, His-EJ and GST-KS expressed in Escherichia coli, and His-PL-7 and His-OJ expressed in Hi-5 cells. After confirming their antigenic activity, with the exception of His-OJ, we developed our ELISA system in which the five recombinant ARSs (without His-OJ were mixed. Efficiency was confirmed using the sera from 526 Japanese patients with connective tissue disease (CTD (IIM n = 250, systemic lupus erythematosus n = 91, systemic sclerosis n = 70, rheumatoid arthritis n = 75, Sjögren's syndrome n = 27 and other diseases n = 13, 168 with idiopathic interstitial pneumonia (IIP and 30 healthy controls collected from eight institutes. IIPs were classified into two groups; idiopathic pulmonary fibrosis (IPF (n = 38 and non-IPF (n = 130. RESULTS were compared with those of RNA immunoprecipitation. RESULTS: Sensitivity and specificity of the ELISA were 97.1% and 99.8%, respectively when compared with the RNA immunoprecipitation assay. Anti-ARS antibodies were detected in 30.8% of IIM, 2.5% of non-myositis CTD, and 10.7% of IIP (5.3% of IPF and 12.3% of non-IPF. Anti-ARS-positive non-IPF patients were younger and more frequently treated with glucocorticoids and/or immunosuppressants than anti-ARS-negative patients. CONCLUSION: A newly established ELISA detected anti-ARS antibodies as efficiently as RNA immunoprecipitation. This system will enable easier and wider use in the detection of anti-ARS antibodies in patients with IIM and IIP.

  17. Comparison of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-TOF and stable isotope dilution assay.

    Science.gov (United States)

    Hälvin, Kristel; Paalme, Toomas; Nisamedtinov, Ildar

    2013-02-01

    The application of LC/MS-TOF method combined with stable isotope dilution assay was studied for determination of thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine in food. Nutritional yeast powder was used as a model food matrix. Acid extraction was compared with various enzymatic treatments in ammonium formate buffer to find a suitable method for the conversion of more complex vitamers into the same forms as the used isotope-labeled internal standards. The enzyme preparations α-amylase, takadiastase, β-glucosidase, and acid phosphatase were all able to liberate thiamine and riboflavin. The diastatic enzyme preparations α-amylase and takadiastase also expressed proteolytic side activities resulting in the formation of small peptides which interfered with the mass spectra of thiamine and riboflavin. Liberation of nicotinamide and pantothenic acid from NAD(+) and CoA, respectively, could not be achieved with any of the studied enzyme preparations. Hydrochloric acid extraction at 121 °C for 30 min was found to be destructive to pantothenic acid, but increased the liberation of pyridoxal.

  18. Development of stable isotope dilution assays for the simultaneous quantitation of biogenic amines and polyamines in foods by LC-MS/MS.

    Science.gov (United States)

    Mayr, Christine M; Schieberle, Peter

    2012-03-28

    Microbial amino acid metabolism may lead to substantial amounts of biogenic amines in either spontaneously fermented or spoiled foods. For products manufactured with starter cultures, it has been suggested that certain strains may produce higher amounts of such amines than others; however, to support efforts of food manufacturers in mitigating amine formation, reliable methods for amine quantitation are needed. Using 10 isotopically labeled biogenic amines as the internal standards, stable isotope dilution assays were developed for the quantitation of 12 biogenic amines and of the 2 polyamines, spermine and spermidine, in one LC-MS/MS run. Application of the method to several foods revealed high concentrations of, for example, tyramine and putrescine in salami and fermented cabbage, whereas histamine was highest in Parmesan cheese and fermented cabbage. On the other hand, ethanolamine was highest in red wine and Parmesan cheese. The results suggest that different amino acid decarboxylases are active in the respective foods depending on the microorganisms present. The polyamine spermine was highest in salami and tuna.

  19. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders.

    Directory of Open Access Journals (Sweden)

    Péter Monostori

    Full Text Available Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA, methylmalonic acidemias and combined remethylation disorders (MMACBL. This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS as in primary screening (second-tier test. We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn

  20. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders.

    Science.gov (United States)

    Monostori, Péter; Klinke, Glynis; Richter, Sylvia; Baráth, Ákos; Fingerhut, Ralph; Baumgartner, Matthias R; Kölker, Stefan; Hoffmann, Georg F; Gramer, Gwendolyn; Okun, Jürgen G

    2017-01-01

    Increased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL. The assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients. Reliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid. A liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening

  1. Development of a new pentaplex real-time PCR assay for the identification of poly-microbial specimens containing Staphylococcus aureus and other staphylococci, with simultaneous detection of staphylococcal virulence and methicillin resistance markers.

    Science.gov (United States)

    Okolie, Charles E; Wooldridge, Karl G; Turner, David P; Cockayne, Alan; James, Richard

    2015-06-01

    Staphylococcus aureus strains harbouring genes encoding virulence and antibiotic resistance are of public health importance. In clinical samples, pathogenic S. aureus is often mixed with putatively less pathogenic coagulase-negative staphylococci (CoNS), both of which can harbour mecA, the gene encoding staphylococcal methicillin-resistance. There have been previous attempts at distinguishing MRSA from MRCoNS, most of which were based on the detection of one of the pathognomonic markers of S. aureus, such as coa, nuc or spa. That approach might suffice for discrete colonies and mono-microbial samples; it is inadequate for identification of clinical specimens containing mixtures of S. aureus and CoNS. In the present study, a real-time pentaplex PCR assay has been developed which simultaneously detects markers for bacteria (16S rRNA), coagulase-negative staphylococcus (cns), S. aureus (spa), Panton-Valentine leukocidin (pvl) and methicillin resistance (mecA). Staphylococcal and non-staphylococcal bacterial strains (n = 283) were used to validate the new assay. The applicability of this test to clinical samples was evaluated using spiked blood cultures (n = 43) containing S. aureus and CoNS in mono-microbial and poly-microbial models, which showed that the 5 markers were all detected as expected. Cycling completes within 1 h, delivering 100% specificity, NPV and PPV with a detection limit of 1.0 × 10(1) to 3.0 × 10(1) colony forming units (CFU)/ml, suggesting direct applicability in routine diagnostic microbiology. This is the most multiplexed real-time PCR-based PVL-MRSA assay and the first detection of a unique marker for CoNS without recourse to the conventional elimination approach. There was no evidence that this new assay produced invalid/indeterminate test results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Development and validation of a liquid chromatography-mass spectrometric assay for simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue.

    Science.gov (United States)

    Kirresh, Tatian; Tuteja, Sony; Russo, D; Brophy, P D; Murry, D J

    2017-03-20

    A sensitive and robust LC-MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5mg/kg and 2mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6μm C18 100Å, 100×2.1mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25ng/ml and the calibration curves covered a concentration range from 0.25 to 50ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89ng/ml and 1.50ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Revival of physostigmine - a novel HPLC assay for simultaneous determination of physostigmine and its metabolite eseroline designed for a pharmacokinetic study of septic patients.

    Science.gov (United States)

    Pinder, Nadine; Zimmermann, Johannes B; Hofer, Stefan; Brenner, Thorsten; Weigand, Markus A; Gubbe, Ute; Hoppe-Tichy, Torsten; Swoboda, Stefanie

    2015-07-01

    Physostigmine, commonly used as an antidote in anticholinergic poisoning, is reported to have additional pharmacological effects, such as activation of the cholinergic anti-inflammatory pathway in sepsis models. Due to the narrow therapeutic range of physostigmine and its metabolite eseroline, however, the plasma concentrations of these substances need to be determined so as to understand their effect and ensure safety in the treatment of septic patients. To determine physostigmine and its metabolite eseroline, a rapid and sensitive high performance liquid chromatography (HPLC) method has been developed and validated. Spiked plasma samples were cleaned up and concentrated using a simple liquid-liquid extraction (LLE) procedure with N-methylphysostigmine as internal standard. Separation was achieved using reversed-phase HPLC on a Kinetex C18 column with gradient elution and fluorescence detection (254 nm excitation/355 nm emission). LLE produced clean extracts and a mean recovery of 80.3% for eseroline and 84.9% for physostigmine. The HPLC assay revealed a limit of detection (LOD) of 0.025 ng/mL and a lower limit of quantification (LLOQ) of 0.05 ng/mL for both analytes. Linearity was observed at 0.05-10.0 ng/mL (r²>0.999). Intra- and inter-day precision ranged from 0.7% to 6.6%, and intra- and inter-day accuracy 97.5%-110.0%. The presented method is useful for human drug level monitoring of physostigmine and eseroline in accordance with current guidelines. Remarkably low plasma concentrations can be quantified after LLE with gradient elution and fluorescence detection, making this a suitable method for pharmacokinetic studies in a clinical setting.

  4. A simple dilute and shoot approach incorporated with pentafluorophenyl (PFP) column based LC-MS/MS assay for the simultaneous determination of trimethylamine N-oxide and trimethylamine in spot urine samples with high throughput.

    Science.gov (United States)

    Li, Xiaoguang Sunny; Li, Shu; Kellermann, Gottfried

    2017-11-01

    Determination of trimethylamine N-oxide (TMAO) and trimethylamine (TMA) in biological and environmental samples has drawn great attention recently due to their increasing association with human health and disease. It remains a challenge to simultaneously quantify TMAO and TMA in a simple, fast and cost-effective manner due to pre-analytical and analytical constraints. For the first time, we describe a dilute and shoot approach combined with LC-MS/MS detection for the simultaneous measurement of the analytes in spot urine samples with high throughput. Compared to the existing methods, the merits of the proposed assay include the use of a simple dilute and shoot approach (100-fold), small sample volume (10μL), short LC run on a PFP column (4.0min) and multi-analyte MS detection without sample cleanup, derivatization, evaporation and a HILIC column. Dilution, LC and MS parameters were optimized in detail. Method validation yielded a wide linearity for TMAO (1.0-400μg/mL) and TMA (0.025-10μg/mL) with a respective limit of quantitation of 1.0 and 0.025μg/mL. The quantitation was not affected by 41 major urinary components, structurally-related drugs and metabolites. The intra- and inter-day assay precisions were ≤3.6% and recoveries were 93.3%-103.3% for spiked quality control samples. The clinical utility of the alternative spot urine sampling approach compared to conventional 24h urine collection was supported by a significant correlation between the two sampling strategies (n=20, p<0.0001, r=0.757-0.862; slope=0.687-1.170) and no statistical difference in day-to-day biological variability (n=20). The applicability and reliability of the assay was verified by the assessment of reference intervals in a cohort of 118 healthy people. The proposed assay would be beneficial for the rapid and accurate determination of the increasingly important TMAO and TMA demanded in clinical, environmental, pharmaceutical and nutritional fields. Copyright © 2017 Elsevier B.V. All

  5. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

    Science.gov (United States)

    Bengsch, Bertram; Ohtani, Takuya; Herati, Ramin Sedaghat; Bovenschen, Niels; Chang, Kyong-Mi; Wherry, E John

    2018-02-01

    The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56 hi versus CD56 dim defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation

  6. Cytotoxicity of Southeast Asian snake venoms

    Directory of Open Access Journals (Sweden)

    A Jamunaa

    2012-01-01

    Full Text Available Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50 under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes.

  7. A Stable Isotope Dilution Nanoflow Liquid Chromatography Tandem Mass Spectrometry Assay for the Simultaneous Detection and Quantification of Glyoxal-Induced DNA Cross-Linked Adducts in Leukocytes from Diabetic Patients.

    Science.gov (United States)

    Chen, Hauh-Jyun Candy; Chang, Ya-Lang; Teng, Yi-Chun; Hsiao, Chiung-Fong; Lin, Tsai-Shiuan

    2017-12-19

    Glyoxal (gx) is a bifunctional electrophile capable of cross-linking DNA. Although it is present in foods and from the environment, endogenous formation of glyoxal occurs through metabolism of carbohydrates and oxidation of lipids and nucleic acids. Plasma concentrations of glyoxal are elevated in in diabetes mellitus patients compared to nondiabetics. The most abundant 2'-deoxyribonucleoside adducts cross-linked by glyoxal are dG-gx-dC, dG-gx-dA, and dG-gx-dG. These DNA cross-links can be mutagenic by damaging the integrity of the DNA structure. Herein, we developed a highly sensitive and specific assay for the simultaneous detection and quantification of the dG-gx-dC and dG-gx-dA cross-links based on stable isotope dilution (SID) nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the highly selected reaction monitoring mode and using a triple quadrupole mass spectrometer. The entire assay procedure involved addition of the stable isotope standards [15N5]dG-gx-dC and [15N5]dG-gx-dA as internal standards, enzyme hydrolysis to release the cross-links as nucleosides, enrichment by a reversed-phase solid-phase extraction column, and nanoLC-NSI/MS/MS analysis. The detection limit is 0.19 amol for dG-gx-dC and 0.89 amol for dG-gx-dA, which is 400 and 80 times more sensitive, respectively, than capillary LC-NSI/MS/MS assay of these adducts. The lower limit of quantification was 94 and 90 amol for dG-gx-dC and dG-gx-dA, respectively, which is equivalent to 0.056 and 0.065 adducts in 108 normal nucleotides in 50 μg of DNA. In type 2 diabetes mellitus (T2DM) patients (n = 38), the levels of dG-gx-dC and dG-gx-dA in leukocyte DNA were 1.94 ± 1.20 and 2.10 ± 1.77 in 108 normal nucleotides, respectively, which were significantly higher than those in nondiabetics (n = 39: 0.83 ± 0.92 and 1.05 ± 0.99 in 108 normal nucleotides, respectively). Excluding the factor of smoking, an exogenous source of glyoxal, levels of these

  8. Sulforhodamine B assay and chemosensitivity.

    Science.gov (United States)

    Voigt, Wieland

    2005-01-01

    The sulforhodamine B (SRB) assay was developed by Skehan and colleagues to measure drug-induced cytotoxicity and cell proliferation for large-scale drug-screening applications. Its principle is based on the ability of the protein dye sulforhodamine B to bind electrostatically and pH dependent on protein basic amino acid residues of trichloroacetic acid-fixed cells. Under mild acidic conditions it binds to and under mild basic conditions it can be extracted from cells and solubilized for measurement. Results of the SRB assay were linear with cell number and cellular protein measured at cellular densities ranging from 1 to 200% of confluence. Its sensitivity is comparable with that of several fluorescence assays and superior to that of Lowry or Bradford. The signal-to-noise ratio is favorable and the resolution is 1000-2000 cells/well. It performed similarly compared to other cytotoxicity assays such as MTT or clonogenic assay. The SRB assay possesses a colorimetric end point and is nondestructive and indefinitely stable. These practical advances make the SRB assay an appropriate and sensitive assay to measure drug-induced cytotoxicity even at large-scale application.

  9. Antimicrobial activity, cytotoxicity, and phytochemical screening of Voacanga globosa (Blanco) Merr. leaf extract (Apocynaceae).

    Science.gov (United States)

    Vital, Pierangeli G; Rivera, Windell L

    2011-10-01

    To determine the antibacterial, antifungal, antiprotozoal, cytotoxic, and phytochemical properties of ethanol extracts of leaves of Voacanga globosa (Blanco) Merr. (V. globosa). The extracts were tested against bacteria and fungus through disc diffusion assay; against protozoa through growth curve determination, antiprotozoal and cytotoxicity assays. The extract revealed antibacterial activities, inhibiting the growth of Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Micrococcus luteus, and Salmonella typhimurium. Antifungal assay showed that it inhibited Candida albicans. The antiprotozoal assay against Trichomonas vaginalis and Entamoeba histolytica showed that V. globosa can inhibit the parasites, wherein the action can be comparable to metronidazole. With the in situ cell death detection kit, Trichomonas vaginalis and Entamoeba histolytica exposed to V. globosa leaf extract was observed to fluoresce simultaneously in red and yellow signals signifying apoptotic-like changes. Preliminary phytochemical screening revealed the chemical composition of plant extract containing alkaloids, saponins, 2-deoxysugars, and hydrolysable tannins. Thus, this study provides scientific evidence on the traditional use of V. globosa leaf extract in treating microbial diseases. Further, the leaf extract can possibly be used to produce alternative forms of antimicrobials. Copyright © 2011 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. Cytotoxicity, mutagenicity, and antimutagenicity of the gentisic acid on HTC cells.

    Science.gov (United States)

    Cavalcante, Flavia Maria Lima; Almeida, Igor Vivian; Düsman, Elisângela; Mantovani, Mário Sérgio; Vicentini, Veronica Elisa Pimenta

    2017-05-16

    Gentisic acid (GA) exhibits antioxidant, anti-inflammatory, and antibiotic activities. This substance can be found in citrus fruits, grapes, olive oil, and peas. Considering that there are few studies in the literature on the toxicity of GA, the present work aimed to investigate its cytotoxic, mutagenic, and antimutagenic activities on HTC cells. GA was diluted in culture medium at the final concentration of 0.08, 0.16, 0.8, 1.6, and 8 μg/mL. The cytotoxicity was determined by the MTT assay and Trypan Blue exclusion method, with methyl methanesulfonate and doxorubicin as positive controls, respectively. The cytokinesis-block micronucleus assay determined the mutagenic/antimutagenic activity with benzo[a]pyrene as positive control. Negative control received culture medium only. GA (0.08-8 μg/mL) was not cytotoxic to HTC cells by the MTT assay nor the Trypan Blue exclusion method as no statistical difference was observed when compared to the control. Concentration of 0.08 and 0.8 μg/mL showed no mutagenic or clastogenic effects, as no significant micronuclei inductions were observed, different from 8 μg/mL, that was mutagenic. Furthermore, none of the concentrations presented an antiproliferative activity. The antimutagenic activity of GA (0.08 μg/mL) was observed at the simultaneous treatment, as it reduced the frequency of micronuclei by 76% (24 h) and 79% (48 h). Although pre- and post-treatments were not statistically different from the mutagen, they reduced the induced-damage by 11% and 21%, respectively. The present study indicated the absence of cytotoxicity and antiproliferative activities of GA, in addition to their antimutagenic/protective effects that may contribute to human health.

  11. Pharmacognostic profile and in vitro cytotoxic activity of Adenema ...

    African Journals Online (AJOL)

    Chloroform, ethyl acetate and ethanolic extracts were investigated for in vitro cytotoxic activity against nine different types of human cancer panel of 60 different strains of tumor cell lines using the sulforhodamine-B (SRB) binding assay. The chloroform extract showed potent cytotoxicity against non small cell lung cancer cell, ...

  12. Kinetin (N 6 -furfuryladenine): Cytotoxicity against MCF-7 breast ...

    African Journals Online (AJOL)

    Kinetin (N 6 -furfuryladenine): Cytotoxicity against MCF-7 breast cancer cell line and interaction with bovine serum albumin. ... African Journal of Biotechnology ... The cytotoxicity effect of kinetin on MCF-7 breast cancer cell lines was measured by the sulforhodamine B (SRB) assay and the results showed that kinetin could ...

  13. Cytotoxicity of Sambucus ebulus on cancer cell lines and protective ...

    African Journals Online (AJOL)

    DR. TONUKARI NYEROVWO

    2013-05-22

    May 22, 2013 ... Also, protective effects of vitamins C and E against SEE-induced cytotoxicity on normal cell lines were studied. Cytotoxicity of SEE on cancer (HepG2 and CT26) and normal (CHO and rat fibroblast) cell lines was evaluated by MTT assay. IC50 of SEE on the cell lines was assessed. Furthermore, IC50 of ...

  14. Cytotoxic and Antioxidant Activities of Antidesma thwaitesianum Müll ...

    African Journals Online (AJOL)

    Purpose: To investigate the cytotoxic and antioxidant activities of the fruit and fruit waste (residue and marc) extracts of Antidesma thwaitesianum Müll. Arg., known as mamao in Thai, using chemical and cell-based assays. Methods: The cytotoxicity of mamao fruit and fruit waste extracts obtained by expression, maceration ...

  15. A simple assay for the simultaneous determination of rosuvastatin acid, rosuvastatin-5S-lactone, and N-desmethyl rosuvastatin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    Science.gov (United States)

    Macwan, Joyce S; Ionita, Ileana A; Akhlaghi, Fatemeh

    2012-01-01

    A simple and sensitive assay was developed and validated for the simultaneous quantification of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST), in buffered human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All the three analytes and the corresponding deuterium-labeled (d6) internal standards were extracted from 50 μL of buffered human plasma by protein precipitation. The analytes were chromatographically separated using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase comprised of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). The analytes were separated at baseline within 6.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode. The calibration curves for all three analytes were linear (R ≥ 0.9964, n = 3) over the concentration range of 0.1-100 ng/mL for RST and RST-LAC, and 0.5-100 ng/mL for DM-RST. Mean extraction recoveries ranged within 88.0-106%. Intra- and inter-run mean percent accuracy were within 91.8-111% and percent imprecision was ≤15%. Stability studies revealed that all the analytes were stable in matrix during bench-top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at -80°C for 1 month. The method was successfully applied in a clinical study to determine the concentrations of RST and the lactone metabolite over 12-h post-dose in patients who received a single dose of rosuvastatin.

  16. Cytotoxicity of activated monocytes on endothelial cells.

    Science.gov (United States)

    Peri, G; Chiaffarino, F; Bernasconi, S; Padura, I M; Mantovani, A

    1990-02-15

    Unstimulated human monocytes did not express appreciable levels of cytotoxicity on normal human umbilical vein endothelial cells (EC) in a 24-48 hr TdR release assay. On activation with IFN-gamma and LPS, monocytes had appreciable cytotoxicity on EC. Monocyte cytotoxicity on EC was not dependent on the presence of contaminating lymphoid cells. Recombinant TNF, IL-1, and IL-6 as well as monocyte supernatants did not exert a cytotoxic effect on EC. Moreover, anti-TNF, anti-IL-1, and anti-IL-6 antibodies, as well as scavengers of reactive oxygen intermediates, did not affect the cytotoxicity of activated monocytes on EC. Antibodies against the beta-chain (CD18) of leukocyte integrins inhibited the adhesion and cytotoxicity of activated monocytes on EC. Pretreatment of EC with IL-1 augmented the adhesion of monocytes on EC. Normal monocytes were not cytotoxic on IL-1-pretreated EC and IL-1 treatment did not increase the susceptibility of EC to activated monocytes. Thus adhesion is necessary but not sufficient for monocyte killing of EC. Anti-alpha L (LFA-1) antibodies markedly reduced monocyte cytotoxicity on EC, although anti-alpha X (p150) antibodies had only a modest effect. Anti-alpha M (Mac-1/CR3) antibodies were intermediate inhibitors of EC killing by activated monocytes. Thus, alpha L, beta 2 (LFA-1), and, to a lesser extent, alpha M, beta 2 (Mac-1/CR3) and alpha X, beta 2 (p 150, 95) integrins are the main adhesive structures involved in the cytotoxic interaction of activated monocytes with EC. Monocyte-mediated damage of EC could play a role as a mechanism of tissue injury under conditions of local or systemic activation of mononuclear phagocytes.

  17. Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

    Science.gov (United States)

    Chun, Song-I; Mun, Chi-Woong

    2015-01-01

    A reference reagent, 3-(trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium (TSP), has been used frequently in nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells. A human osteosarcoma cell line (MG-63) was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM) of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding. In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation) and cell viability. High concentrations of TSP (from 10 to 30 mM) reduced both cell proliferation and viability (to 30% of the control after one week of exposure), but no such effects were found using low concentrations of TSP (0-10 mM). This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

  18. A novel functional assay for simultaneous determination of total fatty acid beta-oxidation flux and acylcarnitine profiling in human skin fibroblasts using (2)H(31)-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry

    NARCIS (Netherlands)

    Law, Lap-Kay; Tang, Nelson Leung-Sang; Hui, Joannie; Ho, Chung-Shun; Ruiter, Jos; Fok, Tai-Fai; Wanders, Ronald J. A.; Lam, Christopher Wai-Kei

    2007-01-01

    BACKGROUND: Two separate and complementary assays, total mitochondrial fatty acid beta-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by

  19. The cytotoxicity study of praziquantel enantiomers

    Directory of Open Access Journals (Sweden)

    Sun Q

    2016-06-01

    Full Text Available Qian Sun, Ruifeng Mao, Dongling Wang, Changyan Hu, Yang Zheng, Dequn Sun Department of Pharmacy, Marine College, Shandong University, Weihai, People’s Republic of China Abstract: Praziquantel (PZQ is prescribed as a racemic mixture (racemic-PZQ, rac-PZQ, which is composed of (R-PZQ and (S-PZQ. In this work, the cytotoxicity of rac-PZQ and its two enantiomers (R-PZQ and (S-PZQ on eight cell lines (L-02, HepG2, prf-plc-5, SH-SY5Y, HUVEC, A549, HCT-15, Raw264.7 was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays. The morphology of apoptotic cells was studied by fluorescence microscope using Hoechst 33342 staining, and the cytotoxicity of the compounds was also tested by lactate dehydrogenase assay. Results revealed that (R-PZQ had negligible cytotoxicity against L-02, SH-SY5Y, HUVEC, A549, HCT-15, and Raw264.7 cells but selectively inhibited tumor cell lines (prf-plc-5 and HepG2. However, in contrast to (R-PZQ, the (S-isomer showed higher cytotoxicity against L-02 cells and lower inhibition on prf-plc-5 and HepG2 cells. Besides, (R-PZQ showed lower cytotoxicity on SH-SY5Y cells than (S-PZQ. Meanwhile, (R-PZQ at <80 µM concentration could promote proliferation of macrophage cells (Raw264.7. Our research revealed that (R-PZQ has lower cytotoxicity than (S-PZQ and has similar cytotoxicity with rac-PZQ. (S-PZQ is the principal enantiomer to cause side effects on human definitive hosts. These findings gave the reasonable reasons for World Health Organization to produce (R-PZQ as a replacement for rac-PZQ for the treatment of schistosomiasis. Keywords: isomer, MTT, selectivity, (R-PZQ

  20. Cytotoxic chalcones from some Indonesian Cryptocarya

    Science.gov (United States)

    Kurniadewi, F.; Syah, Y. M.; Juliawaty, L. D.; Hakim, E. H.; Koyama, K.; Kinoshita, K.

    2017-07-01

    Malignant tumors are one of the main causes of death in the world. Until now the search for cytotoxic (antitumor) compounds from nature, particularly from plants, is being a continuation activities. One group of plants that produce potential cytotoxic compounds is the Cryptocarya, one of the large genera of the Lauraceae family. As a part of our chemical and cytotoxic evaluation of the Cryptocarya species, we examined three species of Indonesian Cryptocarya. The sample of the wood of C. konishii hayata was collected from Cibodas Botanical Garden, West Java while the stem bark of C. phoebeopsis and C. cagayanensis were obtained from Sorong, Papua. Our investigation of flavonoid constituents on these species afforded three chalcone compounds i.e. desmethylinfectocaryone (1), infectocaryone (2) and cryptocaryone (3). The molecular structures of the isolated compounds were determined based on spectroscopic data, including UV, IR, 1D and 2D NMR. Cytotoxic effects of the compounds were evaluated using MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. Compound 1, 2 and 3 displayed strong cytotoxic properties (IC50 properties against HCT116 (colon cancer). Cryptocaryone (3) also showed moderate cytotoxic properties (IC50 < 10 μg/mL) towards A549 (human lung adenocarcinoma epithelial) cells.

  1. Bioassay-guided studies on the cytotoxic and in vitro trypanocidal ...

    African Journals Online (AJOL)

    All the above pure isolates, and the crude extracts of Z. chalybeum had neither biological activity nor cytotoxicity in the brine shrimp assay. A cytotoxic sesquiterpine, characterized as muzigadial, was isolated from W. ugandensis. It was highly toxic in the brine shrimp assay and also had in vitro trypanocidal activity against IL ...

  2. Clinical validation of a novel real-time human papillomavirus assay for simultaneous detection of 14 high-risk HPV type and genotyping HPV type 16 and 18 in China.

    Science.gov (United States)

    Yang, Hui; Li, Lie-Jun; Xie, Long-Xu; Luo, Zhao-Yun; Lu, Min; Lin, Min; Zheng, Xiang-Bin; Huang, Yue; Yang, Li-Ye

    2016-02-01

    In the present study, we describe the laboratory workflow and the clinical validation of a novel multiplex real-time PCR-based HPV assay in China. The cross-sectional validation analysis showed that this assay worked well for detection of 14 HR-HPV types and identification of HPV 16 and 18 in a single sensitive assay that is suitable for both clinical usage and high-throughput cervical screening purposes. We predict that this accurate, high-throughput and low-cost HPV assay can greatly reduce the heavy economic burden of HPV detection in China.

  3. Cytotoxicity, Total Phenolic Contents and Antioxidant Activity of the ...

    African Journals Online (AJOL)

    The leaves of Annona muricata were extracted using ethanol and the extracts were evaluated for cytotoxicity using Brine Shrimp Lethality Assay, total phenolic content (TPC) and antioxidant activity using DPPH radical scavenging assay. The crude extract showed 73.33 % mortality at 1000 μg/mL concentration and its ...

  4. Studies on cytotoxicity and antitumor screening of red and white ...

    African Journals Online (AJOL)

    The cytotoxicity study with cancer cell line A-549 indicated that the methanol insoluble fraction of crude red forms of Abrus precatorius is toxic (CTC50 175,100 mg/ml) in microculture tetrazolium assay and sulphorhodamine B assays, whereas crude and fractions of the red and white forms exhibited toxicity at still higher ...

  5. The Protective Effect of Silybin against Lasalocid Cytotoxic Exposure on Chicken and Rat Cell Lines

    Directory of Open Access Journals (Sweden)

    Lidia Radko

    2013-01-01

    Full Text Available Lasalocid, an ionophore coccidiostat, extensive use implies a risk of toxicological impacts. Protective effects of silybin, a herbal compound of Silybum marianum, are reported elsewhere. The aim of this study was to compare effects of the combined use of lasalocid and silybin in chicken hepatoma cells (LMH and rat myoblasts (L6 cell lines cultures. The cytoprotective effect resulting from an interaction of both pharmaceuticals was measured with the help of MTT reduction and, coomassie brilliant blue binding (CBB and LDH release assays. Isobolography and the combination index (CI estimated the nature and scale of interaction. In all performed tests, the lowest lasalocid EC50-values were obtained for chicken hepatocytes. In the rat myoblasts cultures, the lowest lasalocid EC50-values were found with LDH test. Simultaneously, a lack of silybin cytotoxic effect was proven for the studied cell lines. An interaction between both substances led to a considerable decrease of lasalocid cytotoxicity. The isobolograms and combination index showed a significant antagonistic nature of silybin effect in the course of lasalocid cytotoxicity. It is concluded that the mechanism of cytoprotection results from complex reaction at biochemical and biophysical endpoints during chicken hepatocytes and rat myoblasts cell lines exposure to silybin and lasalocid co-action.

  6. Cytotoxic abietane diterpenes from Hyptis martiusii Benth.

    Science.gov (United States)

    da Araújo, Edigênia Cavalcante Cruz; Lima, Mary Anne S; Montenegro, Raquel C; Nogueira, Marcelle; Costa-Lotufo, Letícia V; Pessoa, Cláudia; de Moraes, Manoel Odorico; Silveira, Edilberto R

    2006-01-01

    From roots of Hyptis martiusii Benth. two tanshinone diterpenes were isolated, the new 7beta-hydroxy-11,14-dioxoabieta-8,12-diene (1) in addition to the known 7alpha-acetoxy-12-hydroxy-11,14-dioxoabieta-8,12-diene (7alpha-acetoxyroyleanone) (2). Structures of 1 and 2 were established by spectroscopic means. The cytotoxic activity against five cancer cell lines was evaluated. Compounds 1 and 2 displayed considerable cytotoxic activity against several cancer cell lines with IC50 values in the range of 3.1 to 11.5 microg/ml and 0.9 to 7.6 microg/ml, respectively. The cytotoxic activity seemed to be related to inhibition of DNA synthesis, as revealed by the reduction of 5-bromo-2'-deoxyuridine incorporation and induction of apoptosis, as indicated by the acridine orange/ethidium bromide assay and morphological changes after 24 h of incubation in leukemic cells.

  7. A new bioassay for the immunocytokine L19-IL2 for simultaneous analysis of both functional moieties.

    Science.gov (United States)

    Winter, Jonas; Barbin, Karin; Bacci, Camilla; Bunte, Thomas

    2011-01-05

    Currently, cancer directed new biological entities (NBEs) in the pharmaceutical R&D pipelines are derived from monoclonal antibodies in various formats, such as immunocytokines. Generally, immunocytokines are bi-functional molecules that consist of a specific targeting antibody-based portion and a linked cytokine. To confirm the quality of the drug product both moieties have to be characterized using appropriate techniques. Until now, the binding capacity of antibodies is usually examined by ligand binding assays whereas the biological activity of the linked cytokine is determined by cell-based potency assays. However, the simultaneous analysis of both functional moieties in a single assay format has not been described so far. In this paper we present a newly designed bioassay format for the anti-cancer immunocytokine L19-IL2, comprising of the human vascular targeting single-chain Fv L19 and human interleukin 2 (IL2). This new potency assay allows simultaneous analysis of both moieties, thus specific L19 binding capacity and the ability of IL2 to induce the proliferation of the detector cytotoxic T-cell line CTLL-2. Assay development was performed with special focus on application of different fitting models for the sigmoid dose-response curves to evaluate the influence of model optimization on the validity of assay results. For assay validation generally accepted characteristics were determined. Assay specificity was shown by testing L19-IL2 related compounds. All other validation parameters were derived from 25 batch runs using five nominal L19-IL2 concentrations, covering a range from 60% to 140% of the standard's potency. Accuracy ranged from -3.4% to -6.9% relative error (%RE). Interbatch precision ranged from 6.1% to 10.6% coefficient of variation (%CV). For assay linearity a coefficient of determination (R(2)) of 0.9992 was found. Assay robustness was shown with L19-IL2 samples after three freeze-thaw cycles and also with different cell passages of the used

  8. Cytotoxicity of Dental Adhesives In Vitro

    Science.gov (United States)

    Koulaouzidou, Elisabeth A.; Helvatjoglu-Antoniades, Maria; Palaghias, George; Karanika-Kouma, Artemis; Antoniades, Dimitrios

    2009-01-01

    Objectives The purpose of this study was to evaluate the cytotoxic effect of six dental adhesives (Admira Bond, Clearfil Liner Bond 2V, ED Primer II, Fuji Bond LC, Gluma Comfort Bond, and NanoBond) applied to cell cultures. Methods The experiments were performed on two cell lines, rat pulp cells (RPC-C2A) and human lung fibroblasts (MRC5). Samples of the adhesives were light-cured and placed in culture medium for 24 hours. The extraction media was applied on the RPC-C2A and the MRC5 cells. Complete medium was used as a control. Cytotoxicity was evaluated with a modified sulforhodamine B (SRB) assay after 24 hours of exposure. Results The cell survival of RPC-C2A cells exposed to Fuji Bond LC, NanoBond, Clearfil Liner Bond 2V, ED Primer II, Admira Bond and Gluma Comfort Bond was 73%, 67%, 50%, 20%, 18% and 5% respectively, relative to the cell survival with the control medium. In the MRC5 cell line, the relative survival was 98%, 80%, 72%, 41%, 19% and 7% after exposure to NanoBond, Fuji Bond LC, Clearfil Liner Bond 2V, ED Primer II, Admira Bond and Gluma Comfort Bond, respectively. Conclusions Different types of dental adhesives showed different cytotoxic effects on cells in vitro. The self-etch adhesives were superior in terms of cytotoxicity. The different cytotoxic effects of dental adhesives should be considered when selecting an appropriate adhesive for operative restorations. PMID:19262725

  9. Cytotoxicity of dental adhesives in vitro.

    Science.gov (United States)

    Koulaouzidou, Elisabeth A; Helvatjoglu-Antoniades, Maria; Palaghias, George; Karanika-Kouma, Artemis; Antoniades, Dimitrios

    2009-01-01

    The purpose of this study was to evaluate the cytotoxic effect of six dental adhesives (Admira Bond, Clearfil Liner Bond 2V, ED Primer II, Fuji Bond LC, Gluma Comfort Bond, and NanoBond) applied to cell cultures. The experiments were performed on two cell lines, rat pulp cells (RPC-C2A) and human lung fibroblasts (MRC5). Samples of the adhesives were light-cured and placed in culture medium for 24 hours. The extraction media was applied on the RPC-C2A and the MRC5 cells. Complete medium was used as a control. Cytotoxicity was evaluated with a modified sulforhodamine B (SRB) assay after 24 hours of exposure. The cell survival of RPC-C2A cells exposed to Fuji Bond LC, NanoBond, Clearfil Liner Bond 2V, ED Primer II, Admira Bond and Gluma Comfort Bond was 73%, 67%, 50%, 20%, 18% and 5% respectively, relative to the cell survival with the control medium. In the MRC5 cell line, the relative survival was 98%, 80%, 72%, 41%, 19% and 7% after exposure to NanoBond, Fuji Bond LC, Clearfil Liner Bond 2V, ED Primer II, Admira Bond and Gluma Comfort Bond, respectively. Different types of dental adhesives showed different cytotoxic effects on cells in vitro. The self-etch adhesives were superior in terms of cytotoxicity. The different cytotoxic effects of dental adhesives should be considered when selecting an appropriate adhesive for operative restorations.

  10. Cytotoxic Activities of Phytochemicals from Ferula Species

    Directory of Open Access Journals (Sweden)

    Seied Mojtaba Valiahdi

    2013-05-01

    Full Text Available Background:Ferula species are reputed in folk medicine for the treatment of a variety of disorders. There have been sporadic reports on the chemopreventive and chemosensitizing activities of some terpenoid coumarin derivatives from the genus Ferula. The present study investigated the cytotoxic activity of 11 phytochemicals (conferone, farnesiferol A, acantrifoside E, mogoltadone, diversin, galbanic acid, herniarin, 7-isopentenyloxycoumarin, umbelliprenin, stylosin and tschimgine from Ferula species together with a newly synthesized prenylated derivative of curcumin (gercumin II.Methods:Cytotoxic activity of phytochemicals was evaluated against ovarian carcinoma (CH1, lung cancer (A549 and melanoma (SK-MEL-28 cell lines using MTT assay.Results and conclusion:Overall, moderate cytotoxic activity was observed from the tested compounds with IC50 values in the micromolar range. The highest activity against CH1 and A549 lines was from conferone while stylosin and tschimgine were the most potent compounds against SK-MEL-28 line. In conclusion, the findings of the present investigation did not support a potent cytotoxic activity of the tested phytochemicals against CH1, A549 and SK-MEL-28 cell lines. With respect to previous reports, the beneficial impact of these phytochemicals in cancer therapy may be more attributable to their chemopreventive or chemosensitizing activity rather than direct cytotoxic effects.

  11. Enzyme assays

    OpenAIRE

    Bisswanger, Hans

    2014-01-01

    The essential requirements for enzyme assays are described and frequently occurring errors and pitfalls as well as their avoidance are discussed. The main factors, which must be considered for assaying enzymes, are temperature, pH, ionic strength and the proper concentrations of the essential components like substrates and enzymes. Standardization of these parameters would be desirable, but the diversity of the features of different enzymes prevents unification of assay conditions. Neverthele...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

    2015-01-15

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

  13. Cytotoxic limonoids from Melia azedarach.

    Science.gov (United States)

    Yuan, Chun-Mao; Zhang, Yu; Tang, Gui-Hua; Li, Yan; He, Hong-Ping; Li, Shi-Fei; Hou, Li; Li, Xing-Yao; Di, Ying-Tong; Li, Shun-Lin; Hua, Hui-Ming; Hao, Xiao-Jiang

    2013-01-01

    Five new compounds (1-5), including two limonoids, one triterpenoid, one steroid, and one sesquiterpenoid, along with nine known limonoids (6-14), were isolated from the bark of Melia azedarach. The structures of the new compounds were elucidated by 2D NMR spectroscopy and mass spectrometry. The isolated compounds as well as three acetylated derivatives of 9 were evaluated for their cytotoxicities against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) by an MTT assay. Seven limonoids (1, 6, 7, 8, 9, 9b, and 9c) showed significant inhibitory activities against tested cell lines with IC50 values ranging from 0.003 to 0.555 µM, and their preliminary structure-activity relationships are also discussed. Georg Thieme Verlag KG Stuttgart · New York.

  14. Evaluation of the new AmpliSens multiplex real-time PCR assay for simultaneous detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis.

    Science.gov (United States)

    Rumyantseva, Tatiana; Golparian, Daniel; Nilsson, Christian S; Johansson, Emma; Falk, My; Fredlund, Hans; Van Dam, Alje; Guschin, Alexander; Unemo, Magnus

    2015-10-01

    In this study, we performed an evaluation of the new CE-marked multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay compared to APTIMA tests, i.e., APTIMA COMBO 2 assay, APTIMA Trichomonas vaginalis assay (FDA-approved), and two different APTIMA Mycoplasma genitalium assays (research use only; one of them only used for discrepancy analysis). Vaginal swabs (n = 209) and first-void urine (FVU) specimens from females (n = 498) and males (n = 554), consecutive attendees (n = 1261) at a dermatovenerological clinic in Sweden, were examined. The sensitivity of the AmpliSens PCR assay for detection of C. trachomatis (6.3% prevalence), M. genitalium (5.7% prevalence), N. gonorrhoeae (0.3% prevalence), and T. vaginalis (0.08% prevalence) was 97.5% (95% confidence interval (CI): 91.2-99.6%), 81.9% (95% CI: 70.7-89.7%), 100% (95% CI: 40.2-100%) and 100% (95% CI: 16.5-100%), respectively. The specificity of the AmpliSens PCR assay was 100% (95% CI: 99.6-100%) for all agents. The analytical sensitivity and specificity for N. gonorrhoeae detection was excellent, i.e., 55 international gonococcal strains detected and 135 isolates of 13 non-gonococcal Neisseria species were negative. In conclusion, the multiplex real-time AmpliSens N.gonorrhoeae/C.trachomatis/M.genitalium/T.vaginalis-MULTIPRIME-FRT PCR assay demonstrated high sensitivity and excellent specificity for the detection of C. trachomatis, N. gonorrhoeae, and T. vaginalis, and excellent specificity but suboptimal sensitivity for M. genitalium detection. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  15. Cytotoxicity of methanol extracts of Elaeis guineensis on MCF-7 and Vero cell lines.

    Science.gov (United States)

    Vijayarathna, Soundararajan; Sasidharan, Sreenivasan

    2012-10-01

    To investigate the cytotoxic effect of Elaeis guineensis methanol extract on MCF-7 and Vero cell. In vitro cytotoxicity was evaluated in by MTT assay. Cell morphological changes were observed by using light microscope. The MTT assay indicated that methanol extract of the plant exhibited significant cytotoxic effects on MCF-7. Morphological alteration of the cell lines after exposure with Elaeis guineensis extract were observed under phase contrast microscope in the dose dependent manner. The results suggest the probable use of the Elaeis guineensis methanol extract in preparing recipes for cancer-related ailments. Further studies on isolation of metabolites and their in vivo cytotoxicity are under investigation.

  16. Aloe QDM complex enhances specific cytotoxic T lymphocyte killing in vivo in metabolic disease mice.

    Science.gov (United States)

    Lee, Youngjoo; Kim, Jiyeon; An, Jinho; Lee, Heetae; Kong, Hyunseok; Song, Youngcheon; Shin, Eunju; Do, Seon-Gil; Lee, Chong-Kil; Kim, Kyungjae

    2017-03-01

    We developed spontaneous diet-induced metabolic disease in mice by feeding them a high-fat diet for 23 weeks and administered Aloe QDM complex for 16 weeks to examine its restorative effect on immune disorders and metabolic syndrome. A series of immune functional assays indicated Aloe QDM complex enhanced lymphocyte proliferation and antigen-specific immunity as determined by the restored functions of cytotoxic T lymphocytes (CTL) and IgG production. The elevated serum TNF-α level was also regulated by Aloe QDM complex treatment, which suggested its complex therapeutic potential. As for metabolic phenotypes, oral administration of Aloe QDM complex significantly improved diabetic symptoms, including high fasting glucose levels and glucose tolerance, and distinctly alleviated lipid accumulation in adipose and hepatic tissue. The simultaneous restoration of Aloe QDM complex on metabolic syndrome and host immune dysfunction, especially on the specific CTL killing was first elucidated in our study.

  17. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Cytotoxicity study of plant Aloe vera (Linn

    Directory of Open Access Journals (Sweden)

    Atul N Chandu

    2012-01-01

    Full Text Available Background: The objective of this study has been to evaluate the in-vitro antitumor activity of Aloe vera extract of in cultured B16F10 melanoma cell line by measuring cell viability using "Trypan blue exclusion assay" method. Aim: To find out such kind of anticancer drug which is a cheap, safe, less toxic, and more potent drug compared to chemotherapy drug. Materials and Methods: In-vitro antitumor activity cell culture1, drug treatment (standard and test extract and Trypan blue exclusion assay growth and viability test 1 were used. Treatment of Aloe vera extract against B16F10 melanoma cell line, in all concentration range, showed decrease in percent cell viability, as compared to that of negative when examined by "Trypan blue exclusion assay". Results: In overall variation of test samples, Aloe vera extract showed its best activity in the concentration of 300 μg/ml, which was approximately equal to the activity of standard drug doxorubicin. Evaluation of in-vitro antitumor activity revealed that Aloe vera extract exhibits good cytotoxic activity. The best cytotoxic activity by Aloe vera was shown at 200 μg/ml concentration. Conclusion: The study of cytoprotection against normal cells by micronucleus assay has shown that the herbal extracts have less toxic effects to the normal blood lymphocytes, as compared to that of standard anticancer drug.

  19. DETECTION OF BACTERIAL CYTOTOXIC ACTIVITIES FROM WATER-DAMAGED CEILING TILE MATERIAL FOLLOWING INCUBATION ON BLOOD AGAR

    Science.gov (United States)

    Samples of ceiling tiles with high levels of bacteria exhibited cytotoxic activities on a HEP-2 tissue culture assay. Ceiling tiles containing low levels of bacterial colonization did not show cytotoxic activities on the HEP-2 tissue culture assay. Using a spread plate procedure ...

  20. Cytotoxic activity of four Mexican medicinal plants.

    Science.gov (United States)

    Vega-Avila, Elisa; Espejo-Serna, Adolfo; Alarcón-Aguilar, Francisco; Velasco-Lezama, Rodolfo

    2009-01-01

    Ibervillea sonorae Greene, Cucurbita ficifolia Bouché, Tagetes lucida Cav and Justicia spicigera Scheltdd are Mexican native plants used in the treatment of different illnesses. The ethanolic extract of J. spicigera and T. lucida as well as aqueous extracts from I. sonorae, C. ficifolia, T. lucida and J. spicigera were investigated using sulforhodamine B assay. These extracts were assessed using two cell line: T47D (Human Breast cancer) and HeLa (Human cervix cancer). Colchicine was used as the positive control. Data are presented as the dose that inhibited 50% control growth (ED50). All of the assessed extracts were cytotoxic (ED50 < 20 microg/ml) against T47D cell line, meanwhile only the aqueous extract from T. lucida and the ethanolic extract from J. spicigera were cytotoxic to HeLa cell line. Ethanolic extract from J. spicigera presented the best cytotoxic effect. The cytotoxic activity of J. spicigera correlated with one of the popular uses, the treatment of cancer.

  1. Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells.

    Science.gov (United States)

    McKenna, Declan J; Gallus, Massimo; McKeown, Stephanie R; Downes, C Stephen; McKelvey-Martin, Valerie J

    2003-08-12

    The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 microg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 microg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet-FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.

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

    Science.gov (United States)

    Basu, Amar S

    2017-08-01

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

  3. Chemical and enzymatic reductive activation of acylfulvene to isomeric cytotoxic reactive intermediates

    OpenAIRE

    Pietsch, Kathryn E.; Neels, James F.; Yu, Xiang; Gong, Jiachang; Sturla, Shana J.

    2011-01-01

    Acylfulvenes, a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic towards cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capa...

  4. In Vitro Cytotoxic Activity of the Essential Oil Extracted from Artemisia Absinthium

    OpenAIRE

    Mahboubeh Taherkhani

    2014-01-01

    Background: Essential oils are found to have multiple active components which can show in vitro cytotoxic action against various cancerous cell lines. This study reports the in vitro cytotoxic effects of the essential oil from Artemisia absinthium L. (Asteraceae) growing wild in Iran. Methods: Water-distilled essential oil of A. absinthium collected from Ardabil, north-western Iran, was examined for its cytotoxic effects using a modified MTT assay. Air-dried aerial parts of A. absinthium w...

  5. [Cytotoxicity of the strontium-substituted hydroxyapatite evaluated by MTT colorimetry].

    Science.gov (United States)

    Fu, Y; Chen, D; Zhang, J

    2001-09-01

    This study was conducted to evaluate the cytotoxicity of strontium-substituted hydroxyapatite by methyl thiazolyl tetrazolium (MTT) colorimetry. We used the MTT method to assay the cytotoxicity of the strontium-substituted hydroxyapatite containing different strontium concentrations (1%, 5%, 10%, 100% Sr2+) and the pure hydroxyapatite. The results showed that the cytotoxicity scores of the different materials were grade 0 or grade 1. These led us to the conclusion that strontium-substituted hydroxyapatite has good biocompatibility.

  6. Structures, chemotaxonomic significance, cytotoxic and Na(+),K(+)-ATPase inhibitory activities of new cardenolides from Asclepias curassavica.

    Science.gov (United States)

    Zhang, Rong-Rong; Tian, Hai-Yan; Tan, Ya-Fang; Chung, Tse-Yu; Sun, Xiao-Hui; Xia, Xue; Ye, Wen-Cai; Middleton, David A; Fedosova, Natalya; Esmann, Mikael; Tzen, Jason T C; Jiang, Ren-Wang

    2014-11-28

    Five new cardenolide lactates (1–5) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (6–16 and 18–21) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 1–3 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (17–21) and cardenolide lactates (1–5) provided unique chemotaxonomic markers for this genus. Compounds 1–21 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.

  7. Transport of active flavonoids, based on cytotoxicity and lipophilicity: an evaluation using the blood-brain barrier cell and Caco-2 cell models.

    Science.gov (United States)

    Yang, Yuya; Bai, Lu; Li, Xiaorong; Xiong, Jie; Xu, Pinxiang; Guo, Chenyang; Xue, Ming

    2014-04-01

    This in vitro study aims to evaluate and compare transmembrane transport of eight cardio-cerebrovascular protection flavonoids including puerarin, rutin, hesperidin, quercetin, genistein, kaempferol, apigenin and isoliquiritigenin via the rat blood-brain barrier cell and Caco-2 cell monolayer models, based on the data of cytotoxicity and lipophilicity. The cytotoxicity of the flavonoids to rat brain microvessel endothelial cell was determined by the MTT assay. The apparent permeability coefficients (Papp) of the flavonoids were calculated from the unilateral transport assays in Transwell system with simultaneous determination using a high performance liquid chromatography. The results showed that the cytotoxicity and oil-water partition coefficient of the flavonoids modified by the number and position of the glycoside and hydroxyl group were the key determinant for the transmembrane transport. The Papp values of the flavonoids reduced adversely when the numbers of glycoside and hydroxyl groups of the flavonoids increased accordingly. The tested flavonoids exhibited time-dependent Papp values in these models. The efflux mechanism related with P-glycoprotein also existed with the polar flavonoids; verapamil could enhance the permeation of rutin and quercetin via inhibition of P-glycoprotein. We propose that genistein and isoliquiritigenin with the permeation priority in vitro Caco-2 and BBB cell model could be better as the drug candidates for cardio-cerebral vascular protection. These findings provided important information for establishing the transport relationship for the flavonoid compounds and evaluating the potential oral bioavailability and brain distribution of the flavonoids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Cytotoxicity assays for mycotoxins produced by Fusarium strains: a review

    NARCIS (Netherlands)

    Gutleb, A.C.; Morrison, E.; Murk, A.J.

    2002-01-01

    Mycotoxins are naturally occurring toxic secondary metabolites of fungi that may be present in food and feed. Several of these mycotoxins have been associated with human and animal diseases. Fusarium species, found worldwide in cereals and other food types for human and animal consumption, are the

  9. Cytotoxic diterpenoids from Jatropha curcas cv. nigroviensrugosus CY Yang Roots.

    Science.gov (United States)

    Liu, JieQing; Yang, YuanFeng; Xia, JianJun; Li, XuYang; Li, ZhongRong; Zhou, Lin; Qiu, MingHua

    2015-09-01

    An investigation of phytochemicals from the roots of Jatropha curcas cv. nigroviensrugosus resulted in the isolation of twenty diterpenoids, including lathyranlactone, an unusual diterpenoid lactone possessing a 5/13/3 tricyclic skeleton, jatrocurcasenones A-E and jatrophodiones B-E, as well as 10 known analogues. All isolates were evaluated for cytotoxicity against the HL-60, SMMC-772, A-549, MCF-7 and SW480 human tumor cell lines using the MTS viability assay. Four of the known analogues showed cytotoxic activity in these cell lines, with IC50 values ranging from 2.0 to 23.0 μM. Moreover, the assessment of their cytotoxic structure-activity relationships showed the epoxy ring between C-5 and C-6 and the hydroxyl group at C-2 were the key functionalities for cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Performance evaluation of the Bio-Rad Laboratories GS HIV Combo Ag/Ab EIA, a 4th generation HIV assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma.

    Science.gov (United States)

    Bentsen, Christopher; McLaughlin, Lisa; Mitchell, Elizabeth; Ferrera, Carol; Liska, Sally; Myers, Robert; Peel, Sheila; Swenson, Paul; Gadelle, Stephane; Shriver, M Kathleen

    2011-12-01

    A multi-center study was conducted to evaluate the Bio-Rad GS HIV Combo Ag/Ab EIA, a 4th generation HIV-1/HIV-2 assay for the simultaneous detection of HIV p24 antigen and antibodies to HIV-1 (groups M and O) and HIV-2 in human serum or plasma in adult and pediatric populations. The objectives of the study were to assess assay performance for the detection of acute HIV infections; sensitivity in known HIV positive samples; percent agreement with HIV status; specificity in low and high risk individuals of unknown HIV status; and to compare assay performance to a 3rd generation HIV assay. The evaluation included testing 9150 samples at four U.S. clinical trial sites, using three kit lots. Unlinked samples were from routine testing, repositories or purchased from vendors. GS HIV Combo Ag/Ab EIA detection in samples from individuals in two separate populations with acute HIV infection was 95.2% (20/21) and 86.4% (38/44). Sensitivity was 100% (1603/1603) in known antibody positive [HIV-1 Groups M and O, and HIV-2] samples. HIV p24 antigen detection was 100% (53/53) in HIV-1 culture supernatants. HIV-1 seroconversion panel detection improved by a range of 0-20 days compared to a 3rd generation HIV test. Specificity was 99.9% (5989/5996) in low risk, 99.9% (959/960) in high risk and 100% (100/100) in pediatric populations. The GS HIV Combo Ag/Ab EIA significantly reduced the diagnostic window when compared to the 3rd generation screening assay, enabling earlier diagnosis of HIV infection. The performance parameters of the Bio-Rad GS HIV Combo Ag/Ab EIA are well suited for use in HIV diagnostic settings. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Manool, a Salvia officinalis diterpene, induces selective cytotoxicity in cancer cells

    National Research Council Canada - National Science Library

    de Oliveira, Pollyanna Francielli; Munari, Carla Carolina; Nicolella, Heloiza Diniz; Veneziani, Rodrigo Cassio Sola; Tavares, Denise Crispim

    2016-01-01

    Manool, a diterpene isolated from Salvia officinalis, was evaluated by the XTT colorimetric assay for cytotoxicity and selectivity against different cancer cell lines: B16F10 (murine melanoma), MCF-7...

  12. Phytochemistry, cytotoxicity and antiviral activity of Eleusine indica (sambau)

    Science.gov (United States)

    Iberahim, Rashidah; Yaacob, Wan Ahmad; Ibrahim, Nazlina

    2015-09-01

    Goose grass also known as Eleusine indica (EI) is a local medicinal plant that displays antioxidant, antimicrobial and anticancer activities. The present study is to determine the phytochemical constituents, cytotoxicity and antiviral activities for both crude extract and fraction obtained from the plant. The crude extract contained more secondary metabolites compared to the hexane fraction as gauged using standard phytochemical tests. Cytotoxicity screening against Vero cells using MTT assay showed that the CC50 values for crude extract and hexane fraction were 2.07 and 5.62 mg/ml respectively. The antiviral activity towards Herpes Simplex Virus type 1 (HSV-1) was determined using plaque reduction assay. The selective indices (SI = CC50 / EC50) for both methanol extract and hexane fraction were 12.2 and 6.2 respectively. These results demonstrate that the extract prepared from E. indica possesses phytochemical compound that was non cytotoxic to the cell with potential antiviral activity.

  13. Cytotoxicity Analysis of Three Bacillus thuringiensis Subsp. israelensis δ-Endotoxins towards Insect and Mammalian Cells

    Science.gov (United States)

    Teixeira Corrêa, Roberto Franco; Ardisson-Araújo, Daniel Mendes Pereira; Monnerat, Rose Gomes; Ribeiro, Bergmann Morais

    2012-01-01

    Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL. PMID:23029407

  14. Kinetics and mechanism of antibacterial activity and cytotoxicity of Ag-RGO nanocomposite.

    Science.gov (United States)

    Moghayedi, Marjan; Goharshadi, Elaheh K; Ghazvini, Kiarash; Ahmadzadeh, Hossein; Ranjbaran, Laleh; Masoudi, Raheleh; Ludwig, Ralf

    2017-08-04

    Nowadays, nanomaterials with remarkable antibacterial activity and low cytotoxicity attract much interest in research. By considering the antibacterial activity of Ag and graphene oxide (GO), the Ag-RGO nanocomposite was prepared by a one-pot and facile technique and it was used to evaluate its antibacterial activity and cytotoxicity against Escherichia coli and glioblastoma cancer cells (U87MG), respectively. The antibacterial activity was studied by micro-dilution and colony counting methods to investigate cell viability. The viability of glioblastoma cells was determined using MTT assay. Since MIC and MBC values of the nanocomposite are 20 and 40μg/mL, respectively, it acts as a bactericidal agent. The antibacterial properties of nanocomposite are time and concentration dependent. The kinetics and mechanism of the antibacterial activity of the nanocomposite were investigated. The antibacterial activity for Ag-RGO nanocomposite is induced by capturing-killing process. From the results, we concluded that Ag-RGO nanocomposite can simultaneously induce apoptosis. Our results bring up a new plan for the use of silver nanoparticles in the form of nanocomposite with reduced graphene oxide in antibacterial applications. Also, Ag-RGO nanocomposite can reduce the viability of U87MG in a dose dependent manner which may show its anticancer potential. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Cytotoxicity analysis of three Bacillus thuringiensis subsp. israelensis δ-endotoxins towards insect and mammalian cells.

    Science.gov (United States)

    Teixeira Corrêa, Roberto Franco; Ardisson-Araújo, Daniel Mendes Pereira; Monnerat, Rose Gomes; Ribeiro, Bergmann Morais

    2012-01-01

    Three members of the δ-endotoxin group of toxins expressed by Bacillus thuringiensis subsp. israelensis, Cyt2Ba, Cry4Aa and Cry11A, were individually expressed in recombinant acrystalliferous B. thuringiensis strains for in vitro evaluation of their toxic activities against insect and mammalian cell lines. Both Cry4Aa and Cry11A toxins, activated with either trypsin or Spodoptera frugiperda gastric juice (GJ), resulted in different cleavage patterns for the activated toxins as seen by SDS-PAGE. The GJ-processed proteins were not cytotoxic to insect cell cultures. On the other hand, the combination of the trypsin-activated Cry4Aa and Cry11A toxins yielded the highest levels of cytotoxicity to all insect cells tested. The combination of activated Cyt2Ba and Cry11A also showed higher toxic activity than that of toxins activated individually. When activated Cry4Aa, Cry11A and Cyt2Ba were used simultaneously in the same assay a decrease in toxic activity was observed in all insect cells tested. No toxic effect was observed for the trypsin-activated Cry toxins in mammalian cells, but activated Cyt2Ba was toxic to human breast cancer cells (MCF-7) when tested at 20 µg/mL.

  16. A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry.

    Directory of Open Access Journals (Sweden)

    Srinivas S Somanchi

    Full Text Available Natural killer (NK cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the "gold standard" for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

  17. Urtica dioica Induces Cytotoxicity in Human Prostate Carcinoma ...

    African Journals Online (AJOL)

    Purpose: To evaluate the cytotoxic mechanisms of an extract from the leaves of the Urtica dioica (UD) plant in LNCaP prostate cancer cells. Methods: LNCaP cells were exposed to the UD extract for 24hrs and cell viability assessed using the. MTT assay. Reactive oxygen species generation was assessed using the NBT ...

  18. Cytotoxic effect of betulinic acid and betulinic acid acetate isolated ...

    African Journals Online (AJOL)

    GREGORY

    2010-09-20

    Sep 20, 2010 ... Key words: Betulinic acid, HL 60, cytotoxicity, MTT assay, DNA laddering, Cell cycle PI. INTRODUCTION. Betulinic acid ... Chemical structure of betulinic acid and its derivatives. (Fulda et al., 1999) and leukemia cells ... feature makes betulinic acid unique in comparison to compounds that are currently used ...

  19. In vitro anticancer activity and cytotoxicity of some papaver alkaloids ...

    African Journals Online (AJOL)

    Materials and Methods: The Vero and HeLa cell lines were treated with various concentrations (1-300 μg/mL) of alkaloids for 48 h. Values for cytotoxicity measured by MTT assay were expressed as the concentration that causes a 50% decrease in cell viability (IC50) (μg/mL). Results: Berberine and macranthine were the ...

  20. In vitro cytotoxic study for partially purified Lasparaginase from fresh ...

    African Journals Online (AJOL)

    ... there is enough evidence to support the claim that Lasparaginase from W. somnifera may be considered chemotherapeutic agent against cancer, such as acute lymphoblastic leukemia and lymphosarcoma. Keywords: Acute lymphocyte leukemia (ALL), chronic lymphocyte leukemia (CLL), L-asparaginase, cytotoxic assay.

  1. Antibacterial activity and cytotoxicity of essential oil of Lantana ...

    African Journals Online (AJOL)

    The essential oil was tested for antibacterial activity against 6 strains, using disc diffusion method, and for cytotoxicity using brine-shrimp lethality assay. The oil showed moderate activity against Candida albican, Bacillus subtilis, Staphylococcus typhi, Pseudomonas aeruginosa and Bacillus aureus. These activities support ...

  2. Urtica dioica Induces Cytotoxicity in Human Prostate Carcinoma ...

    African Journals Online (AJOL)

    Purpose: To evaluate the cytotoxic mechanisms of an extract from the leaves of the Urtica dioica (UD) plant in LNCaP prostate cancer cells. Methods: LNCaP cells were exposed to the UD extract for 24hrs and cell viability assessed using the MTT assay. Reactive oxygen species generation was assessed using the NBT ...

  3. Comparing the cytotoxic potential of Withania somnifera water and ...

    African Journals Online (AJOL)

    The current research evaluated and compared the cytotoxic potential of water and methanol extracts of W. somnifera using a combined crystal violet MTT and Neutral Red assay. MRC-5 cells, a human embryonic lung-derived diploid fibroblast cell line, were the cells of choice. We found that the three lowest concentrations ...

  4. Volatile compounds analysis and antioxidant, antimicrobial and cytotoxic activities of Mindium laevigatum

    Directory of Open Access Journals (Sweden)

    Abdolrasoul Haghir Ebrahimabadi

    2016-12-01

    Full Text Available Objective(s: Mindium laevigatum is an endemic plant of Iran and Turkey and is widely used as blood purifier, antiasthma and antidyspnea in traditional medicine. Chemical composition of volatile materials of the plant and its antioxidant, antimicrobial and cytotoxic activities were reported in this study. Materials and Methods: Simultaneous distillation-extraction (SDE and GC-Mass-FID analysis were used for the plant volatile materials chemical composition identification and quantification. Several antioxidant tests including DPPH radical scavenging, hydrogen peroxide scavenging, reducing power determination, β-carotene-linoleic acid and total phenolic content tests were used for antioxidant activity evaluation. Antimicrobial and anticancer activities were also estimated using microbial strains, cancer cell lines and brine shrimp larva. Results: GC-Mass-FID analysis of volatile samples showed a total of 74 compounds of which palmitic acid (7.4-33.7%, linoleic acid (6.6-18.6%, heneicosane (1.3-9.6% and myristic acid (1.4-6.0% were detected as main volatile components. Moderate to good results were recorded for the plant in              β-carotene-linoleic acid test. Total phenolic content of the extracts as gallic acid equivalents were estimated in the range of 15.7 to 79.6 μg/mg. Some microbial strains showed moderate sensitivities to plant extracts. Brine shrimp lethality test and cytotoxic cancer cell line assays showed mild cytotoxic activities for the plant. Conclusion: Moderate to good antioxidant activities in β-carotene-linoleic acid test and presence of considerable amounts of unsaturated hydrocarbons may explain the plant traditional use in asthma and dyspnea. These findings also candidate it as a good choice for investigating its possible modern medical applications.

  5. Cell-mediated immunity to Plasmodium falciparum infection: evidence against the involvement of cytotoxic lymphocytes

    DEFF Research Database (Denmark)

    Theander, T G; Andersen, B J; Pedersen, B K

    1988-01-01

    by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were...... done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized...

  6. Validation of a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds.

    Science.gov (United States)

    Else, Laura; Watson, Victoria; Tjia, John; Hughes, Andrew; Siccardi, Marco; Khoo, Saye; Back, David

    2010-06-01

    Clinical pharmacokinetic studies of antiretrovirals require accurate and precise measurement of plasma drug concentrations. Here we describe a simple, fast and sensitive HPLC-MS/MS method for determination of the commonly used protease inhibitors (PI) amprenavir, atazanavir, darunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, as well as the more recent antiretrovirals, the CCR5 antagonist maraviroc and the "second generation" NNRTI etravirine and rilpivirine. An internal standard (quinoxalone; QX) was added to plasma aliquots (100 microl) prior to protein precipitation with acetonitrile (500 microl) followed by centrifugation and addition of 0.05% formic acid (200 microl) to the supernatant. Chromatographic separation was achieved using a gradient (acetonitrile and 0.05% formic acid) mobile phase on a reverse-phase C18 column. Detection was via selective reaction monitoring (SRM) operating in positive ionization mode on a triple-quadrupole mass spectrometer. All compounds eluted within a 5 min run time. Calibration curves were validated over concentration ranges reflecting therapeutic concentrations observed in HIV-infected patients from pharmacokinetic data reported in the literature. Correlation coefficients (r2) exceeded 0.998. Inter- and intra-assay variation ranged between 1% and 10% and % recovery exceeded 90% for all analytes. The method described is being successfully applied to measure plasma antiretroviral concentrations from samples obtained from clinical pharmacokinetic studies. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Dual fluorescence resonance energy transfer assay between tunable upconversion nanoparticles and controlled gold nanoparticles for the simultaneous detection of Pb²⁺ and Hg²⁺.

    Science.gov (United States)

    Wu, Shijia; Duan, Nuo; Shi, Zhao; Fang, Congcong; Wang, Zhouping

    2014-10-01

    In this work, we presented a novel dual fluorescence resonance energy transfer (FRET) system for the simultaneous detection of Pb(2+) and Hg(2+). This system employed two color upconversion nanoparticles (UCNPs) as the donors, and controlled gold nanoparticles (AuNPs) as the acceptors. The two donor-acceptor pairs were fabricated by hybridizing the aptamers and their corresponding complementary DNA. Thus, the green and red upconversion fluorescence could be quenched because of a good overlap between the UCNPs fluorescence emission and the AuNPs absorption spectrum. In the presence of Pb(2+) and Hg(2+), the aptamers preferred to bind to their corresponding analytes and formed a G-quadruplexes structure for Pb(2+) and the hairpin-like structure for Hg(2+). As a result, the dual FRET was disrupted, and the green and red upconversion fluorescence was restored. Under optimized experimental conditions, the relative fluorescence intensity increased as the metal ion concentrations were increased, allowing for the quantification of Pb(2+) and Hg(2+). The relationships between the fluorescence intensity and plotting logarithms of ion concentrations were linear in the range from 0.1 to 100 nM for Pb(2+) and 0.5 to 500 nM for Hg(2+), and the detection limits of Pb(2+) and Hg(2+) were 50 pM and 150 pM, respectively. As a practical application, the aptasensor was used to monitor Pb(2+) and Hg(2+) levels in naturally contaminated samples and human serum samples. Ultimately, this type of dual FRET could be used to detect other metal ions or contaminants in food safety analysis and environment monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Simultaneous detection and differentiation of dengue virus serotypes 1-4, Japanese encephalitis virus, and West Nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Yin Jianhua

    2011-07-01

    Full Text Available Abstract Background Rapid identification and differentiation of mosquito-transmitted flaviviruses in acute-phase sera of patients and field-caught vector mosquitoes are important for the prediction and prevention of large-scale epidemics. Results We developed a flexible reverse-transcription loop-mediated isothermal amplification (RT-LAMP unit for the detection and differentiation of dengue virus serotypes 1-4 (DENV1-4, Japanese encephalitis virus (JEV, and West Nile virus (WNV. The unit efficiently amplified the viral genomes specifically at wide ranges of viral template concentrations, and exhibited similar amplification curves as monitored by a real-time PCR engine. The detection limits of the RT-LAMP unit were 100-fold higher than that of RT-PCR in 5 of the six flaviviruses. The results on specificity indicated that the six viruses in the assay had no cross-reactions with each other. By examining 66 viral strains of DENV1-4 and JEV, the unit identified the viruses with 100% accuracy and did not cross-react with influenza viruses and hantaviruses. By screening a panel of specimens containing sera of 168 patients and 279 pools of field-caught blood sucked mosquitoes, results showed that this unit is high feasible in clinical settings and epidemiologic field, and it obtained results 100% correlated with real-time RT-PCR. Conclusions The RT-LAMP unit developed in this study is able to quickly detect and accurately differentiate the six kinds of flaviviruses, which makes it extremely feasible for screening these viruses in acute-phase sera of the patients and in vector mosquitoes without the need of high-precision instruments.

  9. Gold Nanoparticles Inhibit Matrix Metalloproteases without Cytotoxicity.

    Science.gov (United States)

    Hashimoto, M; Sasaki, J I; Yamaguchi, S; Kawai, K; Kawakami, H; Iwasaki, Y; Imazato, S

    2015-08-01

    Nanoparticles (NPs) are currently the focus of considerable attention for dental applications; however, their biological effects have not been fully elucidated. The long-term, slow release of matrix metalloproteases (MMPs) digests collagen fibrils within resin-dentin bonds. Therefore, MMP inhibitors can prolong the durability of resin-dentin bonds. However, there have been few reports evaluating the combined effect of MMP inhibition and the cytotoxic effects of NPs for dentin bonding. The aim of this study was to evaluate MMP inhibition and cytotoxic responses to gold (AuNPs) and platinum nanoparticles (PtNPs) stabilized by polyvinylpyrrolidone (PVP) in cultured murine macrophages (RAW264) by using MMP inhibition assays, measuring cell viability and inflammatory responses (quantitative reverse transcription polymerase chain reaction [RT-qPCR]), and conducting a micromorphological analysis by fluorescence and transmission electron microscopy. Cultured RAW264 cells were exposed to metal NPs at various concentrations (1, 10, 100, and 400 µg/mL). AuNPs and PtNPs markedly inhibited MMP-8 and MMP-9 activity. Although PtNPs were cytotoxic at high concentrations (100 and 400 µg/mL), no cytotoxic effects were observed for AuNPs at any concentration. Transmission electron microscopy images showed a significant nonrandom intercellular distribution for AuNPs and PtNPs, which were mostly observed to be localized in lysosomes but not in the nucleus. RT-qPCR analysis demonstrated inflammatory responses were not induced in RAW264 cells by AuNPs or PtNPs. The cytotoxicity of nanoparticles might depend on the core metal composition and arise from a "Trojan horse" effect; thus, MMP inhibition could be attributed to the surface charge of PVP, which forms the outer coating of NPs. The negative charge of the surface coating of PVP binds to Zn(2+) from the active center of MMPs by chelate binding and results in MMP inhibition. In summary, AuNPs are attractive NPs that effectively

  10. Global assays of fibrinolysis.

    Science.gov (United States)

    Ilich, A; Bokarev, I; Key, N S

    2017-10-01

    Fibrinolysis is an important and integral part of the hemostatic system. Acting as a balance to blood coagulation, the fibrinolytic system protects the body from unwanted thrombus formation and occlusion of blood vessels. As long as blood coagulation and fibrinolysis remain in equilibrium, response to injury, such as vessel damage, is appropriately regulated. However, alterations in this balance may lead to thrombosis or bleeding. A variety of methods have been proposed to assess fibrinolytic activity in blood or its components, but due to the complexity of the system, the design of a "gold standard" assay that reflects overall fibrinolysis has remained an elusive goal. In this review, we describe the most commonly used methods that have been described, such as thromboelastography (TEG and ROTEM), global fibrinolytic capacity in plasma and whole blood, plasma turbidity methods, simultaneous thrombin and plasmin generation assays, euglobulin clot lysis time and fibrin plate methods. All of these assays have strengths and limitations. We suggest that some methods may be preferable for detecting hypofibrinolytic conditions, whereas others may be better for detecting hyperfibrinolytic states. © 2017 John Wiley & Sons Ltd.

  11. Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: an in vitro analysis.

    Science.gov (United States)

    Mara Serpeloni, Juliana; Mazzaron Barcelos, Gustavo Rafael; Prates Mori, Mateus; Yanagui, Karina; Vilegas, Wagner; Aparecida Varanda, Eliana; de Syllos Cólus, Ilce Mara

    2011-07-01

    The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 μg/mL, M. cabucu up to 40 μg/mL, M. albicans up to 40 μg/mL and M. stenostachya up to 60 μg/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 μg/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity. Copyright © 2010 Elsevier GmbH. All rights reserved.

  12. Cytotoxicity and degree of conversion of orthodontic adhesives.

    Science.gov (United States)

    Jagdish, Nithya; Padmanabhan, Sridevi; Chitharanjan, Arun B; Revathi, J; Palani, Gunasekaran; Sambasivam, Mohana; Sheriff, Khaleefathullah; Saravanamurali, K

    2009-11-01

    To test the hypothesis that there is no difference in the cytotoxicity related to the modes of polymerization of five commercially available orthodontic bonding resins, with and without an oxygen-inhibited layer (OIL), and to evaluate the degree of conversion (DC) of these resins and correlate this to cytotoxicity. Five commercially available orthodontic bonding resins were tested for cytotoxicity and DC. Thirty-six disks of standardized dimensions, for each resin, were used for cytotoxicity assessment. Half of them were washed with 99% acetone to remove the OIL (washed resins), and the remaining disks were left intact (intact resins). Glass disks were used as a control. Vero cells were exposed to intact and washed resins on day 1. Cell viability was determined by tetrazolium bromide reduction assay 1, 3, and 6 days after exposure. The DC of the adhesive specimens of each resin, prepared with a procedure identical to the clinical bonding process, was assessed by Fourier transform infrared spectroscopy. Single-cured systems were comparatively less cytotoxic than dual-cured systems. With removal of the OIL, increased cell viability was noted only with two resins on all three days. Resins tested showed differences in DC. A positive correlation was demonstrated by two resins. The hypothesis is rejected. Single-cured systems are superior to dual-cured systems in exhibiting comparatively less toxicity and higher DC. A significant positive correlation was not established between cytotoxicity and DC.

  13. Phytochemical and Cytotoxic Investigations of Curcuma mangga Rhizomes

    Directory of Open Access Journals (Sweden)

    Sri Nurestri A. Malek

    2011-05-01

    Full Text Available Investigations on the cytotoxic effects of the crude methanol and fractionated extracts (hexane, ethyl acetate C. mangga against six human cancer cell lines, namely the hormone-dependent breast cell line (MCF-7, nasopharyngeal epidermoid cell line (KB, lung cell line (A549, cervical cell line (Ca Ski, colon cell lines (HCT 116 and HT-29, and one non-cancer human fibroblast cell line (MRC-5 were conducted using an in-vitro neutral red cytotoxicity assay. The crude methanol and fractionated extracts (hexane and ethyl acetate displayed good cytotoxic effects against MCF-7, KB, A549, Ca Ski and HT-29 cell lines, but exerted no damage on the MRC-5 line. Chemical investigation from the hexane and ethyl acetate fractions resulted in the isolation of seven pure compounds, namely (E-labda-8(17,12-dien-15,16-dial (1, (E-15,16-bisnor-labda-8(17,11-dien-13-on (2, zerumin A (3, β-sitosterol, curcumin, demethoxycurcumin and bis-demethoxycurcumin. Compounds 1 and 3 exhibited high cytotoxic effects against all six selected cancer cell lines, while compounds 2 showed no anti-proliferative activity on the tested cell lines. Compound 1 also demonstrated strong cytotoxicity against the normal cell line MRC-5. This paper reports for the first time the cytotoxic activities of C. mangga extracts on KB, A549, Ca Ski, HT-29 and MRC-5, and the occurrence of compound 2 and 3 in C. mangga.

  14. Long-term cytotoxic effects of contemporary root canal sealers

    Directory of Open Access Journals (Sweden)

    Emmanuel Joao Nogueira Leal da SILVA

    2013-03-01

    Full Text Available Objectives The aim of the present study was to investigate the effects of root canal sealers on the cytotoxicity of 3T3 fibroblasts during a period of 5 weeks. Material and Methods Fibroblasts (3T3, 1×105 cells per well were incubated with elutes of fresh specimens from eight root canal sealers (AH Plus, Epiphany, Endomethasone N, EndoREZ, MTA Fillapex, Pulp Canal Sealer EWT, RoekoSeal and Sealapex and with elutes of the same specimens for 5 succeeding weeks after immersing in simulated body fluid. The cytotoxicity of all root canal sealers was determined using the MTT assay. Data were analyzed using ANOVA and Tukey's test. Results RoekoSeal was the only sealer that did not show any cytotoxic effects (p<0.05. All the other tested sealers exhibited severe toxicity initially (week 0. MTA Fillapex remained moderately cytotoxic after the end of experimental period. Toxicity of the other tested sealers decreased gradually over time. The evaluated root canal sealers presented varying degrees of cytotoxicity, mainly in fresh mode. Conclusions RoekoSeal had no cytotoxic effect both freshly mixed and in the other tested time points. MTA Fillapex was associated with significantly less cell viability when compared to the other tested root canal sealers.

  15. Antitubercular and Cytotoxic Constituents from Goniothalamus gitingensis

    Directory of Open Access Journals (Sweden)

    Allan Patrick G. Macabeo

    2014-01-01

    Full Text Available Phytochemical studies on the leaves of the Philippine endemic Annonaceae plant, Goniothalamus gitingensis enabled the isolation and identification of four secondary metabolites corresponding to three styryllactones, isoaltholactone (1, altholactone (2 and goniopypyrone (3, and the alkaloid liriodenine (4. Structure identification was facilitated through various spectroscopic experiments such as NMR ( 1H, 13C, COSY, HSQC, HMBC and NOESY, LR-EIMS, X-ray and through comparison with literature values. Our study accounts the first report of all compounds in this plant species. The extracts together with the isolated compounds (1-4 were tested for antituberculosis activity using MABA and cytotoxic activity using the CellTiter-Blue1 assay. The majority of the samples tested indicated good inhibitory activity against Mycobacterium tuberculosis H 37Rv (MIC up to 16 m g/mL. Liriodenine (4 showed the most excellent antimycobacterial activity (MIC=16 m g/mL followed by 1 and the 5:4 mixture of 1 and 2. Their cytotoxicity against three human cancer cell lines, human umbilical vein endothelial cell line (HUVEC, human leukemia cell line (K-562 and HeLa cells were also assessed. The crude extract, alkaloid extract, petroleum ether sub-extract and EtOAc sub-extract showed moderate cytotoxicity against the cancer cell-lines. In addition, the results showed that compounds 1, 4 and the mixture of 1 and 2 exhibited highest cytotoxicity against HUVEC, K-562 and HeLa cell lines with GI 50 values up to 4 μg/mL (vs. HUVEC and/or K-562 and CC 50 values up to 25.1 μg/mL (vs. HeLa.

  16. Cytotoxic effects exerted by Tritrichomonas foetus pseudocysts.

    Science.gov (United States)

    Pereira-Neves, Antonio; Nascimento, Ligia Ferreira; Benchimol, Marlene

    2012-07-01

    The protozoan parasite Tritrichomonas foetus displays a pear-shaped form and a pseudocyst stage. However, little is known about the biology of the pseudocyst. The aim of this work was to assess whether pseudocysts exert cytotoxic effects during their interaction with MDCK cells (an epithelial kidney canine cell line) and compare their behavior to that of the pear-shaped parasites. Pseudocysts and pear-shaped parasites from both cultured and freshly isolated T. foetus were used. Electron microscopy revealed that the epithelial cells exhibited more signs of injury, such as depletion of microvilli, retraction from neighboring cells and swollen mitochondria with loss of electron density in the matrix, when the pseudocysts were used in interaction experiments. In addition, during the co-incubation with MDCK cells, pseudocysts exhibited a more intense amoeboid transformation than that found in pear-shaped parasites. The MTT viability assay demonstrated that the pseudocysts were more cytotoxic when in contact with host cells as compared to the flagellated pear-shaped parasites. The JC-1 viability assay revealed that pseudocysts induced a higher loss of mitochondrial membrane potential compared to pear-shaped parasites. Pseudocysts undergoing a budding process were observed after 2.5h of co-incubation with MDCK cells. Our results suggest that the T. foetus pseudocyst might be a more aggressive form. Copyright © 2011 Elsevier GmbH. All rights reserved.

  17. Synergistic cytotoxicity induced by α-solanine and α-chaconine.

    Science.gov (United States)

    Yamashoji, Shiro; Matsuda, Takako

    2013-11-15

    α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Sesquiterpene lactones from Ferula oopoda and their cytotoxic properties.

    Science.gov (United States)

    Kasaian, Jamal; Iranshahy, Milad; Masullo, Milena; Piacente, Sonia; Ebrahimi, Fatemeh; Iranshahi, Mehrdad

    2014-01-01

    Two new sesquiterpene lactones, namely feruhodin A and feruhodin B, together with six known compounds, daucoeudesmanolactone, dehydrooopodin, oopodin, badkhysin, 7-demethylplastochromenol, and scoparone, were isolated from the roots of Ferula oopoda. The structures of these compounds were elucidated by 1D and 2D NMR techniques as well as high-resolution mass spectrometry. Cytotoxic effects of these compounds were evaluated against two human cancer cell lines including MCF7and K562 using the Alamar blue assay. The results showed that dehydrooopodin possessed significant cytotoxic effects with IC₅₀ values of 15 and 5 μM against MCF7 and K562, respectively.

  19. Propofol Enhances Hemoglobin-Induced Cytotoxicity in Neurons

    Science.gov (United States)

    Yuan, Jing; Cui, Guiyun; Li, Wenlu; Zhang, Xiaoli; Wang, Xiaoying; Zheng, Hui; Zhang, Jian; Xiang, Shuanglin; Xie, Zhongcong

    2016-01-01

    BACKGROUND It has been increasingly suggested that propofol protects against hypoxic-/ischemic-induced neuronal injury. As evidenced by hemorrhage-induced stroke, hemorrhage into the brain may also cause brain damage. Whether propofol protects against hemorrhage-induced brain damage remains unknown. Therefore, in this study, we investigated the effects of propofol on hemoglobin-induced cytotoxicity in cultured mouse cortical neurons. METHODS Neurons were prepared from the cortex of embryonic 15-day-old mice. Hemoglobin was used to induce cytotoxicity in the neurons. The neurons were then treated with propofol for 4 hours. Cytotoxicity was determined by lactate dehydrogenase release assay. Caspase-3 activation was examined by Western blot analysis. Finally, the free radical scavenger U83836E was used to examine the potential involvement of oxidative stress in propofol’s effects on hemoglobin-induced cytotoxicity. RESULTS We found that treatment with hemoglobin induced cytotoxicity in the neurons. Propofol enhanced hemoglobin-induced cytotoxicity. Specifically, there was a significant difference in the amount of lactate dehydrogenase release between hemoglobin plus saline (19.84% ± 5.38%) and hemoglobin plus propofol (35.79% ± 4.41%) in mouse cortical neurons (P = 0.00058, Wilcoxon Mann-Whitney U test, n = 8 in the control group or the treatment group). U83836E did not attenuate the enhancing effects of propofol on hemoglobin-induced cytotoxicity in the neurons, and propofol did not significantly affect caspase-3 activation induced by hemoglobin. These data suggested that caspase-3 activation and oxidative stress might not be the underlying mechanisms by which propofol enhanced hemoglobin-induced cytotoxicity. Moreover, these data suggested that the neuroprotective effects of propofol would be dependent on the condition of the brain injury, which will need to be confirmed in future studies. CONCLUSIONS These results from our current proof-of-concept study should

  20. Gold Nanoparticles Cytotoxicity

    Science.gov (United States)

    Mironava, Tatsiana

    Over the last two decades gold nanoparticles (AuNPs) have been used for many scientific applications and have attracted attention due to the specific chemical, electronic and optical size dependent properties that make them very promising agents in many fields such as medicine, imagine techniques and electronics. More specifically, biocompatible gold nanoparticles have a huge potential for use as the contrast augmentation agent in X-ray Computed Tomography and Photo Acoustic Tomography for early tumor diagnostic as well these nanoparticles are extensively researched for enhancing the targeted cancer treatment effectiveness such as photo-thermal and radiotherapy. In most biomedical applications biocompatible gold nanoparticles are labeled with specific tumor or other pathology targeting antibodies and used for site specific drug delivery. However, even though gold nanoparticles poses very high level of anti cancer properties, the question of their cytotoxicity ones they are released in normal tissue has to be researched. Moreover, the huge amount of industrially produced gold nanoparticles raises the question of these particles being a health hazard, since the penetration is fairly easy for the "nano" size substances. This study focuses on the effect of AuNPs on a human skin tissue, since it is fall in both categories -- the side effects for biomedical applications and industrial workers and users' exposure during production and handling. Therefore, in the present project, gold nanoparticles stabilized with the biocompatible agent citric acid were generated and characterized by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). The cytotoxic effect of AuNPs release to healthy skin tissue was modeled on 3 different cell types: human keratinocytes, human dermal fibroblasts, and human adipose derived stromal (ADS) cells. The AuNPs localization inside the cell was found to be cell type dependent. Overall cytotoxicity was found to be dependent

  1. Cytotoxic constituents of Bursera permollis.

    Science.gov (United States)

    Wickramaratne, D B; Mar, W; Chai, H; Castillo, J J; Farnsworth, N R; Soejarto, D D; Cordell, G A; Pezzuto, J M; Kinghorn, A D

    1995-02-01

    Four cytotoxic lignans were isolated from the stem bark of Bursera permollis (Burseraceae), namely, deoxypodophyllotoxin (1), beta-peltatin methyl ether (2), picro-beta-peltatin methyl ether (3), and dehydro-beta-peltatin methyl ether (4). Also isolated was the inactive lignan, nemerosin (5). Compounds 1 and 2 were potently cytotoxic when evaluated against a panel of human cancer cell lines.

  2. Cytotoxicity and genotoxicity of urban particulate matter in mammalian cells.

    Science.gov (United States)

    Dumax-Vorzet, Audrey F; Tate, M; Walmsley, Richard; Elder, Rhod H; Povey, Andrew C

    2015-09-01

    Ambient air particulate matter (PM)-associated reactive oxygen species (ROS) have been linked to a variety of altered cellular outcomes. In this study, three different PM samples from diesel exhaust particles (DEPs), urban dust standard reference material SRM1649a and air collected in Manchester have been tested for their ability to oxidise DNA in a cell-free assay, to increase intracellular ROS levels and to induce CYP1A1 gene expression in mammalian cells. In addition, the cytotoxicity and genotoxicity of PM were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and alkaline comet assay, respectively. All PM samples catalysed the Fenton reaction in a cell-free assay, but only DEP resulted in the generation of ROS as measured by dichlorodihydrofluorescein diacetate oxidation in mammalian cells. However, there was no evidence that increased ROS was a consequence of polycyclic aromatic hydrocarbon metabolism via CYP1A1 induction as urban dust, the Manchester dust samples but not DEP-induced CYP1A1 expression. Urban dust was more cytotoxic in murine embryonic fibroblasts (MEFs) than the other PM samples and also induced expression of GADD45a in the GreenScreen Human Cell assay without S9 activation suggesting the presence of a direct-acting genotoxicant. Urban dust and DEP produced comparable levels of DNA damage, as assessed by the alkaline comet assay, in MEFs at higher levels than those induced by Manchester PM. In conclusion, results from the cytotoxic and genotoxic assays are not consistent with ROS production being the sole determinant of PM-induced toxicity. This suggests that the organic component can contribute significantly to this toxicity and that further work is required to better characterise the extent to which ROS and organic components contribute to PM-induced toxicity. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

  3. Epitope Prediction Assays Combined with Validation Assays Strongly Narrows down Putative Cytotoxic T Lymphocyte Epitopes

    NARCIS (Netherlands)

    Ip, Peng Peng; Nijman, Hans W; Daemen, Toos

    2015-01-01

    Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different

  4. In vitro studies on the cytotoxic potential of surface sealants.

    Science.gov (United States)

    Zingler, S; Matthei, B; Kohl, A; Saure, D; Ludwig, B; Diercke, K; Lux, C J; Erber, R

    2015-01-01

    The objective of this in vitro study was an initial screening of the cytotoxic potential of widely used smooth enamel surface sealants. A total of 20 products were allocated to four groups based on their chemical composition: (1) filled resin-based sealants, (2) unfilled resin-based sealants, (3) a resin-modified, glass ionomer-based sealant, and (4) silicone-based sealants. All materials were applied to human enamel slices both in accordance with manufacturers' instructions and in additional experiments applying 50% undercuring and 50% overcuring. An agar overlay assay was then used to test the specimens following ISO 10933. The cytotoxic potential of each material was interpreted based on a reaction index that summarized the decolorization and lysis scores obtained. The cytotoxic potential decreased as follows: unfilled resin-based sealants > filled resin-based sealants > resin-modified, glass ionomer-based sealant > silicone-based sealants. In 75% of the resin-based products, deliberate undercuring was associated with more extensive decolorization zones, leading to higher rates of cytotoxic potential in two of those products. Overcuring, by contrast, was associated with a tendency for smaller decolorization zones in 50% of the resin-based products. Surface sealants derived from resin monomers exhibited cytotoxic potential in the agar overlay assay. There is also evidence of a possible association with curing, as undercuring can increase the cytotoxic potential, whereas normal curing (as per manufacturers' instructions) or overcuring may help minimize such effects. More research into the biological implications of these materials is needed, especially with regard to their potential impact on the adjacent gingiva.

  5. Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening

    Science.gov (United States)

    Xia, Menghang; Huang, Ruili; Witt, Kristine L.; Southall, Noel; Fostel, Jennifer; Cho, Ming-Hsuang; Jadhav, Ajit; Smith, Cynthia S.; Inglese, James; Portier, Christopher J.; Tice, Raymond R.; Austin, Christopher P.

    2008-01-01

    Background The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects. Objective The National Toxicology Program and the National Institutes of Health Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicologic evaluation. Methods A collection of 1,408 compounds previously tested in one or more traditional toxicologic assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics. Results qHTS of these compounds produced robust and reproducible results, which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited species- or cell type–specific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time dependence in kinetic studies, consistent with known mechanisms of toxicity. Conclusions The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicologic evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response. PMID:18335092

  6. Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis

    Directory of Open Access Journals (Sweden)

    Mariana Belén Joray

    2015-01-01

    Full Text Available The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2′,4′-dihydroxychalcone (1, isoliquiritigenin (2, pinocembrin (3, 7-hydroxyflavanone (4, and 7,4′-dihydroxy-3′-methoxyflavanone (5. Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1–5 was determined by MTT assay on acute lymphoblastic leukemia (ALL and chronic myeloid leukemia (CML cell lines including their multidrug resistant (MDR phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6–9.9 μM and a lower effect against CML cells (IC50 = 27.5–30.0 μM. Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

  7. TREK-1 mediates isoflurane-induced cytotoxicity in astrocytes.

    Science.gov (United States)

    Guo, Haiyun; Peng, Zhengwu; Yang, Liu; Liu, Xue; Xie, Yaning; Cai, Yanhui; Xiong, Lize; Zeng, Yi

    2017-09-05

    There are growing concerns that anaesthetic exposure can cause extensive apoptotic degeneration of neurons and the impairment of normal synaptic development and remodelling. However, little attention has been paid to exploring the possible cytotoxicity of inhalation anaesthetics, such as isoflurane, in astrocytes. In this research, we determined that prolonged exposure to an inhalation anaesthetic caused cytotoxicity in astrocytes, and we identified the underlying molecular mechanism responsible for this process. Astrocytes were exposed to isoflurane, and astrocytic survival was then measured via LDH release assays, MTT assays, and TUNEL staining. TWIK-related potassium (K+) channel-1 (TREK-1) over-expression and knockdown models were also created using lentiviruses. The levels of TREK-1 and brain-derived neurotrophic factor (BDNF) were measured via Western blot and qRT-PCR. Prolonged exposure to isoflurane decreased primary astrocytic viability in a dose- and time-dependent manner. Moreover, with prolonged exposure to isoflurane, the TREK-1 level increased, and the BDNF level was reduced. TREK-1 knockdown promoted the survival of astrocytes and increased BDNF expression following isoflurane exposure. Overdoses of and prolonged exposure to isoflurane induce cytotoxicity in primary astrocytes. TREK-1 plays an important role in isoflurane-induced cultured astrocytic cytotoxicity by down-regulating the expression of BDNF.

  8. Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity.

    Science.gov (United States)

    Zou, L; Jankovic, J; Rowe, D B; Xie, W; Appel, S H; Le, W

    1999-01-01

    Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4-100 microM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D3 antagonist U-99194 A or D2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.

  9. Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Christian Michel

    Full Text Available Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1 in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay, oxidative stress (H2DCF-DA assay, and metabolic activity (MTT assay were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i. natural mineral particles can be cytotoxic to gill epithelial cells, (ii. their cytotoxic potential differs between mineral

  10. Cytotoxic activity of plants from East Azarbaijan province, Iran

    Directory of Open Access Journals (Sweden)

    M. Irani

    2017-11-01

    Full Text Available Background and objectives: Due to the high cancer mortality rates and side effects of different types of cancer treatments, discovering effective treatments without or with fewer side effects is the main purpose of many researchers all around the world. Plants play an important role in the discovery of new drugs. Iran owns rich and varied vegetation but the majority of these plants have not yet undergone chemical, pharmacological and toxicological studies. In the present study, some species from East Azarbaijan province of Iran were evaluated for cytotoxicity effects. Methods: Total methanol extract of 29 plants from 18 families were screened for their cytotoxic activities. The inhibition of cell growth for these extracts was evaluated against MCF-7, A-549, Hep-G2, HT-29 and MDBK cell lines. Their 50% inhibitions of growth (IC50 were determined by MTT assay. Moreover, cytotoxic evaluation of different fractions (ether de petrol, chloroform and methanol of the most potent species was performed. Results: Total extracts and fractions of Bryonia aspera, Centaurea salicifolia, Cuscuta chinensis, Ecbalium elaterium, Gypsophila ruscifolia, Ononis spinosa exhibited potent cytotoxic activity against one or more of the cell lines. Three of the mentioned total extracts presented cytotoxicity effects exclusively against HT-29 cells. Also three fractions (one ether de petrol and two chloroform fractions demonstrated selective cytotoxicity effects against MCF-7cells. Conclusion: It was concluded that these 6 potent species were proper candidates for identification and isolation of active ingredients with cytotoxic effects  and further studies about these species are recommended.

  11. Mangostanaxanthone VII, a new cytotoxic xanthone from Garcinia mangostana.

    Science.gov (United States)

    Ibrahim, Sabrin R M; Mohamed, Gamal A; Elfaky, Mahmoud A; Zayed, Mohamed F; El-Kholy, Amal A; Abdelmageed, Osama H; Ross, Samir A

    2017-11-08

    Garcinia mangostana L. (the queen of fruits, mangosteen, family Guttiferae) is a wealthy source of xanthones. The CHCl3 soluble fraction of the air-dried pericarps of G. mangostana provided a new xanthone: mangostanaxanthone VII (5), along with four known xanthones: mangostanaxanthones I (1) and II (2), gartanin (3) and γ-mangostin (4). The structural verification of these metabolites was achieved by different spectral techniques, including UV, IR, 1D and 2D NMR and HRESIMS. The new metabolite was assessed for cytotoxic potential, using sulforhodamine B (SRB) assay towards the A549 and MCF-7 cancer cell lines. Moreover, its antimicrobial effects were evaluated against various bacterial and fungal strains, using agar disc diffusion assay. Mangostanaxanthone VII showed moderate cytotoxic activity against the A549 and MCF7 cell lines with IC50s 26.1 and 34.8 μM, respectively, compared with doxorubicin (0.74 and 0.41 μM, respectively).

  12. Evaluation of boron neutron capture effects in cell culture using sulforhodamine-B assay and a colony assay.

    Science.gov (United States)

    Wittig, A; Sauerwein, W; Pöller, F; Fuhrmann, C; Hideghéty, K; Streffer, C

    1998-06-01

    The purpose of this study was to find an in vitro method for determining the cytotoxicity of boronated drugs as well as their potential suitability for neutron capture therapy. The survival of human melanoma cells has been determined by a colony assay and the sulforhodamine-B assay after X-irradiation and irradiation with fast d(14) + Be-neutrons using the boronated compound borocaptate sodium (BSH). The cytotoxic effects of BSH have been studied using both methods. Under well-defined experimental conditions, and after a sufficient amount of time for the expression of radiation damage, the results of the sulforhodamine-B assay are qualitatively comparable with the results of the colony assay. The sulforhodamine-B assay is suitable for the screening of compounds for potential use in neutron capture therapy because it is a fast and efficient method that is reproducible and technically advantageous.

  13. Multiplex polymerase chain reaction (PCR) assay for simultaneous ...

    African Journals Online (AJOL)

    user

    2011-02-28

    Feb 28, 2011 ... 3Research Microbial Resistance Center, Tehran University of Medical Sciences. Accepted 7 December, 2010 ... coli (EPEC) and enteroinvasive E. coli (EIEC) in children with diarrhea in Iran a total 300 stool specimens from .... DNA was extracted by bolling 5 colonies of overnight bacterial culture in 200 µl ...

  14. Cytotoxicity effects of alkoxy

    Directory of Open Access Journals (Sweden)

    Wan M. Khairul

    2017-05-01

    Full Text Available In this study, the effort was to design and synthesize five new members of alkoxy substituted thiourea derivatives (3a–3e featuring general formula of A-ArC(ONHC(SNHAr-D in which A represents the methoxy group and D as –OCnH2n+1 (alkoxyl group, where n = 6,7,8,9, and 10 have been successfully designed, prepared, characterized, and evaluated for anti-amoebic activities. They were spectroscopically characterized by 1H and 13C Nuclear Magnetic Resonance (NMR, Fourier Transform Infrared (FT-IR spectroscopy, and Ultraviolet–visible (UV–vis spectroscopy analysis. In turn, they were used to investigate the cytotoxicity effect on Acanthamoeba sp. at their IC50 values and membrane permeability. Compounds 3a and 3b revealed to have good activity towards Acanthamoeba sp. compared to other compounds of 3c, 3d, and 3e. The observation under fluorescence microscopy by AOPI (Acridine-orange/Propidium iodide staining indicated that treated amoeba cells by 3a–3e show loss of their membrane permeability.

  15. Cytotoxic and antioxidant activities of selected Lamiales species from Mexico.

    Science.gov (United States)

    Moreno-Escobar, J A; Bazald A, S; Villarreal, M L; Bonilla-Barbosa, J R; Mendoza, S; Rodríguez-López, V

    2011-12-01

    Methanol extracts obtained from eight species belonging to four families of the Lamiales order native to Mexico were investigated for biological action. Cytotoxicity and antioxidant activity of methanol extracts have been investigated. Cytotoxic activity was evaluated by the sulphorhodamine B protein staining assay against KB (nasopharyngeal), HEp-2 (larynx), HF-6 (colon), MCF7 (breast), PC-3 (prostate), and Ca Ski (cervix) carcinoma cell lines. To analyze the antioxidant activity, common stable radicals chromogens, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS+) and DPPH (1,1-diphenyl-2-picrylhydrazyl) were used. The ferric reducing antioxidant power (FRAP) and the total phenolic content of the samples were also determined. Some of the extracts, such as Limosella aquatica L. (Scrophulariaceae), Mimulus glabratus Kunth. (Phrymaceae), Pedicularis mexicana Zucc. ex Benth. (Orobanchaceae), and Penstemon campanulatus (Cav.) Willd. (Plantaginaceae) displayed remarkably selective cytotoxic activity. However, the extract from Veronica americana (Raf.) Schwein (Plantaginaceae) showed the highest activity with IC₅₀ values of 1.46 and 0.169 g/ μL on PC-3 and HF-6 cells, respectively. With the exception of M. glabratus, all the extracts showed different degrees of antioxidant activity with IC₅₀ values from 0.89 up to 1.8 in the ABTS assay; from 0.49 up to 1.25 mg/mL in the DPPH assay and with the FRAP evaluation, 36 to 68 equivalents in mM of FeSO₄. V. americana also showed the highest antoxidant activity with IC₅₀ values from 0.491 and 0.892 mg/mL, on DPPH and ABTS assays, respectively. These findings demonstrated that the species studied have great potential cytotoxic and antioxidant activity.

  16. Cytotoxic activity of the Proteus hemolysin HpmA.

    OpenAIRE

    Swihart, K G; Welch, R. A.

    1990-01-01

    We previously showed that hpmA is the hemolysin determinant most commonly found among Proteus isolates. To assess the potential contribution of HpmA to virulence, we first characterized the toxic activities of this hemolysin. Hemolytic activity was present in total cell cultures and cell-free supernatants of Proteus clinical isolates as well as Escherichia coli containing cloned hpm genes. HpmA also possesses cytotoxic activity which was detected by a chromium release assay against a variety ...

  17. Online Measurement of Real-Time Cytotoxic Responses Induced by Multi-Component Matrices, such as Natural Products, through Electric Cell-Substrate Impedance Sensing (ECIS

    Directory of Open Access Journals (Sweden)

    Adyary Fallarero

    2015-11-01

    Full Text Available Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract’s pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.

  18. Unperturbed Cytotoxic Lymphocyte Phenotype and Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients.

    Science.gov (United States)

    Theorell, Jakob; Bileviciute-Ljungar, Indre; Tesi, Bianca; Schlums, Heinrich; Johnsgaard, Mette Sophie; Asadi-Azarbaijani, Babak; Bolle Strand, Elin; Bryceson, Yenan T

    2017-01-01

    Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a debilitating disorder linked to diverse intracellular infections as well as physiological stress. Cytotoxic lymphocytes combat intracellular infections. Their function is attenuated by stress. Despite numerous studies, the role of cytotoxic lymphocytes in ME/CFS remains unclear. Prompted by advances in the understanding of defects in lymphocyte cytotoxicity, the discovery of adaptive natural killer (NK) cell subsets associated with certain viral infections, and compelling links between stress, adrenaline, and cytotoxic lymphocyte function, we reassessed the role of cytotoxic lymphocytes in ME/CFS. Forty-eight patients from two independent cohorts fulfilling the Canada 2003 criteria for ME/CFS were evaluated with respect to cytotoxic lymphocyte phenotype and function. Results were compared to values from matched healthy controls. Reproducible differences between patients and controls were not found in cytotoxic lymphocyte numbers, cytotoxic granule content, activation status, exocytotic capacity, target cell killing, or cytokine production. One patient expressed low levels of perforin, explained by homozygosity for the PRF1 p.A91V variant. However, overall, this variant was present in a heterozygous state at the expected population frequency among ME/CFS patients. No single patient displayed any pathological patterns of cellular responses. Increased expansions of adaptive NK cells or deviant cytotoxic lymphocyte adrenaline-mediated inhibition were not observed. In addition, supervised dimensionality reduction analyses of the full, multidimensional datasets did not reveal any reproducible patient/control discriminators. In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide useful biomarkers

  19. Unperturbed Cytotoxic Lymphocyte Phenotype and Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

    Directory of Open Access Journals (Sweden)

    Jakob Theorell

    2017-06-01

    Full Text Available Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS is a debilitating disorder linked to diverse intracellular infections as well as physiological stress. Cytotoxic lymphocytes combat intracellular infections. Their function is attenuated by stress. Despite numerous studies, the role of cytotoxic lymphocytes in ME/CFS remains unclear. Prompted by advances in the understanding of defects in lymphocyte cytotoxicity, the discovery of adaptive natural killer (NK cell subsets associated with certain viral infections, and compelling links between stress, adrenaline, and cytotoxic lymphocyte function, we reassessed the role of cytotoxic lymphocytes in ME/CFS. Forty-eight patients from two independent cohorts fulfilling the Canada 2003 criteria for ME/CFS were evaluated with respect to cytotoxic lymphocyte phenotype and function. Results were compared to values from matched healthy controls. Reproducible differences between patients and controls were not found in cytotoxic lymphocyte numbers, cytotoxic granule content, activation status, exocytotic capacity, target cell killing, or cytokine production. One patient expressed low levels of perforin, explained by homozygosity for the PRF1 p.A91V variant. However, overall, this variant was present in a heterozygous state at the expected population frequency among ME/CFS patients. No single patient displayed any pathological patterns of cellular responses. Increased expansions of adaptive NK cells or deviant cytotoxic lymphocyte adrenaline-mediated inhibition were not observed. In addition, supervised dimensionality reduction analyses of the full, multidimensional datasets did not reveal any reproducible patient/control discriminators. In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide

  20. Unperturbed Cytotoxic Lymphocyte Phenotype and Function in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

    Science.gov (United States)

    Theorell, Jakob; Bileviciute-Ljungar, Indre; Tesi, Bianca; Schlums, Heinrich; Johnsgaard, Mette Sophie; Asadi-Azarbaijani, Babak; Bolle Strand, Elin; Bryceson, Yenan T.

    2017-01-01

    Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a debilitating disorder linked to diverse intracellular infections as well as physiological stress. Cytotoxic lymphocytes combat intracellular infections. Their function is attenuated by stress. Despite numerous studies, the role of cytotoxic lymphocytes in ME/CFS remains unclear. Prompted by advances in the understanding of defects in lymphocyte cytotoxicity, the discovery of adaptive natural killer (NK) cell subsets associated with certain viral infections, and compelling links between stress, adrenaline, and cytotoxic lymphocyte function, we reassessed the role of cytotoxic lymphocytes in ME/CFS. Forty-eight patients from two independent cohorts fulfilling the Canada 2003 criteria for ME/CFS were evaluated with respect to cytotoxic lymphocyte phenotype and function. Results were compared to values from matched healthy controls. Reproducible differences between patients and controls were not found in cytotoxic lymphocyte numbers, cytotoxic granule content, activation status, exocytotic capacity, target cell killing, or cytokine production. One patient expressed low levels of perforin, explained by homozygosity for the PRF1 p.A91V variant. However, overall, this variant was present in a heterozygous state at the expected population frequency among ME/CFS patients. No single patient displayed any pathological patterns of cellular responses. Increased expansions of adaptive NK cells or deviant cytotoxic lymphocyte adrenaline-mediated inhibition were not observed. In addition, supervised dimensionality reduction analyses of the full, multidimensional datasets did not reveal any reproducible patient/control discriminators. In summary, employing sensitive assays and analyses for quantification of cytotoxic lymphocyte differentiation and function, cytotoxicity lymphocyte aberrances were not found among ME/CFS patients. These assessments of cytotoxic lymphocytes therefore do not provide useful biomarkers

  1. Evaluation of Cytotoxicity and Genotoxicity of Acacia aroma Leaf Extracts

    Science.gov (United States)

    Mattana, C. M.; Cangiano, M. A.; Alcaráz, L. E.; Sosa, A.; Escobar, F.; Sabini, C.; Sabini, L.; Laciar, A. L.

    2014-01-01

    Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE) and ethanolic extract (EE) of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings. PMID:25530999

  2. Evaluation of Cytotoxicity and Genotoxicity of Acacia aroma Leaf Extracts

    Directory of Open Access Journals (Sweden)

    C. M. Mattana

    2014-01-01

    Full Text Available Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE and ethanolic extract (EE of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings.

  3. The in vitro cytotoxicity of urine from patients with interstitial cystitis

    DEFF Research Database (Denmark)

    Beier-Holgersen, R; Hermann, G G; Mortensen, S O

    1994-01-01

    Urine from patients with interstitial cystitis has been reported to be more cytotoxic than urine from healthy subjects when tested in vitro against cells from a normal urothelial cell line. The purpose of the present study was to develop a method to measure urinary cytotoxicity and so make...... it possible to estimate the toxicity of urine from interstitial cystitis patients. The study included 10 women with interstitial cystitis and 10 healthy controls. Urine specimens were obtained from both groups and urine cytotoxicity was measured by a modified 51Cr-release assay: A range of urine dilutions...... in urine cytotoxicity between interstitial cystitis patients and healthy controls. Urine cytotoxicity was increased by dilution in both groups....

  4. Antioxidant and cytotoxic activities of three different fractions of Lonicera quinquelocularis (Translucent Honeysuckle plant

    Directory of Open Access Journals (Sweden)

    MUHAMMAD IMRAN K.

    2014-08-01

    Full Text Available The cytotoxic and antioxidant activities of a plant count for its effectiveness against various lethal and sever diseases such as cancer, Parkinson Alzheimer and Diabetics etc. Free radicals in the form of reactive oxygen species and reactive nitrogen species may be the main cause of these severe health issues. Medicinal plants with antioxidant and cytotoxic properties have been proved to be the most reliable method of treatment of the mentioned diseases. In the present study antioxidant and cytotoxic potential of three different fractions i.e. ethanol, chloroform and ethyl acetate extracted from Lonicera quinquelocularis (Translucent Honeysuckle plant were tested. The ethanolic and ethyl acetate fraction showed highest scavenging ability than chloroform fraction against DPPH and ABTS free radicals. In cytotoxic assays, chloroform fraction showed highest lethal effects than ethanolic and ethyl acetate fraction. The data obtained from the experimental work revealed that all the three fractions isolated from Lonicera quinquelocularis (Translucent Honeysuckle possess antioxidant and cytotoxic properties. Key Words:

  5. Cytotoxic effect of a dentin bonding agent: AdheSE

    Directory of Open Access Journals (Sweden)

    Banava S.

    2007-05-01

    Full Text Available Background and Aim: An important requirement for a dentin bonding agent is biological compatibility. Since dentin bonding agents are placed in cavity preparations with subgingival extensions, with direct contact to gingival and mucosal tissues, tissue response to these materials must be investigated. The aim of this study was to examine the cytotoxicity of AdheSE, a self etching adhesive, on human gingival fibroblasts."nMaterials and Methods: In this experimental in vitro study, primary human gingival fibroblasts were exposed to different dilutions of primer & bond of AdheSE (Vivadent, Liechtenstein. The toxicity of the primer was tested in 30 seconds, 300 seconds and 24 hours. The cytotoxicity of the bond was analyzed in uncured mode after 20 seconds, 5 minutes and 24 hours. In cured mode, tested materials were analyzed after 24 and 48 hours. Cytotoxic effects were evaluated using MTT, cell counting and DNA condensation assays. Data were analyzed by two way repeated measure ANOVA with p<0.05 as the level of significance."nResults: MTT Assay revealed that uncured AdheSE Bond was toxic only in 10-1 dilution and the difference with control group was significant (P<0.05. By increasing the time to 300sec. and 24h, dilutions of 10-2 and 10-4 were the most cytotoxic respectively. Cytotoxicity of uncured primer after 30 sec. and 300 sec. began from 10-2 and after 24h began from 10-2 and reached to 10-1. AdheSE in cured mode showed significant difference with control group in 1:2 (P<0.001,1:4 & 1:6 (P<0.01 dilutions. In cell counting assay only the 1:2 dilution was significantly more toxic than control group. Apoptosis (a morphological and biochemical distinct form of cell death that regulates cell turnover comprised in less than 5% of total death in both cured and uncured adhesives."nConclusions: Based on the results of this study, by increasing the exposure time, smaller amounts of bonding could be cytotoxic. Cytotoxicity was related to material

  6. Evaluation of cytotoxicity and gelatinases activity in 3T3 fibroblast cell by root repair materials

    OpenAIRE

    Varol Basak; Tuna Elif Bahar; Karsli Emine; Kasimoglu Yelda; Koruyucu Mine; Seymen Figen; Nurten Rustem

    2016-01-01

    The aim of this study was to investigate the effects of calcium silicate-based products on cytotoxicity in the 3T3 fibroblast and gelatinolytic activity of matrix metalloproteinases (MMPs). 3T3 fibroblasts were incubated directly with Ortho Mineral trioxide aggregate (MTA), BioAggregate, Biodentine, MTA Plus, MTA Angelus and MTA Cerkamed for 24 hours and seven days. The cytotoxicity was determined using an MTT assay. Supernatants were collected to determine MMP-2 and MMP-9. Data were analysed...

  7. New Cytotoxic Azaphilones from Monascus purpureus-Fermented Rice (Red Yeast Rice)

    OpenAIRE

    Zong-Lian Jin; Jian-Quan Zheng; Ming-Tao Liu; Ling-Ling Li; Xiao-Ya Shang; Jin-Jie Li

    2010-01-01

    Using a cell-based cytotoxicity assay three new cytotoxic azaphilones, including two stereoisomers and designated monapurones A-C (1-3), were isolated from the extract of Monascus purpureus-fermented rice (red yeast rice). Their structures were elucidated by detailed interpretation of spectroscopic and chemical data. The relative configurations were assigned on the basis of analysis of NOE data, and the absolute configurations were determined by direct comparison of their CD spectra with thos...

  8. Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.

    Science.gov (United States)

    Pauwels, Bea; Korst, Annelies E C; de Pooter, Christel M J; Pattyn, Greet G O; Lambrechts, Hilde A J; Baay, Marc F D; Lardon, Filip; Vermorken, Jan B

    2003-03-01

    Since there is a growing interest in preclinical research on interactions between radiation and cytotoxic agents, this study focused on the development of an alternative to the very laborious clonogenic assay (CA). The colorimetric sulforhodamine B (SRB) assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines (A549, H292), one colon cancer cell line (HT-29) and one breast cancer cell line (MCF-7). In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both assays. The dose-response curves obtained with the SRB assay and the CA were very similar up to 6 Gy. The radiosensitivity parameters (SF(2), alpha, beta, MID and ID(50)) obtained from the SRB assay and the CA were not significantly different between H292, A549 and MCF-7 cells. The radiation dose-response curves for A549 and H292 cells pretreated with 4 n M gemcitabine for 24 h clearly showed a radiosensitizing effect with both assays. The dose-enhancement factors obtained with the SRB assay and the CA were 1.80 and 1.76, respectively, for A549 cells, and 1.52 and 1.41 for H292 cells. The SRB assay was shown to be as useful as the more traditional CA for research on chemotherapy/radiotherapy interactions in cell lines with moderate radiosensitivity. This assay will be used for more extensive in vitro research on radiosensitizing compounds in these cell lines.

  9. One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells

    OpenAIRE

    Weldon, David J.; Saulsbury, Marilyn D.; Goh, Joshua; Rowland, Leah; Campbell, Petreena; Robinson, Laijia; Miller, Calvin; Christian, Joshua; Amis, Louisa; Taylor, Nia; Davis, Willie; Evans, Stanley L.; Brantley, Eileen

    2014-01-01

    A series of cinnamylideneacetophenones were synthesized via a modified Claisen-Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50 = 71 and 1.9 nM) respectively. Derivative 17 also displayed sub-micromolar (IC50 = 780 nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cy...

  10. COMPARATIVE ANALYSIS OF CYTOTOXIC AND ANTIOXIDANT ACTIVITIES OF LEAF AND BARK EXTRACTS OF CLERODENRUM VISCOSUM AND CLERODENDRUM PHLOMIDIS

    OpenAIRE

    Bhatnagar, Sunita; Pattanaik, Soumya Ranjan

    2012-01-01

    Cytotoxic and antioxidant potential of leaf and bark extracts of two Clerodendrum sps namely C. phlomidis and  C. viscosum was investigated. Solvent extracts   hexane, chloroform, acetone and methanol were tested for their cytotoxic potential using brine shrimp motility assay and antioxidant potential was ascertained using DPPH and FRAP assays.             Cytotoxic activity of all the solvent extracts was tested at four doses 25, 50,100 and 200µl/ml. All the extracts showed d...

  11. Flavonoids of Calligonum polygonoides and their cytotoxicity.

    Science.gov (United States)

    Ahmed, Hayam; Moawad, Abeer; Owis, Asmaa; AbouZid, Sameh; Ahmed, Osama

    2016-10-01

    Context Calligonum polygonoides L. subsp. comosum L' Hér. (Polygonaceae), locally known as "arta", is a slow-growing small leafless desert shrub. Objective Isolation, structure elucidation and evaluation of cytotoxic activity of flavonoids from C. polygonoides aerial parts. Materials and methods Flavonoids in the hydroalcoholic extract of the of C. polygonoides were isolated and purified using column chromatography and preparative HPLC. The structures of the isolated flavonoids were elucidated on the basis of spectroscopic data including 2D NMR techniques. The cytotoxic activity of the isolated flavonoids (6.25, 25, 50 and 100 μg/mL) was evaluated against liver HepG2 and breast MCF-7 cancer cell lines using sulphorhodamine-B assay. Results A new flavonoid, kaempferol-3-O-β-D-(6″-n-butyl glucuronide) (1), and 13 known flavonoids, quercetin 3-O-β-D-(6″-n-butyl glucuronide) (2), kaempferol-3-O-β-D-(6″-methyl glucuronide) (3), quercetin-3-O-β-D-(6″-methyl glucuronide) (4), quercetin-3-O-glucuronide (5), kaempferol-3-O-glucuronide (6), quercetin-3-O-α-rhamnopyranoside (7), astragalin (8), quercetin-3-O-glucopyranoside (9), taxifolin (10), (+)-catechin (11), dehydrodicatechin A (12), quercetin (13), and kaempferol (14), were isolated from the aerial parts of C. polygonoides. Quercetin showed significant cytotoxic activity against HepG2 and MCF-7 cell lines with IC50 values of 4.88 and 0.87 μg/mL, respectively. Structure-activity relationships were analyzed by comparing IC50 values of several pairs of flavonoids differing in one structural element. Discussion and conclusion The activity against breast cancer cell lines decreased by glycosylation at C-3. The presence of 2,3-double bond in ring C, carbonyl group at C-4 and 3',4'-dihydroxy substituents in ring B are essential structural requirements for the cytotoxic activity against breast cancer cells.

  12. Different responses of vanillic acid, a phenolic compound, in HTC cells: cytotoxicity, antiproliferative activity, and protection from DNA-induced damage.

    Science.gov (United States)

    Almeida, I V; Cavalcante, F M L; Vicentini, V E P

    2016-12-19

    The consumption of healthy and natural foods has increased over the last few years, primarily because these foods are rich in substances with biological properties of interest, such as exerting anticancer effects and decreasing oxidative stress in living tissues. These foods support adequate nutrition, maintain health, and improve quality of life. Vanillic acid (VA) is a phenolic compound used widely in the food industry as a flavoring, preservative, and food additive. VA can be found in various cereals, whole grains, fruits, herbs, green tea, juices, beers, and wines and possesses antioxidant, hepatoprotective, cardioprotective, and antiapoptotic activities. Studying the cytotoxicity as well as the mutagenic and antimutagenic effects of different concentrations of VA in Rattus norvegicus hepatoma cells (HTC) can identify new cellular activities of this substance. Concentrations up to 100 µM VA are not cytotoxic to HTC cells in a MTT [3-(4,5-dimethilthiazol-2-yl)-2,5-diphenil tetrazolium bromide] assay after 96-h exposure; therefore, VA does not compromise mitochondrial activity. Similarly, concentrations up to 500 µM do not compromise plasma membrane integrity. VA at 10 and 50 µM showed no mutagenic/clastogenic effects, as no significant micronuclei induction was observed. VA 10 µM presented no antiproliferative activity and reduced the cytotoxicity induced by benzo[a]pyrene. The antimutagenic activity of 10 µM VA was observed by the simultaneous, pre-, and post-treatments, as the phenolic compound significantly reduced the frequency of micronuclei induced by the mutagen. These results indicate that VA exerts different responses in HTC cells. Low concentrations present no cytotoxic, mutagenic, or antiproliferative effects and protect cells from DNA damage.

  13. Cytotoxic, antitumour-promoting and inhibition of protein denaturation effects of flavonoids, isolated from Potentilla evestita Th. Wolf.

    Science.gov (United States)

    Rauf, Abdur; Khan, Rehan; Khan, Haroon; Tokuda, Harukuni

    2015-01-01

    This study was designed to evaluate the isolated flavonoids (chrysin 1, and umbelliferone 2) from Potentilla evestita for cytotoxic, antitumour-promoting and inhibition of protein denaturation activities. The results showed marked cytotoxic effect of compounds 1 and 2 in brine shrimp cytotoxic assay at various concentrations with LD50 of 34.5 and 31.8 mg/mL, respectively. In Epstein-Barr-virus early antigen activation assay, both compounds 1 and 2 illustrated significant antitumour-promoting effect with IC50 values of 462 and 308 mol ratio/32 pmol TPA, respectively. The cytotoxic and antitumour-promoting effects of compounds were strongly supported by inhibition of protein denaturation activity with IC50 of 119 and 112 μg/mL, respectively. In conclusion, both compounds possess strong cytotoxic, antitumour-promoting and inhibition of protein denaturation activities.

  14. Optimization of the sulforhodamine B colorimetric assay.

    Science.gov (United States)

    Papazisis, K T; Geromichalos, G D; Dimitriadis, K A; Kortsaris, A H

    1997-10-27

    Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.

  15. Cytotoxic potential of selected medicinal plants in northeast Brazil.

    Science.gov (United States)

    da Silva, Thiago B C; Costa, Cinara O D'Sousa; Galvão, Alexandre F C; Bomfim, Larissa M; Rodrigues, Ana Carolina B da C; Mota, Mauricio C S; Dantas, Alex A; Dos Santos, Tiago R; Soares, Milena B P; Bezerra, Daniel P

    2016-07-08

    Great biodiversity is a highlight of Brazilian flora. In contrast, the therapeutic potentialities of most species used in folk medicine remain unknown. Several of these species are commonly used to treat cancer. In this study, we investigated the cytotoxic activity of 18 plants from 16 families that are found in the northeast region of Brazil. The following species were studied: Byrsonima sericea DC. (Malpighiaceae), Cupania impressinervia Acev. Rodr. var. (revoluta) Radlk (Sapindaceae), Duranta repens Linn. (Verbenaceae), Helicostylis tomentosa (Poepp. & Endl) Rusby (Moraceae), Himatanthus bracteatus (A.DC.) Woodson (Apocynaceae), Ipomoea purga (Wender.) Hayne (Convolvulaceae), Ixora coccinea Linn. (Rubiaceae), Mabea piriri Aubl. (Euphorbiaceae), Miconia minutiflora (Melastomataceae), Momordica charantia L. (Cucurbitaceae), Ocotea glomerata (Nees) Mez (Lauraceae), Ocotea longifolia Kunth (Oreodaphne opifera Mart. Nees) (Lauraceae), Pavonia fruticosa (Mill.) Fawc. & Rendle (Malvaceae), Psychotria capitata Ruiz & Pav. (Rubiaceae), Schefflera morototoni (Aubl.) Maguire, Steyerm. & Frodin (Araliaceae), Solanum paludosum Moric. (Solanaceae), Xylopia frutescens Aubl. (Annonaceae) and Zanthoxylum rhoifolium Lam. (Rutaceae). Their dried leaves, stems, flowers or fruits were submitted to different solvent extractions, resulting in 55 extracts. After incubating for 72 h, the cytotoxicity of each extract was tested against tumor cell lines using the alamar blue assay. The B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata extracts demonstrated the most potent cytotoxic activity. The chloroform soluble fractions of D. repens flowers and the hexane extract of I. coccinea flowers led to the isolation of quercetin and a mixture of α- and β-amyrin, respectively, and quercetin showed moderate cytotoxic activity. The B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata plants were

  16. Cytotoxic triterpenoid saponins from Lysimachia foenum-graecum.

    Science.gov (United States)

    Dai, Lu-Mei; Huang, Ri-Zhen; Zhang, Bin; Hua, Jing; Wang, Heng-Shan; Liang, Dong

    2017-04-01

    Eleven oleanane-type triterpenoid saponins, foegraecumosides A-K, and eight known ones, were isolated from the aerial parts of Lysimachia foenum-graecum. Their structures were elucidated by spectroscopic data analyses and chemical methods. All isolated saponins were evaluated for their cytotoxicity against four human cancer cell lines (NCI-H460, MGC-803, HepG2, and T24). Seven saponins containing the aglycone cyclamiretin A exhibited moderate cytotoxicity against all tested human cancer cell lines, with IC 50 values of 9.3-24.5 μM. Simultaneously, the cytotoxic activities of foegraecumosides A and B, lysichriside A, ardisiacrispins A and B, cyclaminorin, and 3-O-α-L-rhamnopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 4)-α-l-arabinopyranosyl-cyclamiretin A were tested on drug-resistant lung cancer cell lines (A549 and A549/CDDP, respectively). Ardisiacrispin B displayed moderate cytotoxicity against A549/CDDP, with an IC 50 value of 8.7 μM and a resistant factor (RF) of 0.9. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. REFINEMENT OF AN INDIRECT IMMUNOTOXIN ASSAY OF MONOCLONAL-ANTIBODIES RECOGNIZING THE HUMAN SMALL-CELL LUNG-CANCER CLUSTER-2 ANTIGEN

    NARCIS (Netherlands)

    DERBYSHIRE, EJ; DELEIJ, L; WAWRZYNCZAK, EJ

    Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line

  18. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  19. In vitro cytotoxicity studies of 20 plants used in Nigerian antimalarial ethnomedicine.

    Science.gov (United States)

    Ajaiyeoba, E O; Abiodun, O O; Falade, M O; Ogbole, N O; Ashidi, J S; Happi, C T; Akinboye, D O

    2006-03-01

    Twenty plants identified and selected from Southwest and Middle belt Nigerian antimalarial ethnopharmacology were evaluated for in vitro cytotoxicity using the brine shrimp lethality assay. The methanol extracts of 20 plant samples from 11 plant families were subjected to the assay. Of the studied plants, Lippia multiflora and Morinda lucida bark were found to be cytotoxic, with LC(50) values of 1.1 and 2.6 microg/ml, respectively. The least toxic plant extract was Bridelia micrantha (LC(50) value >9.0 x 10(6) microg/ml). Most of the plants were found to be relatively non-toxic.

  20. Cytotoxic coumarins from Toddalia asiatica.

    Science.gov (United States)

    Phatchana, Ratchanee; Yenjai, Chavi

    2014-06-01

    Three new coumarins and 13 known compounds were isolated from the stem bark of Toddalia asiatica. Compounds 1, 3, 8, and 9 showed cytotoxicity against the NCI-H187 cell line with IC50 values ranging from 6 to 9 µg/mL. Compounds 4 and 9 exhibited cytotoxicity against the MCF-7 cell line with IC50 values of 3.17 and 9.79 µg/mL, respectively. Compound 9 also displayed cytotoxic activity against KB cells with an IC50 value of 8.63 µg/mL. In addition, compound 14 showed antimalarial activity against Plasmodium falciparum with an IC50 value of 3.66 µg/mL. Compounds 5, 9, and 16 exhibited antituberculosis activity against Mycobacterium tuberculosis with MIC values of 50, 50, and 25 µg/mL, respectively. Georg Thieme Verlag KG Stuttgart · New York.

  1. Phytochemicals and Cytotoxicity of Launaea procumbens on Human Cancer Cell Lines.

    Science.gov (United States)

    Rawat, Preeti; Saroj, Lokesh M; Kumar, Anil; Singh, Tryambak D; Tewari, S K; Pal, Mahesh

    2016-07-01

    The plant Launaea procumbens belongs to the family Asteraceae and traditionally used in the treatment rheumatism, kidney, liver dysfunctions and eye diseases. In the present study Phytochemical analysis and fractions of methanolic extract of L. procumbens leaves were tested in vitro for their cytotoxicity. Phytochemical analysis and cytotoxic activity of methanolic extract and fractions of Launaea procumbens against four cancer cell lines K562, HeLa, MIA-Pa-Ca-2 and MCF-2 by SRB assay. Powdered leaves of Launaea procumbens were extracted sequentially with hexane, ethyl acetate, butanol and water by cold extraction. Phytochemical analysis and cytotoxicity assay were carried out for these fractions using SRB assay against four human cancer cell lines, namely leukemia (K562), cervix (HeLa), pancreatic (MIA-Pa-Ca-2) and breast (MCF-7). Ethyl acetate extract exerts potent cytotoxicity against human leukemia (K562), cervix (HeLa) and breast (MCF-7) cell lines IC50 value of 25.30±0.50, 19.80±0.10 and 36.90±4.90 μg/ml respectively. Moderately cytotoxic effect found in hexane extract IC50 value of 41±8 and 48.20±0.50 μg/ml against leukemia (K562), and breast (MCF-7) cancer cell line respectively. The Chemical composition analyzed by GC-MS showed considerable differences in solvent fractions of Launaea procumbens. This study revealed the cytotoxic potential of ethyl acetate and hexane fractions of L. procumbens leaves on different cancer cell lines. Ethyl acetate and Hexane fractions of Launaea procumbens plant exhibit cytotoxicity. Among the different fractions Ethyl acetate showed relatively higher cytotoxicity.Ethyl acetate found more cytotoxic against leukemia (K 562), cervix (HeLa) and breast (MCF-7) cancer cell lines. Moderete cytotoxicity found in hexane fraction against leukemia (K 562) and breast (MCF-7) cancer cell line.GC-MS results showed L. procumbens is a rich source of 1-H- pyrazole, 1-H-imidazole, β -amyrin, α -amyrin and lupeol. These compounds may

  2. Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage.

    Directory of Open Access Journals (Sweden)

    Jui-Hua Hsieh

    Full Text Available Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2 using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo while the other evaluates cell membrane integrity (i.e., cell death, flor. Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our

  3. Simultaneous Determination of Famotidine and Flurbiprofen by High ...

    African Journals Online (AJOL)

    Purpose: To develop a selective, sensitive and accurate simultaneous High Performance Liquid Chromatography (HPLC) method for the analysis of flurbiprofen and famotidine tablet dosage form and excipients. Methods: A simultaneous method for the determination of the two drugs was employed. The assay consisted of ...

  4. Lucigenin chemiluminescence assay as an adjunctive tool for ...

    Indian Academy of Sciences (India)

    A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronic in vivo pulmonary reactions could have potential value as predictors of fibrogenicity ...

  5. Lucigenin chemiluminescence assay as an adjunctive tool for ...

    Indian Academy of Sciences (India)

    Unknown

    *Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health,. Morgantown, WV 26505, USA. †Corresponding author (Fax, 304-293-5449; Email, kvandyke@hsc.wvu.edu). A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have.

  6. Efficiency of immunotoxin cytotoxicity is modulated by the intracellular itinerary.

    Directory of Open Access Journals (Sweden)

    Lori L Tortorella

    Full Text Available Pseudomonas exotoxin-based immunotoxins, including LMB-2 (antiTac(F(v-PE38, are proposed to traffic to the trans-Golgi network (TGN and move by a retrograde pathway to the endoplasmic reticulum, where they undergo translocation to the cytoplasm, a step that is essential for cytotoxicity. The retrograde transport pathways used by LMB-2 are not completely understood, so it is unclear if transit through specific organelles is critical for maximal cytotoxic activity. In this study, we used Chinese hamster ovary (CHO cell lines that express chimeric constructs of CD25, the Tac antigen, attached to the cytoplasmic domain of the TGN-targeted transmembrane proteins, TGN38 and furin. These chimeras are both targeted to the TGN, but the itineraries they follow are quite different. LMB-2 was incubated with the two cell lines, and the efficiency of cell killing was determined using cell viability and cytotoxicity assays. LMB-2 that is targeted through the endocytic recycling compartment to the TGN via Tac-TGN38 kills the cells more efficiently than immunotoxins delivered through the late endosomes by Tac-furin. Although the processing to the 37 kDa active fragment was more efficient in Tac-furin cells than in Tac-TGN38 cells, this was not associated with enhanced cytotoxicity - presumably because the toxin was also degraded more rapidly in these cells. These data indicate that trafficking through specific organelles is an important factor modulating toxicity by LMB-2.

  7. An efficient analysis of nanomaterial cytotoxicity based on bioimpedance

    Energy Technology Data Exchange (ETDEWEB)

    Kandasamy, Karthikeyan; Kim, Sanghyo [College of Bionanotechnology, Kyungwon University, Gyeonggi-do (Korea, Republic of); Choi, Cheol Soo, E-mail: samkim@kyungwon.ac.kr [Lee Gil Ya Diabetes and Cancer Institute, Gachon University of Medicine and Science, Incheon (Korea, Republic of)

    2010-09-17

    In the emerging nanotechnology field, there is an urgent need for the development of a significant and sensitive method that can be used to analyse and compare the cytotoxicities of nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs), since such materials can be applied as contrast agents or drug delivery carriers. The bioimpedance system possesses great potential in many medical research fields including nanotechnology. Electric cell-substrate impedance sensing (ECIS) is a particular bioimpedance system that offers a real-time, non-invasive, and quantitative measurement method for the cytotoxicity of various materials. The present work compared the cytotoxicity of AuNPs to that of purchased single-walled carbon nanotubes (SWCNTs). The size-controlled and monodispersed AuNPs were synthesized under autoclaved conditions and reduced by ascorbic acid (AA) whereas the purchased SWCNTs were used without any surface modifications. Bioimpedance results were validated by conventional WST-1 and trypan blue assays, and transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) were performed to examine nanomaterials inside the VERO cells. This research evaluates the ability of the ECIS system compared to those of conventional methods in analyzing the cytotoxicity of AuNPs and SWCNTs with higher sensitivity under real-time conditions.

  8. Cytotoxic sesquiterpene lactones from the aerial parts of Inula aucheriana.

    Directory of Open Access Journals (Sweden)

    Ahmad Reza Gohari

    2015-06-01

    Full Text Available Inula aucheriana DC is a member of the family Asteraceae which is known to produce cytotoxic secondary metabolites noted as sesquiterpene lactones. In the present study, sesquiterpene lactones inuchinenolide B, 6-deoxychamissonolide (stevin and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were isolated from I. aucheriana. Inuchinenolide B and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10,11(13-dien-12,8α-olide were further evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay to demonstrate cytotoxic activity with IC50 values of (56.6, 19.0, (39.0, 11.8, and (55.7, 15.3 μg/mL against HepG-2, MCF-7 and A-549 cells, respectively. The cytotoxic activity of the two evaluated sesquiterpene lactones partly explains the cytotoxic activity that was previously observed for the extracts of Inula aucheriana. The isolated compounds could be further investigated in cancer research studies.

  9. Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity.

    Science.gov (United States)

    Lee, Hyunkyoung; Jung, Eun-sun; Kang, Changkeun; Yoon, Won Duk; Kim, Jong-Shu; Kim, Euikyung

    2011-09-01

    The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Cytotoxic sesquiterpene lactones from the aerial parts of Inula aucheriana.

    Science.gov (United States)

    Gohari, Ahmad Reza; Mosaddegh, Mahmoud; Naghibi, Farzaneh; Eslami-Tehrani, Bahara; Pirani, Atefeh; Hamzeloo-Moghadam, Maryam; Read, Roger W

    2015-01-01

    Inula aucheriana DC is a member of the family Asteraceae which is known to produce cytotoxic secondary metabolites noted as sesquiterpene lactones. In the present study, sesquiterpene lactones inuchinenolide B, 6-deoxychamissonolide (stevin) and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10),11(13)-dien-12,8α-olide were isolated from I. aucheriana. Inuchinenolide B and 14-acetoxy-1β,5α,7αH-4β-hydroxy-guai-9(10),11(13)-dien-12,8α-olide were further evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay to demonstrate cytotoxic activity with IC50 values of (56.6, 19.0), (39.0, 11.8), and (55.7, 15.3) μg/mL against HepG-2, MCF-7 and A-549 cells, respectively. The cytotoxic activity of the two evaluated sesquiterpene lactones partly explains the cytotoxic activity that was previously observed for the extracts of Inula aucheriana. The isolated compounds could be further investigated in cancer research studies.

  11. An efficient analysis of nanomaterial cytotoxicity based on bioimpedance

    Science.gov (United States)

    Kandasamy, Karthikeyan; Choi, Cheol Soo; Kim, Sanghyo

    2010-09-01

    In the emerging nanotechnology field, there is an urgent need for the development of a significant and sensitive method that can be used to analyse and compare the cytotoxicities of nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs), since such materials can be applied as contrast agents or drug delivery carriers. The bioimpedance system possesses great potential in many medical research fields including nanotechnology. Electric cell-substrate impedance sensing (ECIS) is a particular bioimpedance system that offers a real-time, non-invasive, and quantitative measurement method for the cytotoxicity of various materials. The present work compared the cytotoxicity of AuNPs to that of purchased single-walled carbon nanotubes (SWCNTs). The size-controlled and monodispersed AuNPs were synthesized under autoclaved conditions and reduced by ascorbic acid (AA) whereas the purchased SWCNTs were used without any surface modifications. Bioimpedance results were validated by conventional WST-1 and trypan blue assays, and transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) were performed to examine nanomaterials inside the VERO cells. This research evaluates the ability of the ECIS system compared to those of conventional methods in analyzing the cytotoxicity of AuNPs and SWCNTs with higher sensitivity under real-time conditions.

  12. Altered effector function of peripheral cytotoxic cells in COPD

    Directory of Open Access Journals (Sweden)

    Corne Jonathan M

    2009-06-01

    Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

  13. Genotoxic and cytotoxic effects of storax in vitro.

    Science.gov (United States)

    Karadeniz, Bulent; Ulker, Zeynep; Alpsoy, Lokman

    2013-03-01

    The aim of this study is to investigate the effects of the storax balsam, which is a kind of sweet gum obtained from the Liquidambar orientalis Mill trees, on cell viability, cytotoxicity and genotoxicity in human lymphocyte in vitro. We studied the genotoxic effects of the extract of storax balsam (SE) using sister chromatid exchange (SCE) test system. Also the cytotoxic and inhibitory effects on cell proliferation of SE were evaluated using lactate dehydrogenase (LDH) assay and cell proliferation (WST-1) assay. The SCE frequency was increased when the cells were treated with 1.6 and 4.0 µg/mL SE concentrations (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly depleted the cell number at 24th and 48th hours and elevated the LDH levels (p < 0.05) at 48th hour. These results suggest that SE can be used as an alternative antibacterial and antipathogenic agent due to its cytotoxic and genotoxic effects.

  14. Antimicrobial and cytotoxic effects of Mexican medicinal plants.

    Science.gov (United States)

    Jacobo-Salcedo, Maria del Rosario; Alonso-Castro, Angel Josabad; Salazar-Olivo, Luis A; Carranza-Alvarez, Candy; González-Espíndola, Luis Angel; Domínguez, Fabiola; Maciel-Torres, Sandra Patricia; García-Lujan, Concepción; González-Martínez, Marisela del Rocio; Gómez-Sánchez, Maricela; Estrada-Castillón, Eduardo; Zapata-Bustos, Rocio; Medellin-Milán, Pedro; García-Carrancá, Alejandro

    2011-12-01

    The antimicrobial effects of the Mexican medicinal plants Guazuma ulmifolia, Justicia spicigera, Opuntia joconostle, O. leucotricha, Parkinsonia aculeata, Phoradendron longifolium, P. serotinum, Psittacanthus calyculatus, Tecoma stans and Teucrium cubense were tested against several human multi-drug resistant pathogens, including three Gram (+) and five Gram (-) bacterial species and three fungal species using the disk-diffusion assay. The cytotoxicity of plant extracts on human cancer cell lines and human normal non-cancerous cells was also evaluated using the MTT assay. Phoradendron longifolium, Teucrium cubense, Opuntia joconostle, Tecoma stans and Guazuma ulmifolia showed potent antimicrobial effects against at least one multidrug-resistant microorganism (inhibition zone > 15 mm). Only Justicia spicigera and Phoradendron serotinum extracts exerted active cytotoxic effects on human breast cancer cells (IC50 < or = 30 microg/mL). The results showed that Guazuma ulmifolia produced potent antimicrobial effects against Candida albicans and Acinetobacter lwoffii, whereas Justicia spicigera and Phoradendron serotinum exerted the highest toxic effects on MCF-7 and HeLa, respectively, which are human cancer cell lines. These three plant species may be important sources of antimicrobial and cytotoxic agents.

  15. Brine shrimp cytotoxic activities of Hippophae rhamnoides Linn leaves extracts

    Directory of Open Access Journals (Sweden)

    Javid Ali

    2015-04-01

    Full Text Available Objective: To evaluate brine shrimp lethality assay of solvent extracts (aqueous, methanol, ethanol, acetone, ethyl acetate, chloroform and n-hexane of Hippophae rhamnoides (H. rhamnoides leaves. Methods: Brine shrimp cytotoxicity assay was used to assess the cytotoxic potential of H. rhamnoides leaves extracts. Three vials for concentration of each extract were made and 10 shrimps per vial (30 shrimps per dilution were transferred to specific concentration of each extract. Results: The mortality of aqueous extract was 46.7%, methanol extract was 46.7%, ethanolic extract was 50.0%, ethyl acetate was 26.7%, acetone extract was 33.3%, chloroform extract was 40.0% and n-hexane extract was 33.3%. The lowest LD50 was found in methanol extracts (1199.97 µg/mL. Brine shrimp cytotoxicity of tested extracts of H. rhamnoides showed that mortality rate was concentration dependent. Conclusions: It is concluded that bioactive components are present in all leaves extracts of H. rhamnoides, which could be accounted for its pharmacological effects. Thus, the results support the uses of this plant species in traditional medicine.

  16. Antioxidant Capacity, Cytotoxicity, and Acute Oral Toxicity of Gynura bicolor

    Directory of Open Access Journals (Sweden)

    Wuen Yew Teoh

    2013-01-01

    Full Text Available Gynura bicolor (Compositae which is widely used by the locals as natural remedies in folk medicine has limited scientific studies to ensure its efficacy and nontoxicity. The current study reports the total phenolic content, antioxidant capacity, cytotoxicity, and acute oral toxicity of crude methanol and its fractionated extracts (hexane, ethyl acetate, and water of G. bicolor leaves. Five human colon cancer cell lines (HT-29, HCT-15, SW480, Caco-2, and HCT 116, one human breast adenocarcinoma cell line (MCF7, and one human normal colon cell line (CCD-18Co were used to evaluate the cytotoxicity of G. bicolor. The present findings had clearly demonstrated that ethyl acetate extract of G. bicolor with the highest total phenolic content among the extracts showed the strongest antioxidant activity (DPPH radical scavenging assay and metal chelating assay, possessed cytotoxicity, and induced apoptotic and necrotic cell death, especially towards the HCT 116 and HCT-15 colon cancer cells. The acute oral toxicity study indicated that methanol extract of G. bicolor has negligible level of toxicity when administered orally and has been regarded as safe in experimental rats. The findings of the current study clearly established the chemoprevention potential of G. bicolor and thus provide scientific validation on the therapeutic claims of G. bicolor.

  17. Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

    KAUST Repository

    Rodriguez, Jose E.

    2013-05-01

    Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

  18. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    Science.gov (United States)

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  19. Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Zor, Kinga; Montini, Lucia

    2015-01-01

    In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes......, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study....... The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time...

  20. Aminosilane Functionalization and Cytotoxicity Effects of Upconversion Nanoparticles Y2O3 and Gd2O3 Co-doped with Yb3+and Er3+

    Directory of Open Access Journals (Sweden)

    Dalia Holanda Chavez

    2016-01-01

    trazolium. The assays show that some concentrations of bare UCNPs were cytotoxic for cervical adenocarcinoma cells (HeLa; however, for human colorectal adenocarcino‐ ma all UCNPs are non-cytotoxic. After UCNPs functional‐ ization with silica-aminosilane (APTES/TEOS, all of the nanoparticles tested were found to be non-cytotoxic for both cell lines. The UCNPs functionalized in this work can be further conjugated with specific ligands and used as biolabels for detection of cancer cells.

  1. Stability, cytotoxicity and cell uptake of water-soluble dendron–conjugated gold nanoparticles with 3, 12 and 17 nm cores† †Electronic supplementary information (ESI) available: Additional characterization methods and procedures in addition to the data for the characterization of glutathione-capped gold nanoparticles and dendron-conjugated gold nanoparticles including FT-IR spectra (Fig. S1 and S2), UV-vis spectra (Fig. S3 and S6), TEM images (Fig. S4), MALDI-TOF/TOF spectra (Fig. S5), fluorescence spectra (Fig. S6 and S7), In vitro cytotoxic assay results (Fig. S9) and ICP-MS results (Tables 1 and 2). DOI: 10.1039/c5tb00608b Click here for additional data file.

    Science.gov (United States)

    Deol, Suprit; Weerasuriya, Nisala

    2015-01-01

    This article describes the synthesis of water-soluble dendron–conjugated gold nanoparticles (Den–AuNPs) with various average core sizes and the evaluation of stability, cytotoxicity, cell permeability and uptake of these materials. The characterization of Den–AuNPs using various techniques including transmission electron microscopy (TEM), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), 1H NMR, FT-IR, and UV-vis spectroscopy confirms the dendron conjugation to the glutathione-capped gold nanoparticles (AuNPs). The stability of AuNPs and Den–AuNPs in solutions of different pH and salt concentration is determined by monitoring the changes in surface plasmon bands of gold using UV-vis spectroscopy. The stability of Den–AuNPs at different pH remained about the same compared to that of AuNPs. In comparison, the Den–AuNPs are found to be more stable than the precursor AuNPs maintaining their solubility in the aqueous solution with the salt concentration of up to 100 mM. The improved stability of Den–AuNPs suggests that the post-functionalization of thiol-capped gold nanoparticle surfaces with dendrons can further improve the physiological stability and biocompatibility of gold nanoparticle-based materials. Cytotoxicity studies of AuNPs and Den–AuNPs with and without fluorophores are also performed by examining cell viability for 3T3 fibroblasts using a MTT cell proliferation assay. The conjugation of dendrons to the AuNPs with a fluorophore is able to decrease the cytotoxicity brought about by the fluorophore. The successful uptake of Den–AuNPs in mouse fibroblast 3T3 cells shows the physiological viability of the hybrid materials. PMID:26366289

  2. Linking genotoxic responses with cytotoxic and behavioural or physiological consequences: differential sensitivity of echinoderms (Asterias rubens) and marine molluscs (Mytilus edulis).

    Science.gov (United States)

    Canty, Martin N; Hutchinson, Thomas H; Brown, Rebecca J; Jones, Malcolm B; Jha, Awadhesh N

    2009-08-13

    Integrated laboratory studies addressed multiple biomarker responses in the sea star (Asterias rubens) and the blue mussel (Mytilus edulis) exposed to a range of concentrations of direct and indirect acting genotoxins: methyl methane sulfonate (MMS) and cyclophosphamide (CP; an environmentally relevant anti-cancer pharmaceutical), respectively, in order to determine if the expressed genotoxicity has knock-on effects at the higher levels of biological organisation. The experimental design aimed to concurrently evaluate biomarkers of behavioural and physiological conditions (i.e. 'righting time' and 'clearance rate' for sea stars and mussels, respectively) in addition to cytotoxicity (neutral red retention assay), induction of micronuclei (Mn) and DNA strand breaks (as determined by the Comet assay). The protocol also included the determination of the maximum tolerated concentration (MTC), prior to genotoxic evaluation. The 3d MTC, as determined by the survival of the organisms, showed sea stars to be more sensitive than mussels to MMS (18 and 32 mg L(-1), respectively) and CP (56 and 180 mg L(-1), respectively). For both species and chemicals, cytotoxicity was not found to be significantly different compared to controls. Apart from the MMS exposure to sea stars (which showed 100% mortality at higher concentrations after 5d exposure), clear dose-response relationships were observed for both genotoxicity endpoints in each species. Following exposure to CP, good correlations were also found between the behavioural and physiological responses and genetic damage in each species (sea stars-MN vs. RT: R=0.73; Comet vs. RT: R=0.91; mussels-MN vs. CR: R=0.69; Comet vs. CR: R=0.72). This integrated approach, applying non-invasive assays to simultaneously determine the responses at different levels of biological organisation, indicates the potential value of behavioural and physiological measures in determining the toxicity of chemicals to marine organisms and highlights also

  3. Simultaneous Cake Cutting

    DEFF Research Database (Denmark)

    Balkanski, Eric; Branzei, Simina; Kurokawa, David

    2014-01-01

    We introduce the simultaneous model for cake cutting (the fair allocation of a divisible good), in which agents simultaneously send messages containing a sketch of their preferences over the cake. We show that this model enables the computation of divisions that satisfy proportionality — a popular...

  4. Personality and Simultaneous Speech.

    Science.gov (United States)

    Feldstein, Stanley; And Others

    The purpose of the study was to examine the relationship of the limitation and outcome of simultaneous speech to those dimensions of personality indexes by Cattell's 16PF Questionnaire. More than 500 conversations of 24 female college students were computer-analyzed for instances of simultaneous speech, and the frequencies with which they…

  5. Liquid chromatographic-tandem mass spectrometric assay for ...

    African Journals Online (AJOL)

    Liquid chromatographic-tandem mass spectrometric assay for simultaneous quantitation of tofacitinib, cabozantinib and afatinib in human plasma and urine. Adnan A. Kadi, Ali S. Abdelhameed, Hany W. Darwish, Mohamed W. Attwa, Ahmed H. Bakheit ...

  6. Cholinesterase assay by an efficient fixed time endpoint method

    Directory of Open Access Journals (Sweden)

    Mónica Benabent

    2014-01-01

    The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.

  7. Cytotoxic Mechanisms of Tumor Specific Antibodies

    National Research Council Canada - National Science Library

    Clynes, Raphael

    2001-01-01

    ... and effector-cell-mediated cytotoxicity, we have demonstrated (Clynes RA, Towers TL, Presta LG, Ravetch JV, Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Medicine 6:443-446 (2000...

  8. Cytotoxic Mechanisms of Tumor Specific Antibodies

    National Research Council Canada - National Science Library

    Clynes, Raphael

    2000-01-01

    ... and effector-cell-mediated cytotoxicity, we have demonstrated (clynes RA, Towers TL, Presta LG, Ravetch JV, Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Medicine 6:443-446 (2000...

  9. Enhancement of human natural cytotoxicity by Plasmodium falciparum antigen activated lymphocytes.

    Science.gov (United States)

    Theander, T G; Pedersen, B K; Bygbjerg, I C; Jepsen, S; Larsen, P B; Kharazmi, A

    1987-12-01

    Mononuclear cells (MNC) isolated from malaria immune donors and from donors never exposed to malaria were stimulated in vitro with soluble purified Plasmodium falciparum antigens (SPag) or PPD. After 7 days of culture the proliferative response and the cytotoxic activity against the natural killer cell (NK cell) sensitive cell line, K562, were measured. It was found that SPag stimulation enhanced cytotoxic activity of MNC from donors whose lymphocytes exhibited a strong proliferative response to the antigen. MNC with low proliferative responsiveness showed increased cytotoxic activity if the MNC were preincubated with interleukin 2 (IL-2) for one hour before the start of the cytotoxic assay. SPag activation did not enhance the cytotoxic activity of MNC which did not respond to the antigen in the proliferation assay, and preincubation of these cells with IL-2 did not increase the activity. PPD stimulation enhanced the cytotoxic activity and induced strong proliferative responses in all MNC preparations. The role of NK cells in the protection against malaria is unknown, but they play a role in the protection against virus infection and in the immune surveillance against cancer. Our findings indicate that malaria antigens either directly or through the activation of immunoregulatory cells enhance the NK cell activity.

  10. Cytotoxic, genotoxic/antigenotoxic and mutagenic/antimutagenic effects of the venom of the wasp Polybia paulista.

    Science.gov (United States)

    Hoshina, Márcia M; Santos, Lucilene D; Palma, Mario S; Marin-Morales, Maria A

    2013-09-01

    Hymenoptera venoms are constituted by a complex mixture of chemically or pharmacologically bioactive agents, such as phospholipases, hyaluronidases and mastoparans. Venoms can also contain substances that are able to inhibit and/or diminish the genotoxic or mutagenic action of other compounds that are capable of promoting damages in the genetic material. Thus, the present study aimed to assess the effect of the venom of Polybia paulista, a neotropical wasp, by assays with HepG2 cells maintained in culture. The cytotoxic potential of the wasp venom, assessed by the methyl thiazolyl tetrazolium assay (MTT assay), was tested for the concentrations of 10 μg/mL, 5 μg/mL and 1 μg/mL. As these concentrations were not cytotoxic, they were used to evaluate the genotoxic (comet assay) and mutagenic potential (micronucleus test) of the venom. In this study, it was verified that these concentrations induced damages in the DNA of the exposed cells, and it was necessary to test lower concentrations until it was found those that were not considered genotoxic and mutagenic. The concentrations of 1 ng/mL, 100 pg/mL and 10 pg/mL, which did not induce genotoxicity and mutagenicity, were used in four different treatments (post-treatment, pre-treatment, simultaneous treatment with and without incubation), in order to evaluate if these concentrations were able to inhibit or decrease the genotoxic and mutagenic action of methyl methanesulfonate (MMS). None of the concentrations was able to inhibit and/or decrease the MMS activity. The genotoxic and mutagenic activity of the venom of P. paulista could be caused by the action of phospholipase, mastoparan and hyaluronidase, which are able to disrupt the cell membrane and thereby interact with the genetic material of the cells or even facilitate the entrance of other compounds of the venom that can act on the DNA. Another possible explanation for the genotoxicity and mutagenicity of the venom can be the presence of substances able

  11. In vitro bioactivity and cytotoxicity of chemically treated glass fibers

    Directory of Open Access Journals (Sweden)

    Ângela Leão Andrade

    2004-12-01

    Full Text Available Samples of a commercial glass fiber FM® (Fiber Max were used to test the efficacy of a chemical sol-gel surface treatment to enhance their bioactivity. After treatment with tetraethoxysilane (TEOS, individual fiber samples were soaked into a simulated body fluid (SBF solution, from which they were removed at intervals of 5 and 10 days. Micrographs obtained by scanning electron microscopy (SEM analysis of samples chemically treated with TEOS revealed the formation of a hydroxyapatite (HA coating layer after 5 days into SBF solution. Fourier transform infrared spectroscopic (FTIR analyses confirmed that the coating layer has P-O vibration bands characteristic of HA. The in vitro cytotoxicity was evaluated using a direct contact test, minimum essential medium elution test (ISO 10993-5 and MTT assay. Fibers immersed in SBF and their extracts exhibited lower cytotoxicity than the controls not subjected to immersion, suggesting that SBF treatment improves the biocompatibility of the fiber.

  12. Cytotoxic Biscembranoids from the Soft Coral Sarcophyton pauciplicatum.

    Science.gov (United States)

    Nam, Nguyen Hoai; Tung, Pham The; Ngoc, Ninh Thi; Hanh, Tran Thi Hong; Thao, Nguyen Phuong; Thanh, Nguyen Van; Cuong, Nguyen Xuan; Thao, Do Thi; Huong, Tran Thu; Thung, Do Cong; Kiem, Phan Van; Kim, Young Ho; Minh, Chau Van

    2015-01-01

    Ten biscembranoids (1-10), including the two new compounds sarcophytolides M and N (1 and 2), were isolated from the methanol extract of the Vietnamese soft coral Sarcophyton pauciplicatum. Their structures were elucidated by spectroscopic methods including one dimensional (1D)- and 2D-NMR, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and circular dichroism (CD). The in vitro cytotoxic activity of all isolated compounds against a panel of eight human cancer cell lines including HepG2 (hepatoma cancer), HL-60 (acute leukemia), KB (epidermoid carcinoma), LNCaP (prostate cancer), LU-1 (lung cancer), MCF7 (breast cancer), SK-Mel2 (melanoma), and SW480 (colon adenocarcinoma) was evaluated using the sulforhodamine B colorimetric assay. Among the isolated biscembranoids, 1, 3, 4, 7, 9, and 10 exhibited significant cytotoxic effects and may be selected for further studies of their anticancer activity.

  13. Pretreatment Mitochondrial Priming Correlates with Clinical Response to Cytotoxic Chemotherapy

    Science.gov (United States)

    Chonghaile, Triona Ni; Sarosiek, Kristopher A.; Vo, Thanh-Trang; Ryan, Jeremy A.; Tammareddi, Anupama; Moore, Victoria Del Gaizo; Deng, Jing; Anderson, Ken; Richardson, Paul; Tai, Yu-Tzu; Mitsiades, Constantine S.; Matulonis, Ursula A.; Drapkin, Ronny; Stone, Richard; DeAngelo, Daniel J.; McConkey, David J.; Sallan, Stephen E.; Silverman, Lewis; Hirsch, Michelle S.; Carrasco, Daniel Ruben; Letai, Anthony

    2011-01-01

    Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the pre-treatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from pro-apoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents. PMID:22033517

  14. Evaluation of cell cytotoxic effect on herbal extracts mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Soo; Gwon, Hui Jeong; Choi, Bo Ram; Lim, Youn Mook; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-12-15

    Herbal extracts (HE) such as Houttuynia cordata Thunb., Eucommia ulimoides, Plantago asiatica var., Morus alba L., and Ulmus davidiana var., are known to suppress an atopic dermatitis like skin lesions. In this study, to evaluate the cell cytotoxicity effect on L929, HaCaT and HMC-1 cell by the HE, the herbs were extracted with distilled water (at 75 .deg. C) and then the HE mixtures were freeze-dried for 5 days and sterilized with {gamma}-rays. The cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay. The result showed that the HE mixtures did not significantly affect cell viability and had no toxicity on the cells. These findings indicate that the HE mixtures can be used as a potential therapeutic agent.

  15. Screening of fullerene toxicity by hemolysis assay.

    Science.gov (United States)

    Tramer, Federica; Da Ros, Tatiana; Passamonti, Sabina

    2012-01-01

    Fullerene is a compound formed during carbon burst that has been produced synthetically starting from the 1990s. The spherical shape and the characteristic carbon bonds of this allotrope (C(60)) have made it a suitable molecule for many applications. During the last decade, the low aqueous solubility of this molecule has been improved by chemical functionalization allowing the use of fullerene derivatives in biological fluids. The characterization of the toxicity potential of fullerenes is therefore of growing interest for any biomedical application. Intravenous injection is one of the possible routes of their administrations and therefore red blood cells are among the first targets of fullerene cytotoxicity. Human red blood cells are easily available and separated from plasma. Membrane disruption by toxic compounds is easily detected in red blood cells as release of hemoglobin in the cell medium, which can be assayed spectrophotometrically at λ = 415 nm. Due to the high molar extinction coefficient of hemoglobin, the assay can be performed on a small amount of both red blood cells and the test compounds, which might be available only in small quantities. So, the hemolysis assay is a simple screening test, whose results can guide further investigations on cytotoxicity in more complex experimental models.

  16. Cytotoxic effects and morphological changes of silver nanoparticles in CHO-K1 cells

    Directory of Open Access Journals (Sweden)

    Masoumeh Heshmati

    2015-11-01

    Full Text Available Background: Silver nanoparticles are of interest to be used as antimicrobial agents in medical care, wound dressings and cosmetics. Despite the fact that AgNPs are among the most commercialized nano materials owing to their specific antimicrobial properties, there is limited information about their risk assessment and possible hazards to human health and environment. Thus, this study was carried out to evaluate the cytotoxicity and morphological changes of different concentrations of two samples of commercialized silver nanoparticles (A and B in CHO-K1 cells using MTT assay. Methods: The cytotoxicity effect of silver nanoparticles AgNP-A (19.6 nm in diameter and AgNP-B (15 nm in diameter on CHO-K1 was evaluated in the range of 0.005-500 µg/ml after 24 hours of treatment using MTT assay. The morphological changes of treated cells were examined by light microscopy. Results: Based on the results of cytotoxicity by MTT assay, reduced viability of cells and cytotoxicity were observed. The 50% inhibitory concentration (IC50 of silver nanoparticles was recorded at 100 and 10 µg/ml for AgNP-A and AgNP-B, respectively. Moreover, microscopic observations indicated clear morphological changes of treated cells. Conclusion: Concentration and physicochemical properties of silver nanoparticles like size, shape, surface area, coating and zeta potential play a role in cytotoxicity and morphological changes of treated cells.

  17. Cytotoxic and apoptotic effects of chalcone derivatives of 2-acetyl thiophene on human colon adenocarcinoma cells.

    Science.gov (United States)

    de Vasconcelos, Alana; Campos, Vinicius Farias; Nedel, Fernanda; Seixas, Fabiana Kömmling; Dellagostin, Odir A; Smith, Kevin R; de Pereira, Cláudio Martin Pereira; Stefanello, Francieli Moro; Collares, Tiago; Barschak, Alethéa Gatto

    2013-06-01

    Recent studies report that chalcones exhibit cytotoxicity to human cancer cell lines. Typically, the form of cell death induced by these compounds is apoptosis. In the context of the discovery of new anticancer agents and in light of the antitumour potential of several chalcone derivatives, in the present study, we synthesized and tested the cytotoxicity of six chalcone derivatives on human colon adenocarcinoma cells. Six derivatives of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one were prepared and characterized on the basis of their (1) H and (13) C NMR spectra. HT-29 cells were treated with synthesized chalcones on two concentrations by three different incubation times. Cells were evaluated by cell morphology, Tetrazolium dye (MTT) colorimetric assay, live/dead, flow cytometry (annexin V) and gene expression analyses to determine the cytotoxic way. Chalcones 3-(4-bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C06) and 3-(2-nitrophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C09) demonstrated higher cytotoxicity than other chalcones as shown by cell morphology, live/dead and MTT assays. In addition, C06 induced apoptosis on flow cytometry annexin V assay. These data were confirmed by a decreased expression of anti-apoptotic genes and increased pro-apoptotic genes. Our findings indicate in summary that the cytotoxic activity of chalcone C06 on colorectal carcinoma cells occurs by apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Toxin content and cytotoxicity of algal dietary supplements

    Energy Technology Data Exchange (ETDEWEB)

    Heussner, A.H.; Mazija, L. [Human and Environmental Toxicology, University of Konstanz, 78457 Konstanz (Germany); Fastner, J. [Federal Environmental Agency, Section II 3.3—Drinking-water resources and treatment, Berlin (Germany); Dietrich, D.R., E-mail: daniel.dietrich@uni-konstanz.de [Human and Environmental Toxicology, University of Konstanz, 78457 Konstanz (Germany)

    2012-12-01

    Blue-green algae (Spirulina sp., Aphanizomenon flos-aquae) and Chlorella sp. are commercially distributed as organic algae dietary supplements. Cyanobacterial dietary products in particular have raised serious concerns, as they appeared to be contaminated with toxins e.g. microcystins (MCs) and consumers repeatedly reported adverse health effects following consumption of these products. The aim of this study was to determine the toxin contamination and the in vitro cytotoxicity of algae dietary supplement products marketed in Germany. In thirteen products consisting of Aph. flos-aquae, Spirulina and Chlorella or mixtures thereof, MCs, nodularins, saxitoxins, anatoxin-a and cylindrospermopsin were analyzed. Five products tested in an earlier market study were re-analyzed for comparison. Product samples were extracted and analyzed for cytotoxicity in A549 cells as well as for toxin levels by (1) phosphatase inhibition assay (PPIA), (2) Adda-ELISA and (3) LC–MS/MS. In addition, all samples were analyzed by PCR for the presence of the mcyE gene, a part of the microcystin and nodularin synthetase gene cluster. Only Aph. flos-aquae products were tested positive for MCs as well as the presence of mcyE. The contamination levels of the MC-positive samples were ≤ 1 μg MC-LR equivalents g{sup −1} dw. None of the other toxins were found in any of the products. However, extracts from all products were cytotoxic. In light of the findings, the distribution and commercial sale of Aph. flos-aquae products, whether pure or mixed formulations, for human consumption appear highly questionable. -- Highlights: ► Marketed algae dietary supplements were analyzed for toxins. ► Methods: Phosphatase inhibition assay (PPIA), Adda-ELISA, LC-MS/MS. ► Aph. flos-aquae products all tested positive for microcystins. ► Products tested negative for nodularins, saxitoxins, anatoxin-a, cylindrospermopsin. ► Extracts from all products were cytotoxic.

  19. Cytotoxic Constituents from the Leaves of Zanthoxylum schinifolium

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zhe; Min, Byung Sun; Kim, Ae Kyong; Woo, Mi Hee [Catholic Univ. of Daegu, Gyeongsan (Korea, Republic of); Jun, Do Youn; Kim, Young Ho [Kyungpook National Univ., Daegu (Korea, Republic of)

    2010-04-15

    The roots, stems, pericarps, and seeds of Z. schinifolium were each extracted with MeOH, and the leaves were extracted with 80% MeOH and concentrated. These extracts were examined on MTT for cytotoxicity against Jurkat T cell clone E6.1. The results showed that the leaves extract had the strongest MTT cytotoxicity. The MeOH extract of Z. schinifolium leaves was subsequently fractionated into four parts: methylene chloride, ethyl acetate, n-butanol and water. These fractions were examined on MTT for cytotoxicity. The results showed that the methylene chloride fraction exhibited the strongest MTT cytotoxicity. Chromatographic separation of the methylene chloride and butanol fractions had yielded a quinolin (1), three phenylpropanoids (2, 3, 12), four coumarins (4 ∼ 7), three triterpenoids (8 ∼ 10), an alkaloid (11), an alcohol glucoside (13) and three monoterpene glucosides (14, 15, 16). One of these compounds were identified as new threo-6-amino-5-hydroxy-5-methyl-1,3-oxazinan-4-one (11) together with fifteen known, 3-heptyl-2-methylisoquinolin-1(2H)-one (1), integrifoliodiol (2), cuspidiol (3), bergapten (4), aurapten (5), 8-hydroxy-7-methoxy-chromen-2-one (6), 6,7-dimethoxy-2H-naphthalen-1-one (7), lupeol (8), lupeone (9), β-sitosterol (10), syringin (12), 2-propyl alchol β-D-glucopyranoside (13), vomifoliol-9-O-β-D-glucopyranoside (14), betulalbuside A (15) and cnidioside C (16) on the basis of spectroscopic and chemical evidences. All of the compounds were isolated for the first time from this plant except 5 and 7. In the MTT cytotoxicity assay against Jurkat T cell clone E6.1, IC{sub 50} values of cuspidiol (3) and auraptene (5) were obtained at 7.3 μg/mL and 16.5 μg/mL, respectively.

  20. Differential cytotoxicity of copper ferrite nanoparticles in different human cells.

    Science.gov (United States)

    Ahmad, Javed; Alhadlaq, Hisham A; Alshamsan, Aws; Siddiqui, Maqsood A; Saquib, Quaiser; Khan, Shams T; Wahab, Rizwan; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Akhtar, Mohd Javed; Ahamed, Maqusood

    2016-10-01

    Copper ferrite nanoparticles (NPs) have the potential to be applied in biomedical fields such as cell labeling and hyperthermia. However, there is a lack of information concerning the toxicity of copper ferrite NPs. We explored the cytotoxic potential of copper ferrite NPs in human lung (A549) and liver (HepG2) cells. Copper ferrite NPs were crystalline and almost spherically shaped with an average diameter of 35 nm. Copper ferrite NPs induced dose-dependent cytotoxicity in both types of cells, evident by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide and neutral red uptake assays. However, we observed a quite different susceptibility in the two kinds of cells regarding toxicity of copper ferrite NPs. Particularly, A549 cells showed higher susceptibility against copper ferrite NP exposure than those of HepG2 cells. Loss of mitochondrial membrane potential due to copper ferrite NP exposure was observed. The mRNA level as well as activity of caspase-3 enzyme was higher in cells exposed to copper ferrite NPs. Cellular redox status was disturbed as indicated by induction of reactive oxygen species (oxidant) generation and depletion of the glutathione (antioxidant) level. Moreover, cytotoxicity induced by copper ferrite NPs was efficiently prevented by N-acetylcysteine treatment, which suggests that reactive oxygen species generation might be one of the possible mechanisms of cytotoxicity caused by copper ferrite NPs. To the best of our knowledge, this is the first report showing the cytotoxic potential of copper ferrite NPs in human cells. This study warrants further investigation to explore the mechanisms of differential toxicity of copper ferrite NPs in different types of cells. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity.

    Science.gov (United States)

    Ladd, Brendon; Ackroyd, Jeffrey J; Hicks, J Kevin; Canman, Christine E; Flanagan, Sheryl A; Shewach, Donna S

    2013-12-01

    The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC50 in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Cytotoxic Indole Alkaloids against Human Leukemia Cell Lines from the Toxic Plant Peganum harmala

    Directory of Open Access Journals (Sweden)

    Chunhua Wang

    2015-11-01

    Full Text Available Bioactivity-guided fractionation was used to determine the cytotoxic alkaloids from the toxic plant Peganum harmala. Two novel indole alkaloids, together with ten known ones, were isolated and identified. The novel alkaloids were elucidated to be 2-(indol-3-ylethyl-α-L-rhamnopyranosyl-(1 → 6-β-D-glucopyranoside (2 and 3-hydroxy-3-(N-acetyl-2-aminoethyl-6-methoxyindol-2-one (3. The cytotoxicity against human leukemia cells was assayed for the alkaloids and some of them showed potent activity. Harmalacidine (compound 8, HMC exhibited the highest cytotoxicity against U-937 cells with IC50 value of 3.1 ± 0.2 μmol/L. The cytotoxic mechanism of HMC was targeting the mitochondrial and protein tyrosine kinase signaling pathways (PTKs-Ras/Raf/ERK. The results strongly demonstrated that the alkaloids from Peganum harmala could be a promising candidate for the therapy of leukemia.

  3. Cytotoxicity responses to selected ESAT-6 and CFP-10 peptides in tuberculosis.

    Science.gov (United States)

    Madhan Kumar, M; Raja, Alamelu

    2010-01-01

    Cytotoxicity responses were studied for the ESAT-6 peptides Esp1, Esp6, Esp7, Esp8, and CFP-10 peptides, Cfp6, Cfp7, Cfp8, Cfp9 (synthetic 20-mer peptides) and the recombinant ESAT-6, CFP-10 proteins. Cytolytic molecules perforin, granzymes A and B, granulysin responses in healthy household contacts (HHC) and pulmonary tuberculosis patients (PTB), were studied by intracellular flow cytometry. Functional cytotoxicity was studied in both the groups for the peptides Esp6 and Cfp8 by an enzyme (lactate dehydrogenase) based assay. The results revealed that cytolytic molecule positive CD4+ and CD8+ T cells were increased in HHC in response to Esp1, Esp6, Cfp8 and Cfp9 immunogenic peptides compared to PTB. Functional cytotoxicity results showed higher cytotoxicity (not statistically significant) to be exhibited by the peptide Esp6 than Cfp8 in the HHC. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Phytochemical properties and cytotoxicity evaluation of the aqueous extracts from Rafflesia cantleyi

    Science.gov (United States)

    Bakoush, Sumaia Mohamed Mohamed; Yaacob, Wan Ahmad; Adam, Jumaat; Ibrahim, Nazlina

    2015-09-01

    In the present study, phytochemical properties and cytotoxic evaluation of aqueous extract of Rafflesia cantleyi bud parts were done. Three bud parts including disk, bract and perigone tube were extracted in water to produce crude aqueous extract. Cytotoxic activity of R. cantleyi bud parts was assessed by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against normal cells Vero, 3T3 cell lines and mice peripheral blood mononuclear cells PBMC. Phytochemical analyses revealed the presence of tannins, flavonoids, steroids and alkaloids. The CC50 value against Vero, 3T3 and PBMC cells were equal or more than 125 µg/ml indicating the non-cytotoxic effect of the bud parts extracts. The finding revealed that crude extracts of all the tested bud parts contained potential bioactive compounds which can be used for various biological activities and have no cytotoxicity to selected normal cells.

  5. Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE by membrane vesicles

    Directory of Open Access Journals (Sweden)

    Marilucia Santos Ludovico

    2016-11-01

    Full Text Available Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE. The presence of membrane vesicles (MVs on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM. From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

  6. One-pot synthesis of cinnamylideneacetophenones and their in vitro cytotoxicity in breast cancer cells.

    Science.gov (United States)

    Weldon, David J; Saulsbury, Marilyn D; Goh, Joshua; Rowland, Leah; Campbell, Petreena; Robinson, Laijia; Miller, Calvin; Christian, Joshua; Amis, Louisa; Taylor, Nia; Dill, Cassandra; Davis, Willie; Evans, Stanley L; Brantley, Eileen

    2014-08-01

    A series of cinnamylideneacetophenones were synthesized via a modified Claisen-Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™ assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50=71 and 1.9 nM), respectively. Derivative 17 also displayed sub-micromolar (IC50=780 nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-10A cells. This study provides evidence supporting the continued development of nitro-substituted cinnamylideneacetophenones as small molecules to treat breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Microchip screening platform for single cell assessment of NK cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Karolin eGuldevall

    2016-04-01

    Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  8. Effects of the Absorption Behaviour of ZnO Nanoparticles on Cytotoxicity Measurements

    Directory of Open Access Journals (Sweden)

    Nigar Najim

    2014-01-01

    Full Text Available ZnO absorbs certain wavelengths of light and this behavior is more pronounced for nanoparticles of ZnO. As many toxicity measurements rely on measuring light transmission in cell lines, it is essential to determine how far this light absorption influences experimental toxicity measurements. The main objective was to study the ZnO absorption and how this influenced the cytotoxicity measurements. The cytotoxicity of differently sized ZnO nanoparticles in normal and cancer cell lines derived from lung tissue (Hs888Lu, neuron-phenotypic cells (SH-SY5Y, neuroblastoma (SH-SY5Y, human histiocytic lymphoma (U937, and lung cancer (A549 was investigated. Our results demonstrate that the presence of ZnO affected the cytotoxicity measurements due to the absorption characteristic of ZnO nanoparticles. The data revealed that the ZnO nanoparticles with an average particle size of around 85.7 nm and 190 nm showed cytotoxicity towards U937, SH-SY5Y, differentiated SH-SY5Y, and Hs888Lu cell lines. No effect on the A549 cells was observed. It was also found that the cytotoxicity of ZnO was particle size, concentration, and time dependent. These studies are the first to quantify the influence of ZnO nanoparticles on cytotoxicity assays. Corrections for absorption effects were carried out which gave an accurate estimation of the concentrations that produce the cytotoxic effects.

  9. In Vitro Cytotoxic Activity of the Essential Oil Extracted from Artemisia Absinthium

    Directory of Open Access Journals (Sweden)

    Mahboubeh Taherkhani

    2014-09-01

    Full Text Available Background: Essential oils are found to have multiple active components which can show in vitro cytotoxic action against various cancerous cell lines. This study reports the in vitro cytotoxic effects of the essential oil from Artemisia absinthium L. (Asteraceae growing wild in Iran. Methods: Water-distilled essential oil of A. absinthium collected from Ardabil, north-western Iran, was examined for its cytotoxic effects using a modified MTT assay. Air-dried aerial parts of A. absinthium was subjected to hydrodistillation using a clevenger-type apparatus. Cytotoxicity of the essential oil was measured against Hela and human healthy peripheral blood cells. Results: The 50% cytotoxic concentrations were found to be 48.59 µg/ml and 5456.07 µg/ml for Hela cells and human lymphocytes, respectively. The volatile oil displayed good cytotoxic action against the human tumor cell line. Conclusion: The IC50 shows that cytotoxicity of the oil against human tumor cell line is much higher than that required for healthy human cells. These results indicate low adverse effects for this oil. The findings of this study necessitate the need for further consideration of this essential oil in anti-neoplastic chemotherapy.

  10. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    Science.gov (United States)

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  11. Cytotoxic and antibacterial activity of the mixture of olive oil and lime cream in vitro conditions.

    Science.gov (United States)

    Sumer, Zeynep; Yildirim, Gulay; Sumer, Haldun; Yildirim, Sahin

    2013-01-01

    The mixture of olive oil and lime cream has been traditionally used to treat external burns in the region of Hatay/Antakya and middle Anatolia. Olive oil and lime cream have been employed by many physicians to treat many ailments in the past. A limited number of studies have shown the antibacterial effect of olive oil and that it does not have any toxic effect on the skin. But we did not find any reported studies on the mixture of olive oil and lime cream. The aim of this paper is to investigate the cytotoxic and antibacterial activity of olive oil and lime cream individually or/and in combination in vitro conditions, by using disk-diffusion method and in cell culture. The main purpose in using this mixture is usually to clear burns without a trace. Agar overlay, MTT (Cytotoxicity assay) and antibacterial susceptibility tests were used to investigate the cytotoxic and antibacterial activity of olive oil and lime cream. We found that lime cream has an antibacterial activity but also cytotoxic on the fibroblasts. On the other hand olive oil has limited or no antibacterial effect and it has little or no cytotoxic on the fibroblasts. When we combined lime cream and olive oil, olive oil reduced its cytotoxic impact. These results suggest that mixture of olive oil and lime cream is not cytotoxic and has antimicrobial activity.

  12. Evaluation of the Cytotoxicity of Structurally Correlated p-Menthane Derivatives

    Directory of Open Access Journals (Sweden)

    Luciana Nalone Andrade

    2015-07-01

    Full Text Available Compounds isolated from essential oils play an important role in the prevention and treatment of cancer. Monoterpenes are natural products, and the principal constituents of many essential oils. The aim of this study was to investigate the cytotoxic potential of p-menthane derivatives. Additionally, analogues of perillyl alcohol, a monoterpene with known anticancer activity, were evaluated to identify the molecular characteristics which contribute to their cytotoxicity, which was tested against OVCAR-8, HCT-116, and SF-295 human tumor cell lines, using the MTT assay. The results of this study showed that (−-perillaldehyde 8,9-epoxide exhibited the highest percentage inhibition of cell proliferation (GI = 96.32%–99.89%. Perillyl alcohol exhibited high cytotoxic activity (90.92%–95.82%, while (+-limonene 1,2-epoxide (GI = 58.48%–93.10%, (−-perillaldehyde (GI = 59.28%–83.03%, and (−-8-hydroxycarvotanacetone (GI = 61.59%–94.01% showed intermediate activity. All of the compounds tested were less cytotoxic than perillyl alcohol, except (−-perillaldehyde 8,9-epoxide (IC50 = 1.75–1.03 µL/mg. In general, replacement of C-C double bonds by epoxide groups in addition to the aldehyde group increases cytotoxicity. Furthermore, stereochemistry seems to play an important role in cytotoxicity. We have demonstrated the cytotoxic influence of chemical substituents on the p-menthane structure, and analogues of perillyl alcohol.

  13. Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn

    National Research Council Canada - National Science Library

    Kumar, Harish; Savaliya, Mihir; Biswas, Subhankar; Nayak, Pawan G; Maliyakkal, Naseer; Manjunath Setty, M; Gourishetti, Karthik; Pai, K Sreedhara Ranganath

    2016-01-01

    .... Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB...

  14. Suppression in simultaneous masking.

    Science.gov (United States)

    Fastl, H; Bechly, M

    1983-09-01

    Suppression, i.e., the decrease of masked threshold caused by the addition of a second masker M2 to a first masker M1, is measured for the case of simultaneous masking. The magnitude of suppression decreases with increasing test tone duration; pulsed maskers elicit somewhat more suppression than continuous maskers. In comparison to suppression effects obtained in nonsimultaneous masking (post-masking, pulsation threshold) suppression in simultaneous masking is considerably smaller and was found only at the lower slopes of the two maskers. Suppression in simultaneous masking would not be predicted by those models of suppression which require nonsimultaneous presentation of maskers and test sound.

  15. In vitro cytotoxicity screening of wild plant extracts from Saudi Arabia on human breast adenocarcinoma cells.

    Science.gov (United States)

    Ali, M A; Abul Farah, M; Al-Hemaid, F M; Abou-Tarboush, F M

    2014-05-23

    This study investigated the in vitro anticancer activities of a total of 14 wild angiosperms collected in Saudi Arabia. The cytotoxic activity of each extract was assessed against human breast adenocarcinoma (MCF-7) cell lines by using the MTT assay. Among the plants screened, the potential cytotoxic activity exhibited by the extract of Lavandula dentata (Lamiaceae) was identified, and we analyzed its anticancer potential by testing antiproliferative and apoptotic activity. Our results clearly show that ethanolic extract of L. dentata exhibits promising cytotoxic activity with an IC50 value of 39 μg/mL. Analysis of cell morphological changes, DNA fragmentation and apoptosis (using an Annexin V assay) also confirmed the apoptotic effect of L. dentata extract, and thus, our data call for further investigations to determine the active chemical constituent(s) and their mechanisms of inducing apoptosis.

  16. Goniothalamin, a cytotoxic compound, isolated from Goniothalamus macrophyllus (Blume Hook. f. & Thomson var. macrophyllus

    Directory of Open Access Journals (Sweden)

    Niwat Keawpradub

    2005-08-01

    Full Text Available Bioassay guided fractionation by brine shrimp lethality test led to isolation of a cytotoxic compound, goniothalamin, from the root and stem of Goniothalamus macrophyllus (Blume Hook. f. & Thomson var. macrophyllus. Goniothalamin showed a promising cytotoxicity (SRB assay against colon cancer cell line (IC50 = 0.51±0.02 μg/ml, breast cancer cell lines (IC50= 0.95±0.02 μg/ml and lung carcinoma (IC50 = 3.51± 0.03 μg/ml. LDH assay of goniothalamin suggested that it had no toxicity on cell membrane. Cytotoxic evaluation of goniothalamin to normal cell revealed moderate toxicity against skin fibroblast (IC50 = 26.73± 1.92 μg/ml and human fibroblast (IC50 = 11.99±0.15 μg/ml.

  17. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

    2012-12-15

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  18. New Cytotoxic Azaphilones from Monascus purpureus-Fermented Rice (Red Yeast Rice

    Directory of Open Access Journals (Sweden)

    Zong-Lian Jin

    2010-03-01

    Full Text Available Using a cell-based cytotoxicity assay three new cytotoxic azaphilones, including two stereoisomers and designated monapurones A-C (1-3, were isolated from the extract of Monascus purpureus-fermented rice (red yeast rice. Their structures were elucidated by detailed interpretation of spectroscopic and chemical data. The relative configurations were assigned on the basis of analysis of NOE data, and the absolute configurations were determined by direct comparison of their CD spectra with those of known azaphilones and chemical correlations. In the in vitro assays, monapurones A-C (1-3 showed selective cytotoxicity against human cancer cell line A549 with IC50 values of 3.8, 2.8 and 2.4mM respectively, while exhibiting no significant toxicity to normal MRC-5 and WI-38 cells at the same concentration.

  19. Phytochemical screening, cytotoxicity and antiviral activity of hexane fraction of Phaleria macrocarpa fruits

    Science.gov (United States)

    Ismaeel, Mahmud Yusef Yusef; Yaacob, Wan Ahmad; Tahir, Mariya Mohd.; Ibrahim, Nazlina

    2015-09-01

    Phaleria macrocarpa fruits have been widely used in the traditional medicine for the treatment of several infections. The current study was done to determine the phytochemical content, cytotoxicity and antiviral activity of the hexane fraction (HF) of P. macrocarpa fruits. In the hexane fraction of P. macarocarpa fruits, phytochemical screening showed the presence of terpenoids whereas saponins, alkaloids, tannins and anthraquinones were not present. Evaluation on Vero cell lines by using MTT assay showed that the 50% cytotoxic concentration (CC50) value was 0.48 mg/mL indicating that the fraction is not cytotoxic. Antiviral properties of the plant extracts were determined by plaque reduction assay. The effective concentration (EC50) was 0.18 mg/mL. Whereas the selective index (SI = CC50/EC50) of hexane fraction is 2.6 indicating low to moderate potential as antiviral agent.

  20. Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

    Science.gov (United States)

    Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

    2012-12-01

    Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

  1. Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons

    Science.gov (United States)

    Chowdhury, Sayan Mullick; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Yun; Neville, Kayla; Sitharaman, Balaji

    2012-01-01

    The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10–400 μg/ml) and time-dependent (12–48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 hours, when incubated at 10μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5–25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10μg/ml. The decrease in cell viability was steep with increase in concentration with the CD50 values ≥ 100μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell

  2. Butanol is cytotoxic to Lactococcus lactis while ethanol and hexanol are cytostatic

    DEFF Research Database (Denmark)

    Hviid, Anne-Mette Meisner; Jensen, Peter Ruhdal; Kilstrup, Mogens

    2017-01-01

    resistant lactic acid bacteria. Combined results from alcohol survival rate, live/dead staining, and a novel usage of the beta-galactosidase assay, revealed that while high concentrations of ethanol and hexanol were cytostatic to L. lactis, high concentrations of butanol were cytotoxic, causing irreparable...

  3. Antioxidant and cytotoxic activity of Tecoma stans against lung cancer cell line (A549

    Directory of Open Access Journals (Sweden)

    Jayachandran Philip Robinson

    2017-07-01

    Full Text Available ABSTRACT Human have been constantly using plants and plant products to overcome many diseases. The antioxidant property of the plant sources is studied to obtain an efficacious drug against cancer. The objectives of the present study is to evaluate the antioxidant and cytotoxic activity of the Tecoma stans extracts against lung cancer cell line in comparison with vincristine drug. The antioxidant activity was studied using the standard DPPH assay and the cytotoxic activity using MTT assay. DPPH assay results show that methanolic extract of T. stans in higher concentration show better antioxidant potential than the standard L-ascorbic acid. They exhibited strong antioxidant potential at 20 µg/mL concentration. The absorbance at 517 nm showed that in the range of 0.201-0.0203 compared to that of absorbance of ascorbic acid at 0.023.Cytotoxic activity was studied using MTT assay which showed that the increase in concentration of extract increases the cell death. At 100µg/mL concentration there is an increased cytotoxic activity, i.e., 99% of cell inhibition. The results of antioxidant and anticancerous activity may be positively correlated.

  4. Cytotoxicity and physicochemical properties of hafnium oxide nanoparticles.

    Science.gov (United States)

    Field, James A; Luna-Velasco, Antonia; Boitano, Scott A; Shadman, Farhang; Ratner, Buddy D; Barnes, Chris; Sierra-Alvarez, Reyes

    2011-09-01

    Nano-sized hafnium oxide (HfO(2)) particles are being considered for applications within the semiconductor industry. However, little is known about their cytotoxicity. The objective of this work was to assess several HfO(2) nanoparticles (NPs) samples for their acute cytotoxicity. Dynamic light scattering analysis of the samples indicated that the average particle size of the HfO(2) in aqueous dispersions was in the submicron range with a fraction of particles having nano-dimensions. The media used in the toxicity assays decreased or increased the average particle size of HfO(2) NPs due to dispersion or agglomeration. Static time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed numerous surface contaminants on the NPs. Only one HfO(2) sample caused moderate cytotoxicity to human cell lines. The inhibitory sample caused a 50% response in the Live/Dead assay with HaCaT skin cells at 2200 mg L(-1); and a 50% response in the mitochondrial toxicity test at 300 mg L(-1). A microbial inhibition assay based on methanogenic activity also revealed that another HFO(2) sample caused moderate inhibition. The difference in toxicity between samples could not be attributed to size. Instead the difference in toxicity was likely due to differences in the contaminants of the HfO(2). The ToF-SIMS analysis indicated unique signatures of Br and P in the sample toxic to human cell lines suggesting a distinct synthesis was used for that sample which may have been accompanied by inhibitory impurities. The results taken as a whole indicate that HfO(2) itself is relatively non-toxic. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Metasurfaces: Simultaneous Stokes parameters

    Science.gov (United States)

    Lepetit, Thomas; Kanté, Boubacar

    2015-11-01

    Techniques for determining Stokes parameters, which fully define the polarization state of a wave, require multiple measurements, thus potentially leading to inaccuracies. Researchers now show how to simultaneously determine the parameters for visible light using periodic metal structures.

  6. Simultaneous consolidation and creep

    DEFF Research Database (Denmark)

    Krogsbøll, Anette

    1997-01-01

    Materials that exhibit creep under constant effective stress typically also show rate dependent behavior. The creep deformations and the rate sensitive behavior is very important when engineering and geological problems with large time scales are considered. When stress induced compaction (consol...... (consolidation) is retarded by slow drainage of excess pore pressure it is expected that consolidation and creep occur simultaneously. A constitutive model adressing the problems of rate sensitive behavior and simultaneous consolidation and creep is presented....

  7. Promising cytotoxic activity profile of fermented wheat germ extract (Avemar® in human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Voigt Wieland

    2011-04-01

    Full Text Available Abstract Fermented wheat germ extract (FWGE is currently used as nutrition supplement for cancer patients. Limited recent data suggest antiproliferative, antimetastatic and immunological effects which were at least in part exerted by two quinones, 2-methoxy benzoquinone and 2,6-dimethoxybenzquinone as ingredients of FWGE. These activity data prompted us to further evaluate the in vitro antiproliferative activity of FWGE alone or in combination with the commonly used cytotoxic drugs 5-FU, oxaliplatin or irinotecan in a broad spectrum of human tumor cell lines. We used the sulforhodamine B assay to determine dose response relationships and IC50-values were calculated using the Hill equation. Drug interaction of simultaneous and sequential drug exposure was estimated using the model of Drewinko and potential clinical activity was assessed by the model of relative antitumor activity (RAA. Apoptosis was detected by DNA gel electrophoresis. FWGE induced apoptosis and exerted significant antitumor activity in a broad spectrum of 32 human cancer cell lines. The highest activity was found in neuroblastoma cell lines with an average IC50 of 0.042 mg/ml. Furthermore, IC50-range was very narrow ranging from 0.3 mg/ml to 0.54 mg/ml in 8 colon cancer cell lines. At combination experiments in colon cancer cell lines when FWGE was simultaneously applied with either 5-FU, oxaliplatin or irinotecan we observed additive to synergistic drug interaction, particularly for 5-FU. At sequential drug exposure with 5-FU and FWGE the observed synergism was abolished. Taken together, FWGE exerts significant antitumor activity in our tumor model. Simultaneous drug exposure with FWGE and 5-FU, oxaliplatin or irinotecan yielded in additive to synergistic drug interaction. However, sequential drug exposure of 5-FU and FWGE in colon cancer cell lines appeared to be schedule-dependent (5-FU may precede FWGE. Further evaluation of FWGE as a candidate for clinical combination drug

  8. Cytotoxic prenylxanthones from Garcinia bracteata.

    Science.gov (United States)

    Thoison, O; Fahy, J; Dumontet, V; Chiaroni, A; Riche, C; Tri, M V; Sévenet, T

    2000-04-01

    Bioassay-guided fractionation of a leaf extract of G. bracteata has yielded six new prenylxanthones, bractatin (1), isobractatin (2), 1-O-methylbractatin (3), 1-O-methylisobractatin (4), 1-O-methyl-8-methoxy-8,8a-dihydrobractatin (5), and 1-O-methylneobractatin (6). The structures of these compounds have been elucidated by spectroscopic means (NMR, MS), literature data, and X-ray crystallographic analysis of 2. These compounds possess significant cytotoxicity against the KB cell line.

  9. Triterpenes as Potentially Cytotoxic Compounds

    Directory of Open Access Journals (Sweden)

    Malwina Chudzik

    2015-01-01

    Full Text Available Triterpenes are compounds of natural origin, which have numerously biological activities: anti-cancer properties, anti-inflammatory, anti-oxidative, anti-viral, anti-bacterial and anti-fungal. These substances can be isolated from plants, animals or fungi. Nowadays, when neoplasms are main cause of death, triterpenes can become an alternative method for treating cancer because of their cytotoxic properties and chemopreventive activities.

  10. Simultaneity, relativity and conventionality

    Science.gov (United States)

    Janis, Allen I.

    2008-01-01

    The view of simultaneity presented by Max Jammer is almost breathtaking, encompassing, as the book's subtitle suggests, the period from antiquity to the 21st century. Many interesting things are to be found along the way. For example, what Jammer (p. 49) says "may well be regarded as probably the earliest recorded example of an operational definition of distant simultaneity" is due to St. Augustine (in his Confessions, written in 397 A.D.; for a modern translation, see Augustine, 2006). He was arguing against astrology by presenting the story of two women, one rich and one poor, who gave birth simultaneously. Although the two children thus had precisely the same horoscopes, their lives followed quite different courses. And how was it determined that the births were simultaneous? A messenger went from each birth site to the other, leaving the instant the child was born (and, presumably, traveling with equal speeds). Since the messengers met at the midpoint between the locations of the two births, the births must have been simultaneous. This is, of course, quite analogous to Albert Einstein's definition of simultaneity (given more than 1500 years later), which will be discussed in Section 2.1.

  11. Clinical significance of direct cytotoxicity and toxigenic culture in Clostridium difficile infection.

    Science.gov (United States)

    Reigadas, E; Alcalá, L; Marín, M; Muñoz-Pacheco, P; Catalán, P; Martin, A; Bouza, E

    2016-02-01

    Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Although an optimal diagnosis is crucial, laboratory diagnostics remain challenging. Currently, the reference methods are direct cytotoxicity assay and toxigenic culture; however there is controversy in the interpretation of discordant results of these tests. The aim of our study was to determine the clinical significance of detecting C. difficile only by toxigenic culture with a negative direct cytotoxicity assay. We conducted a prospective study in which patients aged >2 years with CDI were enrolled and monitored at least 2 months after their last episode. Samples were tested by both cytotoxicity assay and toxigenic culture. During the 6-month study period, we identified 169 episodes meeting CDI criteria that had been tested by both assays, out of which 115 were positive for both cytotoxicity assay and toxigenic culture, and 54 CDI episodes (31.9%) were positive only by toxigenic culture. Overall, patients median age was 71.3, 50.9% were male and the most frequent underlying disease was malignancy. The comparison of CDI episodes positive for both assays and by toxigenic culture only revealed the following, respectively: mild CDI (77.4% vs 94.4%; p = 0.008), severe CDI (21.7% vs 5.6%; p = 0.008), severe complicated (0.9% vs 0.0%; p = 1.000), pseudomembranous colitis (1.7% vs 1.9% p = 1.000), recurrence (17.4% vs 14.8%; p = 0.825), overall mortality (8.7% vs 7.4%; p = 1.000) and CDI related mortality (2.6% vs 0%; p = 0.552). CDI episodes positive by cytotoxicity assay were more severe than those positive only by toxigenic culture, however there were a significant proportion of CDI cases (31.9%) that would have been missed if only cytotoxicity had been considered as clinically significant for CDI treatment, including severe CDI cases. Our data suggest that a positive test by toxigenic culture with a negative result for cytotoxicity should

  12. Fully Bayesian Analysis of High-throughput Targeted Metabolomics Assays

    Science.gov (United States)

    High-throughput metabolomic assays that allow simultaneous targeted screening of hundreds of metabolites have recently become available in kit form. Such assays provide a window into understanding changes to biochemical pathways due to chemical exposure or disease, and are usefu...

  13. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells▿

    OpenAIRE

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-01-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barri...

  14. Cytotoxic and antioxidant constituents from the leaves of Psidium guajava.

    Science.gov (United States)

    Feng, Xiao-He; Wang, Zi-Hao; Meng, Da-Li; Li, Xian

    2015-01-01

    Psidium guajava (Myrtaceae) is an evergreen shrub growing extensively throughout the tropical and subtropical areas. Four new compounds, guavinoside C, D, E and F (1-3, 10) were isolated from the leaves of P. guajava, along with six known ones (4-9). Their structures were elucidated by spectroscopic analysis. Compounds 1, 4 and 10 showed significant cytotoxic activities on HeLa, SGC-7901 and A549 cell lines, respectively. Compounds 1 and 4-10 showed antioxidant activities in DPPH, ABTS and FRAP assays, and five of them (1, 4-6, 10) exhibited stronger activities than that of vitamin C. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Comparative cytotoxicity of periodontal bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.H.; Hammond, B.F.

    1988-11-01

    The direct cytotoxicity of sonic extracts (SE) from nine periodontal bacteria for human gingival fibroblasts (HGF) was compared. Equivalent dosages (in terms of protein concentration) of SE were used to challenge HGF cultures. The cytotoxic potential of each SE was assessed by its ability to (1) inhibit HGF proliferation, as measured by direct cell counts; (2) inhibit 3H-thymidine incorporation in HGF cultures; or (3) cause morphological alterations of the cells in challenged cultures. The highest concentration (500 micrograms SE protein/ml) of any of the SEs used to challenge the cells was found to be markedly inhibitory to the HGFs by all three of the criteria of cytotoxicity. At the lowest dosage tested (50 micrograms SE protein/ml); only SE from Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Fusobacterium nucleatum caused a significant effect (greater than 90% inhibition or overt morphological abnormalities) in the HGFs as determined by any of the criteria employed. SE from Capnocytophaga sputigena, Eikenella corrodens, or Wolinella recta also inhibited cell proliferation and thymidine incorporation at this dosage; however, the degree of inhibition (5-50%) was consistently, clearly less than that of the first group of three organisms named above. The SE of the three other organisms tested (Actinomyces odontolyticus, Bacteroides intermedius, and Streptococcus sanguis) had little or no effect (0-10% inhibition) at this concentration. The data suggest that the outcome of the interaction between bacterial components and normal resident cells of the periodontium is, at least in part, a function of the bacterial species.

  16. Experimental considerations on the cytotoxicity of nanoparticles

    Science.gov (United States)

    Kong, Bokyung; Seog, Ji Hyun; Graham, Lauren M; Lee, Sang Bok

    2011-01-01

    Engineered nanoparticles are one of the leading nanomaterials currently under investigation due to their applicability in various fields, including drug and gene delivery, biosensors, cancer treatment and diagnostic tools. Moreover, the number of commercial products containing nanoparticles released on the market is rapidly increasing. Nanoparticles are already widely distributed in air, cosmetics, medicines and even in food. Therefore, the unintended adverse effect of nanoparticle exposure is a growing concern both academically and socially. In this context, the toxicity of nanoparticles has been extensively studied; however, several challenges are encountered due to the lack of standardized protocols. In order to improve the experimental conditions of nanoparticle toxicity studies, serious consideration is critical to obtain reliable and realistic data. The cell type must be selected considering the introduction route and target organ of the nanoparticle. In addition, the nanoparticle dose must reflect the realistic concentration of nanoparticles and must be loaded as a well-dispersed form to observe the accurate size- and shape-dependent effect. In deciding the cytotoxicity assay method, it is important to choose the appropriate method that could measure the toxicity of interest without the false-negative or -positive misinterpretation of the toxicity result. PMID:21793681

  17. Cytotoxic lignan esters from Cinnamomum osmophloeum.

    Science.gov (United States)

    Chen, Tai-Hung; Huang, Yu-Hao; Lin, Jhih-Jhang; Liau, Bing-Chung; Wang, Sheng-Yang; Wu, Yang-Chang; Jong, Ting-Ting

    2010-04-01

    The bark and roots of Cinnamomum osmophloeum are widely used in Taiwan as spice substitutes for C. CASSIA. We have isolated three novel lignan esters, one dibenzylbutane-type ligan ester [9,9'-di-O-feruloyl-(+)-5,5'-dimethoxy secoisolariciresinol (3)] and two cyclolignan esters [(7' S,8' R,8 R) -lyoniresinol-9-O-(E)-feruloyl ester ( 5) and (7' S,8' R,8 R)-lyoniresinol-9,9'-di-O-(E)-feruloyl ester (6)], and several known lignans from the heartwood and roots of C. osmophloeum. We identified these compounds using 1D and 2D NMR spectroscopy and mass spectrometry. Cytotoxicity assays of these novel lignan esters revealed that compound 6 has strong activities against human liver cancer (HepG2 and Hep3B) and oral cancer (Ca9-22) cells, with IC(50) values of 7.87, 4.31, and 2.51 microg/mL, respectively. Georg Thieme Verlag KG Stuttgart.New York.

  18. New and Cytotoxic Components from Antrodia camphorata

    Directory of Open Access Journals (Sweden)

    Tzong-Huei Lee

    2014-12-01

    Full Text Available The solid-state cultured products of Antrodia camphorata as health foods has been blooming for the past few decades in Taiwan. In continuing our studies on the chemical constituents of the solid-state cultured products of this fungus, 6-methoxy-4-methyl-2,3-(methylenedioxyphenol (1 and 4,4'-(ethane-1,2-diylbis(2,3,6-trimethoxyphenol(2 together with 2,3,6-trimethoxy-4-methylphenol (3, 1(10→6abeo-ergosta-5,7,9,22-tetraen-3α-ol (4, citreoanthrasteroid B (5 and dankasterones A (6 and B (7 were purified by a series of column chromatography. Their structures were elucidated by spectral data analysis. For bioactivity assay, compounds 4–7 showed significant cytotoxicity toward murine colorectal CT26 and human leukemia K562 cancer cell lines with IC50 values ranging from 6.7 to 15.3 µM and from 12.5 to 23.1 µM, respectively.

  19. Phytochemical screening, antiproliferative and cytotoxic activities of the mosses Rhytidiadelphus triquetrus (Hedw.) Warnst. and Tortella tortuosa (Hedw.) Limpr.

    OpenAIRE

    Muhammet Şamil Yağlıoğlu; Gökhan Abay; Ibrahim DemirtaŞ; Ayşe Şahin Yağlıoğlu

    2017-01-01

    The paper presents information about the phytochemical analysis, antiproliferative and cytotoxic activities of Rhytidiadelphus triquetrus and Tortella tortuosa extracts. The cytotoxic activities of some extracts shows highest antiproliferative activities were detected with Lactate Dehydrogenase Leakage Assay. Sixteen components obtained from hexane extracts were determined by GC/MS. Palmitic acid was identified as the main component. The phenolic components of the other extracts were determin...

  20. Chemical Composition and Cytotoxic and Antibacterial Activities of the Essential Oil of Aloysia citriodora Palau Grown in Morocco

    OpenAIRE

    Moulay Ali Oukerrou; Mounir Tilaoui; Hassan Ait Mouse; Inass Leouifoudi; Abdeslam Jaafari; Abdelmajid Zyad

    2017-01-01

    The aim of this work is to investigate the in vitro cytotoxic and antibacterial effects of the essential oils of Aloysia citriodora Palau, harvested in different regions of Morocco. The chemical profile was established using gas chromatography-mass spectrometry analysis. The cytotoxic activity against P815, MCF7, and VERO cell lines as well as the normal human peripheral blood mononuclear cells (PBMCs) was evaluated using the MTT assay. Standard, ATCC, strains of bacteria (Escherichia coli, S...

  1. Cytotoxicity and trypanocidal activity of nifurtimox encapsulated in ethylcyanoacrylate nanoparticles.

    Science.gov (United States)

    Sánchez, Gittith; Cuellar, Daniela; Zulantay, Ines; Gajardo, Marta; González-Martin, Guillermo

    2002-01-01

    The aim of the present study was to study the trypanocidal activity of nanoparticles loaded with nifurtimox in comparison with the free drug against Trypanosoma cruzi, responsible for Chagas' disease. Ethylcyanoacrylate nanoparticles acted as the delivery system into cells. As the obligate replicative intracellular form is amastigote, in vitro studies were performed on this form of parasite as well as on cell culture derived trypomastigotes. The fluorescence method used here was very useful as it allowed for the simultaneous study of trypanocide activity and cytotoxicity by determining living or dead parasites within living or dead host cells. According to these results, the greatest trypanocide activity on cell culture-derived trypomastigotes was recorded for nifurtimox-loaded nanoparticles with a 50% inhibitory concentration (IC50) twenty times less than that of the free drug. The cytotoxicity of unloaded nanoparticles at low concentrations was similar to that obtained by free drug when evaluated on Vero cells. Furthermore, nifurtimox-loaded nanoparticles showed increased trypanocide activity on intracellular amastigotes with an IC50 thirteen times less than that of nifurtimox. We also observed that the unloaded nanoparticles possess the previously-described trypanocide activity, similar to the standard solution of nifurtimox, although the mechanism for this has not yet been elucidated. In conclusion, it was possible to establish in vitro conditions using nifurtimox encapsulated nanoparticles in order to decrease the doses of the drug and thus to obtain high trypanocidal activity on both free trypomastigotes and intracellular amastigotes with low cytotoxicity for the host cell.

  2. Antioxidant, Anti-Inflammatory, and Cytotoxic Activities of Garcinia nervosa (Clusiaceae

    Directory of Open Access Journals (Sweden)

    N. M. U. Seruji

    2013-01-01

    Full Text Available In our continuing interest on Sarawak Garcinia species, we carried out the evaluation of antioxidant, anti-inflammatory and cytotoxic activities on the methanolic extracts of Garcinia nervosa. The extracts were prepared from its air-dried grounded leaves and barks. The evaluation of antioxidant activities was done using the (2,2-diphenyl-1-picrylhydrazyl DPPH radical scavenging assay and the result showed high radical scavenging activities. Meanwhile, the anti-inflammatory evaluation was performed using the lipoxygenase assay, hyaluronidase assay, and xanthine oxidase assay which showed, both of these extracts exhibited high anti-inflammatory properties. The lipoxygenase assay showed a high inhibition of enzyme activity for the barks extracts and a moderate enzyme activity for the leaves extracts. However, there were low inhibitions for both extracts in the hyaluronidase assay and only the barks extracts exhibited moderate antigout properties in the xanthine oxidase assay. For the cytotoxic assay, the extracts exhibited positive responses against the three cancer cell lines, the HeLa cell lines, MCF-7 cell lines, and HT-29 cell lines. Thus, Garcinia nervosa contains high antioxidativeand anti-inflammation properties, which have great potential in the development of pharmaceutical and dermatological products.

  3. In Vitro Cytotoxic Activity of Origanum vulgare L. on HCT-116 and MDA-MB-231 Cell Lines

    Science.gov (United States)

    Grbović, Filip; Stanković, Milan S.; Ćurčić, Milena; Đorđević, Nataša; Šeklić, Dragana; Topuzović, Marina; Marković, Snežana

    2013-01-01

    In the present investigation, we examined the cytotoxic effect of methanolic extract from Origanum vulgare on HCT-116 and MDA-MB-231 cell line in vitro. In order to determine the cytotoxic effects we used an MTT viability assay. The results showed that cell growth is significantly lower in extract treated cells compared to untreated control. The effect of inhibition of cell growth was higher in the treatment of HCT-116 cell line than in MDA-MB-231. Based on the results it is determined that O. vulgare is a significant source of biologically active substances that have cytotoxic and antiproliferative activity in vitro. PMID:27137381

  4. Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements

    Directory of Open Access Journals (Sweden)

    Donald H. Atha

    2017-11-01

    Full Text Available Abstract Background When evaluating the toxicity of engineered nanomaterials (ENMS it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA-capped InP and CdSe quantum dots (QDs. We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS, the lactate dehydrogenase assay for membrane viability (LDH, the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. Results The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. Conclusions This study can

  5. Cytotoxic Effects of GM1 Ganglioside and Amyloid β-Peptide on Mouse Embryonic Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Makoto Yanagisawa

    2010-01-01

    Full Text Available AD (Alzheimer's disease is a neurodegenerative disease and the most common form of dementia. One of the pathological hallmarks of AD is the aggregation of extracellular Aβs (amyloid β-peptides in senile plaques in the brain. The process could be initiated by seeding provided by an interaction between GM1 ganglioside and Aβs. Several reports have documented the bifunctional roles of Aβs in NSCs (neural stem cells, but the precise effects of GM1 and Aβ on NSCs have not yet been clarified. We evaluated the effect of GM1 and Aβ-(1–40 on mouse NECs (neuroepithelial cells, which are known to be rich in NSCs. No change of cell number was detected in NECs cultured in the presence of either GM1 or Aβ-(1–40. On the contrary, a decreased number of NECs were cultured in the presence of a combination of GM1 and Aβ-(1–40. The exogenously added GM1 and Aβ-(1–40 were confirmed to incorporate into NECs. The Ras–MAPK (mitogen-activated protein kinase pathway, important for cell proliferation, was intact in NECs simultaneously treated with GM1 and Aβ-(1–40, but caspase 3 was activated. NECs treated with GM1 and Aβ-(1–40 were positive in the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, an indicator of cell death. It was found that GM1 and Aβ-(1–40 interacted in the presence of cholesterol and sphingomyelin, components of cell surface microdomains. The cytotoxic effect was found also in NSCs prepared via neurospheres. These results indicate that Aβ-(1–40 and GM1 co-operatively exert a cytotoxic effect on NSCs, likely via incorporation into NEC membranes, where they form a complex for the activation of cell death signalling.

  6. UPLC/MS/MS method for quantification and cytotoxic activity of sesquiterpene lactones isolated from Saussurea lappa.

    Science.gov (United States)

    Kumar, Ashish; Kumar, Shiv; Kumar, Dharmesh; Agnihotri, Vijai K

    2014-09-11

    Saussurea lappa (Asteraceae) roots have been reputed for the usage in traditional medicinal systems of India, China and Japan for the treatment of various kinds of disorders such as anti-ulcer, anti-convulsant, anti-cancer, hepatoprotective, anti-arthritic and anti-viral activities. Compounds were isolated using a column chromatographic technique. The root extract, fractions and isolated compounds were tested for cytotoxicity against A549 (human lung carcinoma) and C-6 (rat glioma) cells using the Sulphorhodamine B assay. Chromatographic separations of active sesquiterpene lactones were accomplished on BEH-HSS-T3 column at 25°C. Phytochemical investigation of Saussurea lappa root extract resulted in the isolation of isoalantolactone (1), β-cyclocostunolide (2) α-cyclocostunolide (3), 4-hydroxy-3,5-dimethoxycinnamyl-9-O-β-D-glucopyranoside (4), sucrose (5), and alantolactone (6). Their structures were determined by spectroscopic means. Ethanolic extract, chloroform fraction, compounds 1, 2, 3 and 6 possessed significant activity against both tested cells. The quantification was performed using the transitions of m/z 233/105 for isoalantolactone and m/z 233/105 for alantolactone respectively. Costunolide and dehydrocostus lactone were also characterised by comparison of MS/MS fragmentation pattern. This is the first study on simultaneous quantification of isoalantolactone and alantolactone by the UPLC/MS/MS method in Saussurea lappa. Our study against A549 and C-6 cells showed higher cytotoxicity. It is suggested that roots of Saussurea lappa might be a potential source of anticancer compounds. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Antioxidant, Antitubercular and Cytotoxic Activities of Piper imperiale

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    Sanjib Bhakta

    2012-04-01

    Full Text Available Phenolic compounds are widely distributed in Nature and act as pharmacologically active constituents in many herbal medicines. They have multiple biological properties, most notably antioxidant, antibacterial and cytotoxic activities. In the present study an attempt to correlate the phenolic composition of leaf, flower and wood extracts of Piper imperiale, with antioxidant, antitubercular and cytotoxic activities was undertaken. The total phenol content ranged from 1.98 to 6.94 mg GAE/gDW among ethanolic extracts, and gallic acid, catechin, epicatechin, ferulic acid, resveratrol and quercetin were identified and quantified by HPLC. DPPH and ABTS assays showed high antioxidant activity of the leaf extract (EC50ABTS = 15.6 µg/mL, EC50DPPH = 27.3 µg/mL with EC50 in the same order of magnitude as the hydroxyquinone (EC50ABTS = 10.2 µg/mL, EC50DPPH = 15.7 µg/mL. The flower extract showed strong antimicrobial activity against Mycobacterium tuberculosis H37Rv. All the extracts exhibited dose-dependent cytotoxic effects against MCF-7 cancer cells. This is the first time that a Piper extract has been found to be highly active against M. tuberculosis. This study shows the biological potential of Piper imperiale extracts and gives way to bio-guided studies with well-defined biological activities.

  8. Cytotoxic cells induced after Chlamydia psittaci infection in mice

    Energy Technology Data Exchange (ETDEWEB)

    Lammert, J.K.

    1982-03-01

    The ability of spleen cells from Chlamydia psittaci-infected mice to lyse C. psittaci-infected and uninfected target cell monolayers was studied. The cytotoxicity assay used was a terminal label method in which the number of adherent target cells surviving the interaction with effector cells was determined by measuring the uptake of (3H)uridine by such cells. It was observed that in the first few days postinfection (3 to 5), spleens contained cells that lysed infected and uninfected targets with equal efficiency. Subsequently, infected targets were killed primarily. The activity of effector spleen cells for infected targets continued, although at a reduced level, beyond 21 days postinfection. Intact effector cells were required since a disruption by sonication resulted in a loss of cytotoxicity. The enhanced killing observed with infected targets was also observed when target cells were sensitized with heat- or UV-inactivated C. psittaci. This study suggests that the induction of cytotoxic cells after C. psittaci infection may contribute to the ability of the host to control multiplication of the microorganism.

  9. Antimicrobial and cytotoxic potentials of Buddleja polystachya extracts

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    Ghada Ahmed Fawzy

    2013-06-01

    Full Text Available Most of the species of Buddleja have found applications in folk medicine. This study aimed to evaluate the in vitro antimicrobial and cytotoxic potentials of B. polystachya extracts. Four extracts were prepared A-D (dichloromethane, ethyl acetate, n-butanol, and aqueous extracts, respectively. The antimicrobial activity was evaluated using the broth micro-dilution assay for minimum inhibitory concentrations (MIC. The crystal violet staining method (CVS was used for the evaluation of the cytotoxic activity on HepG-2, MCF-7 and HCT-116 human cell lines. Results showed that the highest antimicrobial activity was given by the ethyl acetate extract followed by the dichloromethane extract, while the n-butanol revealed moderate activity against gram positive bacteria only with no activity against the rest of tested microorganisms. The aqueous extract was totally ineffective against all tested microorganisms at 20 mg/ml. Among the four extracts tested, dichloromethane and ethyl acetate extracts showed the highest cytotoxic activity on all three human cell lines.

  10. Evaluation of the cytotoxic activity of some Brazilian medicinal plants.

    Science.gov (United States)

    Ribeiro, Sandra S; de Jesus, Aline M; dos Anjos, Charlene S; da Silva, Thanany B; Santos, Alan D C; de Jesus, Jemmyson R; Andrade, Moacir S; Sampaio, Tais S; Gomes, Wesley F; Alves, Péricles B; Carvalho, Adriana A; Pessoa, Claudia; de Moraes, Manoel O; Pinheiro, Maria L B; Prata, Ana Paula N; Blank, Arie F; Silva-Mann, Renata; Moraes, Valeria R S; Costa, Emmanoel V; Nogueira, Paulo Cesar L; Bezerra, Daniel P

    2012-09-01

    Plants are promising sources of new bioactive compounds. The aim of this study was to investigate the cytotoxic potential of nine plants found in Brazil. The species studied were: Annona pickelii Diels (Annonaceae), Annona salzmannii A. DC. (Annonaceae), Guatteria blepharophylla Mart. (Annonaceae), Guatteria hispida (R. E. Fr.) Erkens & Maas (Annonaceae), Hancornia speciosa Gomes (Apocynaceae), Jatropha curcas L. (Euphorbiaceae), Kielmeyera rugosa Choisy (Clusiaceae), Lippia gracilis Schauer (Verbenaceae), and Hyptis calida Mart. Ex Benth (Lamiaceae). Different types of extractions from several parts of plants resulted in 43 extracts. Their cytotoxicity was tested against HCT-8 (colon carcinoma), MDA-MB-435 (melanoma), SF-295 (glioblastoma), and HL-60 (promielocitic leukemia) human tumor cell lines, using the thiazolyl blue test (MTT) assay. The active extracts were those obtained from G. blepharophylla, G. hispida, J. curcas, K. rugosa, and L. gracilis. In addition, seven compounds isolated from the active extracts were tested; among them, β-pinene found in G. hispida and one coumarin isolated from K. rugora showed weak cytotoxic activity. In summary, this manuscript contributes to the understanding of the potentialities of Brazilian plants as sources of new anticancer drugs. Georg Thieme Verlag KG Stuttgart · New York.

  11. Antioxidant and Cytotoxic Activities of Centella asiatica (L) Urb.

    Science.gov (United States)

    Pittella, Frederico; Dutra, Rafael C.; Junior, Dalton D.; Lopes, Miriam T. P.; Barbosa, Nádia R.

    2009-01-01

    In the present study, the phenolic (Folin-Dennis) and flavonoid (colorimetric assay) constituents, antioxidant [2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) assay] and cytotoxic activities of an aqueous extract (AE) of Centella asiatica leaves were investigated. The aqueous extract (50 g/L) was obtained by infusion followed by cold maceration for 24 h. The levels of phenolic and flavonoid compounds were 2.86 g/100 g and 0.361 g/100 g, respectively. The AE showed elevated DPPH scavenging activity, with an IC50 value of 31.25 μg/mL. The AE had a promising activity against mouse melanoma (B16F1), human breast cancer (MDA MB-231) and rat glioma (C6) cell lines, with IC50 values of 698.0, 648.0 and 1000.0 μg/mL, respectively. A positive correlation was established between the level of flavonoids, antioxidant and antitumor activities. PMID:19865514

  12. Antioxidant and Cytotoxic Activities of Centella asiatica (L Urb.

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    Frederico Pittella

    2009-08-01

    Full Text Available In the present study, the phenolic (Folin-Dennis and flavonoid (colorimetric assay constituents, antioxidant [2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH assay] and cytotoxic activities of an aqueous extract (AE of Centella asiatica leaves were investigated. The aqueous extract (50 g/L was obtained by infusion followed by cold maceration for 24 h. The levels of phenolic and flavonoid compounds were 2.86 g/100 g and 0.361 g/100 g, respectively. The AE showed elevated DPPH scavenging activity, with an IC50 value of 31.25 μg/mL. The AE had a promising activity against mouse melanoma (B16F1, human breast cancer (MDA MB-231 and rat glioma (C6 cell lines, with IC50 values of 698.0, 648.0 and 1000.0 μg/mL, respectively. A positive correlation was established between the level of flavonoids, antioxidant and antitumor activities.

  13. In vitro cytotoxicity of cyanobacteria from water ecosystems of Serbia.

    Science.gov (United States)

    Cetojevic-Simin, D; Svircev, Z; Baltic, V V

    2009-01-01

    The purpose of this study was to investigate whether water samples from water ecosystems of Serbia, unknown so far with regard to cyanotoxin levels, are the source of toxic compounds originating from the biological activity of cyanobacteria. The growth inhibition activity was evaluated using in vitro toxicity assay in Neuro-2a (mouse neuroblastoma) and MRC-5 (human fetal lung) cell lines, after 48 h of exposure time. Cell growth was evaluated by the colorimetric sulforhodamine B (SRB) assay. Our experiments revealed that some of the investigated water samples are toxigenic and alter cell growth of Neuro-2 and MRC-5 cell lines in vitro. Neuro-2a and MRC-5 cell lines responded to the presence of secondary metabolites of cyanobacteria. Significant cytotoxic effects were detected in the samples from lakes (Ludos and Palic), reservoirs (Zobnatica) and rivers (Krivaja).

  14. A quantitative comet infection assay for influenza virus

    Science.gov (United States)

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  15. Cytotoxicity of Light-Cured Dental Materials according to Different Sample Preparation Methods

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    Myung-Jin Lee

    2017-03-01

    Full Text Available Dental light-cured resins can undergo different degrees of polymerization when applied in vivo. When polymerization is incomplete, toxic monomers may be released into the oral cavity. The present study assessed the cytotoxicity of different materials, using sample preparation methods that mirror clinical conditions. Composite and bonding resins were used and divided into four groups according to sample preparation method: uncured; directly cured samples, which were cured after being placed on solidified agar; post-cured samples were polymerized before being placed on agar; and “removed unreacted layer” samples had their oxygen-inhibition layer removed after polymerization. Cytotoxicity was evaluated using an agar diffusion test, MTT assay, and confocal microscopy. Uncured samples were the most cytotoxic, while removed unreacted layer samples were the least cytotoxic (p < 0.05. In the MTT assay, cell viability increased significantly in every group as the concentration of the extracts decreased (p < 0.05. Extracts from post-cured and removed unreacted layer samples of bonding resin were less toxic than post-cured and removed unreacted layer samples of composite resin. Removal of the oxygen-inhibition layer resulted in the lowest cytotoxicity. Clinicians should remove unreacted monomers on the resin surface immediately after restoring teeth with light-curing resin to improve the restoration biocompatibility.

  16. In vitro cytotoxicity assessment of ulvan, a polysaccharide extracted from green algae.

    Science.gov (United States)

    Alves, Anabela; Sousa, Rui A; Reis, Rui L

    2013-08-01

    Sustainable exploitation and valorization of natural marine resources represents a highly interesting platform for the development of novel biomaterials, with both economic and environmental benefits. In this context, toxicity data is regarded as a crucial and fundamental knowledge prior to any advances in the application development of natural derived polymers. In the present work, cytotoxicity of ulvan extracted from green algae Ulva lactuca was assessed by means of standard in vitro cytotoxicity assays. Fibroblast-like cells were incubated in the presence of this green algae's polysaccharide, and cell viability was assayed through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium test. In addition, double stranded DNA and total protein were quantified in order to assess cell number. In order to establish ulvan's non-cytotoxic behaviour, the effect of this polysaccharide on cellular metabolic activity and cell number was directly compared to hyaluronic acid (HA), used as a non-cytotoxic control material. In this study, ulvan demonstrated promising results in terms of cytotoxicity, comparable to the currently used HA, which suggests that ulvan can be considered as non-toxic in the range of concentrations studied. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Cytotoxicity Effects of Amoora rohituka and chittagonga on Breast and Pancreatic Cancer Cells

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    Leo L. Chan

    2011-01-01

    Full Text Available Chemotherapeutic agents for cancer are highly toxic to healthy tissues and hence alternative medicine avenues are widely researched. Majority of the recent studies on alternative medicine suggested that Amoora rohituka possesses considerable antitumor and antibacterial properties. In this work, rohituka and chittagonga, fractionated with petroleum ether, dichloromethane, and ethanol, were explored for their anticancer potential against two breast cancer (MCF-7 and HTB-126 and three pancreatic cancer (Panc-1, Mia-Paca2, and Capan1. The human foreskin fibroblast, Hs68, was also included. Cytotoxicity of each extract was analyzed using the MTT assay and label-free photonic crystal biosensor assay. A concentration series of each extract was performed on the six cell lines. For MCF-7 cancer cells, the chittagonga (Pet-Ether and CH2Cl2 and rohituka (Pet-Ether extracts induced cytotoxicity; the chittagonga (EtoAC and rohituka (MeOH extracts did not induce cytotoxicity. For HTB126, Panc-1, Mia-Paca2, and Capan-1 cancer cells, only the chittagonga CH2Cl2 extract showed a significant cytotoxic effect. The extracts were not cytotoxic to normal fibroblast Hs68 cells, which may be correlated to the specificity of Amoora extracts in targeting cancerous cells. Based on these results, further examination of the potential anticancer properties Amoora species and the identification of the active ingredients of these extracts is warranted.

  18. Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth.

    Science.gov (United States)

    Pires, C W; Botton, G; Cadoná, F C; Machado, A K; Azzolin, V F; da Cruz, I B M; Sagrillo, M R; Praetzel, J R

    2016-08-01

    To evaluate the cytotoxicity, oxidative stress and genotoxicity in vitro of four iodoform pastes and three calcium hydroxide pastes. Peripheral blood mononuclear cells (PBMCs) and pure calf thymus DNA (dsDNA) were exposed to extracts of the pastes. Cytotoxicity was assessed with the MTT assay. Generation of reactive oxygen species (ROS) was evaluated using a DCFH-DA assay, and lipid peroxidation was evaluated using a TBARS assay. Genotoxicity was evaluated using the alkaline comet assay and Genomodifier capacity assay (GEMO). All tests were performed after 24 h and 72 h of cell exposure, except GEMO. After performing the Kolmogorov-Smirnov test, data were analysed by Kruskal-Wallis and Dunn's post-tests, and anova with Dunnett's post-test, with a significance level established at P  0.05). However, chlorhexidine paste caused DNA damage in dsDNA (P < 0.05). Calcium hydroxide pastes caused DNA damage in both tests (P < 0.05). The pastes varied in their ability to induce cytotoxicity, genotoxicity and oxidative stress. In general, Guedes-Pinto, Maxitrol and neomycin sulphate + bacitracin pastes exhibited better biocompatibility in vitro. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  19. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

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

  20. Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection.

    Science.gov (United States)

    Müller, S; Bühler-Jungo, M; Mueller, C

    2000-02-15

    After systemic infection of mice with 104 PFU of lymphocytic choriomeningitis virus (LCMV), infected cells are detected simultaneously in various organs, including spleen and intestinal mucosa. Most notably, virus-infected cells are also present among CD11c+ dendritic cells in the subepithelial area of the small intestinal mucosa. Some of these virus-infected cells are in close spatial association with intestinal intraepithelial lymphocytes (IEL). Therefore, we compared virus-specific cytotoxic activity of CD8 splenocytes with that of IEL subsets. While ex vivo isolated TCRalphabeta+CD8alphaalpha+ IEL exert only minimal virus-specific cytotoxicity, maximum specific killing mediated by TCRalphabeta+CD8alphabeta+ IEL on day 8 postinfection exceeds maximum cytotoxic activity observed with CD8 splenocytes when assessed in vitro. Maximum cytotoxic activity of IEL is preceded by peak perforin and granzyme B mRNA expression in IEL around day 6 postinfection, suggesting a recent activation in situ. The antivirus cytotoxicity of in vivo primed IEL is further demonstrated by the protection from virus production in the spleen of mice infected with LCMV 10 h before adoptive cell transfer. These data indicate a potent priming of LCMV-specific IEL in situ after systemic LCMV infection and suggest that cytotoxic IEL markedly contribute to the elimination of virus-infected cells in the intestinal mucosa.

  1. Sea-originated cytotoxic substances.

    Science.gov (United States)

    Maru, Norihito; Uemura, Daisuke

    2012-01-01

    Cancer accounted for huge number of deaths, which represents about 13% of all deaths worldwide, and the number of the deaths due to cancer is increasing. Natural products and their synthetic analogs are widely used as antitumor drugs. As represented by these drugs, many anticancer drugs originated from cytotoxic compounds. Marine natural products are a gold mine of strong bioactive compounds with unique structures created in evolution of organisms over hundred million years. However, in the field of drug discovery, most studies have focused on plant essences and bacterial metabolites, and candidate compounds from marine origin are still remaining relatively unexplored. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Cytotoxic Compounds from Aloe megalacantha

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    Negera Abdissa

    2017-07-01

    Full Text Available Phytochemical investigation of the ethyl acetate extract of the roots of Aloe megalacantha led to the isolation of four new natural products—1,8-dimethoxynepodinol (1, aloesaponarin III (2, 10-O-methylchrysalodin (3 and methyl-26-O-feruloyl-oxyhexacosanate (4—along with ten known compounds. All purified metabolites were characterized by NMR, mass spectrometric analyses and comparison with literature data. The isolates were evaluated for their cytotoxic activity against a human cervix carcinoma cell line KB-3-1 and some of them exhibited good activity, with aloesaponarin II (IC50 = 0.98 µM being the most active compound.

  3. A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

    Directory of Open Access Journals (Sweden)

    Bendinelli Mauro

    2009-03-01

    Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

  4. Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

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    Hitoshi Uchiyama

    2014-09-01

    Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

  5. [In vitro cytotoxicity of different self-etching dental adhesive systems].

    Science.gov (United States)

    Wiegand, Annette; Caspar, Caroline; Becker, Klaus; Werner, Carola; Attin, Thomas

    2006-01-01

    The study evaluated the cytotoxicity of five self-etching dentin adhesive systems applied on dentin specimens of different thicknesses. The test materials (A: Adper Prompt-L-Pop, B: Xeno II, C: Clearfil SE Bond, D: One up Bond F, E: Resulcin Aqua Prime & Monobond) and a positive control (35% H202) were applied on 1.0, 1.5 and 2.5 mm thick bovine dentin specimens (each subgroup n = 5) in a dentin barrier test device. The experiments were performed with perfusion (2 ml/h) of the pulpal part of the chamber. The eluates were obtained before (baseline) and 15, 30, 45, 60 and 120 min after application of the adhesives and pipetted onto L-929 fibroblasts. Cytotoxicity of the materials was determined in relation to the baseline value using the MTT assay and statistical analysis was performed by ANOVA. After 15 min perfusion, test materials B-E applied on 1.0 mm and B and E applied on 1.5 mm dentin specimens exhibited cytotoxic potential. However, after 30 min perfusion none of the adhesives showed any toxicity. Cytotoxicity decreased with increasing thickness of the dentin slices and was lower for Adper Prompt-L-Pop compared to adhesives B-E. Self-etching adhesive systems might exhibit cytotoxic potential when applied on dentin of less than 1.5 mm thickness. However, cytotoxicity of the materials decreased with increasing dentin thickness and increasing duration of perfusion.

  6. In vitro cytotoxicity of dental composites based on new and traditional polymerization chemistries.

    Science.gov (United States)

    Brackett, M Goël; Bouillaguet, S; Lockwood, P E; Rotenberg, S; Lewis, J B; Messer, R L W; Wataha, J C

    2007-05-01

    The biological response to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. Several new composite formulations claim to reduce unpolymerized residual mass. The current study assessed the cytotoxic responses to several of these new formations and compared them with more traditional formulations. Our hypothesis predicted that if these new polymerization chemistries reduce unpolymerized residual mass, the cytotoxicity of these materials also should be reduced relative to traditional formulations. Materials (HerculiteXRV, Premise, Filtek Supreme, CeramxDuo, Hermes, and Quixfil) were tested in vitro in direct contact with Balb mouse fibroblasts, initially, then after aging in artificial saliva for 0, 1, 3, 5, or 8 weeks. The toxicity was determined by using the MTT assay to the estimate SDH activity. Knoop hardness of the materials also was measured at 0 and 8 weeks to determine whether surface breakdown of the materials in artificial saliva contributed to cytotoxic responses. Materials with traditional methacrylate chemistries (Herculite, Premise, Filtek Supreme) were severely (>50%) cytotoxic throughout the 8-week interval, but materials with newer chemistries or filling strategies (Hermes, CeramXDuo, and Quixfil) improved over time of aging in artificial saliva. Hermes showed the least cytotoxicity at 8 weeks, and was statistically equivalent to Teflon negative controls. Hardness of the materials was unaffected by exposure to artificial saliva. Newer polymerization and filling strategies for dental composites show promise for reducing the release of unpolymerized components and cytotoxicity.

  7. Cytotoxic activities of some mono and bis Mannich bases derived from acetophenone in brine shrimp bioassay.

    Science.gov (United States)

    Gul, Halise Inci; Gul, Mustafa; Hänninen, Osmo

    2002-01-01

    Some mono Mannich bases (1-phenyl-3-amino-1-propanone salts) and bis Mannich bases (1-phenyl-3-amino-2-amino-methyl-1-propanone salts) derived from acetophenone and a few representative quaternary derivatives were synthesised and their cytotoxicity was tested using the brine shrimp bioassay. This assay may serve as an intermediate test before further in vivo animal experiments in large scale, since brine shrimp nauplii as whole organisms were used in this test. Mono Mannich bases were generally more cytotoxic than their corresponding bis Mannich bases. Mannich bases synthesised were cytotoxic in both brine shrimp bioassay in this study and cell culture tests using Jurkat and Renca cells in a previous study. However, the order of the cytotoxic potency of the compounds were reverse, which may result from faster deamination of bis derivatives than optimal level, and different species and test media used in the two test systems. Faster deamination of bis derivatives might have led to elimination of active metabolites before reaching its target. The cytotoxicity of the compounds might have been altered by amino acids and proteins present in cell culture medium but not in sea water used in brine shrimp bioassay affecting their transport through the cell membrane and metabolism in the cell by binding with the compounds. With higher cytotoxic activity compared with 5-fluorouracil (CAS 51-21-8) in brine shrimp bioassay, mono Mannich base 1 and its quaternary derivative 4 and quaternary bis derivative 8 seem to be candidate compounds for further drug design.

  8. Cytotoxic Activity of Selected Iranian Traditional Medicinal Plants on Colon, Colorectal and Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Leila Mohammad Taghizadeh Kashani

    2014-11-01

    Full Text Available Background: Many natural products from plants have been recognized to exert anticancer activity. In this study, ethanolic extracts of selected medicinal herbs from Iranian flora including Alyssum homolocarpum Fisch. (from seeds, Urtica dioica L. (from aerial parts, Cichorium intybus L. (from roots and Solanum nigrum L. (from fruits, were evaluated for their cytotoxic effect on different cell lines.Methods: Cytotoxic effect of these extracts was studied on three different cancer cell lines; colon carcinoma (HT-29, colorectal adenocarcinoma (Caco-2 and breast ductal carcinoma (T47D. In addition, Swiss mouse embryo fibroblasts (NIH 3T3 were used as normal nonmalignant cells. MTT assay (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide was utilized for calculating the cytotoxicity of extracts on cell lines.Results: Results showed the potent cytotoxic activity of U. dioica ethanolic extract against T47D cell line with IC50 value of 46.14±4.55 µg/ml. Other extracts showed poor activity with IC50>100 µg/ml.Conclusions: Cytotoxic activity recorded in the present study revealed high potential antiproliferative activity of U. dioica ethanolic extract against T47D cell line. The real IC50 values of this extract may be considerably lower than the IC50 measured in our study if its pharmacological active compounds become pure. The results emphasize the importance of studies on U. dioica ethanolic extract to characterize potential components as cytotoxic natural medicines.

  9. Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

    Directory of Open Access Journals (Sweden)

    Paschke Reinhard

    2011-09-01

    Full Text Available Abstract Background Betulinic acid (BA is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas, the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. Methods In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays and protein expression was examined with Western blot analyses. Results Under normoxic conditions, a half maximal inhibitory concentration (IC50 of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively and U343MG cells (p Conclusion Our results suggest that BA is capable of improving the effects of tumor therapy in human malignant glioma cells, particularly under hypoxic conditions. Further investigations are necessary to characterize its potential as a radiosensitizer.

  10. Cytotoxicity and genotoxicity of ingenamine G isolated from the Brazilian marine sponge Pachychalina alcaloidifera.

    Science.gov (United States)

    Cavalcanti, Bruno Coêlho; Sombra, Carla Maria Lima; de Oliveira, Jaine H H L; Berlinck, Roberto Gomes de Souza; de Moraes, Manoel Odorico; Pessoa, Claudia

    2008-05-01

    Marine sponges belonging to the order Haplosclerida are one of the more prolific sources of new natural products possessing various biological activities. The present study examined the cytotoxic and genotoxic potential of ingenamine G, an alkaloid isolated from the Brazilian marine sponge Pachychalina alcaloidifera. Ingenamine G displayed a moderate cytotoxic activity against human proliferating lymphocytes evaluated by the MTT assay (IC(50) 15 microg/mL). The hemolytic assay showed that ingenamine G cytotoxic activity was not related to membrane disruption. The comet assay and chromosome aberration analysis were applied to determine the genotoxic and clastogenic potential of ingenamine G, respectively. Cultured human lymphocytes were treated with 5, 10, 15 and 20 microg/mL of ingenamine G during the G(1), G(1)/S, S (pulses of 1 and 6 h), and G(2) phases of the cell cycle. All tested concentrations were cytotoxic, reduced significantly the mitotic index, and were clastogenic in all phases of the cell cycle, especially in S phase. While an increase in DNA-strand breaks was observed starting with the concentration corresponding to the IC(50). The presence of genotoxicity and polyploidy during interphase and mitosis, respectively, suggests that ingenamine G at high concentrations is clastogenic and indirectly affects the construction of mitotic fuse.

  11. Comparative study of cytotoxicity of ferromagnetic nanoparticles and magnetitecontaining polyelectrolyte microcapsules

    Science.gov (United States)

    Minaeva, O. V.; Brodovskaya, E. P.; Pyataev, M. A.; Gerasimov, M. V.; Zharkov, M. N.; Yurlov, I. A.; Kulikov, O. A.; Kotlyarov, A. A.; Balykova, L. A.; Kokorev, A. V.; Zaborovskiy, A. V.; Pyataev, N. A.; Sukhorukov, G. B.

    2017-01-01

    The cytotoxicity of magnetite nanoparticles (MNP) stabilized with citrate acidand polyelectrolyte multilayer microcapsules containing these particles in the shell is analyzed. Microcapsules were prepared by co-precipitation of iron (II) and (III) chlorides. Polyelectrolyte microcapsules synthesized by the layer-by-layer method from biodegradable polymers polyarginine and dextran sulfate. Cytotoxicity of the synthesized objects was studied on the L929 cells culture and human leucocytes. It was also investigated the phagocytic activity of leukocytes for the MNP and magnetite containing polyelectrolyte microcapsules (MCPM). A set of tests (MTT assay, neutral red uptake assay, lactate dehydrogenase release assay) was used to study the cytotoxicity in vitro. All the tests have shown that the magnetic nanoparticles have a greater cytotoxicity in comparison with microcapsules containing an equivalent amount of magnetite. In contrast to the mouse fibroblast culture, human leukocytes were more resistant to the toxic effects of magnetite. At the concentrations used in our studies no significant reduction in the viability of leukocytes has been registered. Both MNP and MCPM undergo phagocytosis, however, the phagocytic activity of leukocytes for these particles was lower than for the standard objects (latex microparticles).

  12. Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

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    Álcio Coutinho de Paula

    2015-04-01

    Full Text Available Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR was used to evaluate expression of apoptosis-inducing caspases (8 and 3. Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10% or necrotic (<1% activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

  13. Cytotoxic Effect on Cancerous Cell Lines by Biologically Synthesized Silver Nanoparticles

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    Balaji Kulandaivelu

    Full Text Available The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective alternative to chemical and physical methods. Silver nanoparticles are biologically synthesized and characterized were used in the study. The invitro cytotoxic effect of biologically synthesized silver nanoparticles against MCF-7 cancer cell lines were assessed. The cytotoxic effects of the silver nanoparticles could significantly inhibited MCF-7 cancer cell lines proliferation in a time and concentration-dependent manner by MTT assay. Acridine orange, ethidium bromide (AO/EB dual staining, caspase-3 and DNA fragmentation assays were carried out using various concentrations of silver nanoparticles ranging from 1 to 100 μg/mL. At 100 μg/mL concentration, the silver nanoparticles exhibited significant cytotoxic effects and the apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays. Western blot analysis has revealed that nanoparticle was able to induce cytochrome c release from the mitochondria, which was initiated by the inhibition of Bcl-2 and activation of Bax. Thus, the results of the present study indicate that biologically synthesized silver nanoparticles might be used to treat breast cancer. The present studies suggest that these nanoparticles could be a new potential adjuvant chemotherapeutic and chemo preventive agent against cytotoxic cells. However, it necessitates clinical studies to ascertain their potential as anticancer agents.

  14. Suitability of Cell-Based Label-Free Detection for Cytotoxicity Screening of Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Claudia Meindl

    2013-01-01

    Full Text Available Cytotoxicity testing of nanoparticles (NPs by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA and automated microscopy (Cell-IQ compared to formazan bioreduction (MTS assay. For validation of the label-free systems different concentrations of ethanol and of amine (AMI polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (20 nm CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

  15. Suitability of cell-based label-free detection for cytotoxicity screening of carbon nanotubes.

    Science.gov (United States)

    Meindl, Claudia; Absenger, Markus; Roblegg, Eva; Fröhlich, Eleonore

    2013-01-01

    Cytotoxicity testing of nanoparticles (NPs) by conventional screening assays is often complicated by interference. Carbon nanotubes (CNTs) are particularly difficult to assess. To test the suitability of cell-based label-free techniques for this application, a panel of CNTs with different diameters and surface functionalizations was assessed by impedance-based technique (xCELLigence RTCA) and automated microscopy (Cell-IQ) compared to formazan bioreduction (MTS assay). For validation of the label-free systems different concentrations of ethanol and of amine (AMI) polystyrene NPs were used. CNTs were evaluated in various cell lines, but only endothelial EAhy926 cells and L929 and V79 fibroblasts could be evaluated in all systems. Polystyrene particles obtained similar results in all assays. All systems identified thin (20 nm) CNTs, but detection by xCELLigence system was less sensitive to CNT-induced cytotoxicity. Despite advantages, such as continuous monitoring and more detailed analysis of cytotoxic effects, label-free techniques cannot be generally recommended for cytotoxicity screening of NPs.

  16. Absolute nuclear material assay

    Science.gov (United States)

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

    2012-05-15

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

  17. Absolute nuclear material assay

    Science.gov (United States)

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

    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.

  18. Antimicrobial and Cytotoxic Activities of Extracts from Laurus nobilis Leaves

    KAUST Repository

    Felemban, Shaza

    2011-05-01

    The cytotoxic activity and antimicrobial properties of crude extracts from Laurus nobilis were investigated. With the use of the organic solvents, methanol and ethanol, crude extracts were obtained. To determine the availability of active bio‐compounds, an analysis using liquid chromatography was conducted. The crude extract was also tested for antimicrobial activity. The disc diffusion method was used against the bacterium Escherichia coli. The results showed a weak antimicrobial activity against E. coli. For cytotoxicity testing, the crude extract was studied on four cell-­lines: human breast adenocarcinoma, human embryonic kidney, HeLa (human cervical adenocarcinoma), and human lung fibroblast. From the alamarBlue® assay results, the extracts most potently affected the cell-­lines of human breast adenocarcinoma and human embryonic kidney. Using the lactate dehydrogenase (LDH) assay, an effect on human embryonic kidney was most prominent. With these findings, a suggestion that the crude extract of Laurus nobilis may have antiproliferative properties is put forth, with the possibility of this mechanism being induction of apoptosis with the involvement of Nuclear Factor Kappa κB (NF κB).

  19. Honeybee venom: influence of collection on quality and cytotoxicity

    Directory of Open Access Journals (Sweden)

    Allyson Fortunato Abrantes

    2017-08-01

    Full Text Available ABSTRACT: Apitoxin is the venom produced by bees. It is a complex chemical compound, rich in protein substances and with pharmacological effects. This study was carried out with the objective of comparing the quality of apitoxin extracted in an apiary in different parts of the collector in relation to moisture content, protein analysis and cytotoxicity assay with Artemia salina L. Type 1 apitoxin was collected from glass slabs at the entrance to the hive, while type 2 apitoxin was collected from the waste accumulated in the collection rods and treated by rinsing in distilled water. Both apitoxins presented significant differences (P>0.05 in relation to protein profile, with type 1 showing a higher content (77.8% than type 2 (51.9%, and presented polypeptide bands with more than 50% of their nitrogenous components having molecular weight below 10KDa. Regarding cytotoxicity assays, type 1 apitoxin had LD50 of 71.5μg mL-1, while type 2 had LD50 of 191.6μg mL-1. Thus, the region where apitoxin accumulates in the collector does influence the product quality if moisture and protein contents are in accordance with the standards recommended in specific legislation, and so it can be commercialized by the beekeeper.

  20. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells▿

    Science.gov (United States)

    Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

    2011-01-01

    The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 μg/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ≥100 μg/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 μg/ml do not decrease cell viability, intracameral voriconazole concentrations of ≥100 μg/ml may increase the risk of corneal endothelial damage. PMID:21768517

  1. Bioactivity-guided isolation of cytotoxic sesquiterpenes of Rolandra fruticosa

    Science.gov (United States)

    Pan, Li; Lantvit, Daniel D.; Riswan, Soedarsono; Kardono, Leonardus B.S.; Chai, Hee-Byung; Carcache de Blanco, Esperanza J.; Farnsworth, Norman R.; Soejarto, Djaja Doel; Swanson, Steven M.; Kinghorn, A. Douglas

    2010-01-01

    Cytotoxicity-guided fractionation of a methanol extract of the leaves and twigs of Rolandra fruticosa using the HT-29 human colon cancer cell line led to the isolation of seven sesquiterpene lactones, including a new isorolandrolide, 13-methoxyisorolandrolide (1), a new bourbonenolide, 2α,13-diacetoxy-4α-hydroxy-8α-isobutyroyloxybourbonen-12,6α-olide (2), as well as five known compounds, 13-acetoxyrolandrolide (3), 8-desacyl-13-acetoxyrolandrolide-8-O-tiglate (4), 2-epi-glaucolide E (5), 2α,13-diacetoxy-4α-hydroxy-8α-methacryloyloxybourbonen-12,6α-olide (6), and 2α,13-diacetoxy-4α-hydroxy-8α-tigloyloxybourbonen-12,6α-olide (7). The structures of the two new sesquiterpenes were elucidated on the basis of spectroscopic methods. All isolates were evaluated for their cytotoxicity using the HT-29 cell line, and only 13-acetoxyrolandrolide (3) was found to possess a potent inhibitory effect against this cell line. Compounds 3, 5 and 6 were also tested in a NF-κB (p65) inhibition assay, and 3 was assessed in an in vivo hollow fiber assay. PMID:20189206

  2. Antimycobacterial and cytotoxic activity of selected medicinal plant extracts.

    Science.gov (United States)

    Nguta, Joseph M; Appiah-Opong, Regina; Nyarko, Alexander K; Yeboah-Manu, Dorothy; Addo, Phyllis G A; Otchere, Isaac; Kissi-Twum, Abena

    2016-04-22

    Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants. The microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis. Results of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8). The overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  3. Cytotoxicity and in vivo efficacy of a novel antimicrobial peptoid against Staphylococcus pseudintermedius

    DEFF Research Database (Denmark)

    Damborg, Peter Panduro; Skindersø, Mette E; Hansen, Paul Robert

    Objectives: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is emerging in dogs worldwide, and few or no treatment options are available for dogs infected by this pathogen due to its multi-resistance phenotype. The objective of this study was to evaluate cytotoxicity and investigate...... the in vivo efficacy of a novel peptoid (D2) previously shown to be effective against S. pseudintermedius in vitro. Methods: D2 was subjected to an array of cytotoxicity assays targeting proliferation, cellular stress, apoptosis and cell cycle in Jurkat cells. D2 was then formulated into a gel...

  4. Honatisine, a novel diterpenoid alkaloid, and six known alkaloids from Delphinium honanense and their cytotoxic activity.

    Science.gov (United States)

    He, Yang Qing; Ma, Zhan Ying; Wei, Xiao Mei; Liu, Dong Jie; Du, Bao Zhong; Yao, Bing Hua; Gao, Li Ming

    2011-11-01

    A novel diterpene alkaloid named honatisine (1) has been isolated from the whole plants of Delphinium honanense, along with six known alkaloids, siwanine E (2), isoatisine (3), atisine (4), delcorinine (5), uraphine (6), and nordhagenine A (7). Their structures were deduced on the basis of their spectral data. All of them were evaluated by a SRB assay for their cytotoxicity, and compound 1 showed a significant cytotoxic activity (IC(50) =3.16 μM) against the MCF-7 cell line. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.

  5. Cytotoxicity and phytochemical analyses of Orthosiphon stamineus leaves and flower extracts

    Science.gov (United States)

    Alwahid, Alaa Abd; Yusoff, Wan Mohtar Wan; Nor, Norefrina Shafinaz Md.; Ibrahim, Nazlina

    2015-09-01

    Orthosiphon stamineus Benth (Lamiaceae) is a plant with many ethnobotanical uses including antifungal and antibacterial activities. This study is aimed to determine the cytotoxicity and phytochemical content of O. stamineus leaves and flower using ethanol and water as solvents. The cytotoxicity of the extracts towards Vero cell was determined by MTT assay. The CC50 values were between 3.4-7.4 mg/ml and can be considered as nontoxic. Phytochemical screening revealed terpenes, alkaloid and phenolic were present in the leaves and flower of O. stamineus that might pose as the bioactive compound.

  6. Prolonged cytotoxic effect of aqueous extracts from dried viscum album on bladder cancer cells.

    Science.gov (United States)

    Hunziker-Basler, N; Zuzak, T J; Eggenschwiler, J; Rist, L; Simões-Wüst, A P; Viviani, A

    2007-03-01

    Aqueous extracts from whole dried mistletoe (Viscum album L., Iscucin) are often used in anti-cancer treatment. We studied the effect of extracts obtained from mistletoe bushes that grew on different host trees on bladder cancer cells by means of MTT-colorimetric cell proliferation/survival assays. The extracts possessed concentration-dependent cytotoxic properties whose extent varied with the host tree, but did not always correlate with the corresponding mistletoe lectin content. A 2-hours treatment of bladder cancer cells triggered a later, strong cytotoxic effect. This prolonged effect suggests that instillation with Iscucin has therapeutic potential for bladder cancer patients.

  7. Fibril fragmentation enhances amyloid cytotoxicity.

    Science.gov (United States)

    Xue, Wei-Feng; Hellewell, Andrew L; Gosal, Walraj S; Homans, Steve W; Hewitt, Eric W; Radford, Sheena E

    2009-12-04

    Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.

  8. Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Ying Miao

    2014-02-01

    Full Text Available AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells in vitro and cat corneal endothelial cells(CCE cells in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5-dimethylthiahiazo (-z-y1-3,5-di-phenytetrazoliumromide (MTT assay, acridine orange (AO/ethidium bromide (EB double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM. The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM, and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis

  9. Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

    Directory of Open Access Journals (Sweden)

    Angela Filomena

    2017-10-01

    Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

  10. Adjuvant antiproliferative and cytotoxic effect of aloin in irradiated HeLaS3 cells

    Science.gov (United States)

    Nićiforović, A.; Adžić, M.; Zarić, B.; Radojčić, M. B.

    2007-09-01

    Naturally occurring phytoanthracycline, aloin, was used to radiosensitize HeLaS3 human cervix carcinoma cells. The results indicated that the cytotoxic adjuvant effect of aloin was synergistic with gammaionizing radiation at all drug concentrations and comparable to the cytotoxicity of 5-10 Gy ionizing radiation alone. Radiosensitization of HeLaS3 cells was achieved by 60 μM aloin, which reduced the IC50 dose of ionizing radiation from 3.4 to 2 Gy. Ionizing radiation and aloin alone or in combination are shown to cause perturbation of the HeLaS3 cell-cycle and increase the percentage of cells in the DNA synthesis (S) phase of the cell cycle. While either of the agents applied alone causes programmed cell death by apoptosis, the simultaneous cell damage by both agents through the altered redox balance compromised cell capacity to conduct this program and led to synergic cytotoxic cell death by necrosis.

  11. A cytotoxic substance from Sangre de Grado.

    Science.gov (United States)

    Itokawa, H; Ichihara, Y; Mochizuki, M; Enomori, T; Morita, H; Shirota, O; Inamatsu, M; Takeya, K

    1991-04-01

    Taspine has been isolated as a cytotoxic substance from Sangre de Grado, sap of Croton palanostigma (Euphorbiaceae), by bioassay guided fractionation. The cytotoxicity (IC50) of taspine was found to be 0.39 microgram/ml against KB cells and 0.17 microgram/ml against V-79 cells.

  12. Cytotoxic sesquiterpene lactones from Inula britannica.

    Science.gov (United States)

    Park, E J; Kim, J

    1998-12-01

    Cytotoxicity-guided fractionation of the flowers of Inula britannica led to the isolation of four sesquiterpene lactones, 4 alpha, 6 alpha-dihydroxyeudesman-8 beta, 12-olide (1), ergolide (2), 8-epi-helenalin (3), and bigelovin (4). Compound 1 was isolated as a new natural product. These compounds showed cytotoxicity against human tumor cell lines.

  13. Cytotoxicity and Genotoxicity Reporter Systems Based on the Use of Mammalian Cells

    Science.gov (United States)

    Baumstark-Khan, Christa; Hellweg, Christine E.; Reitz, Günther

    With the dramatic increase in the number of new agents arising from the chemical, pharmaceutical, and agricultural industries, there is an urgent need to develop assays for rapid evaluation of potential risks to man and environment. The panel of conventional tests used for cytotoxicity and genotoxicity and the strategies to progress from small scale assays to high content screening in toxicology are discussed. The properties of components necessary as sensors and reporters for new reporter assays, and the application of genetic strategies to design assays are reviewed. The concept of cellular reporters is based on the use of promoters of chemical stress-regulated genes ligated to a suitable luminescent or fluorescent reporter gene. Current reporter assays designed from constructs transferred into suitable cell lines are presented.

  14. Digital holographic microscopy overcomes the limitations of in vitro nanomaterial cytotoxicity testing

    Science.gov (United States)

    Mues, Sarah; Ketelhut, Steffi; Kemper, Björn; Schnekenburger, Jürgen

    2017-02-01

    The cytotoxic potential of nanomaterials is commonly evaluated by different cellular endpoints as reactive oxygen species formation, cell viability or cell death. Usually these parameters are determined by intensity based optical readouts that are often influenced by nanomaterial-based interferences. Here we present Digital Holographic Microscopy (DHM) as a multimodal optical method, which overcomes the limitations of conventional in vitro assays based on color or fluorescence read outs. Using cell viability WST8- and cell death LDH-assay we investigated the toxic effects of two representative silver nanomaterials. Therefore, we used a matrix of four cell lines representing different organ functions. Compared to conventional toxicity assays DHM allows time resolved proliferation monitoring which is free of assay system interactions. Also, information about time-dependent mechanisms can be obtained. Additionally, we have analyzed single macrophages for refractive index, cell volume and dry mass after the incubation to cytotoxic silver spheres. The refractive index decreased dose dependent, while cell volume and dry mass stayed constant. We therefore suggest the evaluation of these parameters in cytotoxicity assessment for further evaluation of their relevance under the applied conditions. This demonstrates DHM as valuable label-free tool for nanomaterial toxicity analysis.

  15. Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Utke, K.; Bergmann, S.; Lorenzen, Niels

    2007-01-01

    Mammalian cytotoxic T cells as part of the adaptive immune system recognize virus-infected target cells by binding of their T-cell receptors (TCR) to classical MHC class I molecules loaded with viral peptides. Our previous studies have shown that the allele of the single dominant polymorphic...... classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood...... level of the CD8 alpha gene which is a typical marker for mammalian cytotoxic T cells. Concurrently, the expression of the natural killer cell enhancement factor (NKEF)-like gene was enhanced as measured by real-time RT-PCR. Taken together, these results suggest that both innate and adaptive cell...

  16. Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer

    DEFF Research Database (Denmark)

    Hermann, G G; Petersen, K R; Steven, K

    1990-01-01

    The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those...... determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51Cr-release assays. The PBMC subsets...... that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients....

  17. Caryophyllene-rich essential oil of Didymocarpus tomentosa: chemical composition and cytotoxic activity.

    Science.gov (United States)

    Gowda, Prameela Javarayi; Ramakrishnaiah, Hanumanthaiah; Krishna, Venkatarangaiah; Narra, Sivakrishna; Jagannath, Nataraj

    2012-11-01

    The essential oil from the leaves of Didymocarpus tomentosa was extracted by hydrodistillation and analyzed by GC/FID and GC/MS. Twenty five constituents amounting to 81.6% of the oil were identified. The leaf oil contained 78.7% sesquiterpenes and 2.9% monoterpenes. The leaf essential oil of D. tomentosa is a unique caryophyllene-rich natural source containing beta-caryophyllene, caryophyllene oxide, a-humulene and humulene oxide. The cytotoxic activity of the oil was determined by the BSLT using shrimp larva and the MTT assay using HeLa tumor cell line. The oil showed significant cytotoxic activity with LC50 and IC50 values of 12.26 and 11.4 microg/mL, respectively. This is the first report on the chemical composition and cytotoxic activity of the essential oil of D. tomentosa.

  18. A partially purified Acinetobacter baumannii phage preparation exhibits no cytotoxicity in 3T3 mouse fibroblast cells

    Directory of Open Access Journals (Sweden)

    Alexandra Henein

    2016-08-01

    Full Text Available A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. The cytotoxic effect of a single Acinetobacter baumannii phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 x 109 pfu/mL showed little to no impact on the viability of 3T3 cells after 24h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required.

  19. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells.

    Science.gov (United States)

    Henein, Alexandra E; Hanlon, Geoffrey W; Cooper, Callum J; Denyer, Stephen P; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 10(9) pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required.

  20. Cytotoxicity studies of AZ31D alloy and the effects of carbon dioxide on its biodegradation behavior in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiali, E-mail: wangjialicsu@yahoo.cn [Center for Translational Medicine Research and Development, Institute of Biomedical and Health Engineering, Chinese Academy of Sciences, Shenzhen 518055 (China); Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong SAR (China); Qin, Ling [Center for Translational Medicine Research and Development, Institute of Biomedical and Health Engineering, Chinese Academy of Sciences, Shenzhen 518055 (China); Musculoskeletal Research Laboratory, Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong SAR (China); Wang, Kai [School of Humanities and Social Sciences, Hunan University of Chinese Medicine, Changsha 410208 (China); Wang, Jue; Yue, Ye [Center for Translational Medicine Research and Development, Institute of Biomedical and Health Engineering, Chinese Academy of Sciences, Shenzhen 518055 (China); Li, Yangde [Guangdong Innovation Team for Biodegradable Magnesium and Medical Implants, E-ande, Dongguan 523660 (China); Tang, Jian [Center for Translational Medicine Research and Development, Institute of Biomedical and Health Engineering, Chinese Academy of Sciences, Shenzhen 518055 (China); Li, Weirong [Guangdong Innovation Team for Biodegradable Magnesium and Medical Implants, E-ande, Dongguan 523660 (China)

    2013-10-01

    Magnesium alloys have been advocated as potential artificial bone materials due to their biocompatibility and biodegradability. The understanding of their corrosive mechanism in physiological environments is therefore essential for making application-orientated designs. Thus, this in vitro study was designed to assess the effects of CO{sub 2} on corrosive behavior of AZ31D to mimic in vivo special ingredient. Electrochemical technologies accompanied with Scanning electron microscope, Fourier transform infrared, X-ray diffraction, Energy dispersive spectroscopy and hydrogen evolution measurement were employed to analyze corrosive rates and mechanisms of AZ31D. Moreover, the biocompatibility of AZ31D was assessed with a direct cell attachment assay and an indirect cytotoxicity test in different diluted extracts. The ion concentrations in extracts were measured using inductively coupled plasma mass spectrometry to offer explanations on the differences of cell viability in the indirect test. The results of the direct cytotoxicity assay showed that the corrosive rate of AZ31D was too rapid to allow for cell adhesion. Extracts diluted less than 20 times would cause adverse effects on cell proliferation, likely due to excessive ions and gas release. Moreover, the presence of CO{sub 2} did not cause significant differences on corrosive behavior of AZ31D according to the results of electrochemical testing and hydrogen evolution measurement. This might be caused by the simultaneous process of precipitation and dissolution of MgCO{sub 3} due to the penetration role of CO{sub 2}. This analysis of corrosive atmospheres on the degradation behavior of magnesium alloys would contribute to the design of more scientific in vitro testing systems in the future. - Highlights: • We evaluate the effects of CO{sub 2} on corrosion behavior of magnesium alloys. • We assess the feasibility of commercial AZ31D alloy as potential implants. • CO{sub 2} is not the key factor to minimize

  1. Lateral flow assays.

    Science.gov (United States)

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

    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. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  2. Lateral flow assays

    Science.gov (United States)

    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

  3. Tube-Forming Assays.

    Science.gov (United States)

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

    2016-01-01

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

  4. Simultaneous Inference in Regression

    CERN Document Server

    Liu, Wei

    2010-01-01

    The use of simultaneous confidence bands in linear regression is a vibrant area of research. This book presents an overview of the methodology and applications, including necessary background material on linear models. A special chapter on logistic regression gives readers a glimpse into how these methods can be used for generalized linear models. The appendices provide computational tools for simulating confidence bands. The author also includes MATLAB[registered] programs for all examples on the web. With many numerical examples and software implementation, this text serves the needs of rese

  5. Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Chueh, Pin Ju [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medicine, China Medical University, Taichung 40402, Taiwan (China); Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan (China); Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Liang, Ruei-Yue; Lee, Yi-Hui; Zeng, Zih-Ming [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China); Chuang, Show-Mei, E-mail: smchuang@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan (China)

    2014-01-15

    Highlights: • AuNPs induce apoptosis in Vero cells. • AuNPs-induced attenuation of cell growth in NIH3T3 cells through autophagy. • Cell-cycle delay was associated with the resistance to AuNPs in MRC-5 cells. • Cell growth was continuously monitored using the measurement of cell impedance. -- Abstract: Gold nanoparticles (AuNPs) possess unique properties that have been exploited in several medical applications. However, a more comprehensive understanding of the environmental safety of AuNPs is imperative for use of these nanomaterials. Here, we describe the impacts of AuNPs in various mammalian cell models using an automatic and dye-free method for continuous monitoring of cell growth based on the measurement of cell impedance. Several well-established cytotoxicity assays were also used for comparison. AuNPs induced a concentration-dependent decrease in cell growth. This inhibitory effect was associated with apoptosis induction in Vero cells but not in MRC-5 or NIH3T3 cells. Interestingly, cDNA microarray analyses in MRC-5 cells supported the involvement of DNA damage and repair responses, cell-cycle regulation, and oxidative stress in AuNP-induced cytotoxicity and genotoxicity. Moreover, autophagy appeared to play a role in AuNPs-induced attenuation of cell growth in NIH3T3 cells. In this study, we present a comprehensive overview of AuNP-induced cytotoxicity in a variety of mammalian cell lines, comparing several cytotoxicity assays. Collectively, these assays offer convincing evidence of the cytotoxicity of AuNPs and support the value of a systematic approach for analyzing the toxicology of nanoparticles.

  6. Cytotoxicity and antiangiogenic effects of Rhus coriaria, Pistacia vera and Pistacia khinjuk oleoresin methanol extracts.

    Science.gov (United States)

    Mirian, M; Behrooeian, M; Ghanadian, M; Dana, N; Sadeghi-Aliabadi, H

    2015-01-01

    Angiogenesis, formation of new blood vessels, play an important role in some diseases such as cancer and its metastasis. Using angiogenesis inhibitors, therefore, is one of the ways for cancer treatment and prevention of metastasis. Medicinal plants have been shown to play a major role in the treatment of a variety of cancers. In this direction, cytotoxic and angiogenic effects of oleo gum resin extracts of Rhus coriaria, Pistacia vera and Pistacia khinjuk from Anacardiaceae family were studied. For IC50 values, cytotoxic effects of the plant extracts were evaluated at different concentrations (1, 10, 20, 40, 80,100 μg/ml) against human umbilical vein endothelial normal cell (HUVEC) and Y79 cell lines using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In vitro tube formation on matrigel base was used to evaluate angiogenic effects in the presence of increasing concentrations (50, 100, 250 μg/ml) of the extracts. Vascular endothelium growth factor was used as angiogenesis stimulator. Gas chromatography results showed that α-pinene and β-pinene were the major essential oils constituents of all plant extracts. According to the MTT assay results, the R. coriaria resin extract was more cytotoxic than those of P. vera and P. khinjuk extracts (IC50, 9.1 ± 1.6 vs 9.8 ± 2.1 and 12.0 ± 1.9, respectively; P<0.05). Cytotoxic effects of all extracts against Y79 cell line was significantly higher than those of HUVEC used as a normal cell line (P<0.05). Tube formation assay also showed that extract of R. coriaria resin inhibited angiogenesis