Sodium chloride and potassium sorbate: a synergistic combination against Enterococcus faecalis biofilms: an in vitro study.

PubMed

van der Waal, Suzette V; Jiang, Lei-Meng; de Soet, Johannes J; van der Sluis, Lucas W M; Wesselink, Paul R; Crielaard, Wim

2012-10-01

Incomplete disinfection of the root canal system is a major cause of post-treatment disease. This study aimed to investigate the disinfecting property of organic acid salts and sodium chloride (NaCl), in a double-hurdle strategy, on Enterococcus faecalis biofilms. First of all, the high-throughput resazurin metabolism assay (RMA) was used to test a range of organic acid salts. Then, to gain more insight into the efficacy of sorbate salt solutions, 48-h E. faecalis biofilms were evaluated in colony-forming unit (CFU) assays. Chlorhexidine (CHX) and calcium hydroxide [Ca(OH)(2) ] were tested in parallel as controls. Sorbate salt produced the largest and most significant reduction of fluorescence intensity in the RMA assay. Neither NaCl nor potassium sorbate (KS) alone induced a clinically relevant reduction of CFU counts after 1 h. Surprisingly, the combination of the two in a single solution had a synergistic effect on the inactivation of E. faecalis. Potassium sorbate amplified the efficacy of NaCl. Of the salts tested, NaCl with KS eradicated E. faecalis biofilms within 1 h. This study showed that the double-hurdle strategy indeed leads to synergistic efficacy and is a possible next step in the complete disinfection of endodontic infections. © 2012 Eur J Oral Sci.

Study on the mechanism of antibacterial action of magnesium oxide nanoparticles against foodborne pathogens

USDA-ARS?s Scientific Manuscript database

Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have strong antibacterial activities against several important foodborne pathogens. Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. Th...

Optical sensor for rapid microbial detection

NASA Astrophysics Data System (ADS)

Al-Adhami, Mustafa; Tilahun, Dagmawi; Rao, Govind; Kostov, Yordan

2016-05-01

In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes' signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

PubMed

Singh, Upasana; Akhtar, Shamim; Mishra, Abhishek; Sarkar, Dhiman

2011-02-01

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. Copyright © 2010 Elsevier B.V. All rights reserved.

Catalytic activity of various pepsin reduced Au nanostructures towards reduction of nitroarenes and resazurin

NASA Astrophysics Data System (ADS)

Sharma, Bhagwati; Mandani, Sonam; Sarma, Tridib K.

2015-01-01

Pepsin, a digestive protease enzyme, could function as a reducing as well as stabilizing agent for the synthesis of Au nanostructures of various size and shape under different reaction conditions. The simple tuning of the pH of the reaction medium led to the formation of spherical Au nanoparticles, anisotropic Au nanostructures such as triangles, hexagons, etc., as well as ultra small fluorescent Au nanoclusters. The activity of the enzyme was significantly inhibited after its participation in the formation of Au nanoparticles due to conformational changes in the native structure of the enzyme which was studied by fluorescence, circular dichroism (CD), and infra red spectroscopy. However, the Au nanoparticle-enzyme composites served as excellent catalyst for the reduction of p-nitrophenol and resazurin, with the catalytic activity varying with size and shape of the nanoparticles. The presence of pepsin as the surface stabilizer played a crucial role in the activity of the Au nanoparticles as reduction catalysts, as the approach of the reacting molecules to the nanoparticle surface was actively controlled by the stabilizing enzyme.

Paper microfluidic-based enzyme catalyzed double microreactor.

PubMed

Ferrer, Ivonne M; Valadez, Hector; Estala, Lissette; Gomez, Frank A

2014-08-01

We describe a paper microfluidic-based enzyme catalyzed double microreactor assay using fluorescent detection. Here, solutions of lactate dehydrogenase (LDH) and diaphorase (DI) were directly spotted onto the microfluidic paper-based analytical device (μPAD). Samples containing lactic acid, resazurin, and nicotinamide adenine dinucleotide oxidized form (NAD(+) ), potassium chloride (KCl), and BSA, in MES buffer were separately spotted onto the μPAD and MES buffer flowed through the device. A cascade reaction occurs upon the sample spot overlapping with LDH to form pyruvate and nicotinamide adenine dinucleotide reduced form (NADH). Subsequently, NADH is used in the conversion of resazurin to fluorescent resorufin by DI. The μPAD avoids the need of surface functionalization or enzyme immobilization steps. These microreactor devices are low cost and easy to fabricate and effect reaction based solely on buffer capillary action. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Vitro Cell Death Discrimination and Screening Method by Simple and Cost-Effective Viability Analysis.

PubMed

Helm, Katharina; Beyreis, Marlena; Mayr, Christian; Ritter, Markus; Jakab, Martin; Kiesslich, Tobias; Plaetzer, Kristjan

2017-01-01

For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H2O2 and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments. © 2017 The Author(s)Published by S. Karger AG, Basel.

High Throughput Assay for Bacterial Adhesion on Acellular Dermal Matrices and Synthetic Surgical Materials

PubMed Central

Nyame, Theodore T.; Lemon, Katherine P.; Kolter, Roberto; Liao, Eric C.

2013-01-01

Background There has been increasing use of various synthetic and biologically derived materials in surgery. Biologic surgical materials are used in many plastic surgery procedures, ranging from breast reconstruction to hernia repairs. In particular, acellular dermal matrix (ADM) material has gained popularity in these applications. There is a paucity of data on how ADM compares to other surgical materials as a substrate for bacterial adhesion, the first step in formation biofilm, which occurs in prosthetic wound infections. We have designed a high throughput assay to evaluate Staphylococcus aureus adherence on various synthetic and biologically derived materials. Methods Clinical isolates of Staphylococcus aureus (strains SC-1 and UAMS-1) were cultured with different materials and bacterial adherence was measured using a resazurin cell vitality reporter microtiter assay. Four materials that are commonly utilized in reconstructive procedures were evaluated: prolene mesh, vicryl mesh, and two different ADM preparations (AlloDerm®, FlexHD®). We were able to develop a high throughput and reliable assay for quantifying bacterial adhesion on synthetic and biologically derived materials. Results The resazurin vitality assay can be reliably used to quantify bacterial adherence to acellular dermal matrix material, as well as synthetic material. S. aureus strains SC-1 and UAMS-1 both adhered better to ADM materials (AlloDerm® vs. FlexHD®) than to the synthetic material prolene. S. aureus also adhered better to vicryl than to prolene. Strain UAMS-1 adhered better to vicryl and ADM materials than did strain SC-1. Conclusion Our results suggest that S. aureus adheres more readily to ADM material than to synthetic material. We have developed an assay to rapidly test bacterial formation on surgical materials, using two S. aureus bacterial strains. This provides a standard method to evaluate existing and new materials with regard to bacterial adherence and potential propensity for infection. This assay is particularly important in the clinical context of the severe sequelae of post-operative infection. PMID:22030489

Graphene oxide as a photocatalytic material

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnamoorthy, Karthikeyan; Mohan, Rajneesh; Kim, S.-J.

2011-06-13

The photocatalytic characteristics of graphene oxide (GO) nanostructures synthesized by modified Hummer's method were investigated by measuring reduction rate of resazurin (RZ) into resorufin (RF) as a function of UV irradiation time. The progress of the photocatalytic reaction was monitored by change in color from blue (RZ) into pink (RF) followed by absorption spectra. It exhibited excellent photocatalytic activity, leading to the reduction of RZ in UV irradiation. The fitting of absorbance maximum versus time suggests that the reduction of RZ follow the pseudo first-order reaction kinetics. These results indicate that GO have great potential for use as a photocatalyst.

Rapid Detection of Microbial Contamination Using a Microfluidic Device.

PubMed

Al-Adhami, Mustafa; Tilahun, Dagmawi; Rao, Govind; Gurramkonda, Chandrasekhar; Kostov, Yordan

2017-01-01

A portable kinetics fluorometer is developed to detect viable cells which may be contaminating various samples. The portable device acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye and plots it. The slope of the plot depends on the number of colony forming units per milliliter. The device uses resazurin as the indicator dye. Viable cells reduce resazurin to resorufin, which is more fluorescent. Photodiode is used to detect fluorescence change. The photodiode generated current proportional to the intensity of the light that reached it, and an op-amp is used in a transimpedance differential configuration to ensure amplification of the photodiode's signal. A microfluidic chip is designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the resazurin reduction rate. In tests, the E. coli-containing media are injected into the microfluidic chip and the device is able to detect the presence of E. coli in LB media based on the fluorescence change that occurred in the indicator dye. The device provides fast, accurate, and inexpensive means to optical detection of the presence of viable cells and could be used in the field in place of more complex methods, i.e., loop-meditated isothermal amplification of DNA (LAMP) to detect bacteria in pharmaceutical samples (Jimenez et al., J Microbiol Methods 41(3):259-265, 2000) or measuring the intrinsic fluorescence of the bacterial or yeast chromophores (Estes et al., Biosens Bioelectron 18(5):511-519, 2003).

Synthesis and characterization of silver nanoparticles from Alpinia calcarata by Green approach and its applications in bactericidal and nonlinear optics

NASA Astrophysics Data System (ADS)

Pugazhendhi, S.; Kirubha, E.; Palanisamy, P. K.; Gopalakrishnan, R.

2015-12-01

Development of green route for the synthesis of nanoparticles with plant extracts plays a very important role in nanotechnology without any toxicity chemicals. Herein we report a new approach to synthesize silver nanoparticles (AgNPs) using aqueous extract of Alpinia calcarata root as a reducing as well as stabilizing agent. The crystal structure and purity of the synthesized AgNPs were studied using Powder X-ray Diffraction analysis. The Surface Plasmon Resonance bands of synthesized silver nanoparticles have been obtained and monitored using UV-Visible spectrum. The morphologies of the AgNPs were analyzed using High resolution transmission electron microscopy (HRTEM). The elements present in the A. calcarata extract were determined by the inductively coupled plasma-optical emission Spectrometry (ICP-OES) and Fourier transform infrared spectroscopy (FTIR). Silver nanoparticles from A. calcarata possess very good antimicrobial activity which was confirmed by resazurin dye reduction assay method and thus it is a potential source of antimicrobial agent. The synthesized Ag nanoparticles exhibit good optical nonlinearity and the nonlinear optical studies have been carried out by Z-scan technique.

Pathogenic features and characteristics of food borne pathogens biofilm: Biomass, viability and matrix.

PubMed

Lin, Shiqi; Yang, Ling; Chen, Gu; Li, Bing; Chen, Dingqiang; Li, Lin; Xu, Zhenbo

2017-10-01

Biofilm is a ubiquitous growth pattern of bacterial species survival but is notorious for its threat on public health and food contamination. Extensive studies of the biofilm structure, formation, quantification, quorum sensing system and underlying control strategies have been reported during the past decades. Insightful elucidation of the pathogenic features and characteristic of bacterial biofilm can facilitate in devising appropriate control strategies for biofilm eradication. Therefore, this review mainly summarized the pathogenic features of biofilms from food borne microorganisms, including the biomass (which could be quantified using crystal violet and fluorogenic dye Syto9 assays), viability (which could be determined by tetrazolium salts, fluorescein diacetate, resazurin staining and alamar blue assays) and matrix (which are commonly detected by dimethyl methylene blue and wheat germ agglutinin assays). In addition, three features were further compared with its particular benefits in specific application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Allium sativum Constituents Exhibit Anti-tubercular Activity In vitro and in RAW 264.7 Mouse Macrophage Cells Infected with Mycobacterium tuberculosis H37Rv

PubMed Central

Nair, Swapna S.; Gaikwad, Sujay S.; Kulkarni, Savita P.; Mukne, Alka Pravin

2017-01-01

Background: Long duration of treatment, side-effects of currently used anti-tubercular drugs and emergence of drug-resistant forms of Mycobacterium tuberculosis (MTB) warrants the need to develop new drugs to tackle the scourge of tuberculosis (TB). Garlic is an edible plant reported to have anti-tubercular activity. However, previous researches on anti-tubercular effect of garlic were focused mostly on preliminary in vitro screening. Objective: To identify constituents responsible for anti-tubercular activity of thiosulfinate-derivative rich extract of garlic (GE) and to evaluate activity of the most active constituent in RAW 264.7 mouse macrophage cells infected with M. tuberculosis H37Rv (MTBH). Materials and Methods: In the present study, we have isolated eight compounds from GE by flash chromatography. The isolated compounds were characterized by 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry and Fourier transform infrared spectroscopy. Individual isolates and GE were screened for activity against MTBH by Resazurin Microtitre Plate Assay (REMA). Results: Anti-tubercular activity of GE was superior to that of isolates when evaluated by REMA, possibly due to synergism amongst the constituents of GE. Cytotoxicity of GE was evaluated in RAW 264.7 mouse macrophage cells and it was observed that GE had a favorable selectivity index (>10). Therefore, anti-tubercular activity of GE was further evaluated by intracellular macrophage infection model. GE demonstrated concentration-dependent activity in macrophages infected with MTBH. Conclusion: This is the first report on intracellular anti-tubercular activity of any extract of garlic or its components. Appreciable intracellular anti-tubercular activity of GE in macrophages combined with low cytotoxicity makes it a suitable candidate for further development as an anti-tubercular agent. SUMMARY Thiosulfinate-derivative rich extract of Allium sativum showed better activity than its isolated constituents against Mycobacterium tuberculosis H37Rv.(MTBH) when evaluated by Resazurin Microtitre Plate AssayThe extract showed least cytotoxic potential against RAW 264.7 mouse macrophage cells as compared to rifampicin, isoniazid and ethambutol when evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The extract had an appreciable selectivity indexExtract showed appreciable activity in RAW 264.7 mouse macrophage cells infected with MTBH, indicating its potential to be developed further as an anti-tubercular agent that can be administered as an adjunct to the existing anti-tubercular drug regimen. Abbreviations used: TB: Tuberculosis, MTB: Mycobacterium tuberculosis, MTBH: Mycobacterium tuberculosis H37Rv, GE: Thiosulfinate-derivative rich extract of garlic, REMA: Resazurin Microtitre Plate Assay, VD: Vinyldithiin, CFU: Colony forming unit, 1H NMR: 1H nuclear magnetic resonance spectroscopy, FT-IR: Fourier transform-infrared spectroscopy, LC-MS: Liquid chromatography-mass spectrometry, IC50: Concentration required to inhibit the cells by 50%, ANOVA: Analysis of variance. PMID:28808382

The effect of Fe2NiO4 and Fe4NiO4Zn magnetic nanoparticles on anaerobic digestion activity.

PubMed

Chen, Jian Lin; Steele, Terry W J; Stuckey, David C

2018-06-11

Two types of magnetic nanoparticles (MNPs), i.e. Ni ferrite nanoparticles (Fe 2 NiO 4 ) and Ni Zn ferrite nanoparticles (Fe 4 NiO 4 Zn) containing the trace metals Ni and Fe, were added to the anaerobic digestion of synthetic municipal wastewater at concentrations between 1 and 100 mg Ni L -1 in order to compare their effects on biogas (methane) production and sludge activity. Using the production of methane over time as a measure, the assays revealed that anaerobic digestion was stimulated by the addition of 100 mg Ni L -1 in Fe 2 NiO 4 NPs, while it was inhibited by the addition of 1-100 mg Ni L -1 in Fe 4 NiO 4 Zn NPs. Especially at 100 mg Ni L -1 , Fe 4 NiO 4 Zn NPs resulted in a total inhibition of anaerobic digestion. The metabolic activity of the anaerobic sludge was tested using the resazurin reduction assay, and the assay clearly revealed the negative effect of Fe 4 NiO 4 Zn NPs and the positive effect of Fe 2 NiO 4 NPs. Re-feeding fresh synthetic medium reactivated the NPs added to the anaerobic sludge, except for the experiment with 100 mg Ni L -1 addition of Fe 4 NiO 4 Zn NPs. The findings in this present study indicate a possible new strategy for NPs design to enhance anaerobic digestion. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Comparison of effects of cryopreservation diluents on the ability of Ram sperm to reduce resazurin dye

USDA-ARS?s Scientific Manuscript database

Resazurin dye is an effective way to test the metabolism of sperm. As sperm move, they create metabolic waste which is detected by the dye. Another way sperm are evaluated is by Computer-Assisted Sperm Analysis (CASA). CASA detects motility, progression, curvilinear velocity, lateral head amplitude,...

Delamanid susceptibility testing of Mycobacterium tuberculosis using the resazurin microtitre assay and the BACTEC™ MGIT™ 960 system.

PubMed

Schena, Elisa; Nedialkova, Lubov; Borroni, Emanuele; Battaglia, Simone; Cabibbe, Andrea Maurizio; Niemann, Stefan; Utpatel, Christian; Merker, Matthias; Trovato, Alberto; Hofmann-Thiel, Sabine; Hoffmann, Harald; Cirillo, Daniela Maria

2016-06-01

The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Poly(propylene glycol) and urethane dimethacrylates improve conversion of dental composites and reveal complexity of cytocompatibility testing.

PubMed

Walters, Nick J; Xia, Wendy; Salih, Vehid; Ashley, Paul F; Young, Anne M

2016-02-01

To determine the effects of various monomers on conversion and cytocompatibility of dental composites and to improve these properties without detrimentally affecting mechanical properties, depth of cure and shrinkage. Composites containing urethane dimethacrylate (UDMA) or bisphenol A glycidyl methacrylate (Bis-GMA) with poly(propylene glycol) dimethacrylate (PPGDMA) or triethylene glycol dimethacrylate (TEGDMA) were characterized using the following techniques: conversion (FTIR at 1 and 4mm depths), depth of cure (BS EN ISO 4049:2009 and FTIR), shrinkage (BS EN ISO 17304:2013 and FTIR), strength and modulus (biaxial flexural test) and water sorption. Cytocompatibility of composites and their liquid phase components was assessed using three assays (resazurin, WST-8 and MTS). UDMA significantly improved conversion, BFS and depth of cure compared to Bis-GMA, without increasing shrinkage. UDMA was cytotoxic at lower concentrations than Bis-GMA, but extracts of Bis-GMA-containing composites were less cytocompatible than of those containing UDMA. PPGDMA improved conversion and depth of cure compared to TEGDMA, without detrimentally affecting shrinkage. TEGDMA was shown by all assays to be highly toxic. Resazurin, but not WST-8 and MTS, suggested that PPGDMA exhibited improved cytocompatibility compared to TEGDMA. The use of UDMA and PPGDMA results in composites with excellent conversion, depth of cure and mechanical properties, without increasing shrinkage. Composites containing UDMA appear to be slightly more cytocompatible than those containing Bis-GMA. These monomers may therefore improve the material properties of dental restorations, particularly bulk fill materials. The effect of diluent monomer on cytocompatibility requires further investigation. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Low cytotoxicity of anisotropic gold nanoparticles coated with lysine on peripheral blood mononuclear cells "in vitro".

PubMed

Avila-Alejo, Jorge O; González-Palomo, Ana K; Plascencia-Villa, Germán; José-Yacamán, Miguel; Navarro-Contreras, Hugo R; Pérez-Maldonado, Iván N

2017-12-01

The aim of this study was to evaluate the cytotoxic effects of anisotropic (non spherical morphologies) gold nanoparticles coated with the amino acid Lysine (Lys) on peripheral blood mononuclear cells (PBMC) "in vitro". Gold (Au) nanoparticles tested in this study were synthesized by a seed-mediated growth using Lys as a structure and shape directing agent. Cytotoxic effects were evaluated by cell viability (resazurin assay), reactive oxygen species (ROS) induction (2',7'-dichlorofluorescein diacetate assay), DNA damage (comet assay) and apoptosis/necrosis (AnnexinV/propidium iodide assay) after PBMC were exposed to increasing concentrations (10, 25, 50, 100, and 250μM) of AuNPs coated with Lys (AuNPs-Lys) at different exposure times (3, 6, 12, and 24h). The results demonstrated that AuNPs-Lys exhibited low cytotoxicity towards PBMC, (high cell viability), with low levels of ROS, DNA damage and apoptosis/necrosis detected after treatment. These data suggest that AuNPs-Lys, might be viable for biomedical application subject to further investigations. Copyright © 2017 Elsevier B.V. All rights reserved.

Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170.

PubMed

de Sousa Araújo, Pedro Soares; de Oliveira, Simone Santiago Carvalho; d'Avila-Levy, Claudia Masini; Dos Santos, André Luis Souza; Branquinha, Marta Helena

2018-05-04

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.

Effects of Fungicides on Aquatic Fungi and Bacteria

NASA Astrophysics Data System (ADS)

Conners, D. E.; Rosemond, A. D.; Black, M. C.

2005-05-01

Aquatic microorganisms play an important role in conditioning leaf litter that enters streams and serves as an important base of production for consumers. Contamination of streams by fungicides may adversely affect microorganisms and alter leaf litter processing rates. Unfortunately, microorganisms are rarely used in acute toxicity tests for fungicide evaluation and registration. We adapted the resazurin reduction assay, which is used in medical microbiology, to assess the acute toxicity of four fungicides (azoxystrobin, trifloxystrobin, kresoxim-methyl and chlorothalonil) to aquatic fungi (Articulospora tetracladia) and bacteria (Cytophaga spp.), and investigated the ability of the toxicants to inhibit leaf breakdown in microcosms. Fungi were more sensitive to fungicides than many standard test organisms (cladocerans, green algae, trout), while bacteria were often the least sensitive. All of the fungicides except kresoxim-methyl, when added to microcosms at concentrations that inhibited the fungi by 90 percent in acute tests, reduced leaf breakdown rates by an average of 14.7 percent. Thus, aquatic fungi and their associated functions in streams may be relatively sensitive to fungicides applied terrestrially that enter streams through non-point sources. These data highlight the importance of including aquatic fungi in safety assessments of pesticides for protection of microbial function.

Development of L-lactate dehydrogenase biosensor based on porous silicon resonant microcavities as fluorescence enhancers.

PubMed

Jenie, S N Aisyiyah; Prieto-Simon, Beatriz; Voelcker, Nicolas H

2015-12-15

The up-regulation of L-lactate dehydrogenase (LDH), an intracellular enzyme present in most of all body tissues, is indicative of several pathological conditions and cellular death. Herein, we demonstrate LDH detection using porous silicon (pSi) microcavities as a luminescence-enhancing optical biosensing platform. Non-fluorescent resazurin was covalently attached onto the pSi surface via thermal hydrocarbonisation, thermal hydrosylilation and acylation. Each surface modification step was confirmed by means of FTIR and the optical shifts of the resonance wavelength of the microcavity. Thermal hydrocarbonisation also afforded excellent surface stability, ensuring that the resazurin was not reduced on the pSi surface. Using a pSi microcavity biosensor, the fluorescence signal upon detection of LDH was amplified by 10 and 5-fold compared to that of a single layer and a detuned microcavity, respectively, giving a limit of detection of 0.08 U/ml. The biosensor showed a linear response between 0.16 and 6.5 U/ml, covering the concentration range of LDH in normal as well as damaged tissues. The biosensor was selective for LDH and did not produce a signal upon incubation with another NAD-dependant enzyme L-glutamic dehydrogenase. The use of the pSi microcavity as a sensing platform reduced reagent usage by 30% and analysis time threefold compared to the standard LDH assay in solution. Copyright © 2015 Elsevier B.V. All rights reserved.

Diaphorase Coupling Protocols for Red-Shifting Dehydrogenase Assays

PubMed Central

Davis, Mindy I.; Shen, Min; Simeonov, Anton

2016-01-01

Abstract Dehydrogenases are an important target for the development of cancer therapeutics. Dehydrogenases either produce or consume NAD(P)H, which is fluorescent but at a wavelength where many compounds found in chemical libraries are also fluorescent. By coupling dehydrogenases to diaphorase, which utilizes NAD(P)H to produce the fluorescent molecule resorufin from resazurin, the assay can be red-shifted into a spectral region that reduces interference from compound libraries. Dehydrogenases that produce NAD(P)H, such as isocitrate dehydrogenase 1 (IDH1), can be read in kinetic mode. Dehydrogenases that consume NAD(P)H, such as mutant IDH1 R132H, can be read in endpoint mode. Here, we report protocols for robust and miniaturized 1,536-well assays for WT IDH1 and IDH1 R132H coupled to diaphorase, and the counterassays used to further detect compound interference with the coupling reagents. This coupling technique is applicable to dehydrogenases that either produce or consume NAD(P)H, and the examples provided here can act as guidelines for the development of high-throughput screens against this enzyme class. PMID:27078681

Resazurin as a Proxy for Estimating Stream Respiration

NASA Astrophysics Data System (ADS)

Gonzalez Pinzon, R. A.; Haggerty, R.; Argerich, A.; Briggs, M.; Lautz, L. K.; Lemke, D.; Hare, D. K.

2010-12-01

Hydrologic retention in stream ecosystems favors the reactions of solutes and nutrients in metabolically active transient storage (MATS) zones. These zones are hot spots where metabolic activity is expected to contribute significantly to ecosystem respiration. We compare the results of a series of coinjections of resazurin (Raz) as a redox sensitive tracer, and NaCl as a conservative tracer to investigate the function of MATS zones. Raz is a dye that undergoes an irreversible reduction to resorufin (Rru) when exposed to aerobic respiration. To characterize the transformation of Raz we measured the BTC of the tracers at the boundary conditions, and during plateau concentrations we sampled the longitudinal profile of surface water. We also used the two-station diel technique to quantify gross primary production (GPP) and community respiration (CR) within the reaches. Injections have been performed in streams with different morphology, streambed composition, and riparian vegetation in Oregon-USA (WS 1 and WS 3 in the HJ Andrews Forest LTER, and Drift Creek), Spain (Riera de Santa Fe del Montseny, Catalonia) and Wyoming-USA (Cherry Creek). The results support the idea that under different ranges of community respiration, the transformation of Raz to Rru is a proxy for quantifying MATS, characterizing spatial heterogeneity in respiration rates, and ultimately, could be used to estimate ecosystem respiration in environments where direct measurement is challenging.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

PubMed

Lim, Kah Tee; Zahari, Zuriati; Amanah, Azimah; Zainuddin, Zafarina; Adenan, Mohd Ilham

2016-03-01

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Combining biofilm matrix measurements with biomass and viability assays in susceptibility assessments of antimicrobials against Staphylococcus aureus biofilms.

PubMed

Skogman, Malena Elise; Vuorela, Pia Maarit; Fallarero, Adyary

2012-09-01

Despite that three types of assays (measuring biofilm viability, biomass, or matrix) are described to assess anti-biofilm activity, they are rarely used together. As infections can easily reappear if the matrix is not affected after antibiotic treatments, our goal was to explore the simultaneous effects of antibiotics on the viability, biomass and matrix of Staphylococcus aureus biofilms (ATCC 25923). Viability and biomass were quantified using resazurin and crystal violet staining sequentially in the same plate, while matrix staining was conducted with a wheat germ agglutinin-Alexa Fluor 488 fluorescent conjugate. Establishment of the detection limits and linearity ranges allowed concluding that all three methods were able to estimate biofilm formation in a similar fashion. In a susceptibility study with 18-h biofilms, two model compounds (penicillin G and ciprofloxacin) caused a reduction on the viability and biomass accompanied by an increase or not changed levels of the matrix, respectively. This response pattern was also proven for S. aureus Newman, S. epidermidis and E. coli biofilms. A classification of antibiotics based on five categories according to their effects on viability and matrix has been proposed earlier. Our data suggests a sixth group, represented by penicillin, causing decrease in bacterial viability but showing stimulatory effects on the matrix. Further, if effects on the matrix are not taken into account, the long-term chemotherapeutic effect of antibiotics can be jeopardized in spite of the positive effects on biofilms viability and biomass. Thus, measuring all these three endpoints simultaneously provide a more complete and accurate picture.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Multimodality molecular imaging and extracellular vesicle release based genetic profiling with porphyrin nanodroplets (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zemp, Roger J.; Paproski, Robert J.

2017-03-01

For emerging tissue-engineering applications, transplants, and cell-based therapies it is important to assess cell viability and function in vivo in deep tissues. Bioluminescence and fluorescence methods are poorly suited to deep monitoring applications with high resolution and require genetically-engineered reporters which are not always feasible. We report on a method for imaging cell viability using deep, high-resolution photoacoustic imaging. We use an exogenous dye, Resazurin, itself weakly fluorescent until it is reduced from blue to a pink color with bright red fluorescence. Upon cell death fluorescence is lost and an absorption shift is observed. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration. We detect colorimetric absorption shifts using multispectral photoacoustic imaging and quantify the fraction of viable cells. SKOV-3 cells with and without ±80oC heat treatment were imaged after Resazurin treatment. High 575nm:620nm ratiometric absorption and photoacoustic signals in viable cells were observed with a much lower ratio in low-viability populations.

Comparison of the antibiotic activities of Daptomycin, Vancomycin, and the investigational Fluoroquinolone Delafloxacin against biofilms from Staphylococcus aureus clinical isolates.

PubMed

Siala, Wafi; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M; Hallin, Marie; Denis, Olivier; Van Bambeke, Françoise

2014-11-01

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726-2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparison of the Antibiotic Activities of Daptomycin, Vancomycin, and the Investigational Fluoroquinolone Delafloxacin against Biofilms from Staphylococcus aureus Clinical Isolates

PubMed Central

Siala, Wafi; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.; Hallin, Marie; Denis, Olivier

2014-01-01

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726–2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH. PMID:25114142

Synthesis and Evaluation of Cytotoxic Activity of Some Pyrroles and Fused Pyrroles.

PubMed

Fatahala, Samar S; Mohamed, Mosaad S; Youns, Mahmoud; Abd-El Hameed, Rania H

2017-01-01

Pyrrole derivatives represent a very interesting class as biologically active compounds. The objective of our study was to investigate the cytotoxic and apoptotic effects and antioxidant activity of the newly synthesized pyrrole derivatives. A series of novel pyrroles and fused pyrroles (tetrahydroindoles, pyrrolopyrimidines, pyrrolopyridines and pyrrolotriazines) were synthesized and characterized using IR, 1H NMR, 13C NMR, MS and elemental analysis techniques. The antiproliferative activity of our synthesized compounds and their modulatory effect apoptotic pathway were investigated. The effect on cellular proliferation and viability was monitored by resazurin assay. Apoptotic effect was evaluated by caspase glo 3/7 assay. Synthesized compounds are then tested for their anticancer activities against three different cell lines representing three different tumor types, namely; the HepG-2 (Human hepatocellular liver carcinoma cell line), the human MCF-7 cell line (breast cancer) and the pancreatic resistant Panc-1 cells. Compounds Ia-e, IIe, and IXc, d showed a promising anti-cancer activity on all tested cell lines. Antioxidant and wound healing invasion assays were examined for promising anticancer candidate compounds. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Preparation of Laponite Bioceramics for Potential Bone Tissue Engineering Applications

PubMed Central

Li, Kai; Ju, Yaping; Li, Jipeng; Zhang, Yongxing; Li, Jinhua; Liu, Xuanyong; Shi, Xiangyang; Zhao, Qinghua

2014-01-01

We report a facile approach to preparing laponite (LAP) bioceramics via sintering LAP powder compacts for bone tissue engineering applications. The sintering behavior and mechanical properties of LAP compacts under different temperatures, heating rates, and soaking times were investigated. We show that LAP bioceramic with a smooth and porous surface can be formed at 800°C with a heating rate of 5°C/h for 6 h under air. The formed LAP bioceramic was systematically characterized via different methods. Our results reveal that the LAP bioceramic possesses an excellent surface hydrophilicity and serum absorption capacity, and good cytocompatibility and hemocompatibility as demonstrated by resazurin reduction assay of rat mesenchymal stem cells (rMSCs) and hemolytic assay of pig red blood cells, respectively. The potential bone tissue engineering applicability of LAP bioceramic was explored by studying the surface mineralization behavior via soaking in simulated body fluid (SBF), as well as the surface cellular response of rMSCs. Our results suggest that LAP bioceramic is able to induce hydroxyapatite deposition on its surface when soaked in SBF and rMSCs can proliferate well on the LAP bioceramic surface. Most strikingly, alkaline phosphatase activity together with alizarin red staining results reveal that the produced LAP bioceramic is able to induce osteoblast differentiation of rMSCs in growth medium without any inducing factors. Finally, in vivo animal implantation, acute systemic toxicity test and hematoxylin and eosin (H&E)-staining data demonstrate that the prepared LAP bioceramic displays an excellent biosafety and is able to heal the bone defect. Findings from this study suggest that the developed LAP bioceramic holds a great promise for treating bone defects in bone tissue engineering. PMID:24955961

Cytotoxicity of compounds from Xylopia aethiopica towards multi-factorial drug-resistant cancer cells.

PubMed

Kuete, Victor; Sandjo, Louis P; Mbaveng, Armelle T; Zeino, Maen; Efferth, Thomas

2015-12-15

Multidrug resistance (MDR) in cancer represent a major hurdle in chemotherapy. Previously, the methanol extract of the medicinal spice Xylopia aethiopica displayed considerable cytotoxicity against multidrug resistant (MDR) cancer cell lines. The present study was designed to assess the cytotoxicity of compounds, 16α-hydroxy-ent-kauran-19-oic acid (2), 3,4',5-trihydroxy-6″,6″-dimethylpyrano[2,3-g]flavone (3), isotetrandrine (5) and trans-tiliroside (6) derived from the methanol crude extract of Xylopia aethiopica against 9 drug-sensitive and -resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. Flavonoid 3 and alkaloid 5 also displayed IC50 values ranging from 2.61 µM (towards leukemia CCRF-CEM cells) to 18.60 µM (towards gliobastoma multiforme U87MG.ΔEGFR cells) and from 1.45 µM (towards HepG2 cells) to 7.28 µM (towards MDA-MB-231-pcDNA cells), respectively. IC50 values ranged from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Compound 3 induced apoptosis in leukemia CCRF-CEM cells mediated by the disruption of the MMP, whilst 5 induced apoptosis mediated by ROS production. Compounds 2 and 5 represent potential cytotoxic phytochemicals that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers. Copyright © 2015 Elsevier GmbH. All rights reserved.

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

PubMed Central

Sykes, Melissa L.; Avery, Vicky M.

2015-01-01

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed. PMID:27120069

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes.

PubMed

Sykes, Melissa L; Avery, Vicky M

2015-12-01

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Novel Assay for Easy and Rapid Quantification of Helicobacter pylori Adhesion.

PubMed

Skindersoe, Mette E; Rasmussen, Lone; Andersen, Leif P; Krogfelt, Karen A

2015-06-01

Reducing adhesion of Helicobacter pylori to gastric epithelial cells could be a new way to counteract infections with this organism. We here present a novel method for quantification of Helicobacter pylori adhesion to cells. Helicobacter pylori is allowed to adhere to AGS or MKN45g cells in a 96-well microtiter plate. Then wells are added saponin, which lyses the cells without affecting the bacteria. After addition of alamarBlue(®) (resazurin) and 1- to 2-hour incubation, fluorescence measurements can be used to quantify the number of adherent bacteria. By use of the method, we demonstrate that adhesion of both a sabA and babA deletion mutant of H. pylori is significantly reduced compared to the wild type. The method offers a number of applications and may be used to compare the adherence potential of different strains of H. pylori to either cells or different materials or to screen for potential anti-adhesive compounds. The results presented here suggest that this easy and reproducible assay is well suited for quantitative investigation of H. pylori adhesion. © 2015 John Wiley & Sons Ltd.

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

PubMed

Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

2016-04-01

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. © 2016 Society for Laboratory Automation and Screening.

Interlab study on nanotoxicology of representative graphene oxide

NASA Astrophysics Data System (ADS)

Durán, Nelson; Martinez, Diego S. T.; Justo, Giselle Z.; de Lima, Renata; Lúcia de Castro, Vera; Umbuzeiro, Gisela A.; Barbieri, Edison; Durán, Marcela; Melo, Patricia S.; Alves, Oswaldo L.; Fávaro, Wagner J.

2015-05-01

The graphene sample GO:Single-layer graphene oxide, purity 99%, thickness 0.7-1.2 nm (AFM); ∼300-800nm X&Y dimensions is the standard size <450 nm & 1-20 μm lateral dimensions. Cheap Tubes Inc., Bratleboro, USA was selected for our study. Exhaustive characterization of GO was afforded. It exhibited thermal stability over 60oC and it was suspended in deionized water after ultrasonication(1 mg/mL) (stable 10 days). All the biological fluids used in the different assays were used as control of the colloidal suspension stability. Then, all the studies were carried out within that stability period. The cytotoxicity assays were carried out by the Alamar Blue (Resazurin) reduction, MTT and flow cytometry assays in mouse embryonic fibroblast cells (3T3), human keratinocytes (HaCaT), colorectal cancer cells (Caco-2/HCT 116), Lewis lung cancer cells (3LL), acute myeloid leukemia cells (KG-1, Jurkat, Kasumi-1) and chronic myeloid leukemia cells (K562, Lucena) and no significant toxicity was found after exposition to 0.1-100 μg/mL for 24 and 48 h. Breast cancer cells, MCF-7, showed a 20% reduction on cell viability at 24 and 48 h. No cytotoxicity were found in lymphocytes, Chinese hamster ovary cells (CHO) and human macrophage cell line (U937) at 0.1-50 μg/mL, but 30-50% survival inhibition was observed at 100 μg/mL. A dose-dependent increase in apoptosis was observed in some cells (Kasumi-1, Jurkatand K562 cells). In the case of CHO and 3T3 cells, greater levels of necrosis with increasing concentrations of GO (>50 μg/mL) were observed. Genotoxic study using the Comet assay showed slight DNA damage in lymphocytes at all concentrations tested, while more significant effects was observed in CHO cells. Econanotoxicity was carried out by lethality assays in the nematode Caenorhabditis elegans, d in the freshwater coelenterate Hydra, Daphania amd in Shrimp with no signs of toxicity at concentrations varying from 0.1-100 μg/mL of GO. However, death and disintegration of Hydra was observed after exposition to 100 μg/mL for 72 h. In in vivo studies, no changes in biochemical parameters of Fischer 344 rats were observed after the i.p. administration of GO. Some black agglomerates were found in the intraperitoneal cavity of rats injected with GO. However, in Fisher 344 rats-bearing prostate tumors, treatment with GO (up to 100 μg/mL) negatively affected the hepatic parameters, whilst in the renal ones, an improvement was observed. Studies are in progress to understand the mechanisms involved in the uptake of GO by RES. GO appears as a potential non-toxic in vitro and in vivo assays at the concentrations used in this interlab experiments.

The prediction of human skin responses by using the combined in vitro fluorescein leakage/Alamar Blue (resazurin) assay.

PubMed

Clothier, Richard; Starzec, Gemma; Pradel, Lionel; Baxter, Victoria; Jones, Melanie; Cox, Helen; Noble, Linda

2002-01-01

A range of cosmetics formulations with human patch-test data were supplied in a coded form, for the examination of the use of a combined in vitro permeability barrier assay and cell viability assay to generate, and then test, a prediction model for assessing potential human skin patch-test results. The target cells employed were of the Madin Darby canine kidney cell line, which establish tight junctions and adherens junctions able to restrict the permeability of sodium fluorescein across the barrier of the confluent cell layer. The prediction model for interpretation of the in vitro assay results included initial effects and the recovery profile over 72 hours. A set of the hand-wash, surfactant-based formulations were tested to generate the prediction model, and then six others were evaluated. The model system was then also evaluated with powder laundry detergents and hand moisturisers: their effects were predicted by the in vitro test system. The model was under-predictive for two of the ten hand-wash products. It was over-predictive for the moisturisers, (two out of six) and eight out of ten laundry powders. However, the in vivo human patch test data were variable, and 19 of the 26 predictions were correct or within 0.5 on the 0-4.0 scale used for the in vivo scores, i.e. within the same variable range reported for the repeat-test hand-wash in vivo data.

Accelerating bacterial growth detection and antimicrobial susceptibility assessment in integrated picoliter droplet platform.

PubMed

Kaushik, Aniruddha M; Hsieh, Kuangwen; Chen, Liben; Shin, Dong Jin; Liao, Joseph C; Wang, Tza-Huei

2017-11-15

There remains an urgent need for rapid diagnostic methods that can evaluate antibiotic resistance for pathogenic bacteria in order to deliver targeted antibiotic treatments. Toward this end, we present a rapid and integrated single-cell biosensing platform, termed dropFAST, for bacterial growth detection and antimicrobial susceptibility assessment. DropFAST utilizes a rapid resazurin-based fluorescent growth assay coupled with stochastic confinement of bacteria in 20 pL droplets to detect signal from growing bacteria after 1h incubation, equivalent to 2-3 bacterial replications. Full integration of droplet generation, incubation, and detection into a single, uninterrupted stream also renders this platform uniquely suitable for in-line bacterial phenotypic growth assessment. To illustrate the concept of rapid digital antimicrobial susceptibility assessment, we employ the dropFAST platform to evaluate the antibacterial effect of gentamicin on E. coli growth. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-06-28

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

Screening of the MMV and GSK open access chemical boxes using a viability assay developed against the kinetoplastid Crithidia fasciculata.

PubMed

Kipandula, Wakisa; Young, Simon A; MacNeill, Stuart A; Smith, Terry K

2018-06-01

Diseases caused by the pathogenic kinetoplastids continue to incapacitate and kill hundreds of thousands of people annually throughout the tropics and sub-tropics. Unfortunately, in the countries where these neglected diseases occur, financial obstacles to drug discovery and technical limitations associated with biochemical studies impede the development of new, safe, easy to administer and effective drugs. Here we report the development and optimisation of a Crithidia fasciculata resazurin viability assay, which is subsequently used for screening and identification of anti-crithidial compounds in the MMV and GSK open access chemical boxes. The screening assay had an average Z' factor of 0.7 and tolerated a maximum dimethyl sulfoxide concentration of up to 0.5%. We identified from multiple chemical boxes two compound series exhibiting nanomolar potency against C. fasciculata, one centred around a 5-nitrofuran-2-yl scaffold, a well-known moiety in several existing anti-infectives, and another involving a 2-(pyridin-2-yl) pyrimidin-4-amine scaffold which seems to have pan-kinetoplastid activity. This work facilitates the future use of C. fasciculata as a non-pathogenic and inexpensive biological resource to identify mode of action/protein target(s) of potentially pan-trypanocidal potent compounds. This knowledge will aid in the development of new treatments for African sleeping sickness, Chagas disease and leishmaniasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions

PubMed Central

Leelakanok, Nattawut; Fischer, Carol L.; Bates, Amber M.; Guthmiller, Janet M.; Johnson, Georgia K.; Salem, Aliasger K.; Brogden, Kim A.; Brogden, Nicole K.

2015-01-01

Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM ± std err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2–35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and > 40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. PMID:26367466

Anti-infective and cytotoxic properties of Bupleurum marginatum

PubMed Central

2014-01-01

Background Bupleurum marginatum Wall. ex DC (Apiaceae) is a perennial herb widely used in traditional Chinese and Kampo medicine for the treatment of various infectious diseases. The biological activities of B. marginatum have not been fully investigated. This study aims to investigate the antitrypanosomal, antimicrobial and antiviral activities of methanol (ME) and dichloromethane (DCM) extracts of B. marginatum aerial parts and the ability of both extracts to inhibit the growth of different cancer cell lines. Methods Phytochemical characterization of the extracts was performed by LC-MS profiling. The antitrypanosomal activity was evaluated using the resazurin method. The antimicrobial activity was assessed using agar diffusion and microdilution methods, and the minimum inhibitory concentration (MIC) values were determined. The antiviral activity was determined for 6.25, 12.5, and 50 μg/mL doses using a plaque reduction assay. Cytotoxicity was investigated in eight cancer cell lines (Caco-2, CCL-81, CCRF-CEM, COS-7, HL-60, MIA PaCa-2, MCF-7, and PANC-1) using the MTT assay and the caspase 3/7 activity was determined over the range of 62.5–1000 μg/mL. Results Phytochemical analyses resulted in the characterization of 15 components, mainly flavonoids and lignans. The DCM extract showed significant antitrypanosomal activity (IC50: 36.21 μg/mL) and moderate activity against Streptococcus pyogenes (MIC value: 0.25 mg/mL). At a dose of 12.5 μg/mL, the DCM extract inhibited 73.6% of the plaque production by hepatitis A virus. CCRF-CEM cells were the most sensitive to both extracts (IC50: 12.5–22.7 μg/mL). The cytotoxicity was mediated by induction of apoptosis (19-fold increase in the cellular caspase 3/7 level after treatment with the DCM extract at 1 mg/mL). Conclusions ME and DCM extract of B. marginatum showed anti-infective and antiproliferative effects. PMID:24438177

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method.

PubMed

Leong, Cheryl; Buttafuoco, Antonino; Glatz, Martin; Bosshard, Philipp P

2017-06-01

Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC 90 values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals. Copyright © 2017 American Society for Microbiology.

Antifungal Susceptibility Testing of Malassezia spp. with an Optimized Colorimetric Broth Microdilution Method

PubMed Central

Leong, Cheryl; Buttafuoco, Antonino

2017-01-01

ABSTRACT Malassezia is a genus of lipid-dependent yeasts. It is associated with common skin diseases such as pityriasis versicolor and atopic dermatitis and can cause systemic infections in immunocompromised individuals. Owing to the slow growth and lipid requirements of these fastidious yeasts, convenient and reliable antifungal drug susceptibility testing assays for Malassezia spp. are not widely available. Therefore, we optimized a broth microdilution assay for the testing of Malassezia that is based on the CLSI and EUCAST assays for Candida and other yeasts. The addition of ingredients such as lipids and esculin provided a broth medium formulation that enabled the growth of all Malassezia spp. and could be read, with the colorimetric indicator resazurin, by visual and fluorescence readings. We tested the susceptibility of 52 strains of 13 Malassezia species to 11 commonly used antifungals. MIC values determined by visual readings were in good agreement with MIC values determined by fluorescence readings. The lowest MICs were found for the azoles itraconazole, posaconazole, and voriconazole, with MIC90 values of 0.03 to 1.0 μg/ml, 0.06 to 0.5 μg/ml, and 0.03 to 2.0 μg/ml, respectively. All Malassezia spp. were resistant to echinocandins and griseofulvin. Some Malassezia spp. also showed high MIC values for ketoconazole, which is the most widely recommended topical antifungal to treat Malassezia skin infections. In summary, our assay enables the fast and reliable susceptibility testing of Malassezia spp. with a large panel of different antifungals. PMID:28381607

In vitro fibroblast and pre-osteoblastic cellular responses on laser surface modified Ti-6Al-4V.

PubMed

Chikarakara, Evans; Fitzpatrick, Patricia; Moore, Eric; Levingstone, Tanya; Grehan, Laura; Higginbotham, Clement; Vázquez, Mercedes; Bagga, Komal; Naher, Sumsun; Brabazon, Dermot

2014-12-29

The success of any implant, dental or orthopaedic, is driven by the interaction of implant material with the surrounding tissue. In this context, the nature of the implant surface plays a direct role in determining the long term stability as physico-chemical properties of the surface affect cellular attachment, expression of proteins, and finally osseointegration. Thus to enhance the degree of integration of the implant into the host tissue, various surface modification techniques are employed. In this work, laser surface melting of titanium alloy Ti-6Al-4V was carried out using a CO2 laser with an argon gas atmosphere. Investigations were carried out to study the influence of laser surface modification on the biocompatibility of Ti-6Al-4V alloy implant material. Surface roughness, microhardness, and phase development were recorded. Initial knowledge of these effects on biocompatibility was gained from examination of the response of fibroblast cell lines, which was followed by examination of the response of osteoblast cell lines which is relevant to the applications of this material in bone repair. Biocompatibility with these cell lines was analysed via Resazurin cell viability assay, DNA cell attachment assay, and alamarBlue metabolic activity assay. Laser treated surfaces were found to preferentially promote cell attachment, higher levels of proliferation, and enhanced bioactivity when compared to untreated control samples. These results demonstrate the tremendous potential of this laser surface melting treatment to significantly improve the biocompatibility of titanium implants in vivo.

A Novel Polyaminocarboxylate Compound To Treat Murine Pulmonary Aspergillosis by Interfering with Zinc Metabolism.

PubMed

Laskaris, Paris; Vicentefranqueira, Rocío; Helynck, Olivier; Jouvion, Grégory; Calera, José Antonio; du Merle, Laurence; Suzenet, Franck; Buron, Frédéric; de Sousa, Rodolphe Alves; Mansuy, Daniel; Cavaillon, Jean-Marc; Latgé, Jean-Paul; Munier-Lehmann, Hélène; Ibrahim-Granet, Oumaima

2018-06-01

Aspergillus fumigatus can cause pulmonary aspergillosis in immunocompromised patients and is associated with a high mortality rate due to a lack of reliable treatment options. This opportunistic pathogen requires zinc in order to grow and cause disease. Novel compounds that interfere with fungal zinc metabolism may therefore be of therapeutic interest. We screened chemical libraries containing 59,223 small molecules using a resazurin assay that compared their effects on an A. fumigatus wild-type strain grown under zinc-limiting conditions and on a zinc transporter knockout strain grown under zinc-replete conditions to identify compounds affecting zinc metabolism. After a first screen, 116 molecules were selected whose inhibitory effects on fungal growth were further tested by using luminescence assays and hyphal length measurements to confirm their activity, as well as by toxicity assays on HeLa cells and mice. Six compounds were selected following a rescreening, of which two were pyrazolones, two were porphyrins, and two were polyaminocarboxylates. All three groups showed good in vitro activity, but only one of the polyaminocarboxylates was able to significantly improve the survival of immunosuppressed mice suffering from pulmonary aspergillosis. This two-tier screening approach led us to the identification of a novel small molecule with in vivo fungicidal effects and low murine toxicity that may lead to the development of new treatment options for fungal infections by administration of this compound either as a monotherapy or as part of a combination therapy. Copyright © 2018 American Society for Microbiology.

In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA).

PubMed

Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Sy, Jamie Bernadette A; Clavio, Nina Abigail B; Macalino, Stephani Joy Y; Emnacen, Inno A; Lee, Alexandra P; Ko, Paul Kenny L; Concepcion, Gisela P

2017-01-01

Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis ( Mtb ), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 (( Z )- N -(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 μg/mL concentration against the growth of the Mtb H37Ra strain.

In silico discovery and in vitro activity of inhibitors against Mycobacterium tuberculosis 7,8-diaminopelargonic acid synthase (Mtb BioA)

PubMed Central

Billones, Junie B; Carrillo, Maria Constancia O; Organo, Voltaire G; Sy, Jamie Bernadette A; Clavio, Nina Abigail B; Macalino, Stephani Joy Y; Emnacen, Inno A; Lee, Alexandra P; Ko, Paul Kenny L; Concepcion, Gisela P

2017-01-01

Computer-aided drug discovery and development approaches such as virtual screening, molecular docking, and in silico drug property calculations have been utilized in this effort to discover new lead compounds against tuberculosis. The enzyme 7,8-diaminopelargonic acid aminotransferase (BioA) in Mycobacterium tuberculosis (Mtb), primarily involved in the lipid biosynthesis pathway, was chosen as the drug target due to the fact that humans are not capable of synthesizing biotin endogenously. The computational screening of 4.5 million compounds from the Enamine REAL database has ultimately yielded 45 high-scoring, high-affinity compounds with desirable in silico absorption, distribution, metabolism, excretion, and toxicity properties. Seventeen of the 45 compounds were subjected to bioactivity validation using the resazurin microtiter assay. Among the 4 actives, compound 7 ((Z)-N-(2-isopropoxyphenyl)-2-oxo-2-((3-(trifluoromethyl)cyclohexyl)amino)acetimidic acid) displayed inhibitory activity up to 83% at 10 μg/mL concentration against the growth of the Mtb H37Ra strain. PMID:28280303

Development of Iron Doped Silicon Nanoparticles as Bimodal Imaging Agents

PubMed Central

Singh, Mani P.; Atkins, Tonya M.; Muthuswamy, Elayaraja; Kamali, Saeed; Tu, Chuqiao; Louie, Angelique Y.; Kauzlarich, Susan M.

2012-01-01

We demonstrate the synthesis of water-soluble allylamine terminated Fe doped Si (SixFe) nanoparticles as bimodal agents for optical and magnetic imaging. The preparation involves the synthesis of a single source iron containing precursor, Na4Si4 with x% Fe (x = 1, 5, 10), and its subsequent reaction with NH4Br to produce hydrogen terminated SixFe nanoparticles. The hydrogen-capped nanoparticles are further terminated with allylamine via thermal hydrosilylation. Transmission electron microscopy (TEM) indicates that the average particle diameter is ~3.0±1.0 nm. The Si5Fe nanoparticles show strong photoluminescence quantum yield in water (~ 10 %) with significant T2 contrast (r2/r1value of 4.31). Electron paramagnetic resonance (EPR) and Mössbauer spectroscopies indicate that iron in the nanoparticles is in the +3 oxidation state. Analysis of cytotoxicity using the resazurin assay on HepG2 liver cells indicates that the particles have minimal toxicity. PMID:22616623

Synthesis, characterization and biological studies of Schiff bases derived from heterocyclic moiety.

PubMed

Shanty, Angamaly Antony; Philip, Jessica Elizabeth; Sneha, Eeettinilkunnathil Jose; Prathapachandra Kurup, Maliyeckal R; Balachandran, Sreedharannair; Mohanan, Puzhavoorparambil Velayudhan

2017-02-01

Some new Schiff bases (H 1 -H 7 ) have been synthesized by the condensation of 2-aminophenol, 2-amino-4-nitrophenol, 2-amino-4-methylphenol, 2-amino benzimidazole with thiophene-2-carboxaldehyde and pyrrole-2-carboxaldehyde. The structures of newly synthesized compounds were characterized by elemental analysis, FT-IR, 1 H NMR, UV-VIS, and single crystal X-ray crystallography. The in vitro antibacterial activity of the synthesized compounds has been tested against Salmonella typhi, Bacillus coagulans, Bacillus pumills, Escherichia coli, Bacillus circulans, Pseudomonas, Clostridium and Klebsilla pneumonia by disk diffusion method. The quantitative antimicrobial activity of the test compounds was evaluated using Resazurin based Microtiter Dilution Assay. Ampicillin was used as standard antibiotics. Schiff bases individually exhibited varying degrees of inhibitory effects on the growth of the tested bacterial species. The antioxidant activity of the synthesized compounds was determined by the 1,1-diphenyl-2-picrylhydrazyl(DPPH) method. IC 50 value of synthesized Schiff bases were calculated and compared with standard BHA. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of iron-doped silicon nanoparticles as bimodal imaging agents.

PubMed

Singh, Mani P; Atkins, Tonya M; Muthuswamy, Elayaraja; Kamali, Saeed; Tu, Chuqiao; Louie, Angelique Y; Kauzlarich, Susan M

2012-06-26

We demonstrate the synthesis of water-soluble allylamine-terminated Fe-doped Si (Si(xFe)) nanoparticles as bimodal agents for optical and magnetic imaging. The preparation involves the synthesis of a single-source iron-containing precursor, Na(4)Si(4) with x% Fe (x = 1, 5, 10), and its subsequent reaction with NH(4)Br to produce hydrogen-terminated Si(xFe) nanoparticles. The hydrogen-capped nanoparticles are further terminated with allylamine via thermal hydrosilylation. Transmission electron microscopy indicates that the average particle diameter is ∼3.0 ± 1.0 nm. The Si(5Fe) nanoparticles show strong photoluminescence quantum yield in water (∼10%) with significant T(2) contrast (r(2)/r(1) value of 4.31). Electron paramagnetic resonance and Mössbauer spectroscopies indicate that iron in the nanoparticles is in the +3 oxidation state. Analysis of cytotoxicity using the resazurin assay on HepG2 liver cells indicates that the particles have minimal toxicity.

Synthesis and cytotoxicity study of magnesium ferrite-gold core-shell nanoparticles.

PubMed

Nonkumwong, Jeeranan; Pakawanit, Phakkhananan; Wipatanawin, Angkana; Jantaratana, Pongsakorn; Ananta, Supon; Srisombat, Laongnuan

2016-04-01

In this work, the core-magnesium ferrite (MgFe2O4) nanoparticles were prepared by hydrothermal technique. Completed gold (Au) shell coating on the surfaces of MgFe2O4 nanoparticles was obtained by varying core/shell ratios via a reduction method. Phase identification, morphological evolution, optical properties, magnetic properties and cytotoxicity to mammalian cells of these MgFe2O4 core coated with Au nanoparticles were examined by using a combination of X-ray diffraction, scanning electron microscopy, transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, UV-visible spectroscopy (UV-vis), vibrating sample magnetometry and resazurin microplate assay techniques. In general, TEM images revealed different sizes of the core-shell nanoparticles generated from various core/shell ratios and confirmed the completed Au shell coating on MgFe2O4 core nanoparticles via suitable core/shell ratio with particle size less than 100 nm. The core-shell nanoparticle size and the quality of coating influence the optical properties of the products. The UV-vis spectra of complete coated MgFe2O4-Au core-shell nanoparticles exhibit the absorption bands in the near-Infrared (NIR) region indicating high potential for therapeutic applications. Based on the magnetic property measurement, it was found that the obtained MgFe2O4-Au core-shell nanoparticles still exhibit superparamagnetism with lower saturation magnetization value, compared with MgFe2O4 core. Both of MgFe2O4 and MgFe2O4-Au core-shell also showed in vitro non-cytotoxicity to mouse areola fibroblast (L-929) cell line. Copyright © 2015 Elsevier B.V. All rights reserved.

Alamar blue reagent interacts with cell-culture media giving different fluorescence over time: potential for false positives.

PubMed

Munshi, Soumyabrata; Twining, Robert C; Dahl, Russell

2014-01-01

The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence. We have used Opti-MEM, DMEM and 1:1 DMEM:Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test. Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p<0.05). We also show that the time-dependent change in RFU of 1:1 DMEM:Opti-MEM was more gradual compared to that of the other two media. These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

Control of multidrug-resistant planktonic Acinetobacter baumannii: biocidal efficacy study by atmospheric-pressure air plasma

NASA Astrophysics Data System (ADS)

Zhe, RUAN; Yajun, GUO; Jing, GAO; Chunjun, YANG; Yan, LAN; Jie, SHEN; Zimu, XU; Cheng, CHENG; Xinghao, LIU; Shumei, ZHANG; Wenhui, DU; Paul, K. CHU

2018-04-01

In this research, an atmospheric-pressure air plasma is used to inactivate the multidrug-resistant Acinetobacter baumannii in liquid. The efficacy of the air plasma on bacterial deactivation and the cytobiological variations after the plasma treatment are investigated. According to colony forming units, nearly all the bacteria (6-log) are inactivated after 10 min of air plasma treatment. However, 7% of the bacteria enter a viable but non-culturable state detected by the resazurin based assay during the same period of plasma exposure. Meanwhile, 86% of the bacteria lose their membrane integrity in the light of SYTO 9/PI staining assay. The morphological changes in the cells are examined by scanning electron microscopy and bacteria with morphological changes are rare after plasma exposure in the liquid. The concentrations of the long-living RS, such as H2O2, {{{{NO}}}3}-, and O3, in liquid induced by plasma treatment are measured, and they increase with plasma treatment time. The changes of the intracellular ROS may be related to cell death, which may be attributed to oxidative stress and other damage effects induced by RS plasma generated in liquid. The rapid and effective bacteria inactivation may stem from the RS in the liquid generated by plasma and air plasmas may become a valuable therapy in the treatment of infected wounds.

Cryoprotection from lipoteichoic acid

NASA Astrophysics Data System (ADS)

Rice, Charles V.; Middaugh, Amy; Wickham, Jason R.; Friedline, Anthony; Thomas, Kieth J.; Johnson, Karen; Zachariah, Malcolm; Garimella, Ravindranth

2012-10-01

Numerous chemical additives lower the freezing point of water, but life at sub-zero temperatures is sustained by a limited number of biological cryoprotectants. Antifreeze proteins in fish, plants, and insects provide protection to a few degrees below freezing. Microbes have been found to survive at even lower temperatures, and with a few exceptions, antifreeze proteins are missing. Survival has been attributed to external factors, such as the high salt concentration of brine veins and adhesion to particulates or ice crystal defects. We have discovered an endogenous cryoprotectant in the cell wall of bacteria, lipoteichoic acid biopolymers. Adding 1% LTA to bacteria cultures immediately prior to freezing provides 50% survival rate, similar to the results obtained with 1% glycerol. In the absence of an additive, bacterial survival is negligible as measured with the resazurin cell viability assay. The mode of action for LTA cryoprotection is unknown. With a molecular weight of 3-5 kDa, it is unlikely to enter the cell cytoplasm. Our observations suggest that teichoic acids could provide a shell of liquid water around biofilms and planktonic bacteria, removing the need for brine veins to prevent bacterial freezing.

Assessment of in vitro killing assays for detecting praziquantel-induced death in Posthodiplostomum minimum metacercariae.

PubMed

Bader, Chris; Jesudoss Chelladurai, Jeba; Starling, David E; Jones, Douglas E; Brewer, Matthew T

2017-10-01

Control of parasitic infections may be achieved by eliminating developmental stages present within intermediate hosts, thereby disrupting the parasite life cycle. For several trematodes relevant to human and veterinary medicine, this involves targeting the metacercarial stage found in fish intermediate hosts. Treatment of fish with praziquantel is one potential approach for targeting the metacercaria stage. To date, studies investigating praziquantel-induced metacercarial death in fish rely on counting parasites and visually assessing morphology or movement. In this study, we investigate quantitative methods for detecting praziquantel-induced death using a Posthodiplostomum minimum model. Our results revealed that propidium iodide staining accurately identified praziquantel-induced death and the level of staining was proportional to the concentration of praziquantel. In contrast, detection of ATP, resazurin metabolism, and trypan blue staining were poor indicators of metacercarial death. The propidium iodide method offers an advantage over simple visualization of parasite movement and could be used to determine EC 50 values relevant for comparison of praziquantel sensitivity or resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Biofunctional composite coating architectures based on polycaprolactone and nanohydroxyapatite for controlled corrosion activity and enhanced biocompatibility of magnesium AZ31 alloy.

PubMed

Zomorodian, A; Garcia, M P; Moura E Silva, T; Fernandes, J C S; Fernandes, M H; Montemor, M F

2015-03-01

In this work a biofunctional composite coating architecture for controlled corrosion activity and enhanced cellular adhesion of AZ31 Mg alloys is proposed. The composite coating consists of a polycaprolactone (PCL) matrix modified with nanohydroxyapatite (HA) applied over a nanometric layer of polyetherimide (PEI). The protective properties of the coating were studied by electrochemical impedance spectroscopy (EIS), a non-disturbing technique, and the coating morphology was investigated by field emission scanning electron microscopy (FE-SEM). The results show that the composite coating protects the AZ31 substrate. The barrier properties of the coating can be optimized by changing the PCL concentration. The presence of nanohydroxyapatite particles influences the coating morphology and decreases the corrosion resistance. The biocompatibility was assessed by studying the response of osteoblastic cells on coated samples through resazurin assay, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The results show that the polycaprolactone to hydroxyapatite ratio affects the cell behavior and that the presence of hydroxyapatite induces high osteoblastic differentiation. Copyright © 2014 Elsevier B.V. All rights reserved.

Transport of a conservative and "smart" tracers' in a first-order creek: role of transient storage type

USDA-ARS?s Scientific Manuscript database

Quantification of microbial fate and transport in streams has become one of most important topics in studying biogeochemical properties and behavior of stream ecosystems. Using "smart" tracer such as resazurin (Raz) allows assessment of sediment-water interactions and associated biological activity ...

Quantifying spatial differences in metabolism in headwater streams

Treesearch

Ricardo González-Pinzón; Roy Haggerty; Alba Argerich

2014-01-01

Stream functioning includes simultaneous interaction among solute transport, nutrient processing, and metabolism. Metabolism is measured with methods that have limited spatial representativeness and are highly uncertain. These problems restrict development of methods for up-scaling biological processes that mediate nutrient processing. We used the resazurin–resorufin (...

The Influence of Osmolytes on the Antimicrobial Activity of Silver Nanoparticles

NASA Astrophysics Data System (ADS)

Reed-Jones, Neiunna L.

Silver nanoparticles (Ag-NPs) are gaining popularity as antimicrobial agents due to their broad- spectrum activity and lower propensity to develop resistance in bacteria. However, with increased frequency of use, there remains a possibility for bacteria to develop resistance or adaptive mechanisms against Ag-NPs overtime. Mechanisms used by bacteria to resist antimicrobial agents include efflux pumps, heat shock proteins (Hsp) and accumulation of compatible solutes, generally referred to as osmolytes. The latter mechanism is typically employed when bacteria are under osmotic stress they accumulate osmolytes either through de novo synthesis or exogenously. Osmolytes act to stabilize the bacterial cell membrane by maintaining the native protein structure, while at the same time, ensuring compatibility with other cellular structures and functions. The most common osmolytes accumulated by bacteria are glycine betaine, proline, carnitine, choline, trehalose and glutamate. Since Ag-NPs target the cell membrane it is conceivable that osmolytes may suppress its bactericidal activity. In the present study, we assess the antimicrobial efficacy of Ag-NPs in the presence of glycine betaine and proline. Exponential phase cultures (106 cfu/ml) of Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to a predetermined the minimum inhibitory concentration (MIC) of 0.4 mM of citrate-stabilized Ag-NPs and incubated at 37°C for 48 h. This was replicated with the addition of 1 mM of either glycine betaine or proline. Growth was monitored by optical density, standard plate count, resazurin assay and LIVE/DEAD analyses. The result showed that Ag-NPs had no detectable effect on osmolyte treated cells. The average plate count of cultures supplemented with either glycine betaine or proline ranged from 108 to 109 cfu/ml after 48 h. Resazurin assay also showed that there was a significant increase in the cells supplemented with glycine betaine, 90% for E.coli O157:H7 and 44.3% for L. monocytogenes. No detectable metabolic activity was observed in the cells exposed to Ag-NPs alone. These results were corroborated by LIVE/DEAD analyses, which revealed over 86% cell viability in L. monocytogenes and E.coli O157:H7 treated with the osmolytes compared to 0.3% viability in those treated with Ag-NPs alone. SDS-PAGE analysis of the periplasmic proteins of the cells treated with Ag-NP and glycine betaine showed no qualitative difference in the protein profiles compared to the control cells grown in Trypticase Soy Broth alone. However, protein bands of cells treated with proline showed detectable difference relative to the control. The present findings suggest that glycine betaine and proline can suppress the antimicrobial activity of Ag-NPs.

Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment

PubMed Central

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J.; Chatterjee, Aditi; Prasad, T. S. Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division. PMID:25874956

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

PubMed

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J; Chatterjee, Aditi; Prasad, T S Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

In vitro antimicrobial effects and mechanism of atmospheric-pressure He/O2 plasma jet on Staphylococcus aureus biofilm

NASA Astrophysics Data System (ADS)

Xu, Zimu; Shen, Jie; Cheng, Cheng; Hu, Shuheng; Lan, Yan; Chu, Paul K.

2017-03-01

The antimicrobial effects and associated mechanism of inactivation of Staphylococcus aureus (S. aureus) NCTC-8325 biofilms induced by a He/O2 atmospheric-pressure plasma jet (APPJ) are investigated in vitro. According to CFU (colony forming units) counting and the resazurin-based assay, the 10 min He/O2 (0.5%) APPJ treatment produces the optimal inactivation efficacy (>5 log10 ml-1) against the S. aureus biofilm and 5% of the bacteria enter a viable but non-culturable (VBNC) state. Meanwhile, 94% of the bacteria suffer from membrane damage according to SYTO 9/PI counterstaining. Scanning electron microscopy (SEM) reveals that plasma exposure erodes the extracellular polymeric substances (EPS) and then the cellular structure. The H2DCFDA-stained biofilms show larger concentrations of intracellular reactive oxygen species (ROS) in membrane-intact bacteria with increasing plasma dose. The admixture of oxygen in the working gas highly contributes to the deactivation efficacy of the APPJ against S. aureus and the plasma-induced endogenous ROS may work together with the discharge-generated ROS to continuously damage the bacterial membrane structure leading to deactivation of the biofilm microbes.

In vitro synergistic antibacterial activity of the essential oil from Zingiber cassumunar Roxb against extensively drug-resistant Acinetobacter baumannii strains.

PubMed

Boonyanugomol, Wongwarut; Kraisriwattana, Kairin; Rukseree, Kamolchanok; Boonsam, Kraisorn; Narachai, Panchaporn

In this study, we determined the antibacterial and synergistic activities of the essential oil from Zingiber cassumunar against the extensively drug-resistant (XDR) Acinetobacter baumannii strains. The antibacterial and synergistic properties of the essential oil from Z. cassumunar were examined by agar disc diffusion tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated by broth microdilution using the resazurin assay. The in vitro time-kill antibacterial kinetics was analyzed using the plate count technique. We found that the essential oil from Z. cassumunar had antibacterial activity against A. baumannii, with MIC and MBC ranging from 7.00 to 9.24mg/ml. The essential oil could completely inhibit A. baumannii at 1h, and coccoid-shaped bacteria were found after treatment. In addition, the essential oil had a synergistic effect when combined with antibiotics, e.g., aminoglycosides, fluoroquinolones, tetracyclines, and folate pathway inhibitors. Thus, the essential oil from Z. cassumunar has strong antibacterial and synergistic activities against XDR A. baumannii, which may provide the basis for the development of a new therapy against drug-resistant bacteria. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Interferometry as a tool for evaluating effects of antimicrobial doses on Mycobacterium bovis growth.

PubMed

Machado, Rachel R P; Dutra, Rafael C; Raposo, Nádia R B; Lesche, Bernhard; Gomes, Marlei S; Duarte, Rafael S; Soares, Geraldo Luiz G; Kaplan, Maria Auxiliadora C

2015-12-01

Interferometry was used together with the conventional microplate resazurin assay to evaluate the antimycobacterial properties of essential oil (EO) from fruits of Pterodon emarginatus and also of rifampicin against Mycobacterium bovis. The aim of this work is not only to investigate the potential antimycobacterial activity of this EO, but also to test the interferometric method in comparison with the conventional one. The Minimum Inhibitory Concentration (MIC) values of EO (625 μg/mL) and rifampicin (4 ng/mL) were firstly identified with the microplate method. These values were used as parameters in Drug Susceptibility Tests (DST) with interferometry. The interferometry confirmed the MIC value of EO identified with microplate and revealed a bacteriostatic behavior for this concentration. At 2500 μg/mL interferometry revealed bactericidal activity of the EO. Mycobacterial growth was detected with interferometry at 4 ng/mL of rifampicin and even at higher concentrations. One important difference is that the interferometric method preserves the sample, so that after weeks of quantitative observation, the sample can be used to evaluate the bactericidal activity of the tested drug. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA damage induced by hydroquinone can be prevented by fungal detoxification.

PubMed

Pereira, Pedro; Enguita, Francisco J; Ferreira, João; Leitão, Ana Lúcia

2014-01-01

Hydroquinone is a benzene metabolite with a wide range of industrial applications, which has potential for widespread human exposure; however, the toxicity of hydroquinone on human cells remains unclear. The aims of this study are to investigate the cytotoxicity and genotoxicity of hydroquinone in human primary fibroblasts and human colon cancer cells (HCT116). Low doses of hydroquinone (227-454 μM) reduce the viability of fibroblasts and HCT116 cells, determined by resazurin conversion, and induce genotoxic damage (DNA strand breaks), as assessed by alkaline comet assays. Bioremediation may provide an excellent alternative to promote the degradation of hydroquinone, however few microorganisms are known that efficiently degrade it. Here we also investigate the capacity of a halotolerant fungus, Penicillium chrysogenum var. halophenolicum , to remove hydroquinone toxicity under hypersaline condition. The fungus is able to tolerate high concentrations of hydroquinone and can reverse these noxious effects via degradation of hydroquinone to completion, even when the initial concentration of this compound is as high as 7265 μM. Our findings reveal that P. chrysogenum var. halophenolicum efficiently degrade hydroquinone under hypersaline conditions, placing this fungus among the best candidates for the detoxification of habitats contaminated with this aromatic compound.

Cytotoxicity, genotoxicity and mechanism of action (via gene expression analysis) of the indole alkaloid aspidospermine (antiparasitic) extracted from Aspidosperma polyneuron in HepG2 cells.

PubMed

Coatti, Giuliana Castello; Marcarini, Juliana Cristina; Sartori, Daniele; Fidelis, Queli Cristina; Ferreira, Dalva Trevisan; Mantovani, Mário Sérgio

2016-08-01

Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 μM) and genotoxic (starting at 50 μM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 μM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 μM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 μM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended.

In vitro effects of Panax ginseng in aristolochic acid-mediated renal tubulotoxicity: apoptosis versus regeneration.

PubMed

Bunel, Valérian; Antoine, Marie-Hélène; Nortier, Joëlle; Duez, Pierre; Stévigny, Caroline

2015-03-01

This in vitro study aimed to determine the effects of a Panax ginseng extract on aristolochic acid-mediated toxicity in HK-2 cells. A methanolic extract of ginseng (50 µg/mL) was able to reduce cell survival after treatment with 50 µM aristolochic acid for 24, 48, and 72 h, as evidenced by a resazurin reduction assay. This result was confirmed by a flow cytometric evaluation of apoptosis using annexin V-PI staining, and indicated higher apoptosis rates in cells treated with aristolochic acid and P. ginseng extract compared with aristolochic acid alone. However, P. ginseng extract by itself (5 and 50 µg/mL) increased the Ki-67 index, indicating an enhancement in cellular proliferation. Cell cycle analysis excluded a P. ginseng extract-mediated induction of G2/M cell cycle arrest such as the one typically observed with aristolochic acid. Finally, β-catenin acquisition was found to be accelerated when cells were treated with both doses of ginseng, suggesting that the epithelial phenotype of renal proximal tubular epithelial cells was maintained. Also, ginseng treatment (5 and 50 µg/mL) reduced the oxidative stress activity induced by aristolochic acid after 24 and 48 h. These results indicate that the ginseng extract has a protective activity towards the generation of cytotoxic reactive oxygen species induced by aristolochic acid. However, the ginseng-mediated alleviation of oxidative stress did not correlate with a decrease but rather with an increase in aristolochic acid-induced apoptosis and death. This deleterious herb-herb interaction could worsen aristolochic acid tubulotoxicity and reinforce the severity and duration of the injury. Nevertheless, increased cellular proliferation and migration, along with the improvement in the epithelial phenotype maintenance, indicate that ginseng could be useful for improving tubular regeneration and the recovery following drug-induced kidney injury. Such dual activities of ginseng certainly warrant further in vivo studies. Georg Thieme Verlag KG Stuttgart · New York.

Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water

PubMed Central

Shin, Gwy-Am; Sobsey, Mark D.

2003-01-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone. PMID:12839770

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water.

PubMed

Shin, Gwy-Am; Sobsey, Mark D

2003-07-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.

Personal care product preservatives: risk assessment and mixture toxicities with an industrial wastewater.

PubMed

Carbajo, Jose B; Perdigón-Melón, Jose A; Petre, Alice L; Rosal, Roberto; Letón, Pedro; García-Calvo, Eloy

2015-04-01

The aquatic toxicity of eight preservatives frequently used in personal care products (PCPs) (iodopropynyl butylcarbamate, bronopol, diazolidinyl urea, benzalkonium chloride, zinc pyrithione, propylparaben, triclosan and a mixture of methylchloroisothiazolinone and methylisothiazolinone) was assessed by means of two different approaches: a battery of bioassays composed of single species tests of bacteria (Vibrio fischeri and Pseudomonas putida) and protozoa (Tetrahymena thermophila), and a whole biological community resazurin-based assay using activated sludge. The tested preservatives showed considerable toxicity in the studied bioassays, but with a marked difference in potency. In fact, all biocides except propylparaben and diazolidinyl urea had EC50 values lower than 1 mg L(-1) in at least one assay. Risk quotients for zinc pyrithione, benzalkonium chloride, iodopropynyl butylcarbamate and triclosan as well as the mixture of the studied preservatives exceeded 1, indicating a potential risk for the process performance and efficiency of municipal sewage treatment plants (STPs). These four single biocides explained more than 95% of the preservative mixture risk in all bioassays. Each individual preservative was also tested in combination with an industrial wastewater (IWW) from a cosmetics manufacturing facility. The toxicity assessment was performed on binary mixtures (preservative + IWW) and carried out using the median-effect principle, which is a special case of the concept of Concentration Addition (CA). Almost 70% of all experiments resulted in EC50 values within a factor of 2 of the values predicted by the median-effect principle (CI values between 0.5 and 2). The rest of the mixtures whose toxicity was mispredicted by CA were assessed with the alternative concept of Independent Action (IA), which showed higher predictive power for the biological community assay. Therefore, the concept used to accurately predict the toxicity of mixtures of a preservative with a complex industrial wastewater depends on degree of biological complexity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lotic ecosystem response to chronic metal contamination assessed by the resazurin-resorufin smart tracer with data assimilation by the Markov chain Monte Carlo method

NASA Astrophysics Data System (ADS)

Stanaway, D. J.; Flores, A. N.; Haggerty, R.; Benner, S. G.; Feris, K. P.

2011-12-01

Concurrent assessment of biogeochemical and solute transport data (i.e. advection, dispersion, transient storage) within lotic systems remains a challenge in eco-hydrological research. Recently, the Resazurin-Resorufin Smart Tracer System (RRST) was proposed as a mechanism to measure microbial activity at the sediment-water interface [Haggerty et al., 2008, 2009] associating metabolic and hydrologic processes and allowing for the reach scale extrapolation of biotic function in the context of a dynamic physical environment. This study presents a Markov Chain Monte Carlo (MCMC) data assimilation technique to solve the inverse model of the Raz Rru Advection Dispersion Equation (RRADE). The RRADE is a suite of dependent 1-D reactive ADEs, associated through the microbially mediated reduction of Raz to Rru (k12). This reduction is proportional to DO consumption (R^2=0.928). MCMC is a suite of algorithms that solve Bayes theorem to condition uncertain model states and parameters on imperfect observations. Here, the RRST is employed to quantify the effect of chronic metal exposure on hyporheic microbial metabolism along a 100+ year old metal contamination gradient in the Clark Fork River (CF). We hypothesized that 1) the energetic cost of metal tolerance limits heterotrophic microbial respiration in communities evolved in chronic metal contaminated environments, with respiration inhibition directly correlated to degree of contamination (observational experiment) and 2) when experiencing acute metal stress, respiration rate inhibition of metal tolerant communities is less than that of naïve communities (manipulative experiment). To test these hypotheses, 4 replicate columns containing sediment collected from differently contaminated CF reaches and reference sites were fed a solution of RRST, NaCl, and cadmium (manipulative experiment only) within 24 hrs post collection. Column effluent was collected and measured for Raz, Rru, and EC to determine the Raz Rru breakthrough curves (BTC), subsequently modeled by the RRADE and thereby allowing derivation of in situ rates of metabolism. RRADE parameter values are estimated through Metropolis Hastings MCMC optimization. Unknown prior parameter distributions (PD) were constrained via a sensitivity analysis, except for the empirically estimated velocity. MCMC simulations were initiated at random points within the PD. Convergence of target distributions (TD) is achieved when the variance of the mode values of the six RRADE parameters in independent model replication is at least 10^{-3} less than the mode value. Convergence of k12, the parameter of interest, was more resolved, with modal variance of replicate simulations ranging from 10^{-4} less than the modal value to 0. The MCMC algorithm presented here offers a robust approach to solve the inverse RRST model and could be easily adapted to other inverse problems.

Quantitative measurement of stream respiration using the resazurin-resorufin system

NASA Astrophysics Data System (ADS)

Gonzalez Pinzon, R. A.; Acker, S.; Haggerty, R.; Myrold, D.

2011-12-01

After three decades of active research in hydrology and stream ecology, the relationship between stream solute transport, metabolism and nutrient dynamics is still unresolved. These knowledge gaps obscure the function of stream ecosystems and how they interact with other landscape processes. To date, measuring rates of stream metabolism is accomplished with techniques that have vast uncertainties and are not spatially representative. These limitations mask the role of metabolism in nutrient processing. Clearly, more robust techniques are needed to develop mechanistic relationships that will ultimately improve our fundamental understanding of in-stream processes and how streams interact with other ecosystems. We investigated the "metabolic window of detection" of the Resazurin (Raz)-Resorufin (Rru) system (Haggerty et al., 2008, 2009). Although previous results have shown that the transformation of Raz to Rru is strongly correlated with respiration, a quantitative relationship between them is needed. We investigated this relationship using batch experiments with pure cultures (aerobic and anaerobic) and flow-through columns with incubated sediments from four different streams. The results suggest that the Raz-Rru system is a suitable approach that will enable hydrologists and stream ecologists to measure in situ and in vivo respiration at different scales, thus opening a reliable alternative to investigate how solute transport and stream metabolism control nutrient processing.

Nonprenylated Xanthones from Gentiana lutea, Frasera caroliniensis, and Centaurium erythraea as Novel Inhibitors of Vascular Smooth Muscle Cell Proliferation.

PubMed

Waltenberger, Birgit; Liu, Rongxia; Atanasov, Atanas G; Schwaiger, Stefan; Heiss, Elke H; Dirsch, Verena M; Stuppner, Hermann

2015-11-13

Aberrant proliferation of vascular smooth muscle cells (VSMC) plays a major role in restenosis, the pathological renarrowing of the blood vessel lumen after surgical treatment of stenosis. Since available anti-proliferative pharmaceuticals produce unfavorable side effects, there is high demand for the identification of novel VSMC proliferation inhibitors. A natural product screening approach using a resazurin conversion assay enabled the identification of gentisin (1) from Gentiana lutea as a novel inhibitor of VSMC proliferation with an IC50 value of 7.84 µM. Aiming to identify further anti-proliferative compounds, 13 additional nonprenylated xanthones, isolated from different plant species, were also tested. While some compounds showed no or moderate activity at 30 µM, 1-hydroxy-2,3,4,5-tetramethoxyxanthone (4), swerchirin (6), and methylswertianin (7) showed IC50 values between 10.2 and 12.5 µM. The anti-proliferative effect of 1, 4, 6, and 7 was confirmed by the quantification of DNA synthesis (BrdU incorporation) in VSMC. Cell death quantification (determined by LDH release in the culture medium) revealed that the compounds are not cytotoxic in the investigated concentration range. In conclusion, nonprenylated xanthones are identified as novel, non-toxic VSMC proliferation inhibitors, which might contribute to the development of new therapeutic applications to combat restenosis.

Antimycobacterial triterpenes from the Canadian medicinal plant Sarracenia purpurea.

PubMed

Morrison, Steven A; Li, Haoxin; Webster, Duncan; Johnson, John A; Gray, Christopher A

2016-07-21

The purple pitcher plant, Sarracenia purpurea, is a medicinal plant used by the Canadian First Nations to treat a wide variety of illnesses. The Mi'kmaq and Wolastoqiyik (Maliseet) peoples of Eastern Canada have traditionally used infusions of S. purpurea for the treatment of tuberculosis-like symptoms. Previous investigations have shown methanolic extracts of S. purpurea to possess antimycobacterial activity. To isolate and identify antimycobacterial constituents from S. purpurea. Methanolic extracts of S. purpurea were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The antimycobacterial constituents were identified by NMR, MS and polarimetry. The triterpenes betulinaldehyde, β-sitosterol, betulinic acid, and ursolic acid were isolated from S. purpurea. Betulinaldehyde, betulinic acid, and ursolic acid exhibited MICs of 450, 950, and 450μM and IC50s of 98, 169, and 93μM against M. tuberculosis H37Ra respectively whilst β-sitosterol was inactive (MIC and IC50 of >1000μM). Betulinaldehyde, betulinic acid, and ursolic acid were identified as the principal constituents responsible for the antimycobacterial activity of S. purpurea. This work is consistent with the ethnopharmacological use of S. purpurea by Canadian First Nations as a treatment against infectious diseases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Antimycobacterial and Antibacterial Activity of Allium sativum Bulbs

PubMed Central

Viswanathan, V.; Phadatare, A. G.; Mukne, Alka

2014-01-01

Tuberculosis is one of the major public health problems faced globally. Resistance of Mycobacterium tuberculosis to antitubercular agents has called for an urgent need to investigate newer drugs to combat tuberculosis. Garlic (Allium sativum) is an edible plant which has generated a lot of curiosity throughout human history as a medicinal plant. Garlic contains sulfur compounds like allicin, ajoene, allylmethyltrisulfide, diallyltrisulfide, diallyldisulphide and others which exhibit various biological properties like antimicrobial, anticancer, antioxidant, immunomodulatory, antiinflammatory, hypoglycemic, and cardiovascular effects. According to various traditional systems of medicine, garlic is one of the established remedies for tuberculosis. The objective of the current study was to investigate in vitro antimycobacterial activity as well as anti-bacterial activity of various extracts rich in specific phytoconstituents from garlic. Preparation of garlic extracts was done based on the chemistry of the constituents and their stability. The estimation of in vitro antimycobacterial activity of different garlic extracts was done using Resazurin microtire plate assay technique whereas activity of garlic oil was evaluated by colony count method. The antibacterial activity of extracts and oil was estimated by zone of inhibition method. Extracts of garlic rich in allicin and ajoene showed appreciable antimycobacterial activity as compared to standard drugs. Garlic oil demonstrated significant antibacterial activity, particularly against methicillin-resistant Staphylococcus aureus. PMID:25035540

Effects of different concentrations of Platelet-rich Plasma and Platelet-Poor Plasma on vitality and differentiation of autologous Adipose tissue-derived stem cells.

PubMed

Felthaus, Oliver; Prantl, Lukas; Skaff-Schwarze, Mona; Klein, Silvan; Anker, Alexandra; Ranieri, Marco; Kuehlmann, Britta

2017-01-01

Autologous fat grafts and adipose-derived stem cells (ASCs) can be used to treat soft tissue defects. However, the results are inconsistent and sometimes comprise tissue resorption and necrosis. This might be due to insufficient vascularization. Platelet-rich plasma (PRP) is a source of concentrated autologous platelets. The growth factors and cytokines released by platelets can facilitate angiogenesis. The simultaneous use of PRP might improve the regeneration potential of fat grafts. The optimal ratio has yet to be elucidated. A byproduct of PRP preparation is platelet-poor plasma (PPP). In this study we investigated the influence of different concentrations of PRP on the vitality and differentiation of ASCs. We processed whole blood with the Arthrex Angel centrifuge and isolated ASCs from the same donor. We tested the effects of different PRP and PPP concentrations on the vitality using resazurin assays and the differentiation of ASCs using oil-red staining. Both cell vitality and adipogenic differentiation increase to a concentration of 10% to 20% PRP. With a PRP concentration of 30% cell vitality and differentiation decrease. Both PRP and PPP can be used to expand ASCs without xenogeneic additives in cell culture. A PRP concentration above 20% has inhibitory effects.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract.

PubMed

Correia, Vanessa Carolina de Sena; Lima, Nathália Oliveira; Oliveira, Flávio Augusto de Souza; Santos, Ana Paula de Azevedo Dos; Teles, Carolina Bioni Garcia; Oliveira, Waldesse Piragé de; Pimenta, And Raphael Sanzio

2016-01-01

Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Taking the Silver Bullet Colloidal Silver Particles for the Topical Treatment of Biofilm-Related Infections.

PubMed

Richter, Katharina; Facal, Paula; Thomas, Nicky; Vandecandelaere, Ilse; Ramezanpour, Mahnaz; Cooksley, Clare; Prestidge, Clive A; Coenye, Tom; Wormald, Peter-John; Vreugde, Sarah

2017-07-05

Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase assay. The antibiofilm activity was assessed in vitro by the resazurin assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1%), and 98% (±1%) biofilm killing of SA, MRSA, and PA, respectively, while significantly reducing mortality of infected nematodes. The in vivo antibiofilm activity was linked to the accumulation of AgNPs in the intestinal tract of C. elegans as observed by 3D X-ray tomography. Quasi-spherical AgNPs were physically stable in suspension for over 6 months with no observed loss in antibiofilm activity. While toxicity and stability limited the utilization of cubic and star-shaped AgNPs, quasi-spherical AgNPs could be rapidly synthesized, were stable and nontoxic, and showed substantial in vitro and in vivo activity against clinically relevant biofilms. Quasi-spherical AgNPs hold potential as pharmacotherapy, for example, as topical treatment for biofilm-related infections.

Peptide-biphenyl hybrid-capped AuNPs: stability and biocompatibility under cell culture conditions

NASA Astrophysics Data System (ADS)

Connolly, Mona; Pérez, Yolanda; Mann, Enrique; Herradón, Bernardo; Fernández-Cruz, María L.; Navas, José M.

2013-07-01

In this study, we explored the biocompatibility of Au nanoparticles (NPs) capped with peptide-biphenyl hybrid (PBH) ligands containing glycine (Gly), cysteine (Cys), tyrosine (Tyr), tryptophan (Trp) and methionine (Met) amino acids in the human hepatocellular carcinoma cell line Hep G2. Five AuNPs, Au[(Gly-Tyr-Met)2B], Au[(Gly-Trp-Met)2B], Au[(Met)2B], Au[(Gly-Tyr-TrCys)2B] and Au[(TrCys)2B], were synthesised. Physico-chemical and cytotoxic properties were thoroughly studied. Transmission electron micrographs showed isolated near-spherical nanoparticles with diameters of 1.5, 1.6, 2.3, 1.8 and 2.3 nm, respectively. Dynamic light scattering evidenced the high stability of suspensions in Milli-Q water and culture medium, particularly when supplemented with serum, showing in all cases a tendency to form agglomerates with diameters approximately 200 nm. In the cytotoxicity studies, interference caused by AuNPs with some typical cytotoxicity assays was demonstrated; thus, only data obtained from the resazurin based assay were used. After 48-h incubation, only concentrations ≥50 μg/ml exhibited cytotoxicity. Such doses were also responsible for an increase in reactive oxygen species (ROS). Some differences were observed among the studied NPs. Of particular importance is the AuNPs capped with the PBH ligand (Gly-Tyr-TrCys)2B showing remarkable stability in culture medium, even in the absence of serum. Moreover, these AuNPs have unique biological effects on Hep G2 cells while showing low toxicity. The production of ROS along with supporting optical microscopy images suggests cellular interaction/uptake of these particular AuNPs. Future research efforts should further test this hypothesis, as such interaction/uptake is highly relevant in drug delivery systems.

Cytotoxicity and modes of action of four Cameroonian dietary spices ethno-medically used to treat cancers: Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense.

PubMed

Kuete, Victor; Sandjo, Louis P; Wiench, Benjamin; Efferth, Thomas

2013-08-26

Echinops giganteus, Imperata cylindrica, Piper capense and Xylopia aethiopica are four medicinal spices used in Cameroon to treat cancers. The above plants previously displayed cytotoxicity against leukemia CCRF-CEM and CEM/ADR5000 cell lines as well as human pancreatic MiaPaCa-2 cells. The present study aims at emphasizing the study of the cytotoxicity and the modes of action of the above plants on a panel of ten cancer cell lines including various sensitive and drug-resistant phenotypes. The study has been extended to the isolation of the bioactive constituents from Echinops giganteus. The cytotoxicity of the extracts was determined using a resazurin reduction assay, whereas the caspase-Glo assay was used to detect the activation of caspases 3/7, caspase 8 and caspase 9 in cells treated with the four extracts. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells, analysis of mitochondrial membrane potential (MMP) as well as measurement of reactive oxygen species (ROS). The four tested extracts inhibited the proliferation of all tested cancer cell lines including sensitive and drug-resistant phenotypes. Collateral sensitivity of cancer cells to the extract of Echinops giganteus was generally better than to doxorubicin. The recorded IC50 ranges were 3.29 µg/mL [against human knockout clones HCT116 (p53(-/-)) colon cancer cells] to 14.32 µg/mL (against human liver hepatocellular carcinoma HepG2 cells) for the crude extract from Echinops giganteus, 4.17 µg/mL (against breast cancer cells transduced with control vector MDA-MB231 cells) to 19.45 µg/mL (against MDA-MB-231 BCRP cells) for that of Piper capense, 4.11 µg/mL (against leukemia CCRF-CEM cells) to 30.60 µg/mL (against leukemia HL60AR cells) for Xylopia aethiopica, 3.28 µg/mL [against HCT116 (p53(-/-)) cells] to 33.43 µg/mL (against HepG2 cells) for Imperata cylindica and 0.11 µg/mL (against CCRF-CEM cells) to 132.47 µg/mL (against HL60AR cells) for doxorubicin. The four tested extracts induced apoptosis in CCRF-CEM cells via the alteration loss of MMP whilst that of Piper capense also enhanced the production of ROS. The studied plants are potential cytotoxic drugs that deserve more detailed exploration in the future, to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Tracer-based characterization of hyporheic exchange and benthic biolayers in streams

NASA Astrophysics Data System (ADS)

Knapp, Julia L. A.; González-Pinzón, Ricardo; Drummond, Jennifer D.; Larsen, Laurel G.; Cirpka, Olaf A.; Harvey, Judson W.

2017-02-01

Shallow benthic biolayers at the top of the streambed are believed to be places of enhanced biogeochemical turnover within the hyporheic zone. They can be investigated by reactive stream tracer tests with tracer recordings in the streambed and in the stream channel. Common in-stream measurements of such reactive tracers cannot localize where the processing primarily takes place, whereas isolated vertical depth profiles of solutes within the hyporheic zone are usually not representative of the entire stream. We present results of a tracer test where we injected the conservative tracer bromide together with the reactive tracer resazurin into a third-order stream and combined the recording of in-stream breakthrough curves with multidepth sampling of the hyporheic zone at several locations. The transformation of resazurin was used as an indicator of metabolism, and high-reactivity zones were identified from depth profiles. The results from our subsurface analysis indicate that the potential for tracer transformation (i.e., the reaction rate constant) varied with depth in the hyporheic zone. This highlights the importance of the benthic biolayer, which we found to be on average 2 cm thick in this study, ranging from one third to one half of the full depth of the hyporheic zone. The reach-scale approach integrated the effects of processes along the reach length, isolating hyporheic processes relevant for whole-stream chemistry and estimating effective reaction rates.

High throughput and miniaturised systems for biodegradability assessments.

PubMed

Cregut, Mickael; Jouanneau, Sulivan; Brillet, François; Durand, Marie-José; Sweetlove, Cyril; Chenèble, Jean-Charles; L'Haridon, Jacques; Thouand, Gérald

2014-01-01

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

Tracer-based characterization of hyporheic exchange and benthic biolayers in streams

USGS Publications Warehouse

Knapp, Julia L.A.; González-Pinzón, Ricardo; Drummond, Jennifer D.; Larsen, Laurel G.; Cirpka, Olaf A.; Harvey, Judson W.

2017-01-01

Shallow benthic biolayers at the top of the streambed are believed to be places of enhanced biogeochemical turnover within the hyporheic zone. They can be investigated by reactive stream tracer tests with tracer recordings in the streambed and in the stream channel. Common in-stream measurements of such reactive tracers cannot localize where the processing primarily takes place, whereas isolated vertical depth profiles of solutes within the hyporheic zone are usually not representative of the entire stream. We present results of a tracer test where we injected the conservative tracer bromide together with the reactive tracer resazurin into a third-order stream and combined the recording of in-stream breakthrough curves with multidepth sampling of the hyporheic zone at several locations. The transformation of resazurin was used as an indicator of metabolism, and high-reactivity zones were identified from depth profiles. The results from our subsurface analysis indicate that the potential for tracer transformation (i.e., the reaction rate constant) varied with depth in the hyporheic zone. This highlights the importance of the benthic biolayer, which we found to be on average 2 cm thick in this study, ranging from one third to one half of the full depth of the hyporheic zone. The reach-scale approach integrated the effects of processes along the reach length, isolating hyporheic processes relevant for whole-stream chemistry and estimating effective reaction rates.

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

PubMed

Brady, Pamlea N; Macnaughtan, Megan A

2015-12-15

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of Colorimetric Assays for Analyzing Reductively Methylated Proteins: Biases and Mechanistic Insights

PubMed Central

Brady, Pamlea N.; Macnaughtan, Megan A.

2015-01-01

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

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

PubMed

Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

2017-12-01

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

A High-Throughput Screening Platform of Microbial Natural Products for the Discovery of Molecules with Antibiofilm Properties against Salmonella

PubMed Central

Paytubi, Sonia; de La Cruz, Mercedes; Tormo, Jose R.; Martín, Jesús; González, Ignacio; González-Menendez, Victor; Genilloud, Olga; Reyes, Fernando; Vicente, Francisca; Madrid, Cristina; Balsalobre, Carlos

2017-01-01

In this report, we describe a High-Throughput Screening (HTS) to identify compounds that inhibit biofilm formation or cause the disintegration of an already formed biofilm using the Salmonella Enteritidis 3934 strain. Initially, we developed a new methodology for growing Salmonella biofilms suitable for HTS platforms. The biomass associated with biofilm at the solid-liquid interface was quantified by staining both with resazurin and crystal violet, to detect living cells and total biofilm mass, respectively. For a pilot project, a subset of 1120 extracts from the Fundación MEDINA's collection was examined to identify molecules with antibiofilm activity. This is the first validated HTS assay of microbial natural product extracts which allows for the detection of four types of activities which are not mutually exclusive: inhibition of biofilm formation, detachment of the preformed biofilm and antimicrobial activity against planktonic cells or biofilm embedded cells. Currently, several extracts have been selected for further fractionation and purification of the active compounds. In one of the natural extracts patulin has been identified as a potent molecule with antimicrobial activity against both, planktonic cells and cells within the biofilm. These findings provide a proof of concept that the developed HTS can lead to the discovery of new natural compounds with antibiofilm activity against Salmonella and its possible use as an alternative to antimicrobial therapies and traditional disinfectants. PMID:28303128

Design, synthesis, and characterization of (1-(4-aryl)- 1H-1,2,3-triazol-4-yl)methyl, substituted phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates against Mycobacterium tuberculosis

PubMed Central

Venugopala, Katharigatta N; Dharma Rao, G B; Bhandary, Subhrajyoti; Pillay, Melendhran; Chopra, Deepak; Aldhubiab, Bandar E; Attimarad, Mahesh; Alwassil, Osama Ibrahim; Harsha, Sree; Mlisana, Koleka

2016-01-01

The novel (1-(4-aryl)-1H-1,2,3-triazol-4-yl)methyl, substituted phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives were synthesized by the click reaction of the dihydropyrimidinones, bearing a terminal alkynyl group, with various substituted aryl azides at room temperature using a catalytic amount of Cu(OAc)2 and sodium ascorbate in a 1:2 ratio of acetone and water as a solvent. The newly synthesized compounds were characterized by a number of spectroscopic techniques, such as infrared, liquid chromatography-mass spectrometry, 1H, and 13C nuclear magnetic resonance along with single crystal X-ray diffraction. The current procedure for the synthesis of 1,2,3-triazole hybrids with dihydropyrimidinones is appropriate for the synthesis of a library of analogs 7a-l and the method accessible here is operationally simple and has excellent yields. The title compounds 7a-l were evaluated for their in vitro antitubercular activity against H37RV and multidrug-resistant strains of Mycobacterium tuberculosis by resazurin microplate assay plate method and it was found that compound 7d was promising against H37RV and multidrug-resistant strains of M. tuberculosis at 10 and 15 μg/mL, respectively. PMID:27601885

Design, synthesis, and characterization of (1-(4-aryl)- 1H-1,2,3-triazol-4-yl)methyl, substituted phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates against Mycobacterium tuberculosis.

PubMed

Venugopala, Katharigatta N; Dharma Rao, G B; Bhandary, Subhrajyoti; Pillay, Melendhran; Chopra, Deepak; Aldhubiab, Bandar E; Attimarad, Mahesh; Alwassil, Osama Ibrahim; Harsha, Sree; Mlisana, Koleka

2016-01-01

The novel (1-(4-aryl)-1H-1,2,3-triazol-4-yl)methyl, substituted phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate derivatives were synthesized by the click reaction of the dihydropyrimidinones, bearing a terminal alkynyl group, with various substituted aryl azides at room temperature using a catalytic amount of Cu(OAc)2 and sodium ascorbate in a 1:2 ratio of acetone and water as a solvent. The newly synthesized compounds were characterized by a number of spectroscopic techniques, such as infrared, liquid chromatography-mass spectrometry, (1)H, and (13)C nuclear magnetic resonance along with single crystal X-ray diffraction. The current procedure for the synthesis of 1,2,3-triazole hybrids with dihydropyrimidinones is appropriate for the synthesis of a library of analogs 7a-l and the method accessible here is operationally simple and has excellent yields. The title compounds 7a-l were evaluated for their in vitro antitubercular activity against H37RV and multidrug-resistant strains of Mycobacterium tuberculosis by resazurin microplate assay plate method and it was found that compound 7d was promising against H37RV and multidrug-resistant strains of M. tuberculosis at 10 and 15 μg/mL, respectively.

Endothelial cell responses in terms of adhesion, proliferation, and morphology to stiffness of polydimethylsiloxane elastomer substrates.

PubMed

Ataollahi, Forough; Pramanik, Sumit; Moradi, Ali; Dalilottojari, Adel; Pingguan-Murphy, Belinda; Wan Abas, Wan Abu Bakar; Abu Osman, Noor Azuan

2015-07-01

Extracellular environments can regulate cell behavior because cells can actively sense their mechanical environments. This study evaluated the adhesion, proliferation and morphology of endothelial cells on polydimethylsiloxane (PDMS)/alumina (Al2 O3 ) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 2.5, 5, 7.5, and 10 wt % Al2 O3 at a curing temperature of 50°C for 4 h. The substrates were then characterized by mechanical, structural, and morphological analyses. The cell adhesion, proliferation, and morphology of cultured bovine aortic endothelial (BAEC) cells on substrate materials were evaluated by using resazurin assay and 1,1'-dioctadecyl-1,3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The composites (PDMS/2.5, 5, 7.5, and 10 wt % Al2 O3 ) exhibited higher stiffness than the pure PDMS substrate. The results also revealed that stiffer substrates promoted endothelial cell adhesion and proliferation and also induced spread morphology in the endothelial cells compared with lesser stiff substrates. Statistical analysis showed that the effect of time on cell proliferation depended on stiffness. Therefore, this study concludes that the addition of different Al2 O3 percentages to PDMS elevated substrate stiffness which in turn increased endothelial cell adhesion and proliferation significantly and induced spindle shape morphology in endothelial cells. © 2014 Wiley Periodicals, Inc.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

PubMed

Liu, Rongxia; Heiss, Elke H; Waltenberger, Birgit; Blažević, Tina; Schachner, Daniel; Jiang, Baohong; Krystof, Vladimir; Liu, Wanhui; Schwaiger, Stefan; Peña-Rodríguez, Luis M; Breuss, Johannes M; Stuppner, Hermann; Dirsch, Verena M; Atanasov, Atanas G

2018-04-01

Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC 50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G 0 /G 1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cytotoxic and Apoptotic Effects of Different Extracts of Artemisia turanica Krasch. on K562 and HL-60 Cell Lines

PubMed Central

Tayarani-Najaran, Zahra; Sareban, Mahla; Gholami, Atefeh; Emami, Seyed Ahmad; Mojarrab, Mahdi

2013-01-01

Artemisia is an important genus of Iranian flora. Cytotoxic activities for some species of the genus have already been reported. In this study, we have investigated the cytotoxic effects of n-hexane, CH2Cl2, EtOAc, EtOH, and EtOH/H2O (1 : 1) extracts of A. turanica Krasch. on two human leukemic cancer cell lines (K562 and HL-60) and J774 as normal cells using alamarBlue (resazurin) assay. PI staining of the fragmented DNA and western blot analysis were used to evaluate the possible apoptotic effect of the extract. The CH2Cl2 extract of A. turanica showed the most antiproliferative effect on cancer cells among all tested extracts with IC50 values of 69 and 104 μg/mL on K562 and HL-60 cells, respectively, whereas the normal cells were not affected significantly by this extract. Sub-G1 peak in the flow cytometry histogram of the cells treated with CH2Cl2 extract of A. turanica and cleavage of PARP protein confirmed the induction of apoptosis with CH2Cl2 extract. Taken together, the findings of the present work suggest the anticancer potential of CH2Cl2 extract of A. turanica on human leukemic cancer cell lines. PMID:24288497

Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

PubMed

Ochoa-Gonzalez, Fatima; Cervantes-Villagrana, Alberto R; Fernandez-Ruiz, Julio C; Nava-Ramirez, Hilda S; Hernandez-Correa, Adriana C; Enciso-Moreno, Jose A; Castañeda-Delgado, Julio E

2016-01-01

Several epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation. HaCaT Cells were exposed to various concentrations of metformin and cell viability was evaluated by a Resazurin assay; Proliferation was also evaluated with a colony formation assay and with CFSE dilution assay by flow cytometry. Cell cycle was also evaluated by flow cytometry by PI staining. An animal model of wound healing was used to evaluate the effect of metformin in wound closure. Also, an analysis of patients receiving metformin treatment was performed to determine the effect of metformin treatment on the outcome and wound area. Statistical analysis was performed on SPSS v. 18 and GraphPad software v.5. Metformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin. Metformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.

The Design, Synthesis, and Characterization of Open Sites on Metal Clusters

NASA Astrophysics Data System (ADS)

Nigra, Michael Mark

Coordinatively unsaturated corner and edge atoms have been hypothesized to have the highest activity of sites responsible for many catalytic reactions on a metal surface. Recent studies have validated this hypothesis in varied reaction systems. However, quantification of different types of coordinatively unsaturated sites, and elucidation of their individual catalytic rates has remained a largely unresolved challenge when understanding catalysis on metal surfaces. Yet such structure-function knowledge would be invaluable to the design of more active and selective metal-surface catalysts in the future. I investigated the catalytic contributions of undercoordinated sites such as corner and edge atoms are investigated in a model reaction system using organic ligands bound to the gold nanoparticle surface. The catalyst consisted of 4 nm gold nanoparticles on a metal oxide support, using resazurin to resorufin as a model reaction system. My results demonstrate that in this system, corner atom sites are the most undercoordinated sites, and are over an order of magnitude more active when compared to undercoordinated edge atom sites, while terrace sites remain catalytically inactive for the reduction reaction of resazurin to resorufin. Catalytic activity has been also demonstrated for calixarene-bound gold nanoparticles using the reduction of 4-nitrophenol. With the 4-nitrophenol reduction reaction, a comparative study was undertaken to compare calixarene phosphine and calixarene thiol bound 4 nm gold particles. The results of the study suggested that a leached site was responsible for catalysis and not sites on the original gold nanoparticles. Future experiments with calixarene bound gold clusters could investigate ligand effects in reactions where the active site is not a leached or aggregated gold species, possibly in oxidation reactions, where electron-rich gold is hypothesized to be a good catalyst. The results that emphasize the enhanced catalytic activity of undercoordinated sites led me to synthesize small gold clusters consisting of a high fraction of coordinatively unsaturated open sites. This was enabled through an approach that utilized bulky calix[4]arene ligands that are bound to a gold core. Since the size of the calix[4]arene ligand is commensurate with the size of the gold cluster core, the calix[4]arene ligand does not pack closely together on the gold cluster surface. This in turn results in areas of accessible gold atom sites between ligands. Additionally, these calix[4]arene ligands prevent cluster aggregation and electronically tune the gold core in a manner conceptually similar to enzymes affecting reactivity through organic side-chains acting as ligands. I quantified the number of open sites that result from this packing problem on the gold cluster surface, using fluorescence probe chemisorption experiments. The results of these chemisorption measurements support the mechanical model of accessibility whereby accessibility is not dependent on the identity of the functional group, whether it be calixarene phosphines or N-heterocyclic carbenes, bound to the gold surface, but rather to the relative radii of curvature of bound ligands and the gold cluster core. Additional materials characterization was completed with transmission electron microscopy in both bright-field imaging of zeolites, in MCM-22 and delaminated ITQ-2 and UCB-1 materials, and in dark field imaging of glucan coatings on oxide particles. These materials could prove to be interesting materials as to use as supports for the calixarene-bound metal clusters described above or for other metal clusters.

Visualisation of gas-liquid mass transfer around a rising bubble in a quiescent liquid using an oxygen sensitive dye

NASA Astrophysics Data System (ADS)

Dietrich, Nicolas; Hebrard, Gilles

2018-02-01

An approach for visualizing and measuring the mass transfer around a single bubble rising in a quiescent liquid is reported. A colorimetric technique, developed by (Dietrich et al. Chem Eng Sci 100:172-182, 2013) using an oxygen sensitive redox dye was implemented. It was based on the reduction of the colorimetric indicator in presence of oxygen, this reduction being catalysed by sodium hydroxide and glucose. In this study, resazurin was selected because it offered various reduced forms with colours ranging from transparent (without oxygen) to pink (in presence of oxygen). These advantages made it possible to visualize the spatio-temporal oxygen mass transfer around rising bubbles. Images were recorded by a CCD camera and, after post-processing, the shape, size, and velocity of the bubbles were measured and the colours around the bubbles mapped. A calibration, linking the level of colour with the dissolved oxygen concentration, enabled colour maps to be converted into oxygen concentration fields. A rheoscopic fluid was used to visualize the wake of the bubbles. A calculation method was also developed to determine the transferred oxygen fluxes around bubbles of two sizes (d = 0.82 mm and d = 2.12 mm) and the associated liquid-side mass transfer coefficients. The results compared satisfactorily with classical global measurements made by oxygen micro-sensors or from the classical models. This study thus constitutes a striking example of how this new colorimetric method could become a remarkable tool for exploring gas-liquid mass transfer in fluids.

Visualisation of gas-liquid mass transfer around a rising bubble in a quiescent liquid using an oxygen sensitive dye

NASA Astrophysics Data System (ADS)

Dietrich, Nicolas; Hebrard, Gilles

2018-07-01

An approach for visualizing and measuring the mass transfer around a single bubble rising in a quiescent liquid is reported. A colorimetric technique, developed by (Dietrich et al. Chem Eng Sci 100:172-182, 2013) using an oxygen sensitive redox dye was implemented. It was based on the reduction of the colorimetric indicator in presence of oxygen, this reduction being catalysed by sodium hydroxide and glucose. In this study, resazurin was selected because it offered various reduced forms with colours ranging from transparent (without oxygen) to pink (in presence of oxygen). These advantages made it possible to visualize the spatio-temporal oxygen mass transfer around rising bubbles. Images were recorded by a CCD camera and, after post-processing, the shape, size, and velocity of the bubbles were measured and the colours around the bubbles mapped. A calibration, linking the level of colour with the dissolved oxygen concentration, enabled colour maps to be converted into oxygen concentration fields. A rheoscopic fluid was used to visualize the wake of the bubbles. A calculation method was also developed to determine the transferred oxygen fluxes around bubbles of two sizes (d = 0.82 mm and d = 2.12 mm) and the associated liquid-side mass transfer coefficients. The results compared satisfactorily with classical global measurements made by oxygen micro-sensors or from the classical models. This study thus constitutes a striking example of how this new colorimetric method could become a remarkable tool for exploring gas-liquid mass transfer in fluids.

In vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against biofilm of Brazilian strains of Burkholderia pseudomallei.

PubMed

Bandeira, Tereza de Jesus Pinheiro Gomes; Moreira, Camila Alencar; Brilhante, Raimunda Sâmia Nogueira; Castelo-Branco, Débora de Souza Collares Maia; Neto, Manoel Paiva de Araújo; Cordeiro, Rossana de Aguiar; Rodrigues, Terezinha de Jesus Santos; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

2013-11-01

This study aimed at investigating the in vitro activities of amoxicillin-clavulanate, doxycycline, ceftazidime, imipenem, and trimethoprim-sulfamethoxazole against Burkholderia pseudomallei in planktonic and biofilm forms, through broth microdilution and resazurin-based viability staining, respectively. In planktonic growth, the strains were susceptible to the drugs, while in biofilm growth, significantly higher antimicrobial concentrations were required, especially for ceftazidime and imipenem, surpassing the resistance breakpoints. These results highlight the importance of the routine evaluation of biofilm antimicrobial susceptibility.

Concomitant administration of fluoxetine and amantadine modulates the activity of peritoneal macrophages of rats subjected to a forced swimming test.

PubMed

Roman, Adam; Rogóz, Zofia; Kubera, Marta; Nawrat, Dominika; Nalepa, Irena

2009-01-01

Recent studies show that administration of a non-competitive NMDA receptor antagonist, amantadine (AMA), potentiates the action of antidepressant drugs. Since antidepressants may modulate functioning of the immune system and activation of a pro-inflammatory response in depressive disorders is frequently reported, the aim of the present study was to examine whether a combined administration of AMA and the antidepressant, fluoxetine (FLU), to rats subsequently subjected to a forced swimming test (FST) modifies the parameters of macrophage activity, directly related to their immunomodulatory functions, i.e., arginase (ARG) activity and synthesis of nitric oxide (NO). We found that 10 mg/kg AMA and 10 mg/kg FLU, ineffective in FST for antidepressant-like activity when administered alone, increased the ARG/NO ratio in macrophages when administered concomitantly. This effect was accompanied by a decrease of cellular adherence. Concurrently, the basal metabolic activity of the cells measured with reduction of resazurin, and intracellular host defense as assessed by a synthesis of superoxide anion, were not affected by such antidepressive treatment. Our data indicate that co-administration of AMA and FLU decreases the pro-inflammatory properties of macrophages and causes a redirection of immune response toward anti-inflammatory activity, as one can anticipate in the case of an effective antidepressive treatment.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

PubMed

Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Rao, Parcha Sreenivasa; Reilly, Gwendolen C

2018-06-01

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

Protective Effects of Pistacia lentiscus L. fruit extract against calcium oxalate monohydrate induced proximal tubular injury.

PubMed

Cheraft-Bahloul, Nassima; Husson, Cécile; Ourtioualous, Meriam; Sinaeve, Sébastien; Atmani, Djebbar; Stévigny, Caroline; Nortier, Joëlle L; Antoine, Marie-Hélène

2017-09-14

The world prevalence of kidney stones is increasing and plants are frequently used to treat urolithiasis. Pistacia lentiscus L, a plant which freely grows around the Mediterranean basin areas, is widely used for various pathologies. P. lentiscus has an important impact as it has economical value on top of its pharmacological interest. Decoctions of its aerial parts and/or resin are used to treat kidney stones. To in vitro assess the potential nephroprotective effect of Pistacia lentiscus ethanolic fruit extract (PLEF) on proximal tubular cells in response to the adhesion of calcium oxalate monohydrate (COM) crystals. Human Kidney [HK]-2 cells were incubated with and without COM in the presence or absence of PLEF. Cell viability was measured by the resazurin assay. The expression of E-cadherin was analyzed by PCR. The extracellular production of H 2 O 2 was measured by Amplex® Red H 2 O 2 Assay. The numbers of detached or non-adherent COM crystals in the presence of PLEF were microscopically captured and counted using ImageJ software. The interaction of PLEF with COM and the effect of PLEF on crystal size were analyzed by flow cytometry. The spectrophotometric measurement of turbidity was performed for assessing the COM concentration. PLEF incubated with COM was able to increase the cell viability. The decrease of E-cadherin expression after incubation with COM was counteracted by PLEF. Overproduction of H 2 O 2 induced by COM was also inhibited by PLEF. Observations using flow cytometry showed that interactions between PLEF and the COM crystals occurred. PLEF was also effective in reducing the particles size and in lowering COM concentration. Our data show that COM tubulotoxicity can be significantly reversed by PLEF -at least in part- via an inhibition of COM crystals adhesion onto the apical membrane. This early beneficial effect of PLEF needs to be further investigated as a useful strategy in nephrolithiasis prevention. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Terrestrial and marine Antarctic fungi extracts active against Xanthomonas citri subsp. citri.

PubMed

Vieira, G; Purić, J; Morão, L G; Dos Santos, J A; Inforsato, F J; Sette, L D; Ferreira, H; Sass, D C

2018-07-01

This study aims to obtain secondary metabolites extracts from filamentous fungi isolated from soil and marine sediments from Antarctica and assess its potential antibacterial activity on Xanthomonas citri subsp. citri, the agent of citrus canker. Metabolites production was conducted in Malt 2% broth at 15°C for 20 days after which intracellular and extracellular extracts were obtained. The extracts were evaluated by cell viability assays through Resazurin Microtitre Assay. From 158 fungal extracts, 33 hampered bacterial growth in vitro. The average inhibition of the extracts obtained from terrestrial (soil) and marine (sediments) fungi was 94 and 97% respectively. These inhibition values were close to the average of 90% cell death for the positive control. MIC90 and MBC for the bioactive extracts were established. Isolates that produced active metabolites against the phytopathogen were identified using molecular taxonomy (ITS-rRNA sequencing) as: Pseudogymnoascus, Penicillium, Cadophora, Paraconiothyrium and Toxicocladosporium. Antarctic fungal strains isolated from terrestrial and marine sediments were able to produce secondary metabolites with antimicrobial activity against X. citri subsp. citri, highlighting the importance of these microbial genetic resources. These metabolites have potential to be used as alternatives for the control of this plant pathogen. This manuscript makes an impact on the study of micro-organisms from extreme habitats and their possible contribution in discovering new active molecules against pathogens of agricultural interest. Studies on the Antarctic continent and its communities have attracted the scientific community due to the long period of isolation and low levels of disturbance that surrounds the region. Knowing the potential of fungi in this region to produce active secondary metabolites, we aim to contribute to the discovery of compounds with antibacterial action in Xanthomonas citri subsp. citri, a plant pathogen present in several regions around the globe. © 2018 The Society for Applied Microbiology.

Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

PubMed

Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

2014-01-01

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected] Copyright © 2014 Elsevier Inc. All rights reserved.

Multifunctional adhesive polymers: Preactivated thiolated chitosan-EDTA conjugates.

PubMed

Netsomboon, Kesinee; Suchaoin, Wongsakorn; Laffleur, Flavia; Prüfert, Felix; Bernkop-Schnürch, Andreas

2017-02-01

The aim of this study was to synthesis preactivated thiolated chitosan-EDTA (Ch-EDTA-cys-2MNA) conjugates exhibiting in particular high mucoadhesive, cohesive and chelating properties. Thiol groups were coupled with chitosan by carbodiimide reaction and further preactivated by attachment with 2-mercaptonicotinic acid (2MNA) via disulfide bond formation. Determinations of primary amino and sulfhydryl groups were performed by TNBS and Ellman's tests, respectively. Cytotoxicity was screened by resazurin assay in Caco-2 cells. Mucoadhesive properties and bivalent cation binding capacity with Mg 2+ and Ca 2+ in comparison to chitosan-EDTA (Ch-EDTA) and thiolated Ch-EDTA (Ch-EDTA-cys) were evaluated. Determination of 2MNA and total sulfhydryl groups indicated that 80% of thiol groups were preactivated. The results from cytotoxicity studies demonstrated that Ch-EDTA-cys and Ch-EDTA-cys-2MNA were not toxic to the cells at the polymer test concentration of 0.25% (w/v) while cell viability decreased by increasing the concentration of Ch-EDTA. Although EDTA molecule was modified by thiolation and preactivation, approximately 50% of chelating properties of the conjugates were maintained compared to Ch-EDTA. Ch-EDTA-cys-2MNA adhered on freshly excised porcine intestinal mucosa up to 6h while Ch-EDTA adhered for just 1h. According to the combination of mucoadhesive and chelating properties of the conjugates synthesized in this study, Ch-EDTA-cys-2MNA might be useful for various mucosal drug delivery systems. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung.

PubMed

Kirchner, Sebastian; Fothergill, Joanne L; Wright, Elli A; James, Chloe E; Mowat, Eilidh; Winstanley, Craig

2012-06-05

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests. Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions. An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a > 128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions. The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods. Several in vitro models have been used previously to study P. aeruginosa biofilms. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung. In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa and affect antibiotic susceptibility. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Evaluation of the anti-mycobacterium tuberculosis activity and in vivo acute toxicity of Annona sylvatic

PubMed Central

2014-01-01

Background The recent emergence of extensively multidrug-resistant Mycobacterium tuberculosis strains has further complicated the control of tuberculosis. There is an urgent need for the development of new molecular candidates antitubercular drugs. Medicinal plants have been an excellent source of leads for the development of drugs. The aim of this study was to evaluate the in vitro activity of 28 alcoholic extracts and essential oils of native and exotic Brazilian plants against Mycobacterium tuberculosis and to further study these extracts through chemical fractionation, the isolation of their constituents, and an evaluation of the in vivo acute toxicity of the active extracts. To the best of our knowledge this is the first chemical characterization, antituberculosis activity and acute toxicity evaluation of Annona sylvatica. Methods The anti-mycobacterial activity of these extracts and their constituent compounds was evaluated using the resazurin reduction microtiter assay (REMA). To investigate the acute toxicity of these extracts in vivo, female Swiss mice were treated with the extracts at doses of 500, 1000 and 2000 mg · kg-1 of body weight. The extracts were characterized by LC-MS, and the constituents were isolated and identified by chromatographic analysis of spectroscopic data. Results Of the 28 extracts, the methanol extract obtained from the leaves of Annona sylvatica showed anti-mycobacterial activity with an minimal inhibitory concentration (MIC) of 184.33 μg/mL, and the ethyl acetate fraction (EAF) resulting from liquid-liquid partitioning of the A. sylvatica extract showed an MIC of 115.2 μg/mL. The characterization of this extract by LC-MS identified flavonoids and acetogenins as its main constituents. The phytochemical study of the A. sylvatica EAF resulted in the isolation of quercetin, luteolin, and almunequin. Conclusions Among the compounds isolated from the EAF, luteolin and almunequin were the most promising, with MICs of 236.8 μg/mL (827.28 μM) and 209.9 μg/mL (328.48 μM), respectively. The acute administration of the EAF fraction in doses of 500, 1000, and 2000 mg · kg-1 of body weight did not cause signs of toxicity in the treated animals. PMID:24974069

Cytotoxicity of 18 Cameroonian medicinal plants against drug sensitive and multi-factorial drug resistant cancer cells.

PubMed

Mbaveng, Armelle T; Manekeng, Hermione T; Nguenang, Gaelle S; Dzotam, Joachim K; Kuete, Victor; Efferth, Thomas

2018-08-10

Recommendations have been made stating that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account if selecting plants for anticancer screening, since these reflect disease states bearing relevance to cancer or cancer-like symptoms. Cameroonian medicinal plants investigated in this work are traditionally used to treat cancer or ailments with relevance to cancer or cancer-like symptoms. In this study, 21 methanol extracts from 18 Cameroonian medicinal plants were tested in leukemia CCRF-CEM cells, and the best extracts were further tested on a panel of human cancer cell lines, including various multi-drug-resistant (MDR) phenotypes. Mechanistic studies were performed with the three best extracts. Resazurin reduction assay was used to evaluate cytotoxicity and ferroptotic effects of methanol extracts from different plants. Flow cytometry was used to analyze cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) of extracts from Curcuma longa rhizomes (CLR), Lycopersicon esculentum leaves (LEL), and Psidium guajava bark (PGB). In a pre-screening of all extracts, 13 out of 21 (61.9%) had IC 50 values below 80 µg/mL. Six of these active extracts displayed IC 50 values below 30 µg/mL: Cola pachycarpa leaves (CPL), Curcuma longa rhizomes (CLR), Lycopersicon esculentum leaves, Persea americana bark (PAB), Physalis peruviana twigs (PPT) and Psidium guajava bark (PGB). The best extracts displayed IC 50 values from 6.25 µg/mL (against HCT116 p53 -/- ) to 10.29 µg/mL (towards breast adenocarcinoma MDA-MB-231-BCRP cells) for CLR, from 9.64 µg/mL (against breast adenocarcinoma MDA-MB-231 cells) to 57.74 µg/mL (against HepG2 cells) for LEL and from 1.29 µg/mL (towards CEM/ADR5000 cells) to 62.64 µg/mL (towards MDA-MB-231 cells) for PGB. CLR and PGB induced apoptosis in CCRF-CEM cells via caspases activation, MMP depletion and increase ROS production whilst LEL induced apoptosis mediated by caspases activation and increase ROS production. The best botanicals tested were CLR and LEL, which are worth to be explored in more detail to fight cancers including MDR phenotypes. Copyright © 2018 Elsevier B.V. All rights reserved.

A field comparison of multiple techniques to quantify groundwater - surface-water interactions

USGS Publications Warehouse

González-Pinzón, Ricardo; Ward, Adam S; Hatch, Christine E; Wlostowski, Adam N; Singha, Kamini; Gooseff, Michael N.; Haggerty, Roy; Harvey, Judson; Cirpka, Olaf A; Brock, James T

2015-01-01

Groundwater–surface-water (GW-SW) interactions in streams are difficult to quantify because of heterogeneity in hydraulic and reactive processes across a range of spatial and temporal scales. The challenge of quantifying these interactions has led to the development of several techniques, from centimeter-scale probes to whole-system tracers, including chemical, thermal, and electrical methods. We co-applied conservative and smart reactive solute-tracer tests, measurement of hydraulic heads, distributed temperature sensing, vertical profiles of solute tracer and temperature in the stream bed, and electrical resistivity imaging in a 450-m reach of a 3rd-order stream. GW-SW interactions were not spatially expansive, but were high in flux through a shallow hyporheic zone surrounding the reach. NaCl and resazurin tracers suggested different surface–subsurface exchange patterns in the upper ⅔ and lower ⅓ of the reach. Subsurface sampling of tracers and vertical thermal profiles quantified relatively high fluxes through a 10- to 20-cm deep hyporheic zone with chemical reactivity of the resazurin tracer indicated at 3-, 6-, and 9-cm sampling depths. Monitoring of hydraulic gradients along transects with MINIPOINT streambed samplers starting ∼40 m from the stream indicated that groundwater discharge prevented development of a larger hyporheic zone, which progressively decreased from the stream thalweg toward the banks. Distributed temperature sensing did not detect extensive inflow of ground water to the stream, and electrical resistivity imaging showed limited large-scale hyporheic exchange. We recommend choosing technique(s) based on: 1) clear definition of the questions to be addressed (physical, biological, or chemical processes), 2) explicit identification of the spatial and temporal scales to be covered and those required to provide an appropriate context for interpretation, and 3) maximizing generation of mechanistic understanding and reducing costs of implementing multiple techniques through collaborative research.

RAPID TETRAZOLIUM DYE REDUCTION ASSAY TO ASSESS THE BACTERICIDAL ACTIVITY OF OYSTER (CRASSOSTREA VIRGINICA) HEMOCYTES AGAINST VIBRIO PARAHAEMOLYTICUS

EPA Science Inventory

An assay was developed to assess the ability of oyster, Crassostrea virginica, hemocytes to kill the human pathogenic bacterium, Vibrio parahaemolyticus (ATCC 17802). Bacterial killing was estimated colorimetrically by the enzymatic reduction of a tetrazolium dye, 3-(4,5-dimethyl...

In vitro methods of assessing ocular biocompatibility using THP-1-derived macrophages.

PubMed

McCanna, David Joseph; Barthod-Malat, Aurore V; Gorbet, Maud B

2015-01-01

Macrophages play an important role in the elimination of infections, the removal of debris and in tissue repair after infection and trauma. In vitro models that assess ocular biomaterials for toxicity typically focus on the effects of these materials on epithelial or fibroblast cells. This investigation evaluated known ocular toxins deposited on model materials for their effects on the viability and activation of macrophages. THP-1-derived macrophages were cultured onto silicone films (used as a base biomaterial) deposited with chemical toxins (benzalkonium chloride (BAK), zinc diethyldithiocarbamate (ZDEC) and lipopolysaccharide (LPS)). Utilizing three fluorescent dyes calcein, ethidium homodimer-1 (EthD-1) and annexin V, the viability of macrophages attached to the biomaterial was determined using confocal microscopy. Propidium iodide (PI) staining and alamarBlue® (resazurin) reduction were used to assess cell death and metabolic activity. CD14, CD16, CD33, CD45, and CD54 expression of adherent macrophages, were also evaluated to detect LPS activation of macrophages using flow cytometry. The sensitivity of this test battery was demonstrated as significant toxicity from treated surfaces with ZDEC (0.001-0.01%), and BAK (0.001%-0.1%) was detected. Also, macrophage activation could be detected by measuring CD54 expression after exposure to adsorbed LPS. These in vitro methods will be helpful in determining the toxicity potential of new ocular biomaterials.

Specific detection of membrane-toxic substances with a conductivity assay.

PubMed

Eich, J; Dürholt, H; Steger-Hartmann, T; Wagner, E

2000-03-01

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds. Copyright 2000 Academic Press.

Molecular characterization and drug susceptibility profile of a Mycobacterium avium subspecies avium isolate from a dog with disseminated infection.

PubMed

Armas, Federica; Furlanello, Tommaso; Camperio, Cristina; Trotta, Michele; Novari, Gianluca; Marianelli, Cinzia

2016-04-01

Mycobacterium avium-intracellulare complex (MAC) infections have been described in many mammalian species, including humans and pets. We isolated and molecularly typed the causative agent of a rare case of disseminated mycobacteriosis in a dog. We identified the pathogen as M. avium subspecies avium by sequencing the partial genes gyrB and rpsA . Considering the zoonotic potential of this infection, and in an attempt to ensure the most effective treatment for the animal, we also determined the drug susceptibility profile of the isolate to the most common drugs used to treat MAC disease in humans. The pathogen was tested in vitro against the macrolide clarithromycin, as well as against amikacin, ciprofloxacin, rifampicin, ethambutol and linezolid, by the resazurin microdilution assay. It was found to be sensitive to all tested drugs apart from ethambutol. Despite the fact that the pathogen was sensitive to the therapies administered, the dog's overall clinical status worsened and the animal died shortly after antimicrobial susceptibility results became available. Nucleotide sequencing of the embB gene, the target gene most commonly associated with ethambutol resistance, showed new missense mutations when compared to sequences available in public databases. In conclusion, we molecularly identified the MAC pathogen and determined its drug susceptibility profile in a relatively short period of time (7 days). We also characterized new genetic mutations likely to have been involved in the observed ethambutol resistance. Our results confirmed the usefulness of both the gyrB and the rpsA genes as biomarkers for an accurate identification and differentiation of MAC pathogens.

Tolerance of Pseudomonas aeruginosa in in-vitro biofilms to high-level peracetic acid disinfection.

PubMed

Akinbobola, A B; Sherry, L; Mckay, W G; Ramage, G; Williams, C

2017-10-01

Biofilm has been suggested as a cause of disinfection failures in flexible endoscopes where no lapses in the decontamination procedure can be identified. To test this theory, the activity of peracetic acid, one of the widely used disinfectants in the reprocessing of flexible endoscopes, was evaluated against both planktonic and sessile communities of Pseudomonas aeruginosa. To investigate the ability of P. aeruginosa biofilm to survive high-level peracetic acid disinfection. The susceptibility of planktonic cells of P. aeruginosa and biofilms aged 24, 48, 96, and 192 h to peracetic acid was evaluated by estimating their viability using resazurin viability and plate count methods. The biomass of the P. aeruginosa biofilms was also quantified using Crystal Violet assay. Planktonic cells of P. aeruginosa were treated with 5-30 ppm concentration of peracetic acid in the presence of 3.0 g/L of bovine serum albumin (BSA) for 5 min. Biofilms of P. aeruginosa were also treated with various peracetic acid concentrations (100-3000 ppm) for 5 min. Planktonic cells of P. aeruginosa were eradicated by 20 ppm of peracetic acid, whereas biofilms showed an age-dependent tolerance to peracetic acid, and 96 h biofilm was only eradicated at peracetic acid concentration of 2500 ppm. Ninety-six-hour P. aeruginosa biofilm survives 5 min treatment with 2000 ppm of peracetic acid, which is the working concentration used in some endoscope washer-disinfectors. This implies that disinfection failure of flexible endoscopes might occur when biofilms build up in the lumens of endoscopes. Copyright © 2017. Published by Elsevier Ltd.

Antibacterial and antifungal activities from Siamese crocodile blood.

PubMed

Leelawongtawon, Ratree; Siruntawineti, Jindawan; Chaeychomsri, Win; Sattaponpan, Chisanucha

2010-12-01

To evaluate the in vitro antimicrobial activity of the Siamese crocodile blood against bacteria and fungi. Thirty Siamese crocodile blood samples including freeze dried whole blood (FDWB), fresh serum (FS), and freeze dried serum (FDS) were evaluated for antimicrobial susceptibility and MIC values against ATCC-registered strains of nine bacterial species and two fungal species and one fungus isolated from a clinical specimen, by using the standard broth microdilution method and a modified resazurin microtiter plate assay. The result showed that FS (80 mg/ml) and FDS (100 mg/ml) inhibited Gram negative bacteria including Enterobacter aerogenes ATCC 13048, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 27736, Salmonella typhimurium ATCC 13311 and Pseudomonas aeruginosa ATCC 27853 with the susceptibility rate at 23.30%, 10.00%, 40.00%, 70.00%, and 86.67%, respectively for FS, and 30.00%, 10.00%, 43.33%, 76.67% and 90.00%, respectively for FDS. The MIC and MBC were in the range of 12.50-100.00 mg/ml and 25.00-100.00 mg/m1 respectively. FS and FDS also inhibited Cryptococcus neoformans 250309 and Aspergillus niger with the susceptibility rate at 90.00% and 80.00%, respectively for FS and 100.00% and 83.33%, respectively for FDS. The MIC was in the range of 25.00-100.00 mg/ml. However, FS and FDS did not inhibit Gram positive bacteria and did not kill fungi. FDWB (100 mg/ml) could neither inhibit bacteria nor fungi. FS and FDS from Siamese crocodile exhibited potential antibacterial and antifungal activities.

Titanium dioxide nanotubes functionalized with Cratylia mollis seed lectin, Cramoll, enhanced osteoblast-like cells adhesion and proliferation.

PubMed

Oliveira, Weslley F; Silva, Germana M M; Cabral Filho, Paulo E; Fontes, Adriana; Oliveira, Maria D L; Andrade, César A S; Silva, Márcia V; Coelho, Luana C B B; Machado, Giovanna; Correia, Maria T S

2018-09-01

An alternative to accelerate the osseointegration on titanium dioxide nanotubes (TNTs) used in osseointegrated implants is through the functionalization of these nanostructured surfaces with biomolecules. In this work, we immobilized a lectin with recognized mitogenic activity, the Cramoll lectin, extracted from Cratylia mollis seeds, on surfaces modified by TNTs. For the immobilization of Cramoll on TNTs surfaces, we used the Layer-by-Layer technique (LbL) by growing five alternate layers of poly(allylamine) hydrochloride (PAH) and poly(acrylic) acid (PAA); lastly we incubated the lectin, at different concentrations, with the TNTs-LbL. Before and after the immobilization procedures, the substrate surfaces were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and, electrochemical impedance spectroscopy (EIS). We also evaluated the Cramoll activity after immobilization on TNTs by using the lectin interaction with ovalbumin. The lectin did not lose its biological activity, even after immobilization onto nanotubular arrays. In addition, we observed an increase osteoblast-like cell adhesion on the TNTs-LbL-Cramoll system when compared to the bare TNTs surfaces. Moreover, a significative cell proliferation was identified on the substrates when Cramoll was immobilized at concentrations of 80, 160 and 320 μg/mL after 48 h of incubation by using the resazurin assay. Our results suggest that Cramoll was efficiently immobilized on a nanotubular array and this new platform presents a great potential to be tested in implantology. Copyright © 2018 Elsevier B.V. All rights reserved.

Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle.

PubMed

Franco, Marília Masello Junqueira; Ribeiro, Márcio Garcia; Pavan, Fernando Rogério; Miyata, Marcelo; Heinemann, Marcos Bryan; de Souza Filho, Antonio Francisco; Cardoso, Rosilene Fressatti; de Almeida, Aryadne Larissa; Sakate, Ricardo Ichiro; Paes, Antonio Carlos

2017-05-01

In developing nations, 10-20% of the human cases of tuberculosis are caused by Mycobacterium bovis. However, this percentage may be underestimated because most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few have the systems in place to identify M. bovis. There are few studies investigating genotypic diversity and drug resistance in M. bovis from animal and/or human infections. The genotypic diversity of M. bovis strains obtained from bovine lymph nodes were investigated by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR). The phenotypic resistance to isoniazid and rifampicin and MIC values of the isolates were determined using the resazurin microtiter assay plate method (REMA). The evaluation of the possible genetic basis for such resistance was performed with GenoType MTBDRplus. Sixty-seven isolates were obtained, of which 11 (16%) were MDR-TB, 8 (12%) were isoniazid-resistant, and 2 (3%) were rifampicin-resistant. Mutations associated with drug resistance were not found. Genotyping techniques enabled the grouping of the strains into 12 clusters and 21 isolates with unique profiles. The high frequency of M. bovis reinforces the impact of the pathogen as a major causal agent of bovine tuberculosis in the study area. The resistance of the strains to drugs used for first-line treatment of human tuberculosis raises public health concerns. Further studies are required to elucidate the basis of drug resistance and genotypic diversity in M. bovis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Self-emulsifying drug delivery systems (SEDDS): Proof-of-concept how to make them mucoadhesive.

PubMed

Leonaviciute, Gintare; Adamovic, Nada Trivic; Lam, Hung Thanh; Rohrer, Julia; Partenhauser, Alexandra; Bernkop-Schnürch, Andreas

2017-03-01

The objective of this study was to provide a proof-of-concept that self-emulsifying drug delivery systems can be made mucoadhesive by the incorporation of hydrophobic mucoadhesive polymers. In order to obtain such a hydrophobic mucoadhesive polymer, Eudragit® S100 was thiolated by covalent attachment of cysteamine. After determination of the thiol group content, in vitro mucoadhesion studies (rotating cylinder and rheological measurements) were performed. Then, synthesized conjugate was incorporated into self-emulsifying drug delivery systems (SEDDS) and their toxic potential as well as that of unmodified and thiolated Eudragit® S100 was examined on Caco-2 cell line. Lastly, the mucoadhesiveness of developed SEDDS on porcine intestinal mucosa was determined. Generated thiolated Eudragit® S100 displaying 235±14μmol of free thiol groups and 878±101μmol of disulfide bonds per gram polymer showed a great improvement in both: dynamic viscosity with mucus and adhesion time on mucosal tissue compared to the unmodified polymer. Resazurin assay revealed that unmodified and thiolated polymers and also SEDDS dispersions were non-toxic over Caco-2 cells. Furthermore, the incorporation of 1.5% (w/w) of such thiomer into SEDDS led to remarkably improved mucoadhesiveness. Blank SEDDS were completely removed from the mucosa within 15min, whereas >60% of SEDDS containing thiolated Eudragit® S100 were still attached to it. These results provide evidence that SEDDS can be made mucoadhesive by the incorporation of hydrophobic mucoadhesive polymers. Copyright © 2016 Elsevier B.V. All rights reserved.

Antimycobacterial activity of medicinal plants used by the Mayo people of Sonora, Mexico.

PubMed

Coronado-Aceves, Enrique Wenceslao; Sánchez-Escalante, José Jesús; López-Cervantes, Jaime; Robles-Zepeda, Ramón Enrique; Velázquez, Carlos; Sánchez-Machado, Dalia Isabel; Garibay-Escobar, Adriana

2016-08-22

Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb), which generates 9 million new cases worldwide each year. The Mayo ethnicity of southern Sonora, Mexico is more than 2000 years old, and the Mayos possess extensive knowledge of traditional medicine. To evaluate the antimycobacterial activity levels of extracts of medicinal plants used by the Mayos against Mtb and Mycobacterium smegmatis (Msm) in the treatment of TB, respiratory diseases and related symptoms. A total of 34 plant species were collected, and 191 extracts were created with n-hexane, dichloromethane, ethyl acetate (EtOAc), methanol and water. Their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against Mtb H37Rv using the microplate alamar blue assay (MABA) and against Msm using the resazurin microplate assay (REMA) at 6 and 2 days of exposure, respectively, and at concentrations of 250-1.9µg/mL (n-hexane extracts) and 1000-7.81µg/mL (extracts obtained with dichloromethane, EtOAc, methanol and water). Rhynchosia precatoria (Willd.) DC. (n-hexane root extract), Euphorbia albomarginata Torr. and A. Gray. (EtOAc shoot extract) and Helianthus annuus L. (n-hexane stem extract) were the most active plants against Mtb H37Rv, with MICs of 15.6, 250, 250µg/mL and MBCs of 31.25, 250, 250µg/mL, respectively. R. precatoria (root) was the only active plant against Msm, with MIC and MBC values of ≥250µg/mL. None of the aqueous extracts were active. This study validates the medicinal use of certain plants used by the Mayo people in the treatment of TB and related symptoms. R. precatoria, E. albomarginata and H. annuus are promising plant sources of active compounds that act against Mtb H37Rv. To our knowledge, this is the first time that their antimycobacterial activity has been reported. Crude extracts obtained with n-hexane, EtOAc and dichloromethane were the most active against Mtb H37Rv. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Microbial toxicity of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites.

PubMed

Liang, Jidong; Olivares, Christopher; Field, Jim A; Sierra-Alvarez, Reyes

2013-11-15

2,4-Dinitroanisole (DNAN) is an insensitive munitions compound considered to replace conventional explosives such as 2,4,6-trinitrotoluene (TNT). DNAN undergoes facile microbial reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN). This study investigated the inhibitory effect of DNAN, MENA, and DAAN toward various microbial targets in anaerobic (acetoclastic methanogens) and aerobic (heterotrophs and nitrifiers) sludge, and the bioluminescent bacterium, Aliivibrio fischeri, used in the Microtox assay. Aerobic heterotrophic and nitrifying batch experiments with DAAN could not be performed because the compound underwent extensive autooxidation in these assays. DNAN severely inhibited methanogens, nitrifying bacteria, and A. fischeri (50% inhibitory concentrations (IC50) ranging 41-57μM), but was notably less inhibitory to aerobic heterotrophs (IC50>390 μM). Reduction of DNAN to MENA and DAAN lead to a marked decrease in methanogenic inhibition (i.e., DNAN>MENA≈DAAN). Reduction of all nitro groups in DNAN also resulted in partial detoxification in assays with A. fischeri. In contrast, reduction of a single nitro group did not alter the inhibitory impact of DNAN toward A. fischeri and nitrifying bacteria given the similar IC50 values determined for MENA and DNAN in these assays. These results indicate that reductive biotransformation could reduce the inhibitory potential of DNAN. Copyright © 2013 Elsevier B.V. All rights reserved.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

PubMed

Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

2016-09-01

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

PERKINSUS-"CIDAL" ACTIVITY OF OYSTER HEMOCYTES USING A TETRAZOLIUM DYE REDUCTION ASSAY: OPTIMIZATION AND APPLICATIONS

EPA Science Inventory

A bactericidal assay developed to assess the ability of oyster (Crassostrea virginica) hemocytes to kill the human pathogen Vibrio parahaemolyticus was optimized to estimate killing of the oyster parasite Perkinsus marinus. Assay variables, temperature, hemocyte:parasite ratio, i...

Rapid assay for microbially reducible ferric iron in aquatic sediments

USGS Publications Warehouse

Lovely, Derek R.; Philips , Elizabeth J.P.

1987-01-01

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.

Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

PubMed

Russell, K A; Koch, T G

2016-03-01

Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles—an in vitro and in vivo study

NASA Astrophysics Data System (ADS)

Grenho, L.; Salgado, C. L.; Fernandes, M. H.; Monteiro, F. J.; Ferraz, M. P.

2015-08-01

Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA-ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections.

Anti-mycobacterial natural products from the Canadian medicinal plant Juniperus communis.

PubMed

Carpenter, Caitlyn D; O'Neill, Taryn; Picot, Nadia; Johnson, John A; Robichaud, Gilles A; Webster, Duncan; Gray, Christopher A

2012-09-28

Common juniper, Juniperus communis, is amongst the plants most frequently used by the indigenous peoples of North America for medicinal purposes. The First Nations of the Canadian Maritimes use infusions of juniper primarily as a tonic and for the treatment of tuberculosis. Previous investigations of extracts derived from the aerial parts of J. communis have shown it to possess anti-mycobacterial activity. The aim of the study is to isolate and identify anti-mycobacterial constituents from the aerial parts of J. communis. Methanolic extracts of J. communis needles and branches were subjected to bioassay guided fractionation using the microplate resazurin assay (MRA) to assess inhibitory activity against Mycobacterium tuberculosis strain H37Ra. The anti-mycobacterial constituents were identified by NMR, MS and polarimetry. The diterpenes isocupressic acid and communic acid and the aryltetralin lignan deoxypodophyllotoxin were isolated from the J. communis extract. Isocupressic acid and communic acid (isolated as an inseparable 3:2 mixture of cis and trans isomers) displayed MICs of 78 μM and 31 μM and IC(50)s of 46 μM and 15 μM against M. tuberculosis H37Ra respectively. Deoxypodophyllotoxin was less active, with a MIC of 1004 μM and an IC(50) of 287 μM. Isocupressic acid, communic acid and deoxypodophyllotoxin were identified as the principal constituents responsible for the anti-mycobacterial activity of the aerial parts of J. communis. Although further research will be required to evaluate the relative activities of the two communic acid isomers, this work validates an ethnopharmacological use of this plant by Canadian First Nations and Native American communities. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

[A preliminary study on the molecular characteristics of D-cycloserine resistance of Mycobacterium tuberculosis].

PubMed

Li, C; Li, G L; Luo, Q; Li, S J; Wang, R B; Lou, Y L; Lyu, J X; Wan, K L

2017-02-10

Objective: To investigate the relationship between D-cycloserine resistance and the gene mutations of alrA , ddlA and cycA of Mycobacterium ( M. ) tuberculosis , as well as the association between D-cycloserine resistance and spoligotyping genotyping. Methods: A total of 145 M. tuberculosis strains were selected from the strain bank. D-cycloserine resistant phenotypes of the strains were determined by the proportion method and the minimal inhibitory concentration was determined by resazurin microtiter assay. PCR amplification and DNA direct sequencing methods were used for the analysis of gene mutations. Relationship between the resistance phenotype and genotype was analyzed by chi -square test. Results: Of the 145 clinically collected strains, 24 (16.6%) of them were D-cycloserine resistant and 121 (83.4%) were sensitive. There were only synonymous mutations noticed on alrA , ddlA and cycA in sensitive strains. Of the 24 D-cycloserine resistant strains, 3 (12.5%) isolates' cycA and 1 (4.2%) isolates' alrA happened to be non-synonymous mutations, in which the codes were 188, 318 and 508 of cycA , and 261 of alrA , respectively. Results on drug sensitivity tests confirmed the minimal inhibitory concentration of the mutant strains were all increased to some degrees. The D-cycloserine resistant rates of 88 Beijing genotype and 57 non-Beijing genotype strains were 20.5% and 10.5% , respectively, but with no statistically significant difference ( χ (2) =2.47, P >0.05). Conclusions: The non-synonymous mutations of alrA and cycA might contribute to one of the mechanisms of M. tuberculosis D-cycloserine resistance. M. tuberculosis Beijing genotype or non-Beijing genotype was not considered to be associated with the D-cycloserine resistance.

High Sensitivity of Giardia duodenalis to Tetrahydrolipstatin (Orlistat) In Vitro

PubMed Central

Hahn, Juliane; Seeber, Frank; Kolodziej, Herbert; Ignatius, Ralf; Laue, Michael; Aebischer, Toni; Klotz, Christian

2013-01-01

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC5024h = 2.8 µM) being more sensitive than WB-C6 (IC5024h = 6.2 µM). The effect was significantly more potent compared to MTZ (IC5024h = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis. PMID:23977083

Stimulated phase-shift acoustic nanodroplets enhance vancomycin efficacy against methicillin-resistant Staphylococcus aureus biofilms.

PubMed

Guo, Hao; Wang, Ziming; Du, Quanyin; Li, Pan; Wang, Zhigang; Wang, Aimin

2017-01-01

Bacterial biofilms on the surface of prostheses are becoming a rising concern in managing prosthetic joint infections. The inherent resistant features of biofilms render traditional antimicrobial therapy unproductive and revision surgery outcomes uncertain. This situation has prompted the exploration of novel antimicrobial strategies. The synergy of ultrasound microbubbles and vancomycin has been proposed as an efficient alternative for biofilm eradication. The purpose of this study was to evaluate the anti-biofilm effect of stimulated phase-shift acoustic nanodroplets (NDs) combined with vancomycin. We fabricated lipid phase-shift NDs with a core of liquid perfluoropentane. A new phase change mode for NDs incorporating an initial unfocused low-intensity pulsed ultrasound for 5 minutes and a subsequent incubation at 37°C into a 24-hour duration was developed. Methicillin-resistant Staphylococcus aureus (MRSA) biofilms were incubated with vancomycin and NDs under the hybrid stimulation. Biofilm morphology following treatment was determined using confocal laser scanning microscopy and scanning electron microscopy. Resazurin assay was used to quantify bactericidal efficacy against MRSA biofilm bacteria. NDs treated sequentially with ultrasound and heating at 37°C achieved gradual and substantial ND vaporization and cavitation in a successive process. NDs after stimulation were capable of generating stronger destruction on biofilm structure which was best characterized by residual circular arc margins and more dead bacteria. Furthermore, NDs combined with vancomycin contributed to significantly decreasing the metabolic activity of bacteria in MRSA biofilms ( P <0.05). Phase-shift acoustic NDs could exert a significant bactericidal effect against MRSA biofilms through a new stimulation mode. Acoustic NDs present advantages over microbubbles for biofilm damage. This anti-biofilm strategy could be used either alone or as an enhancer of traditional antibiotics in the control of prosthetic joint infections.

Antimicrobial activity of natural products from the flora of Northern Ontario, Canada.

PubMed

Vandal, Janique; Abou-Zaid, Mamdouh M; Ferroni, Garry; Leduc, Leo G

2015-06-01

The number of multidrug resistant (MDR) microorganisms is increasing and the antimicrobial resistance expressed by these pathogens is generating a rising global health crisis. In fact, there are only a few antimicrobial agents left that can be used against MDR bacteria and fungi. In this study, the antimicrobial activities of selected natural products from the flora of Northern Ontario against selected microorganisms are reported. Plants were collected from Sault Ste. Marie, Ontario, Canada, and ethanol extracts were prepared using EtOH:H2O (1:1, v/v). Fungal cultures used in this study were Candida albicans ATCC 10231 and Schizosaccharomyces octosporus. Bacterial cultures employed included Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Mycobacterium phlei ATCC 11758, and Streptococcus lactis ATCC 19435. The microplate resazurin assay was used to screen for antimicrobial activity. Extracts of four plant species Chimaphila umbellata L. (Pyrolaceae), Betula papyrifera Marshall (Betulaceae), Rhus typhina L. (Anacardiaceae), and Fraxinus pennsylvanica Marshall (Oleaceae), and six compounds (gallic acid, ethyl gallate, caffeic acid, sinapic acid, gentisic acid, and chlorogenic acid) demonstrated antibacterial or antifungal activities with MICs ranging from 62.5 to 1000 µg/mL, respectively, for a chemical fraction of an extract from Betula papyrifera against the bacterium S. aureus. The present study has shown that certain plant extracts and select fractions and standard chemical compounds exhibit antimicrobial effects. Prince's Pine, Chimaphila umbellate, White Birch, Betula papyrifera, Staghorn Sumac, Rhus typhina, and Green Ash, Fraxinus pennsylvanica were the principal extracts exhibiting notable antibacterial and/or antifungal activities; while gallic acid, ethyl gallate, and caffeic acid demonstrated antibacterial activities and sinapic acid, gentisic acid, and chlorogenic acid demonstrated antifungal activities.

Individual and combined in vitro (anti)androgenic effects of certain food additives and cosmetic preservatives.

PubMed

Pop, Anca; Drugan, Tudor; Gutleb, Arno C; Lupu, Diana; Cherfan, Julien; Loghin, Felicia; Kiss, Béla

2016-04-01

The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT and BHA+BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antimycobacterial activity of methanolic plant extract of Artemisia capillaris containing ursolic acid and hydroquinone against Mycobacterium tuberculosis.

PubMed

Jyoti, Md Anirban; Nam, Kung-Woo; Jang, Woong Sik; Kim, Young-Hee; Kim, Su-Kyung; Lee, Byung-Eui; Song, Ho-Yeon

2016-04-01

In order to protect against Mycobacterium tuberculosis (MTB) infection, novel drugs and new targets should be screened from the vast source of plants. We investigated the potentiality of the herbal plant of Artemisia capillaris extract (AC) against Mycobacterium tuberculosis. In this study, we isolated ursolic acid and hydroquinone by bio-activity guided fractionation from the methanol extracts of AC, and tested the inhibitory effects against several strains of MTB. Anti-mycobacterial evaluation of these compounds was carried out using the MGIT™ 960 and resazurin assay. Mycobacterial morphological changes due to the treatment of these compounds were further evaluated by Transmission electron microscopy (TEM). Ursolic acid (UA) and hydroquinone (HQ) inhibited the growth of both susceptible and resistant strains of M. tuberculosis. The MIC (minimum inhibitory concentration) values of both UA and HQ were 12.5 μg/ml against the susceptible strains of M. tuberculosis. Also both UA and HQ showed 12.5-25 μg/ml of MIC values against MDR/XDR MTB strains. However, against clinical strains of MTB, UA was found sensitive against those strains that are sensitive against both INH and RFP but resistant against those strains that are resistant to INH. On the other hand HQ was sensitive against all clinical strains. TEM image-analysis of the strain H37Ra after treatment with UA revealed cell wall lysis, whereas HQ-treated cells showed deformed cytoplasmic morphology. All these results indicate that AC extracts containing UA and HQ possess promising chemotherapeutic potency against MTB for future use. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Variability of antioxidant and biological activities of Rhus tripartitum related to phenolic compounds

PubMed Central

Ben Miled, Hanène; Saada, Mariem; Jallali, Ines; Ben Barka, Zaineb; Tlili, Mounira; Alimi, Hichem; Sakly, Mohsen; Ben Rhouma, Khémais; Abderrabba, Manef; Abdelmelek, Hafedh; Tebourbi, Olfa; Ksouri, Riadh

2017-01-01

Rhus species are known in traditional medicine for their therapeutic virtue and their extracts showed numerous important properties including antimalarial, antimicrobial, antiviral, and hypoglycemic and anticonvulsant activities. Rhus tripartitum (Ucria) is a medicinal plant widely used in Tunisia folk medicine against chronic diarrhea and gastric ulcer. This study was designed to examine in vitro and ex vivo antioxidant, anti-inflammatory and anticancer activities of four extracts of Rhus tripartitum root cortex with increasing solvent polarity (hexane, dichloromethane, methanol and water). HPLC was used to identify and quantify phenolic compounds in Rhus extract. Water extract showed the highest antioxidant activity using oxygen radical absorbance capacity (ORAC method) with 8.95 ± 0.47 µmol Trolox/mg and a cell based-assay with 0.28 ± 0.12 µmol Trolox/mg as compared to the other fractions. Moreover, methanol extract displayed the strongest anti-cancer activity against human lung carcinoma (A-549) and colon adenocarcinoma cell lines (DLD-1) with an IC50 value of 60.69 ± 2.58 and 39.83 ± 4.56 µg/ml (resazurin test) and 44.52 ± 5.96 and 55.65 ± 6.00 µg/ml (hoechst test), respectively. Besides, the highest anti-inflammatory activity, inhibiting nitric oxide (NO) release, was exhibited by dichloromethane extract with 31.5 % at 160 µg/ml in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The HPLC analysis showed that catechol and kaempferol were the major phenolics. These data suggest the richness of all fractions of Ucria root on interesting bioactive molecules with different polarity and confirm the known traditional therapeutics virtues of this species for the treatment of dysentery, diarrhea and gastric ulcer. PMID:28694749

Variability of antioxidant and biological activities of Rhus tripartitum related to phenolic compounds.

PubMed

Ben Miled, Hanène; Saada, Mariem; Jallali, Ines; Ben Barka, Zaineb; Tlili, Mounira; Alimi, Hichem; Sakly, Mohsen; Ben Rhouma, Khémais; Abderrabba, Manef; Abdelmelek, Hafedh; Tebourbi, Olfa; Ksouri, Riadh

2017-01-01

Rhus species are known in traditional medicine for their therapeutic virtue and their extracts showed numerous important properties including antimalarial, antimicrobial, antiviral, and hypoglycemic and anticonvulsant activities. Rhus tripartitum (Ucria) is a medicinal plant widely used in Tunisia folk medicine against chronic diarrhea and gastric ulcer. This study was designed to examine in vitro and ex vivo antioxidant, anti-inflammatory and anticancer activities of four extracts of Rhus tripartitum root cortex with increasing solvent polarity (hexane, dichloromethane, methanol and water). HPLC was used to identify and quantify phenolic compounds in Rhus extract. Water extract showed the highest antioxidant activity using oxygen radical absorbance capacity (ORAC method) with 8.95 ± 0.47 µmol Trolox/mg and a cell based-assay with 0.28 ± 0.12 µmol Trolox/mg as compared to the other fractions. Moreover, methanol extract displayed the strongest anti-cancer activity against human lung carcinoma (A-549) and colon adenocarcinoma cell lines (DLD-1) with an IC 50 value of 60.69 ± 2.58 and 39.83 ± 4.56 µg/ml (resazurin test) and 44.52 ± 5.96 and 55.65 ± 6.00 µg/ml (hoechst test), respectively. Besides, the highest anti-inflammatory activity, inhibiting nitric oxide (NO) release, was exhibited by dichloromethane extract with 31.5 % at 160 µg/ml in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The HPLC analysis showed that catechol and kaempferol were the major phenolics. These data suggest the richness of all fractions of Ucria root on interesting bioactive molecules with different polarity and confirm the known traditional therapeutics virtues of this species for the treatment of dysentery, diarrhea and gastric ulcer.

Physicochemical and in vitro biological evaluations of furazolidone-based β-cyclodextrin complexes in Leishmania amazonensis.

PubMed

Carvalho, Suzana Gonçalves; Siqueira, Larissa Ataíde; Zanini, Marcos Santos; Dos Santos Matos, Ana Paula; Quaresma, Carla Holandino; da Silva, Luisa Mota; de Andrade, Sérgio Faloni; Severi, Juliana Aparecida; Villanova, Janaina Cecília Oliveira

2018-06-15

Recently, there have been numerous cases of leishmaniasis reported in different Brazilian states. The use of furazolidone (FZD) to treat leishmaniasis has been previously described; however, the drug is associated with adverse effects such as anorexia, weight loss, incoordination, and fatigue in dogs. Thus, in the present study, we prepared and evaluated inclusion complexes between FZD and β-cyclodextrin (β-CD) to guarantee increased drug solubility and reduce the toxicity associated with high doses. The FZD:β-CD complexes were prepared by two different techniques (kneading and lyophilization) prior to incorporation in an oral pharmaceutical dosage form. Formation of the complexes was confirmed using appropriate physicochemical methods. Antileishmanial activity against L. amazonensis was tested in vitro via a microplate assay using resazurin dye and cytotoxicity was determined using the fibroblast L929 lineage. Solubility studies showed the formation of complexes with complexation efficiencies lower than 100%. Physicochemical analysis revealed that FZD was inserted into the β-CD cavity after complexation by both methods. Biological in vitro evaluations demonstrated that free FZD and the FZD:β-CD complexes presented significant leishmanicidal activity against L. amazonensis with IC 50 values of 6.16 μg/mL and 1.83 μg/mL for the complexes prepared by kneading and lyophilization, respectively. The data showed that these complexes reduced the survival of promastigotes and presented no toxicity for tested cells. Our results indicate that the new compounds could be a cost-effective alternative for use in the pharmacotherapy of leishmaniasis in dogs infected with L. amazonensis. Copyright © 2018 Elsevier Ltd. All rights reserved.

BEST Engineered Hyporheic Zones: Enhanced Hyporheic Exchange and Resazurin and Nitrate Cycling in Constructed Stream Experiments

NASA Astrophysics Data System (ADS)

Herzog, S.; McCray, J. E.; Higgins, C. P.

2016-12-01

The hyporheic zone is a hotspot for biogeochemical processing that can attenuate a variety of nonpoint source contaminants in streamwater. However, hyporheic zones in urban and agricultural streams are often degraded and poorly connected with surface water. To increase hyporheic exchange and improve water quality, we introduced engineered streambeds as a stormwater and restoration best management practice. Modifications to streambed hydraulic conductivity and reactivity are termed Biohydrochemical Enhancements for Streamwater Treatment (BEST). BEST are subsurface modules that utilize low-permeability sediments to drive efficient hyporheic exchange, and reactive geomedia to increase reaction rates within the hyporheic zone. This research utilized two artificial stream flumes at the Colorado School of Mines in Golden, CO. Each lined stream flume was 15m long, 0.3m wide, had 0.3m sediment depth, and was continuously dosed with recycled water at 0.25 L/s. One flume served as an all-sand control condition, the other featured BEST modules at 1m spacing with a mixture of 70/30 sand/woodchips (v/v). NaCl breakthrough curves were monitored and analyzed using STAMMT-L, a mobile-immobile exchange model, which showed greater hyporheic exchange and residence times in the BEST stream relative to the control. This result is even more apparent when the calibrated models are used to simulate longer stream reaches. Water quality samples at the reach scale also revealed greater attenuation of nitrate and transformation of the indicator compound resazurin into resorufin. Together these compounds demonstrate that BEST can attenuate contaminants that degrade under anaerobic and aerobic conditions, respectively. These experimental results were also compared to previous numerical simulations to evaluate model accuracy, and show reasonable agreement. Altogether, these results show that BEST may be an effective novel best management practice for improving streamwater quality in urban and agricultural settings.

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. PMID:26863313

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-03-01

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

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

Definition of human rotavirus serotypes by plaque reduction assay.

PubMed Central

Wyatt, R G; Greenberg, H B; James, W D; Pittman, A L; Kalica, A R; Flores, J; Chanock, R M; Kapikian, A Z

1982-01-01

Twenty different human rotavirus reassortants were characterized serologically by a plaque reduction assay as belonging to one of three distinct serotypes. Fourteen were similar if not identical to our prototype Wa strain; two were like the prototype DS-1 strain, and four belonged to a third serotype for which a prototype has not yet been selected. Hyperimmune sera raised against the three serotypes were required to distinguish among them, since postinfection sera had lower titers and were more cross-reactive than hyperimmune sera. These results confirmed the ability of a qualitative cytopathic neutralization test to predict correctly the Wa or DS-1 serotype. A strain of rhesus rotavirus (MMU 18006) was identified as belonging to the newly defined third serotype. Finally, an attempt was made to correlate previously published serotype analysis by neutralization of fluorescent cell-forming units with the results determined by the plaque reduction neutralization assay. PMID:6286487

Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection.

PubMed

Piedra, Pedro A; Hause, Anne M; Aideyan, Letisha

2016-01-01

Assays that measure RSV-specific neutralizing antibody activity are very useful for evaluating vaccine candidates, performing seroprevalence studies, and detecting infection. Neutralizing antibody activity is normally measured by a plaque reduction neutralization assay or by a microneutralization assay with or without complement. These assays measure the functional capacity of serum (or other fluids) to neutralize virus infectivity in cells as compared to ELISA assays that only measure the binding capacity against an antigen. This chapter discusses important elements in standardization of the RSV-specific microneutralization assay for use in the laboratory.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

PubMed

Liu, Bing-Hsien; Lin, Yu-Chen; Ho, Chia-Shin; Yang, Che-Chuan; Chang, Yun-Tsui; Chang, Jui-Feng; Li, Chun-Yuan; Cheng, Cheng-Shun; Huang, Jiun-Yan; Lee, Yen-Fu; Hsu, Ming-Hung; Lin, Feng-Chun; Wang, Hao-Ching; Lo, Chu-Fang; Yang, Shieh-Yueh; Wang, Han-Ching

2015-01-01

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Automated data processing and radioassays.

PubMed

Samols, E; Barrows, G H

1978-04-01

Radioassays include (1) radioimmunoassays, (2) competitive protein-binding assays based on competition for limited antibody or specific binding protein, (3) immunoradiometric assay, based on competition for excess labeled antibody, and (4) radioreceptor assays. Most mathematical models describing the relationship between labeled ligand binding and unlabeled ligand concentration have been based on the law of mass action or the isotope dilution principle. These models provide useful data reduction programs, but are theoretically unfactory because competitive radioassay usually is not based on classical dilution principles, labeled and unlabeled ligand do not have to be identical, antibodies (or receptors) are frequently heterogenous, equilibrium usually is not reached, and there is probably steric and cooperative influence on binding. An alternative, more flexible mathematical model based on the probability or binding collisions being restricted by the surface area of reactive divalent sites on antibody and on univalent antigen has been derived. Application of these models to automated data reduction allows standard curves to be fitted by a mathematical expression, and unknown values are calculated from binding data. The vitrues and pitfalls are presented of point-to-point data reduction, linear transformations, and curvilinear fitting approaches. A third-order polynomial using the square root of concentration closely approximates the mathematical model based on probability, and in our experience this method provides the most acceptable results with all varieties of radioassays. With this curvilinear system, linear point connection should be used between the zero standard and the beginning of significant dose response, and also towards saturation. The importance is stressed of limiting the range of reported automated assay results to that portion of the standard curve that delivers optimal sensitivity. Published methods for automated data reduction of Scatchard plots for radioreceptor assay are limited by calculation of a single mean K value. The quality of the input data is generally the limiting factor in achieving good precision with automated as it is with manual data reduction. The major advantages of computerized curve fitting include: (1) handling large amounts of data rapidly and without computational error; (2) providing useful quality-control data; (3) indicating within-batch variance of the test results; (4) providing ongoing quality-control charts and between assay variance.

Reductive methods for isotopic labeling of antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Champney, W.S.

1989-08-15

Methods for the reductive methylation of the amino groups of eight different antibiotics using {sup 3}HCOH or H{sup 14}COH are presented. The reductive labeling of an additional seven antibiotics by NaB{sub 3}H{sub 4} is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

Characterization of Lactic Acid Bacteria as Poultry Probiotic Candidates with Aflatoxin B1 Binding Activities

NASA Astrophysics Data System (ADS)

Damayanti, E.; Istiqomah, L.; Saragih, J. E.; Purwoko, T.; Sardjono

2017-12-01

Our previous studies have selected lactic acid bacteria (LAB) with antifungal activities from traditional fermented foods made from cassava (G7) and silage feed palm leaf (PDS5 and PDS3). In this study we evaluated their ability to bind aflatoxin B1 (AFB1) and probiotic characteristic. The probiotic characteristic assays of LAB consisted of resistance to acidic conditions (pH 3), gastric juice and bile salts 0.3%. We also carried out an in vitro evaluation of LAB aflatoxin binding ability in viable and non-viable cell for 24 and 48 hours of incubation. The measurement of aflatoxin content was performed by ELISA method using AgraQuant Total Aflatoxin Assay kit. The results showed that all isolates were potential as probiotics and the G7 isolate had the highest viability among other isolates in pH 3 (92.61 %) and the bile salts assay (97.71 %). The percentage of aflatoxin reduction between viable and non-viable cell from each LAB isolate were different. The highest aflatoxin reduction in viable cell assay was performed by G7 isolate (69.11 %) whereas in non-viable cell assay was performed by PDS3 isolate (73.75 %) during incubation time 48 hours. In this study, G7 isolate performed the best probiotic characteristics with the highest viability in acid pH assay, bile salt 0.3% assay and percentage of aflatoxin B1 reduction in viable cell condition. Molecular identification using 16S rRNA sequence analysis showed that G7 isolate had homology with Lactobacillus plantarum (99.9%). It was concluded that Lactobacillus plantarum G7 was potential as probiotic with aflatoxin binding activities.

Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication.

PubMed

Wieten, Rosanne W; Jonker, Emile F F; Pieren, Daan K J; Hodiamont, Caspar J; van Thiel, Pieter P A M; van Gorp, Eric C M; de Visser, Adriëtte W; Grobusch, Martin P; Visser, Leo G; Goorhuis, Abraham

2016-03-04

The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and the Plaque Reduction Neutralization Test) in a group of Dutch immune-compromised travellers with a median of 33 days (IQR [28-49]) after primary YF vaccination. We collected samples of 15 immune-compromised vaccinees vaccinated with the 17D yellow fever vaccine between 2004 and 2012. All samples measured in the plaque reduction neutralization test yielded positive results (>80% virus neutralization with a 1:10 serum dilution). Immune Fluorescence Assay sensitivity was 28% (95% CI [0.12-0.49]). No adverse events were reported. All immune-compromised patients mounted an adequate response with protective levels of virus neutralizing antibodies to the 17-D YF vaccine. No adverse effects were reported. Compared to the plaque reduction neutralization test, the sensitivity of the Immune Fluorescence Assay test was low. Further research is needed to ascertain that 17D vaccination in immune-compromised patients is safe. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

PubMed Central

Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M. Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

2016-01-01

Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

A highly sensitive and versatile virus titration assay in the 96-well microplate format.

PubMed

Borisevich, V; Nistler, R; Hudman, D; Yamshchikov, G; Seregin, A; Yamshchikov, V

2008-02-01

This report describes a fast, reproducible, inexpensive and convenient assay system for virus titration in the 96-well format. The micromethod substantially increases assay throughput and improves the data reproducibility. A highly simplified variant of virus quantification is based on immunohistochemical detection of virus amplification foci obtained without use of agarose or semisolid overlays. It can be incorporated into several types of routine virological assays successfully replacing the laborious and time-consuming conventional methods based on plaque formation under semisolid overlays. The method does not depend on the development of CPE and can be accommodated to assay viruses with substantial differences in growth properties. The use of enhanced immunohistochemical detection enabled a five- to six-fold reduction of the total assay time. The micromethod was specifically developed to take advantage of multichannel pipettor use to simplify handling of a large number of samples. The method performs well with an inexpensive low-power binocular, thus offering a routine assay system usable outside of specialized laboratory setting, such as for testing of clinical or field samples. When used in focus reduction-neutralization tests (FRNT), the method accommodates very small volumes of immune serum, which is often a decisive factor in experiments involving small rodent models.

“Life-like” assessment of antimicrobial surfaces by a new touch transfer assay displays strong superiority of a copper alloy compared to silver containing surfaces

PubMed Central

Tofern, Sabrina; Kunz, Wladimir; Schütze, Sara; Riecke, Michael; Solbach, Werner; Wuske, Thomas

2017-01-01

Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. In this study we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces, with confirmed antibacterial activity by a modified ISO 22196 (JIS Z 2801) assay to test such surfaces under more realistic conditions. Bacteria were taken up from a dry standardized primary contaminated surface (PCS) with disinfected fingers or fingers covered with sterile and moistened cotton gloves. Subsequently, bacteria were transferred by pressing on secondary contaminated surfaces (SCS) with or without potential antibacterial activity and the relative reduction rate was determined after 24 h. A stable transmission rate between PCS and SCS was observed using moistened sterile gloves. A copper containing alloy displayed at least a tenfold reduction of the bacterial load consistently reaching less than 2.5 cfu/cm2. In contrast, no significant reduction of bacterial contamination by silver containing surfaces and matured pure silver was observed in the touch transfer assay. With the touch transfer assay we successfully established a new reproducible method modeling cross contamination. Using the new method we were able to demonstrate that several surfaces with confirmed antimicrobial activity in a modified ISO 22196 (JIS Z 2801) assay lacked effectiveness under defined ambient conditions. This data indicate that liquid based assays like the ISO 22196 should be critically reviewed before claiming antibacterial activity for surfaces in the setting of contamination of dry surfaces by contact to the human skin. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization in hospital environments. PMID:29135999

Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ooko, Edna

Background: Multidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methods: Cytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC{sub 50} values and binding energies. Results: The compounds displayed IC{sub 50} values between 0.7more » ± 0.03 and 20.2 ± 0.25 μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from − 9.00 ± 0.10 to − 6.20 ± 0.02 kcal/mol and pKi values from 0.24 ± 0.04 to 29.17 ± 0.88 μM. At the ATP-binding site of P-gp, lowest binding energies ranged from − 9.78 ± 0.17 to − 6.79 ± 0.01 kcal/mol and pKi values from 0.07 ± 0.02 to 0.03 ± 0.03 μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R = 0.797 and R = 0.794 for training and test sets). Conclusion: Selected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR. - Highlights: • Novel derivatives of curcumin in reversing multidrug resistance (MDR) • Biological and Insilco assays to assess effect on P-glycoprotein (P-gp) • Curcumin synthetic derivatives as possible lead compound against multidrug resistant cancer.« less

Tolerance assays: measuring the unknown.

PubMed

Newell, Kenneth A; Larsen, Christian P

2006-06-15

Distinguishing transplant recipients who will benefit from a reduction in, or even the withdrawal of, immunosuppression from those who require intensive, lifelong immunosuppression will be dependent on developing strategies for immune monitoring. Currently, no assays have been shown to accurately predict the development or presence of donor-specific tolerance in humans after transplantation. In this overview we describe and discuss those assays that we believe may be useful for identifying tolerant transplant recipients. Validation of "tolerance" assays will be critical for the safe development of tolerance regimens in humans.

Establishing Assay Cutoffs for HLA Antibody Screening of Apheresis Donors

PubMed Central

Carrick, Danielle M.; Norris, Philip J.; Endres, Robert O.; Pandey, Suchitra; Kleinman, Steven H.; Wright, David; Sun, Yu; Busch, Michael P.

2011-01-01

BACKGROUND TRALI is the leading cause of transfusion-related deaths. Donor HLA antibodies have been implicated in TRALI cases. Blood centers are implementing TRALI risk reduction strategies based on HLA antibody screening of some subpopulations of ever-pregnant apheresis platelet donors. However, if screening assay cutoffs are too sensitive, donation loss may adversely impact blood availability. STUDY DESIGN Pregnancy history and HLA antibody screening and single antigen bead (SAB) data from blood donors in the REDS-II Leukocyte Antibody Prevalence Study (LAPS) were evaluated for correlations between assay screening values, HLA antibody titer, and number of HLA antigen specificities. The probabilities of matching a cognate antigen in a recipient were calculated and examined in association with total number of specificities observed and screening values. The relative impact of imposing various screening assay cutoffs or pregnancy stratification was examined in relation to detection of HLA antibody reactive donations and loss of donors and donations. RESULTS We provide evidence that higher HLA Ab screening assay values are associated with maintaining higher screening signals upon dilution and an increased breadth of specificities compared with lower screening values; the latter correlated with an increased risk of a cognate antigen match in potential recipients. Depending upon the TRALI risk reduction strategy used, the potential loss of donations ranged between 0.9 and 6.0%. CONCLUSION This analysis should enable blood centers to decide upon a TRALI risk reduction strategy for apheresis platelets that is consistent with how much donation loss the blood center can tolerate. PMID:21332726

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed Central

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results. PMID:3032390

The kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats: calibration to the indirect immunofluorescence assay and computerized standardization of results through normalization to control values.

PubMed

Barlough, J E; Jacobson, R H; Downing, D R; Lynch, T J; Scott, F W

1987-01-01

The computer-assisted, kinetics-based enzyme-linked immunosorbent assay for coronavirus antibodies in cats was calibrated to the conventional indirect immunofluorescence assay by linear regression analysis and computerized interpolation (generation of "immunofluorescence assay-equivalent" titers). Procedures were developed for normalization and standardization of kinetics-based enzyme-linked immunosorbent assay results through incorporation of five different control sera of predetermined ("expected") titer in daily runs. When used with such sera and with computer assistance, the kinetics-based enzyme-linked immunosorbent assay minimized both within-run and between-run variability while allowing also for efficient data reduction and statistical analysis and reporting of results.

Mammalian cell line-based bioassays for toxicological evaluation of landfill leachate treated by Pseudomonas sp. ISTDF1.

PubMed

Ghosh, Pooja; Das, Mihir Tanay; Thakur, Indu Shekhar

2014-01-01

Landfill leachate has become a serious environmental concern because of the presence of many hazardous compounds which even at trace levels are a threat to human health and environment. Therefore, it is important to assess the toxicity of leachate generated and discharge it conforming to the safety standards. The present work examined the efficiency of an earlier reported Pseudomonas sp. strain ISTDF1 for detoxification of leachate collected from Okhla landfill site (New Delhi, India). GC-MS analysis performed after treatment showed the removal of compounds like alpha-limonene diepoxide, brominated dioxin-2-one, Bisphenol A, nitromusk, phthalate derivative, and nitrobenzene originally found in untreated leachate. ICP-AES analysis for heavy metals also showed reduction in concentrations of Zn, Cd, Cr, Fe, Ni, and Pb bringing them within the limit of safety discharge. Methyl tetrazolium (MTT) assay for cytotoxicity, alkaline comet assay for genotoxicity, and 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-like behavior were carried out in human hepato-carcinoma cell line HepG2 to evaluate the toxic potential of treated and untreated leachates. The bacterium reduced toxicity as shown by 2.5-fold reduction of MTT EC50 value, 7-fold reduction in Olive Tail Moment, and 2.8-fold reduction in EROD induction after 240 h of bacterial treatment.

Chronic fatigue syndrome: exercise performance related to immune dysfunction.

PubMed

Nijs, Jo; Meeus, Mira; McGregor, Neil R; Meeusen, Romain; de Schutter, Guy; van Hoof, Elke; de Meirleir, Kenny

2005-10-01

To date, the exact cause of abnormal exercise response in chronic fatigue syndrome (CFS) remains to be revealed, but evidence addressing intracellular immune deregulation in CFS is growing. Therefore, the aim of this cross-sectional study was to examine the interactions between several intracellular immune variables and exercise performance in CFS patients. After venous blood sampling, subjects (16 CFS patients) performed a maximal exercise stress test on a bicycle ergometer with continuous monitoring of cardiorespiratory variables. The following immune variables were assessed: the ratio of 37 kDa Ribonuclease (RNase) L to the 83 kDa native RNase L (using a radiolabeled ligand/receptor assay), RNase L enzymatic activity (enzymatic assay), protein kinase R activity assay (comparison Western blot), elastase activity (enzymatic-colorimetric assay), the percent of monocytes, and nitric oxide determination (for monocytes and lymphocytes; flow cytometry, live cell assay). Forward stepwise multiple regression analysis revealed 1) that elastase activity was the only factor related to the reduction in oxygen uptake at a respiratory exchange ratio (RER) of 1.0 (regression model: R = 0.53, F (1,14) = 15.5, P < 0.002; elastase activity P < 0.002); 2) that the protein kinase R activity was the principle factor related to the reduction in workload at RER = 1.0; and 3) that elastase activity was the principle factor related to the reduction in percent of target heart rate achieved. These data provide evidence for an association between intracellular immune deregulation and exercise performance in patients with CFS. To establish a causal relationship, further study of these interactions using a prospective longitudinal design is required.

Optimization and Validation of the TZM-bl Assay for Standardized Assessments of Neutralizing Antibodies Against HIV-1

PubMed Central

Sarzotti-Kelsoe, Marcella; Bailer, Robert T; Turk, Ellen; Lin, Chen-li; Bilska, Miroslawa; Greene, Kelli M.; Gao, Hongmei; Todd, Christopher A.; Ozaki, Daniel A.; Seaman, Michael S.; Mascola, John R.; Montefiori, David C.

2014-01-01

The TZM-bl assay measures antibody-mediated neutralization of HIV-1 as a function of reductions in HIV-1 Tat-regulated firefly luciferase (Luc) reporter gene expression after a single round of infection with Env-pseudotyped viruses. This assay has become the main endpoint neutralization assay used for the assessment of preclinical and clinical trial samples by a growing number of laboratories worldwide. Here we present the results of the formal optimization and validation of the TZM-bl assay, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay was evaluated for specificity, accuracy, precision, limits of detection and quantitation, linearity, range and robustness. The validated manual TZM-bl assay was also adapted, optimized and qualified to an automated 384-well format. PMID:24291345

A two-fold reduction in measurement time for neutron assay: Initial tests of a prototype dual-gated shift register

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, J.E.; Bourret, S.C.; Krick, M.S.

1996-09-01

Neutron coincidence counting (NCC) is used routinely around the world for nondestructive mass assay of uranium and plutonium in many forms, including waste. Compared with other methods, NCC is generally the most flexible, economic, and rapid. Many applications of NCC would benefit from a reduction in counting time required for a fixed random error. We have developed and tested the first prototype of a dual- gated, shift-register-based electronics unit that offers the potential of decreased measurement time for all passive and active NCC applications.

A two-fold reduction in measurement time for neutron assay: Initial tests of a prototype dual-gated shift register

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, J.E.; Bourret, S.C.; Krick, M.S.

1996-12-31

Neutron coincidence counting (NCC) is used routinely around the world for nondestructive mass assay of uranium and plutonium in many forms, including waste. Compared with other methods, NCC is generally the most flexible, economic, and rapid. Many applications of NCC would benefit from a reduction in counting time required for a fixed random error. The authors have developed and tested the first prototype of a dual-gated, shift-register-based electronics unit that offers the potential of decreased measurement time for all passive and active NCC applications.

Inhibition of Methanogenesis in Marine Sediments by Acetylene and Ethylene: Validity of the Acetylene Reduction Assay for Anaerobic Microcosms

PubMed Central

Oremland, Ronald S.; Taylor, Barrie F.

1975-01-01

Methanogenesis was irreversibly inhibited in sediments by concentrations of acetylene employed in nitrogen fixation assays (1 to 20%, vol/vol). Ethylene, but not ethane, also stopped methane production, and the inhibition was reversed by gassing with hydrogen. PMID:1190767

Paper-based solid-phase nucleic acid hybridization assay using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Shahmuradyan, Anna; Krull, Ulrich J

2013-02-05

A paper-based solid-phase assay is presented for transduction of nucleic acid hybridization using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled oligonucleotide targets provided the proximity required for FRET-sensitized emission from Cy3, which served as an analytical signal. The assay exhibited rapid transduction of nucleic acid hybridization within minutes. Without any amplification steps, the limit of detection of the assay was found to be 300 fmol with the upper limit of the dynamic range at 5 pmol. The implementation of glutathione-coated QDs for the development of nucleic acid hybridization assay integrated on a paper-based platform exhibited excellent resistance to nonspecific adsorption of oligonucleotides and showed no reduction in the performance of the assay in the presence of large quantities of noncomplementary DNA. The selectivity of nucleic acid hybridization was demonstrated by single-nucleotide polymorphism (SNP) detection at a contrast ratio of 19 to 1. The reuse of paper over multiple cycles of hybridization and dehybridization was possible, with less than 20% reduction in the performance of the assay in five cycles. This work provides an important framework for the development of paper-based solid-phase QD-FRET nucleic acid hybridization assays that make use of a ratiometric approach for detection and analysis.

A combined pharmacodynamic quantitative and qualitative model reveals the potent activity of daptomycin and delafloxacin against Staphylococcus aureus biofilms.

PubMed

Bauer, Julia; Siala, Wafi; Tulkens, Paul M; Van Bambeke, Françoise

2013-06-01

Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections.

A Combined Pharmacodynamic Quantitative and Qualitative Model Reveals the Potent Activity of Daptomycin and Delafloxacin against Staphylococcus aureus Biofilms

PubMed Central

Bauer, Julia; Siala, Wafi; Tulkens, Paul M.

2013-01-01

Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections. PMID:23571532

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

PubMed Central

Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

2014-01-01

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig. PMID:26317057

Comparative Inactivation of Murine Norovirus, Human Adenovirus, and Human JC Polyomavirus by Chlorine in Seawater

PubMed Central

de Abreu Corrêa, Adriana; Carratala, Anna; Barardi, Celia Regina Monte; Calvo, Miquel; Bofill-Mas, Sílvia

2012-01-01

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log10 GC reductions and a 2.3- and 2.4-log10 PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log10 GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log10 GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration. PMID:22773637

Analytical, Characterization and Stability Studies of Chemicals, Bulk Drugs and Drug Formulations

DTIC Science & Technology

1997-10-01

assay, Report No. 933. 19. WR-242511AF, BM19356, 8-[(4-amino-l-methylbutyl)amino]-5-(1-hexyloxy)-6-methoxy- 4-methylquinoline DL- tartrate , assay...colorimetric protein assay based on complexation between bicinchoninic acid and cuprous ion, which results from reduction of cupric ion by a protein, was...amino-l-methylbutyl)amino]-5-(1-hexyloxy)-6-methoxy-4- methylquinoline DL- tartrate , shelf life, 3- and 6-month samplings, Report No. 738; 9-, 12-, 18

A Novel, Rapid Assay for Detection and Differentiation of Serotype-Specific Antibodies to Venezuelan Equine Encephalitis Complex Alphaviruses

DTIC Science & Technology

2005-01-01

Research Center Detachment, Lima, Peru Abstract. An epitope-blocking enzyme-linked immunosorbent assay was developed for the rapid differentiation of...subtype and variety of antibodies to VEEV in equines, humans, or rodent reservoir hosts can be critical for determining the potential of a naturally...of human sera from Mexico and Peru using a blocking enzyme-linked immunosorbent assay and plaque reduction neutralization tests* Serum number Country

Optimization and Validation of a Plaque Reduction Neutralization Test for the Detection of Neutralizing Antibodies to Four Serotypes of Dengue Virus Used in Support of Dengue Vaccine Development

PubMed Central

Timiryasova, Tatyana M.; Bonaparte, Matthew I.; Luo, Ping; Zedar, Rebecca; Hu, Branda T.; Hildreth, Stephen W.

2013-01-01

A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype–specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT50 for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT50 was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10. PMID:23458954

Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices.

PubMed

Gargotti, M; Lopez-Gonzalez, U; Byrne, H J; Casey, A

2018-02-01

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in "improved viability levels" or "reduced" toxicity or cellular "resistance" compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

Essential oils: in vitro activity against Leishmania amazonensis, cytotoxicity and chemical composition.

PubMed

Andrade, Milene Aparecida; Azevedo, Clênia Dos Santos; Motta, Flávia Nader; Santos, Maria Lucília Dos; Silva, Camila Lasse; Santana, Jaime Martins de; Bastos, Izabela M D

2016-11-08

The current chemotherapy for cutaneous leishmaniosis (CL) has a series of drug limitations such as toxic side effects, long duration, high costs and drug resistance, which requires the development of new drugs or effective alternatives to the CL treatment. Essential oils (EOs) are complex mixtures of secondary metabolites from various plants. It has been shown that several EOs, or their constituents, have inhibitory activity against protozoa. Thus, this study aims to evaluate the biological activity of different essential oils (EOs) on Leishmania (L.) amazonensis promastigotes forms, as well as their cytotoxicity on mammalian cells and chemical composition. Sixteen EOs were evaluated by mean of IC 50 /24 h and cytotoxicity against L6 cells (CC 50 /24 h) using Resazurin assay. Only those EOs that presented better results for IC 50 /24 h were submitted to GC-MS analysis to determine their chemical constitution. The EO from Cinnamodendron dinisii, Matricaria chamomilla, Myroxylon peruiferum, Salvia sclarea, Bulnesia sarmientoi, Ferula galbaniflua, Siparuna guianensis and Melissa officinalis were the most active against L. amazonensis with IC50/24 h ranging from 54.05 to 162.25 μg/mL. Analysis of EOs by GC-MS showed mainly the presence of β-farnesene (52.73 %) and bisabolol oxide (12.09 %) for M. chamomilla; α-copaene (13.41 %), safrole (8.35 %) and δ-cadinene (7.08 %) for M. peruiferum; linalool (28.80 %) and linalyl acetate (60.08 %) for S. sclarea; guaiol (48.29 %) and 2-undecanone (19.49 %) for B. sarmientoi; ethyl phthalate (13.09 %) and methyl-8-pimaren-18-oate (41.82 %) for F. galbaniflua; and neral (37.18 %) and citral (5.02 %) for M. officinalis. The EO from F. galbaniflua showed to be effective against L. amazonensis promastigotes forms and presented low cytotoxic activity against L6 cells. Thus, it represents a strong candidate for future studies aiming its molecular activity on these pathogenic parasites.

Antimycobacterial potency and cytotoxicity study of three medicinal plants.

PubMed

Tsouh Fokou, Patrick Valere; Appiah-Opong, Regina; Yeboah-Manu, Dorothy; Kissi-Twum, Abena Adomah; Yamthe, Lauve Rachel Tchokouaha; Mokale Kognou, Aristide Laurel; Addo, Phyllis; Boyom, Fabrice Fekam; Nyarko, Alexander Kwadwo

2016-12-01

Mycobacterial infections including tuberculosis, leprosy, and buruli ulcer are among the most prevalent, debilitating, and deadly tropical diseases, especially in Sub-Saharan Africa. The development of drug resistance to the currently available drugs and the poor compliance emphasize the need for new chemotherapeutic agents. This study was designed to evaluate the in vitro activity of Cleistopholis patens, Annona reticulata, and Greenwayodendron suaveolens against Mycobacterium smegmatis. The safety on normal liver cells was also assessed. The crude extracts, fractions, and subfractions were tested against M. smegmatis and for cell cytotoxicity on WRL-68, normal human hepatocyte using microdilution resazurin-based assays. The phytochemical screening was performed using standard methods. Most of the extracts, fractions, and subfractions inhibited the growth of M. smegmatis with minimum inhibitory concentration (MIC) values ranging from 6.25μg/mL to 125μg/mL. The subfractions P12 and P29 from G. suaveolens twig were more potent with MIC values of 6.25μg/mL and 25μg/mL, respectively. Fruit crude extract and root CH 2 Cl 2 fraction from A. reticulata also showed activity with MIC values of 50μg/mL and 25μg/mL, respectively. Crude extracts from the twig and stem bark of C. patens displayed inhibition at MIC values of 125μg/mL and 100μg/mL, respectively. Majority of active extracts showed no cell cytotoxicity, except the extract from C. patens with IC 50 ranging from 41.40μg/mL to 93.78μg/mL. The chemical investigation of the promising extracts revealed the presence of phenols, alkaloids, glycosides, triterpenes, and acetogenins. The results achieved from this preliminary antimycobacterial drug discovery study supported the traditional claims of C. patens, A. reticulata, and G. suaveolens in the treatment of mycobacterial infections. Meanwhile, further fractionation is required to characterize the active ingredients. Copyright © 2016.

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

PubMed

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting. Copyright © 2015 Elsevier GmbH. All rights reserved.

Decolorization of a reactive copper-phthalocyanine dye under methanogenic conditions.

PubMed

Beydili, M I; Matthews, R D; Pavlostathis, S G

2001-01-01

The objective of this research was to assess the biological decolorization of the copper-phthalocyanine dye Reactive Blue 7 (RB7) under methanogenic conditions using a mixed, methanogenic culture in a repetitive dye addition batch assay. The initial rate of decolorization was 13.2 mg/L-d and 5.7 mg/L-d for the first and second dye addition, respectively. For an initial RB7 concentration of ca. 300 mg/L, the extent of decolorization remained constant (about 62%) for each repetitive RB7 addition and resulted in a residual color build up. Declining absorbance ratio values (A664/A620) with increasing incubation time confirmed that the observed color removal was due to transformation as opposed to adsorption on the biomass. Chemical decolorization assays using sodium dithionite as the reducing agent resulted in similar absorbance spectra to that obtained after biological decolorization. In addition, in both the chemical and biological decolorization assays, partial oxidation of the reduced dye solution upon exposure to air resulted in higher residual color, indicating that the reduction and decolorization of RB7 are partially reversible. These results also suggest that RB7 reduction and decolorization both chemically and biologically most likely followed a similar reduction mechanism.

Clover-Tagged Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones for Rapid Detection of Virus Neutralizing Antibodies.

PubMed

Huang, Baicheng; Xiao, Xia; Xue, Biyun; Zhou, En-Min

2018-06-24

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a widespread disease that affects domestic pigs of all ages. Accurate and rapid detection of PRRSV specific neutralizing antibodies levels in a pig herd is beneficial for the evaluation of the herd's immunity to combat the specific viral infection. However, the current methods for viral detection, including fluorescent focus neutralization (FFN) and cytopathic effect (CPE) reduction neutralizing assays, are subjective and time-consuming. Therefore, a Clover-tagged PRRSV virus neutralization assay were developed that instrumentally measures the fluorescence signal of Clover stably expressing by a PRRSV infectious clone for at least 10 passages. Herein, the results showed that the proposed Clover-tagged PRRSV neutralization assay is reliable using instrumental measurements of the fluorescence signal of Clover and allows for rapid detection of neutralizing antibodies against PRRSV. The assay was evaluated by testing swine sera from experimental and field samples, and comparisons were made with the traditional FFN and CPE reduction assays. These results suggest that the Clover-tagged PRRSV infectious clone offers a fast and reliable testing method for neutralizing antibodies and could permit high-throughput screening of new antiviral agents. Copyright © 2018. Published by Elsevier B.V.

Modified in vivo comet assay detects the genotoxic potential of 14-hydroxycodeinone, an α,β-unsaturated ketone in oxycodone.

PubMed

Pant, Kamala; Roden, Nicholas; Zhang, Charles; Bruce, Shannon; Wood, Craig; Pendino, Kimberly

2015-12-01

14-Hydroxycodeinone (14-HC) is an α,β-unsaturated ketone impurity found in oxycodone drug substance and has a structural alert for genotoxicity. 14-HC was tested in a combined Modified and Standard Comet Assay to determine if the slight decrease in % Tail DNA noted in a previously conducted Standard Comet Assay with 14-HC could be magnified to clarify if the response was due to cross-linking activity. One limitation of the Standard Comet Assay is that DNA cross-links cannot be reliably detected. However, under certain modified testing conditions, DNA cross-links and chemical moieties that elicit such cross-links can be elucidated. One such modification involves the induction of additional breakages of DNA strands by gamma or X-ray irradiation. To determine if 14-HC is a DNA crosslinker in vivo, a Modified Comet Assay was conducted using X-ray irradiation as the modification to visualize crosslinking activity. In this assay, 14-HC was administered orally to mice up to 320 mg/kg/day. Results showed a statistically significant reduction in percent tail DNA in duodenal cells at 320 mg/kg/day, with a nonstatistically significant but dose-related reduction in percent tail DNA also observed at the mid dose of 160 mg/kg/day. Similar decreases were not observed in cells from the liver or stomach, and no increases in percent tail DNA were noted for any tissue in the concomitantly conducted Standard Comet Assay. Taken together, 14-HC was identified as a cross-linking agent in the duodenum in the Modified Comet Assay. © 2015 Wiley Periodicals, Inc.

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay

PubMed Central

Logue, James; Tuznik, Kaylie; Follmann, Dean; Grandits, Greg; Marchand, Jonathan; Reilly, Cavan; Sarro, Yeya dit Sadio; Pettitt, James; Stavale, Eric J.; Fallah, Mosoka; Olinger, Gene G.; Bolay, Fatorma K.; Hensley, Lisa E.

2018-01-01

As part of the scientific community’s development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I–III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity. PMID:29481881

Simultaneous titration and phenotypic antiviral drug susceptibility testing for herpes simplex virus 1 and 2.

PubMed

Tardif, Keith D; Jorgensen, Shane; Langer, Janine; Prichard, Mark; Schlaberg, Robert

2014-11-01

Most herpes simplex virus (HSV) isolates from treatment-naïve patients are susceptible to antivirals. However, prolonged antiviral therapy can select for drug-resistant strains, especially in immunocompromised patients. Standard phenotypic methods for antiviral resistance testing are labor and time-intense and molecular resistance determinants are insufficiently understood for routine diagnostic use of genotypic resistance testing. To enable rapid, scalable antiviral susceptibility testing and minimize viral passage, we developed a 7-day, 96-well assay for simultaneous HSV 1/2 titration and phenotypic resistance testing for acyclovir and foscarnet. The assay was optimized and validated by testing clinical isolates and laboratory strains (n=39) with known IC50 for acyclovir (23 resistant) and foscarnet (1 resistant) based on plaque reduction or dye-uptake assays. A chemiluminescent detection reagent is used for quantification of cytopathic effect instead of plaque counting or measuring dye-uptake. Drug concentrations inhibiting 50% of chemiluminescent signal reduction (IC50) were determined concurrently at each of three virus dilutions. Results agree for 92.3% (acyclovir) and 100% (foscarnet) of isolates. For all three discordant samples, results of reference testing by plaque reduction agreed with the chemiluminescent assay. Reproducibility studies showed 100% qualitative agreement and 3-37% coefficient of variation based on IC50. Chemiluminescence detection as a surrogate for cellular viability with an automated plate reader provides improved throughput and workflow, as well as high accuracy and reproducibility for antiviral drug susceptibility testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

PubMed

Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

2011-01-01

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

The root iron reductase assay: an examination of key factors that must be respected to generate meaningful assay results

USDA-ARS?s Scientific Manuscript database

Plant iron researchers have been quantifying root iron reductase activity since the 1970's, using a simple spectrophotometric method based on the color change of a ferrous iron chromophore. The technique was used by Chaney, Brown, and Tiffin (1972) to demonstrate the obligatory reduction of ferric i...

Kinetic tetrazolium microtiter assay

NASA Technical Reports Server (NTRS)

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

1992-01-01

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

Effects of bioirrigation of non-biting midges (Diptera: Chironomidae) on lake sediment respiration

PubMed Central

Baranov, Viktor; Lewandowski, Jörg; Romeijn, Paul; Singer, Gabriel; Krause, Stefan

2016-01-01

Bioirrigation or the transport of fluids into the sediment matrix due to the activities of organisms such as bloodworms (larvae of Diptera, Chironomidae), has substantial impacts on sediment respiration in lakes. However, previous quantifications of bioirrigation impacts of Chironomidae have been limited by technical challenges such as the difficulty to separate faunal and bacterial respiration. This paper describes a novel method based on the bioreactive tracer resazurin for measuring respiration in-situ in non-sealed systems with constant oxygen supply. Applying this new method in microcosm experiments revealed that bioirrigation enhanced sediment respiration by up to 2.5 times. The new method is yielding lower oxygen consumption than previously reported, as it is only sensitive to aerobic heterotrophous respiration and not to other processes causing oxygen decrease. Hence it decouples the quantification of respiration of animals and inorganic oxygen consumption from microbe respiration in sediment. PMID:27256514

Effects of bioirrigation of non-biting midges (Diptera: Chironomidae) on lake sediment respiration.

PubMed

Baranov, Viktor; Lewandowski, Jörg; Romeijn, Paul; Singer, Gabriel; Krause, Stefan

2016-06-03

Bioirrigation or the transport of fluids into the sediment matrix due to the activities of organisms such as bloodworms (larvae of Diptera, Chironomidae), has substantial impacts on sediment respiration in lakes. However, previous quantifications of bioirrigation impacts of Chironomidae have been limited by technical challenges such as the difficulty to separate faunal and bacterial respiration. This paper describes a novel method based on the bioreactive tracer resazurin for measuring respiration in-situ in non-sealed systems with constant oxygen supply. Applying this new method in microcosm experiments revealed that bioirrigation enhanced sediment respiration by up to 2.5 times. The new method is yielding lower oxygen consumption than previously reported, as it is only sensitive to aerobic heterotrophous respiration and not to other processes causing oxygen decrease. Hence it decouples the quantification of respiration of animals and inorganic oxygen consumption from microbe respiration in sediment.

Effects of bioirrigation of non-biting midges (Diptera: Chironomidae) on lake sediment respiration

NASA Astrophysics Data System (ADS)

Baranov, Viktor; Lewandowski, Jörg; Romeijn, Paul; Singer, Gabriel; Krause, Stefan

2016-06-01

Bioirrigation or the transport of fluids into the sediment matrix due to the activities of organisms such as bloodworms (larvae of Diptera, Chironomidae), has substantial impacts on sediment respiration in lakes. However, previous quantifications of bioirrigation impacts of Chironomidae have been limited by technical challenges such as the difficulty to separate faunal and bacterial respiration. This paper describes a novel method based on the bioreactive tracer resazurin for measuring respiration in-situ in non-sealed systems with constant oxygen supply. Applying this new method in microcosm experiments revealed that bioirrigation enhanced sediment respiration by up to 2.5 times. The new method is yielding lower oxygen consumption than previously reported, as it is only sensitive to aerobic heterotrophous respiration and not to other processes causing oxygen decrease. Hence it decouples the quantification of respiration of animals and inorganic oxygen consumption from microbe respiration in sediment.

Graphene oxide nanostructures modified multifunctional cotton fabrics

NASA Astrophysics Data System (ADS)

Krishnamoorthy, Karthikeyan; Navaneethaiyer, Umasuthan; Mohan, Rajneesh; Lee, Jehee; Kim, Sang-Jae

2012-06-01

Surface modification of cotton fabrics using graphene oxide (GO) nanostructures was reported. Scanning electron microscopic (SEM) investigations revealed that the GO nanostructure was coated onto the cotton fabric. The molecular level interaction between the graphene oxide and the cotton fabric is studied in detail using the Fourier transform infra-red (FTIR) spectra. Thermogravimetric analysis (TGA) showed that GO loaded cotton fabrics have enhanced thermal stability compared to the bare cotton fabrics. The photocatalytic activity of the GO-coated cotton fabrics was investigated by measuring the photoreduction of resazurin (RZ) into resorufin (RF) under UV light irradiation. The antibacterial activity was evaluated against both Gram-negative and Gram-positive bacteria and the results indicated that the GO-coated cotton fabrics are more toxic towards the Gram-positive ones. Our results provide a way to develop graphene oxide-based devices for the biomedical applications for improving health care.

Digital antimicrobial susceptibility testing using the MilliDrop technology.

PubMed

Jiang, L; Boitard, L; Broyer, P; Chareire, A-C; Bourne-Branchu, P; Mahé, P; Tournoud, M; Franceschi, C; Zambardi, G; Baudry, J; Bibette, J

2016-03-01

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.

Aerobic growth of Anoxybacillus pushchinoensis K1(T): emended descriptions of A. pushchinoensis and the genus Anoxybacillus

NASA Technical Reports Server (NTRS)

Pikuta, Elena; Cleland, David; Tang, Jane

2003-01-01

In this work, corrections are made to the descriptions of the species Anoxybacillus pushchinoensis corrig. and the genus ANOXYBACILLUS: Experiments to determine the relationship of A. pushchinoensis K1(T) to oxygen showed that it was capable of aerobic growth, but preferred to grow anaerobically. During aerobic growth, the redox indicator resazurin was reduced as a result of hydrogen gas production. The facultatively anaerobic nature of K1(T) was ascertained by cultivation in aerobic liquid medium, where growth began at the bottom of the tube. The anaerobic nature of K1(T) was also indicated by a negative catalase reaction. This work is submitted to correct the description of the species A. pushchinoensis from obligate anaerobe to aerotolerant anaerobe and to emend the description of the genus Anoxybacillus from obligate anaerobes or facultative anaerobes to aerotolerant anaerobes or facultative anaerobes.

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens.

PubMed

Visser, M; Stephan, D; Jaynes, J M; Burger, J T

2012-06-01

Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays.

PubMed

Guillo, Christelle; Ferrance, Jerome P; Landers, James P

2006-04-28

Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

PubMed Central

Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

2015-01-01

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox. PMID:26426117

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

PubMed

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.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

PubMed

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy

PubMed Central

Fei, Ping; Zhang, Qi; Huang, Luling; Xu, Yu; Zhu, Xiong; Tai, Zhengfu; Gong, Bo; Ma, Shi; Yao, Quanyao; Li, Jing; Zhao, Peiquan

2014-01-01

Purpose To investigate the clinical features and disease-causing mutations in two Chinese families with familial exudative vitreoretinopathy (FEVR). Methods Clinical data and genomic DNA were collected for patients with FEVR. The coding exons and adjacent intronic regions of FZD4, LRP5, TSPAN12, and NDP were amplified with PCR, and the resulting amplicons were analyzed with Sanger sequencing. Wild-type and mutant LRP5 proteins were assayed for the Norrin/β-catenin pathway by luciferase reporter assays. Results Two novel heterozygous mutations in the LRP5 gene were identified in two relatives—p.A422T and p.L540P. Typical FEVR fundus change and mild reduced bone mineral density (BMD) was found in the two patients and the affected parent. In the luciferase studies, both p.A422T and p.L540P mutants displayed a significant reduction of the luciferase activity in SuperTopFlash (STF) cells in response to Norrin (87% reduction for p.A422T and 97% reduction for p.L540P). Both patients had an additional LRP5 sequence change (p.Q816P in Patient 1 from the unaffected mother and p.T852M in Patient 2 verified as a new mutation). Luciferase assay showed no reduction for p.Q816P and 94.9% reduction for the new mutation p.T852M, suggesting that p.Q816P may be not pathogenic and p.T852M may be pathogenic. Conclusions Our findings demonstrated two new novel LRP5 mutations in Chinese patients with FEVR and mild reduced BMD. They emphasize the complexity of FEVR mutations and phenotypes. PMID:24715757

Capsaicin-capped silver nanoparticles: its kinetics, characterization and biocompatibility assay

NASA Astrophysics Data System (ADS)

Amruthraj, Nagoth Joseph; Preetam Raj, John Poonga; Lebel, Antoine

2015-04-01

Capsaicin was used as a bio-reductant for the reduction of silver nitrate to form silver nanoparticles. The formation of the silver nanoparticles was initially confirmed by color change and Tyndall effect of light scattering. It was characterized with UV-visible spectroscopy, FTIR and TEM. Hemagglutination (H) test and H-inhibition assay were performed in the presence of AgNPs-capsaicin conjugates. The silver colloid solution after complete reduction turned into pale gray color. The characteristic surface plasmon resonance of silver nanoparticles (SNPs) was observed at 450 nm. Time taken for complete bio-reduction of silver nitrate and capping was found to be 16 hours. The amount of capsaicin required to reduce 20 ml of 1 mM silver nitrate solution was found to be 40 μg approximately. The FTIR results confirmed the capping of capsaicin on the silver metal. The particle size was within the range of 20-30 nm. The hemagglutination and H-inhibition test was negative for all the blood groups. The capsaicin-capped silver nanoparticles were compatible with blood cells in hemagglutination test implying biocompatibility as future therapeutic drug.

Assessment of antioxidant potential, total phenolics and flavonoids of different solvent fractions of monotheca buxifolia fruit.

PubMed

Jan, Shumaila; Khan, Muhammad Rashid; Rashid, Umbreen; Bokhari, Jasia

2013-10-01

This study was conducted to investigate the antioxidant potential of methanol extract and its derived fractions (hexane, ethyl acetate, butanol, and aqueous) of fruits of Monotheca buxifolia (Falc.) Dc., a locally used fruit in Pakistan. Dried powder of the fruit of M. buxifolia was extracted with methanol and the resultant was fractionated with solvents having escalating polarity; n-hexane, chloroform, ethyl acetate, n-butanol and the residual soluble aqueous fraction. Total phenolic and total flavonoid contents were estimated for the methanol and various fractions. These fractions were also subjected to various in vitro assays to estimate the scavenging activity for 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide, hydroxyl, hydrogen peroxide and reductive ability for ferric ions and phosphomolybdate assay. The n-butanol, aqueous and methanol fractions possessed high amount of phenolics and flavonoids compared with other fractions, and subsequently showed a pronounced scavenging activity on DPPH, ABTS, superoxide, hydroxyl and hydrogen peroxide radicals and had a potent reductive ability on ferric ion and phosphomolybdate assay. There was a found significant correlation between total phenolic and flavonoid contents and EC50 of DPPH, superoxide, hydrogen peroxide radical and phosphomolybdate assays, whereas a nonsignificant correlation was found with the hydroxyl radical and ABTS radical assay. M. buxifolia fruit can be used as natural antioxidant source to prevent damage associated with free radicals.

Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves 1

PubMed Central

Klepper, Lowell; Flesher, Donna; Hageman, R. H.

1971-01-01

An in vivo assay of nitrate reductase activity was developed by vacuum infiltration of leaf discs or sections with a solution of 0.2 m KNO3 (with or without phosphate buffer, pH 7.5) and incubation of the infiltrated tissue and medium under essentially anaerobic conditions in the dark. Nitrite production, for computing enzyme activity, was determined on aliquots of the incubation media, removed at intervals. By adding, separately, various metabolites of the glycolytic, pentose phosphate, and citric acid pathways to the infiltrating media, it was possible to use the in vivo assay to determine the prime source of reduced nicotinamide adenine dinucleotide (NADH) required by the cytoplasmically located NADH-specific nitrate reductase. It was concluded that sugars that migrate from the chloroplast to the cytoplasm were the prime source of energy and that the oxidation of glyceraldehyde 3-phosphate was ultimately the in vivo source of NADH for nitrate reduction. This conclusion was supported by experiments that included: inhibition studies with iodoacetate; in vitro studies that established the presence and functionality of the requisite enzymes; and studies showing the effect of light (photosynthate) and exogenous carbohydrate on loss of endogenous nitrate from plant tissue. The level of nitrate reductase activity obtained with the in vitro assay is higher (2.5- to 20-fold) than with the in vivo assay for most plant species. The work done to date would indicate that the in vivo assays are proportional to the in vitro assays with respect to ranking genotypes for nitrate-reducing potential of a given species. The in vivo assay is especially useful in studying nitrate assimilation in species like giant ragweed from which only traces of active nitrate reductase can be extracted. PMID:16657841

Use of the Filovirus Animal Non-Clinical Group (FANG) Ebola virus immuno-assay requires fewer study participants to power a study than the Alpha Diagnostic International assay.

PubMed

Logue, James; Tuznik, Kaylie; Follmann, Dean; Grandits, Greg; Marchand, Jonathan; Reilly, Cavan; Sarro, Yeya Dit Sadio; Pettitt, James; Stavale, Eric J; Fallah, Mosoka; Olinger, Gene G; Bolay, Fatorma K; Hensley, Lisa E

2018-05-01

As part of the scientific community's development of medical countermeasures against Ebola virus disease, optimization of standardized assays for product evaluation is paramount. The recent outbreak heightened awareness to the scarcity of available assays and limited information on performance and reproducibility. To evaluate the immunogenicity of vaccines entering Phase I-III trials and to identify survivors, two enzyme-linked immunosorbent assays, the Filovirus Animal Non-Clinical Group assay and the Alpha Diagnostics International assay, were evaluated for detection of immunoglobulin G against Ebola virus glycoprotein. We found that the Filovirus Animal Nonclinical Group assay produced a wider range of relative antibody concentrations, higher assay precision, larger relative accuracy range, and lower regional background. Additionally, to sufficiently power a vaccine trial, use of the Filovirus Animal Nonclinical Group assay would require one third the number of participants than the Alpha Diagnostics International assay. This reduction in needed study participants will require less money, fewer man hours, and much less time to evaluate vaccine immunogenicity. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The local lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?

PubMed

Kimber, Ian; Dearman, Rebecca J; Betts, Catherine J; Gerberick, G Frank; Ryan, Cindy A; Kern, Petra S; Patlewicz, Grace Y; Basketter, David A

2006-04-01

The local lymph node assay (LLNA), an alternative approach to skin-sensitizing testing, has made a significant contribution to animal welfare by permitting a reduction and refinement of animal use. Although there is clearly an aspiration to eliminate the use of animals in such tests, it is appropriate also to consider other opportunities for refinement and reduction of animal use. We have therefore explored the use of a modified version of the LLNA for screening purposes when there is a need to evaluate the sensitizing activity of a large number of chemicals, as will be the case under the auspices of registration, evaluation and authorization of chemicals (REACH). Using an existing LLNA database of 211 chemicals, we have examined whether a cut-down assay comprising a single high-dose group and a concurrent vehicle control would provide a realistic approach for screening chemicals for sensitizing potential. The analyses reported here suggest this is the case. We speculate that the animal welfare benefits may be enhanced further by reducing the number of animals per experimental group. However, a detailed evaluation will be necessary to provide reassurance that a reduction in group size would provide adequate sensitivity across a range of skin sensitization potencies.

Combination of hyperthermia and photodynamic therapy on mesenchymal stem cell line treated with chloroaluminum phthalocyanine magnetic-nanoemulsion

NASA Astrophysics Data System (ADS)

de Paula, Leonardo B.; Primo, Fernando L.; Pinto, Marcelo R.; Morais, Paulo C.; Tedesco, Antonio C.

2015-04-01

The present study reports on the preparation and the cell viability assay of two nanoemulsions loaded with magnetic nanoparticle and chloroaluminum phthalocyanine. The preparations contain equal amount of chloroaluminum phthalocyanine (0.05 mg/mL) but different contents of magnetic nanoparticle (0.15×1013 or 1.50×1013 particle/mL). The human bone marrow mesenchymal stem cell line was used as the model to assess the cell viability and this type of cell can be used as a model to mimic cancer stem cells. The cell viability assays were performed in isolated as well as under combined magnetic hyperthermia and photodynamic therapy treatments. We found from the cell viability assay that under the hyperthermia treatment (1 MHz and 40 Oe magnetic field amplitude) the cell viability reduction was about 10%, regardless the magnetic nanoparticle content within the magnetic nanoparticle/chloroaluminum phthalocyanine formulation. However, cell viability reduction of about 50% and 60% were found while applying the photodynamic therapy treatment using the magnetic nanoparticle/chloroaluminum phthalocyanine formulation containing 0.15×1013 or 1.50×1013 magnetic particle/mL, respectively. Finally, an average reduction in cell viability of about 66% was found while combining the hyperthermia and photodynamic therapy treatments.

Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

PubMed

Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

2017-10-15

Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans

PubMed Central

Li, De-Dong; Chai, Dong; Huang, Xiao-Wen; Guan, Shao-Xing; Du, Jiang; Zhang, Hao-Yu

2017-01-01

ABSTRACT Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro. A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans. Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole. PMID:28607012

Potent In Vitro Synergism of Fluconazole and Osthole against Fluconazole-Resistant Candida albicans.

PubMed

Li, De-Dong; Chai, Dong; Huang, Xiao-Wen; Guan, Shao-Xing; Du, Jiang; Zhang, Hao-Yu; Sun, Yan; Jiang, Yuan-Ying

2017-08-01

Osthole is a natural coumarin that exhibits wide biological and pharmacological activities such as neuroprotective, osteogenic, immunomodulation, antitumor, and anti-inflammatory effects. In this study, we investigated the antifungal effects of osthole in vitro A checkerboard microdilution assay showed that osthole has significant synergistic effect with fluconazole against fluconazole-resistant Candida albicans Similar results were obtained from a growth curve assay. Meanwhile, XTT reduction assay demonstrated the synergism of fluconazole and osthole against C. albicans biofilm formation. Microarray results showed that the expression of genes involved in the oxidation-reduction process, energy metabolism, and transportation changed significantly after the combined treatment with fluconazole and osthole, and further results showed that endogenous reactive oxygen species (ROS) was significantly increased in the combination group. In conclusion, these results demonstrate the synergism of fluconazole and osthole against fluconazole-resistant C. albicans and indicate that endogenous ROS augmentation might contribute to the synergism of fluconazole and osthole. Copyright © 2017 American Society for Microbiology.

Antiviral activity of polymethoxylated flavones from "Guangchenpi", the edible and medicinal pericarps of citrus reticulata 'Chachi'.

PubMed

Xu, Jiao-Jiao; Wu, Xia; Li, Man-Mei; Li, Guo-Qiang; Yang, Yi-Ting; Luo, Hu-Jie; Huang, Wei-Huang; Chung, Hau Yin; Ye, Wen-Cai; Wang, Guo-Cai; Li, Yao-Lan

2014-03-12

The present study found that the supercritical fluid extract of "Guangchenpi" possessed in vitro antiviral activity against respiratory syncytial virus (RSV). Bioassay-guided isolation and identification of this extract led to obtain five active polymethoxylated flavones (1-5). Cytopathic effect (CPE) reduction assay exhibited that tangeretin (2) and nobiletin (3), two major polymethoxylated flavones in the extract, possessed better anti-RSV effect comparable to the positive control ribavirin. Plaque reduction assay revealed that tangeretin dose-dependently inhibited RSV-induced plaque formation on the HEp-2 cells. This polymethoxylated flavone mainly affected the intracellular replication of RSV, and it also could inhibit RSV entry into the HEp-2 cells. Further investigations with quantitative real-time PCR and confocal and Western blot assays indicated that tangeretin downregulated the expression of RSV phosphoprotein (P protein). Results suggest the potential application of the supercritical fluid extract of "Guangchenpi" and tangeretin in the treatment and the prevention of RSV infection.

Efficacy of individualised diets in patients with irritable bowel syndrome: a randomised controlled trial.

PubMed

Ali, Ather; Weiss, Theresa R; McKee, Douglas; Scherban, Alisa; Khan, Sumiya; Fields, Maxine R; Apollo, Damian; Mehal, Wajahat Z

2017-01-01

Patients with irritable bowel syndrome (IBS) are often placed on diets guided by food intolerance assays, although these have not been validated. We assessed the effects of individualised diets in patients with IBS guided by a leucocyte activation test. This is a parallel-group, double-blind, randomised controlled trial of 58 adults with IBS seen at an academic health centre in Northeast USA. Peripheral venous blood was analysed using a leucocyte activation test; individual foods were reported to produce positive or negative results. Participants were randomised to a 4-week diet with either individualised guidance to eliminate foods with positive assay results and allow foods with negative assay results (intervention), or with individualised guidance, matched in rigour and complexity, to eliminate foods with negative assay results and allow foods with positive assay results (comparison). The primary outcome was between-group differences in the IBS Global Improvement Scale (GIS). Secondary outcomes included reductions in IBS Symptom Severity Scale (SSS) scores and increases in IBS Adequate Relief (AR) and Quality of Life (QOL) scores. An aptamer-based proteomic analysis was conducted in strong responders. The intervention group had significantly greater increases in mean GIS score after 4 weeks (0.86 vs comparison; 95% CI 0.05 to 1.67; p=0.04) and 8 weeks (1.22 vs comparison; 95% CI 0.22 to 2.22; p=0.02). The intervention group also had significantly greater reductions in mean SSS score at 4 weeks (-61.78 vs comparison; 95% CI -4.43 to -119.14; p=0.04) and 8 weeks (-66.42 vs comparison; 95% CI -5.75 to -127.09; p=0.03). There were no significant differences between intervention and comparison groups in mean AR or QOL scores. A reduction in neutrophil elastase concentration was associated with reduced symptoms. Elimination diets guided by leucocyte activation tests reduced symptoms. These findings could lead to insights into the pathophysiology of IBS. NCT02186743.

Evaluation of cytotoxicity, genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay, comet assay and zebrafish embryo test.

PubMed

Yang, Fan; Zhang, Qianqian; Guo, Huarong; Zhang, Shicui

2010-10-01

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC>ZQ>OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparison of an Assay for Dehalococcoides DNA and a Microcosm Study in Predicting Reductive Dechlorination of Chlorinated Ethenes in the field

EPA Science Inventory

The study aims to compare the detection of 16S rRNA gene of Dehalococcoides species and the microcosm study for biotransformation in predicting reductive dechlorination of chlorinated ethylenes in ground water at hazardous waste sites. A total of 72 ground water samples were coll...

A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

PubMed

Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

1984-10-01

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

Monocytes and macrophages in malignant melanoma. III. Reduction of nitroblue tetrazolium by peripheral blood monocytes.

PubMed Central

Hedley, D. W.; Currie, G. A.

1978-01-01

Peripheral-blood monocytes from normal individuals and from patients with malignant melanoma reduce nitroblue tetrazolium (NBT). A quantitative assay for dye reduction was applied to 25 healthy donors and 31 patients with malignant melanoma. NBT reduction expressed as dye reduction per monocyte was significantly impaired in patients with disseminated disease, and they responded poorly to a phagocytic stimulus. Monocytes from patients with micrometastatic disease, however, showed normal resting NBT reduction but, following exposure to a suspension of latex-polystyrene, showed significantly greater NBT reduction than those from normal individuals. Since NBT reduction is an indirect measure of intracellular hexose-monophosphate-shunt activity we conclude that the monocytes from patients with minimal disease are in some way activated. PMID:656304

Characterization of Attenuated Strains of Rift Valley Fever Virus

DTIC Science & Technology

1988-01-01

confirmed as RVF virus by a plaque-reduction neutralization test (PRNT) (Earley et al., 1967) using antibody produced against ZH501. Viral replication in...original exposure. Sera were obtained from surviving hamsters and assayed for RVF virus antibody . The Reed-Muench formula (Reed & Muench, 1938) was used to... antibody production. we obtained sera from surviving hamsters that had been inoculated with the various RVF strains. Virus assays. We evaluated

Glutathione as a promising anti-hydrophobicity agent against Malassezia spp.

PubMed

Sivasankar, Chandran; Ponmalar, Ayyappan; Bhaskar, James P; Pandian, Shunmugiah K

2015-10-01

The genus Malassezia has recently attracted wide attention in medical microbiology and dermatology as a pathogen. They are lipophilic yeasts possessing high level of cell surface hydrophobicity (CSH). L-glutathione (GSH) is a ubiquitous antioxidant which offers protection against microbial infections. This study is intended to investigate the role of GSH as a potential anti-hydrophobicity agent against Malazessia spp. Microbial adherence to hydrocarbon assay was performed to assess the anti-hydrophobicity activity (AHA) of GSH against four Malassezia spp. The assay revealed that GSH at 400 μg ml(-1) concentration inhibited CSH, ranging from 84% to 95% in M. furfur, M. globosa, M. restricta and M. sympodialis without killing the cells. The AHA of GSH was corroborated by auto-aggregation assay and zeta-potential measurement, through which delayed cell aggregation was observed due to reduction in CSH level and not by modification in cell surface charge. In addition, colony-forming unit assay was performed in which 62-93% of CSH reduction was observed in Malassezia spp. tested. Furthermore, GSH treatment enhanced the sensitivity of Malassezia spp. towards human blood at the rate of 64-72%. The AHA was further confirmed through Fourier transform infrared analysis. Thus, this study portrays GSH as a prospective therapeutic alternative for Malassezia-mediated infections. © 2015 Blackwell Verlag GmbH.

Modified RS-LAMP assay and use of lateral flow devices for rapid detection of Leifsonia xyli subsp. xyli.

PubMed

Naidoo, N; Ghai, M; Moodley, K; Mkize, L; Martin, L; McFarlane, S; Rutherford, S

2017-12-01

Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U μl -1 ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities. This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis. © 2017 The Society for Applied Microbiology.

pH and reduction dual-responsive dipeptide cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

PubMed

Liu, Qiang; Su, Rong-Chuan; Yi, Wen-Jing; Zheng, Li-Ting; Lu, Shan-Shan; Zhao, Zhi-Gang

2017-03-31

A series of tocopherol-based cationic lipid 3a-3f bearing a pH-sensitive imidazole moiety in the dipeptide headgroup and a reduction-responsive disulfide linkage were designed and synthesized. Acid-base titration of these lipids showed good buffering capacities. The liposomes formed from 3 and co-lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could efficiently bind and condense DNA into nanoparticles. Gel binding and HPLC assays confirmed the encapsulated DNA could release from lipoplexes 3 upon addition of 10 mM glutathione (GSH). MTT assays in HEK 293 cells demonstrated that lipoplexes 3 had low cytotoxicity. The in vitro gene transfection studies showed cationic dipeptide headgroups clearly affected the transfection efficiency (TE), and arginine-histidine based dipeptide lipid 3f give the best TE, which was 30.4 times higher than Lipofectamine 3000 in the presence of 10% serum. Cell-uptake assays indicated that basic amino acid containing dipeptide cationic lipids exhibited more efficient cell uptake than serine and aromatic amino acids based dipeptide lipids. Confocal laser scanning microscopy (CLSM) studies corroborated that 3 could efficiently deliver and release DNA into the nuclei of HeLa cells. These results suggest that tocopherol-based dipeptide cationic lipids with pH and reduction dual-sensitive characteristics might be promising non-viral gene delivery vectors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Analyses of the genotoxic and mutagenic potential of the products formed after the biotransformation of the azo dye Disperse Red 1.

PubMed

Chequer, Farah Maria Drumond; Lizier, Thiago Mescoloto; de Felício, Rafael; Zanoni, Maria Valnice Boldrin; Debonsi, Hosana Maria; Lopes, Norberto Peporine; Marcos, Ricard; de Oliveira, Danielle Palma

2011-12-01

Azo dyes constitute the largest class of synthetic dyes. Following oral exposure, these dyes can be reduced to aromatic amines by the intestinal microflora or liver enzymes. This work identified the products formed after oxidation and reduction of the dye Disperse Red 1, simulating hepatic biotransformation and evaluated the mutagenic potential of the resultant solution. Controlled potential electrolysis was carried out on dye solution using a Potentiostat/Galvanostat. HPLC-DAD and GC/MS were used to determine the products generated after the oxidation/reduction process. The Salmonella/microsome assay with the strains TA98 and YG1041 without S9, and the mouse lymphoma assay (MLA) using the thymidine kinase (Tk) gene, were used to evaluate the mutagenicity of the products formed. Sulfate 2-[(4-aminophenyl)ethylamino]-ethanol monohydrate, nitrobenzene, 4-nitro-benzamine and 2-(ethylphenylamino)-ethanol were detected. This dye has already being assigned as mutagenic in different cell system. In addition, after the oxidation/reduction process the dye still had mutagenic activity for the Salmonella/microsome assay. Nevertheless, both the original dye Disperse Red 1 and its treated solutions showed negative results in the MLA. The present results suggest that the ingestion of water and food contaminated with this dye may represent human and environmental health problem, due to the generation of harmful compounds after biotransformation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells.

PubMed

Lima, Kelly Goulart; Krause, Gabriele Catyana; Schuster, Aline Daniele; Catarina, Anderson Velasque; Basso, Bruno Souza; De Mesquita, Fernanda Cristina; Pedrazza, Leonardo; Marczak, Elisa Simon; Martha, Bianca Andrade; Nunes, Fernanda Bordignon; Chiela, Eduardo Cremonese Filippi; Jaeger, Natália; Thomé, Marcos Paulo; Haute, Gabriela Viegas; Dias, Henrique Bregolin; Donadio, Márcio Vinícius Fagundes; De Oliveira, Jarbas Rodrigues

2016-12-01

Hepatocellular carcinoma is the most prevalent primary liver tumor and is among the top ten cancer that affect the world population. Its development is related, in most cases, to the existence of chronic liver injury, such as in cirrhosis. The knowledge about the correlation between chronic inflammation and cancer has driven new researches with anti-inflammatory agents that have potential for the development of antitumor drugs. Gallic acid is a phenolic acid found in many natural products and have shown anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant actions. The purpose of this study was to investigate the effect of gallic acid on acute and chronic cell proliferation and inflammatory parameters of hepatocellular carcinoma cells (HepG2), as well as to investigate the mechanisms involved. Results showed that the gallic acid decreased the proliferation of HepG2 cells in a dose-dependent manner (Trypan blue exclusion assay), without causing necrosis (LDH assay). We observed a significant increase in the percentage of small and regular nuclei (Nuclear Morphometric Analysis assay), a significant induction of apoptosis by Annexin V-FITC and PI assay and no interference with the cell cycle using the FITC BrdU Flow Kit. We observed a significant reduction in the levels of IL-8 and increased levels of IL-10 and IL-12 (Cytometric Bead Array Human Inflammation Assay). Furthermore, gallic acid caused no cancer cells regrowth at a long term (Cumulative Population Doubling assay). According to these results, gallic acid showed a strong potential as an anti-tumor agent in hepatocellular carcinoma cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effect of storage temperature on the activity of submitochondrial particles.

PubMed

Doherty, Francis G

2008-12-01

The submitochondrial particle (SMP) assay employs processed mammalian mitchondria to assess the toxicity of chemical contaminants in aqueous solutions. Particles and associated reagents are commercially available to support two individual procedures, the electron transfer (ETr) and reverse electron transfer (RET) assays. The objective of the present study was to assess the effect of storage temperature on SMP activity. One RET and one ETr assay were conducted with sodium dodecylsulfate on each of two vials of particles stored at -20 and -80 degrees C at periodic intervals over a six-month span. Results demonstrated that SMP could remain active in either assay through six months of storage at either temperature. However, there were isolated vials of particles stored at -20 degrees C that exhibited unacceptable reductions in activity for both the ETr and the RET assays that were not related to storage duration. These results were used to develop guidance in assessing the acceptability of particle activity in SMP assays.

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

PubMed

Tsukatani, Tadayuki; Suenaga, Hikaru; Ishiyama, Munetaka; Ezoe, Takatoshi; Matsumoto, Kiyoshi

2011-07-15

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of a Test Method for the Evaluation of DNA Damage in Mouse Spermatogonial Stem Cells

PubMed Central

Jeon, Hye Lyun; Yi, Jung-Sun; Kim, Tae Sung; Oh, Youkyung; Lee, Hye Jeong; Lee, Minseong; Bang, Jin Seok; Ko, Kinarm; Ahn, Il Young; Ko, Kyungyuk; Kim, Joohwan; Park, Hye-Kyung; Lee, Jong Kwon; Sohn, Soo Jung

2017-01-01

Although alternative test methods based on the 3Rs (Replacement, Reduction, Refinement) are being developed to replace animal testing in reproductive and developmental toxicology, they are still in an early stage. Consequently, we aimed to develop alternative test methods in male animals using mouse spermatogonial stem cells (mSSCs). Here, we modified the OECD TG 489 and optimized the in vitro comet assay in our previous study. This study aimed to verify the validity of in vitro tests involving mSSCs by comparing their results with those of in vivo tests using C57BL/6 mice by gavage. We selected hydroxyurea (HU), which is known to chemically induce male reproductive toxicity. The 50% inhibitory concentration (IC50) value of HU was 0.9 mM, as determined by the MTT assay. In the in vitro comet assay, % tail DNA and Olive tail moment (OTM) after HU administration increased significantly, compared to the control. Annexin V, PI staining and TUNEL assays showed that HU caused apoptosis in mSSCs. In order to compare in vitro tests with in vivo tests, the same substances were administered to male C57BL/6 mice. Reproductive toxicity was observed at 25, 50, 100, and 200 mg/kg/day as measured by clinical measures of reduction in sperm motility and testicular weight. The comet assay, DCFH-DA assay, H&E staining, and TUNEL assay were also performed. The results of the test with C57BL/6 mice were similar to those with mSSCs for HU treatment. Finally, linear regression analysis showed a strong positive correlation between results of in vitro tests and those of in vivo. In conclusion, the present study is the first to demonstrate the effect of HU-induced DNA damage, ROS formation, and apoptosis in mSSCs. Further, the results of the current study suggest that mSSCs could be a useful model to predict male reproductive toxicity. PMID:28443181

Field-evaluation of a new lateral flow assay for detection of cellular and humoral immunity against Mycobacterium leprae.

PubMed

Bobosha, Kidist; Tjon Kon Fat, Elisa M; van den Eeden, Susan J F; Bekele, Yonas; van der Ploeg-van Schip, Jolien J; de Dood, Claudia J; Dijkman, Karin; Franken, Kees L M C; Wilson, Louis; Aseffa, Abraham; Spencer, John S; Ottenhoff, Tom H M; Corstjens, Paul L A M; Geluk, Annemieke

2014-05-01

Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.

Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae

PubMed Central

Bobosha, Kidist; Tjon Kon Fat, Elisa M.; van den Eeden, Susan J. F.; Bekele, Yonas; van der Ploeg-van Schip, Jolien J.; de Dood, Claudia J.; Dijkman, Karin; Franken, Kees L. M. C.; Wilson, Louis; Aseffa, Abraham; Spencer, John S.; Ottenhoff, Tom H. M.; Corstjens, Paul L. A. M.; Geluk, Annemieke

2014-01-01

Background Field-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings. Methods The combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs. Results Single and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas. Conclusions Dry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC. PMID:24810599

Use of stimulable bioluminescence from dinoflagellates as a means of detecting toxicity in the marine environment. (Reannouncement with new availability information). Professional paper

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapota, D.; Moskowitz, G.; Grovhoug, J.

1993-03-01

Phytoplankton bioassays have been used as biological tools in assessing environmental contamination. In our laboratory, a simple bioassay has been developed which measures the light output from bioluminescence dinoflagellates for assessment of toxic effects when exposed to a single toxicant or mixture. Successful use of this type of bioassay has provided data on the acute response and has demonstrated the chronic effects, from hours up to 11 days, on dinoflagellate cells of Pyrocystis lunula and Gonyaulax polyedra upon exposure to several metals and storm drain effluent. Dinoflagellate cells were exposed to various concentrations of tributyltin chloride (TBTCI), copper (11) sulfatemore » (CUS04), zinc sulfate (ZnSO4), or storm drain effluent. Stimulable bioluminescence was measured at each test period (3 or 4 h, 24 h, 48 h, 72 h, etc.) following setup for all assays. Cells were kept in the dark for 3 or 4 h prior to testing. Stirring the cells within the chamber stimulated maximum bioluminescence from the dinoflagellates. An IC50 (an estimated concentration that is likely to cause a 50% reduction in light output) was estimated for all assays. The trend of light reduction as a response to increasing dose level of test article was observed in all assays. A reduction in light output was measured from cells exposed to 1.6, 4.2, and 12.8 ug/L TBTCI. The IC50 decreased from 8.5 ug/L at 120 h to 3.0 ug/L at 264 h. The cells exposed to 6.25%, 12.5%, and 25.0% storm drain effluent exhibited a statistically significant (P=0.05) reduction in light output in as little as 3 h exposure....Plankton, Oceanography, Bioluminescence.« less

Effect of variations in the redox potential of Gleysol on barium mobility and absorption in rice plants.

PubMed

Magalhães, Marcio Osvaldo Lima; Sobrinho, Nelson Moura Brasil do Amaral; Zonta, Everaldo; de Carvalho, Michel Miranda; Tolón-Becerra, Alfredo

2012-09-01

Two assays were designed to obtain information about the influence of redox potential variations on barium mobility and bioavailability in soil. One assay was undertaken in leaching columns, and the other was conducted in pots cultivated with rice (Oryza sativa) using soil samples collected from the surface of Gleysol in both assays. Three doses of barium (100,300 mg kg(-1) and 3000 mg kg(-1)-soil dry weight) and two redox potential values (oxidizing and reducing) were evaluated. During the incubation period, the redox potential (Eh) was monitored in columns and pots until values of -250 mV were reached. After the incubation period, geochemical partitioning was conducted on the barium using the European Communities Bureau of Reference (BCR) method. Rainfall of 200 mm d(-1) was simulated in the columns and in the planting of rice seedlings in the pots. The results of the geochemical partitioning demonstrated that the condition of reduction favors increased barium concentrations in the more labile chemical forms and decreased levels in the chemical forms related to oxides. The highest barium concentrations in leached extracts (3.36 mg L(-1)) were observed at the highest dose and condition of reduction at approximately five times above the drinking water standard. The high concentrations of barium in the soil did not affect plant dry matter production. The highest levels and accumulation of barium in roots, leaves, and grains of rice were found at the highest dose and condition of reduction. These results demonstrate that reduction leads to solubilization of barium sulfate, thereby favoring greater mobility and bioavailability of this element. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

Oxygen requirements of yeasts. [Saccharomyces cerevisiae; Candida tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Visser, W.; Scheffers, W.A.; Batenburg-Van Der Vegte, W.H.

1990-12-01

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, suchmore » as Torulaspora delbrueckii and Candida tropicalis, grew poorly ({mu}{sub max}, 0.03 and 0.05 h{sup {minus}1}, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.« less

Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei.

PubMed

Sidrim, José Júlio Costa; Vasconcelos, David Caldas; Riello, Giovanna Barbosa; Guedes, Glaucia Morgana de Melo; Serpa, Rosana; Bandeira, Tereza de Jesus Pinheiro Gomes; Monteiro, André Jalles; Cordeiro, Rossana de Aguiar; Castelo-Branco, Débora de Souza Collares Maia; Rocha, Marcos Fábio Gadelha; Brilhante, Raimunda Sâmia Nogueira

2017-01-01

Efflux pumps are important defense mechanisms against antimicrobial drugs and maintenance of Burkholderia pseudomallei biofilms. This study evaluated the effect of the efflux pump inhibitor promethazine on the structure and antimicrobial susceptibility of B. pseudomallei biofilms. Susceptibility of planktonic cells and biofilms to promethazine alone and combined with antimicrobials was assessed by the broth microdilution test and biofilm metabolic activity was determined with resazurin. The effect of promethazine on 48 h-grown biofilms was also evaluated through confocal and electronic microscopy. The minimum inhibitory concentration (MIC) of promethazine was 780 mg l -1 , while the minimum biofilm elimination concentration (MBEC) was 780-3,120 mg l -1 . Promethazine reduced the MIC values for erythromycin, trimethoprim/sulfamethoxazole, gentamicin and ciprofloxacin and reduced the MBEC values for all tested drugs (p<0.05). Microscopic analyses demonstrated that promethazine altered the biofilm structure of B. pseudomallei, even at subinhibitory concentrations, possibly facilitating antibiotic penetration. Promethazine improves antibiotics efficacy against B. pseudomallei biofilms, by disrupting biofilm structure.

Tungsten Doped TiO2 with Enhanced Photocatalytic and Optoelectrical Properties via Aerosol Assisted Chemical Vapor Deposition

PubMed Central

Sathasivam, Sanjayan; Bhachu, Davinder S.; Lu, Yao; Chadwick, Nicholas; Althabaiti, Shaeel A.; Alyoubi, Abdulrahman O.; Basahel, Sulaiman N.; Carmalt, Claire J.; Parkin, Ivan P.

2015-01-01

Tungsten doped titanium dioxide films with both transparent conducting oxide (TCO) and photocatalytic properties were produced via aerosol-assisted chemical vapor deposition of titanium ethoxide and dopant concentrations of tungsten ethoxide at 500 °C from a toluene solution. The films were anatase TiO2, with good n-type electrical conductivities as determined via Hall effect measurements. The film doped with 2.25 at.% W showed the lowest resistivity at 0.034 Ω.cm and respectable charge carrier mobility (14.9 cm3/V.s) and concentration (×1019 cm−3). XPS indicated the presence of both W6+ and W4+ in the TiO2 matrix, with the substitutional doping of W4+ inducing an expansion of the anatase unit cell as determined by XRD. The films also showed good photocatalytic activity under UV-light illumination, with degradation of resazurin redox dye at a higher rate than with undoped TiO2. PMID:26042724

Use of the DISST Model to Estimate the HOMA and Matsuda Indexes Using Only a Basal Insulin Assay

PubMed Central

Docherty, Paul D.; Chase, J. Geoffrey

2014-01-01

Background: It is hypothesized that early detection of reduced insulin sensitivity (SI) could prompt intervention that may reduce the considerable financial strain type 2 diabetes mellitus (T2DM) places on global health care. Reduction of the cost of already inexpensive SI metrics such as the Matsuda and HOMA indexes would enable more widespread, economically feasible use of these metrics for screening. The goal of this research was to determine a means of reducing the number of insulin samples and therefore the cost required to provide an accurate Matsuda Index value. Method: The Dynamic Insulin Sensitivity and Secretion Test (DISST) model was used with the glucose and basal insulin measurements from an Oral Glucose Tolerance Test (OGTT) to predict patient insulin responses. The insulin response to the OGTT was determined via population based regression analysis that incorporated the 60-minute glucose and basal insulin values. Results: The proposed method derived accurate and precise Matsuda Indices as compared to the fully sampled Matsuda (R = .95) using only the basal assay insulin-level data and 4 glucose measurements. Using a model employing the basal insulin also allows for determination of the 1-day HOMA value. Conclusion: The DISST model was successfully modified to allow for the accurate prediction an individual’s insulin response to the OGTT. In turn, this enabled highly accurate and precise estimation of a Matsuda Index using only the glucose and basal insulin assays. As insulin assays account for the majority of the cost of the Matsuda Index, this model offers a significant reduction in assay cost. PMID:24876431

Fertility of frozen-thawed stallion semen cannot be predicted by the currently used laboratory methods

PubMed Central

Kuisma, P; Andersson, M; Koskinen, E; Katila, T

2006-01-01

The aim of the project was to use current simple and practical laboratory tests and compare results with the foaling rates of mares inseminated with commercially produced frozen semen. In Exp. 1, semen was tested from 27 and in Exp. 2 from 23 stallions; 19 stallions participated in both experiments. The mean number of mares per stallion in both experiments was 37 (min. 7, max. 121). Sperm morphology was assessed and bacterial culture performed once per stallion. In Exp. 1, progressive motility after 0, 1, 2, 3, and 4 h of incubation using light microscopy, motility characteristics measured with an automatic sperm analyzer, plasma membrane integrity using carboxyfluorescein diacetate/propidium iodide (CFDA/PI) staining and light microscopy, plasma membrane integrity using PI staining and a fluorometer, plasma membrane integrity using a resazurin reduction test, and sperm concentration were evaluated. In Exp. 2, the same tests as in Exp. 1 and a hypo-osmotic swelling test (HOST) using both light microscopy and a fluorometer were performed immediately after thawing and after a 3-h incubation. Statistical analysis was done separately to all stallions and to those having ≥ 20 mares; in addition, stallions with foaling rates < 60 or ≥ 60% were compared. In Exp. 1, progressive motility for all stallions after a 2 – 4-h incubation correlated with the foaling rate (correlation coefficients 0.39 – 0.51), (p < 0.05). In stallions with > 20 mares, the artificial insemination dose showed a correlation coefficient of -0.58 (p < 0.05). In Exp. 2, the HOST immediately after thawing showed a negative correlation with foaling rate (p < 0.05). No single test was consistently reliable for predicting the fertilizing capacity of semen, since the 2 experiments yielded conflicting results, although the same stallions sometimes participated in both. This shows the difficulty of frozen semen quality control in commercially produced stallion semen, and on the other hand, the difficulty of conducting fertility trials in horses. PMID:16987393

HPLC-DAD phenolic profile, cytotoxic and anti-kinetoplastidae activity of Melissa officinalis.

PubMed

Cunha, Francisco; Tintino, Saulo R; Figueredo, Fernando; Barros, Luiz; Duarte, Antonia E; Vega Gomez, Maria Celeste; Coronel, Cathia Cecilia; Rolón, Mírian; Leite, Nadghia; Sobral-Souza, Celestina E; Brito, S V; Waczuc, Emily Pansera; Boligon, Aline Augusti; Athayde, Margareth; Kamdem, Jean Paul; Coutinho, Henrique Douglas Melo; Franco, Jéferson

2016-09-01

Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 μg/mL inhibited 80.39 and 54.27% of promastigote (LC50  value = 105.78 μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value  = 110.69 μg/mL) and against epimastigote (LC50 value  = 245.23 μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 μg/mL with 95.41% (LC50  value = 141.01 μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (< 500 μg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.

Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells.

PubMed

Saeed, Mohamed E M; Meyer, Marion; Hussein, Ahmed; Efferth, Thomas

2016-06-20

Traditional medicine plays a major role for primary health care worldwide. Cancer belongs to the leading disease burden in industrialized and developing countries. Successful cancer therapy is hampered by the development of resistance towards established anticancer drugs. In the present study, we investigated the cytotoxicity of 29 extracts from 26 medicinal plants of South-Africa against leukemia cell lines, most of which are used traditionally to treat cancer and related symptoms. We have investigated the plant extracts for their cytotoxic activity towards drug-sensitive parental CCRF-CEM leukemia cells and their multidrug-resistant P-glycoprotein-overexpressing subline, CEM/ADR5000 by means of the resazurin assay. A panel of 60 NCI tumor cell lines have been investigated for correlations between selected phytochemicals from medicinal plants and the expression of resistance-conferring genes (ABC-transporters, oncogenes, tumor suppressor genes). Seven extracts inhibited both cell lines (Acokanthera oppositifolia, Hypoestes aristata, Laurus nobilis, Leonotis leonurus, Plectranthus barbatus, Plectranthus ciliates, Salvia apiana). CEM/ADR5000 cells exhibited a low degree of cross-resistance (3.35-fold) towards the L. leonurus extract, while no cross-resistance was observed to other plant extracts, although CEM/ADR5000 cells were highly resistant to clinically established drugs. The log10IC50 values for two out of 14 selected phytochemicals from these plants (acovenoside A and ouabain) of 60 tumor cell lines were correlated to the expression of ABC-transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS) and tumor suppressors (TP53). Sensitivity or resistance of the cell lines were not statistically associated with the expression of these genes, indicating that multidrug-resistant, refractory tumors expressing these genes may still respond to acovenoside A and ouabain. The bioactivity of South African medicinal plants may represent a basis for the development of strategies to treat multidrug-resistant tumors either by phytotherapeutic approaches with whole plant preparations or by classical drug development with isolated compounds such as acovenoside A or ouabain. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Susceptibility of Salmonella enterica Isolates from Tomato Farm Environments to Fatty Acids Naturally Found on Tomato Fruit.

PubMed

Dev Kumar, Govindaraj; Micallef, Shirley A

2017-05-01

Salmonella enterica subsp. enterica can colonize tomato fruit as it interacts with fruit surface compounds. The exometabolome of tomato fruit contains a mixture of compounds, including fatty acids, which could affect Salmonella fitness. Fatty acids detected in fruit exudates were investigated for Salmonella inhibition. Pelargonic, lauric, myristic, palmitic, margaric, stearic, and oleic acids were suspended in water dissolved in dimethyl sulfoxide (DMSO) or emulsified in water and quillaja saponin to assess how bioavailability impacted Salmonella growth. The minimum inhibitory concentrations of fatty acids were determined using a resazurin assay. Quillaja saponin emulsion and DMSO solution of pelargonic acid were inhibitory to Salmonella at 31.25 mM. Lauric and myristic acid emulsions inhibited growth at 1 M concentrations in quillaja emulsions and 62.5 mM in DMSO. Lauric and myristic acids significantly affected growth of Salmonella Newport, Javiana, and Typhimurium (p ≤ 0.05). Growth curve analysis using the Baranyi model revealed reduced maxima populations for all treatments (p ≤ 0.001) and shorter lag phase durations for Salmonella Newport with lauric acid (p < 0.01) and Salmonella Javiana with lauric (p < 0.001) and myristic (p < 0.001) acids. Salmonella Newport and Javiana exhibited an accelerated growth rate with lauric acid (p < 0.001) as a result of early stationary phase transition (shorter log phase). In myristic acid-amended media, Salmonella Javiana also displayed a faster growth rate (p < 0.001). Pelargonic acid (31.25 mM) treatment of Salmonella cells resulted in a drop in culturable cells to below detection in an hour. Microscopic analysis with Cyto-dye and propidium iodide of bacterial cells treated with pelargonic acid indicated a mixture of live and dead cells, with cell lysis of some cells. A subset of cells exhibited elongation-possibly indicating filament formation, a known antibiotic stress response. The results suggest that fatty acids present in tomato fruit surface exudates may exert a restrictive effect on Salmonella growth on fruit.

Reduced adherence of Candida to silane-treated silicone rubber.

PubMed

Price, C L; Williams, D W; Waters, M G J; Coulthwaite, L; Verran, J; Taylor, R L; Stickler, D; Lewis, M A O

2005-07-01

Silicone rubber is widely used in the construction of medical devices that can provide an essential role in the treatment of human illness. However, subsequent microbial colonization of silicone rubber can result in clinical infection or device failure. The objective of this study was to determine the effectiveness of a novel silane-treated silicone rubber in inhibiting microbial adherence and material penetration. Test material was prepared by a combination of argon plasma discharge treatment and fluorinated silane coupling. Chemicophysical changes were then confirmed by X-ray photoelectron spectroscopy, contact-angle measurement, and atomic force microscopy. Two separate adherence assays and a material penetration assay assessed the performance of the new material against four strains of Candida species. Results showed a significant reduction (p < 0.01) of Candida albicans GDH 2346 adherence to silane-treated silicone compared with untreated controls. This reduction was still evident after the incorporation of saliva into the assay. Adherence inhibition also occurred with Candida tropicalis MMU and Candida krusei NCYC, although this was assay dependent. Reduced penetration of silane-treated silicone by Candida was evident when compared to untreated controls, plaster-processed silicone, and acrylic-processed silicone. To summarize, a novel silicone rubber material is described that inhibits both candidal adherence and material penetration. The clinical benefit and performance of this material remains to be determined. Copyright 2005 Wiley Periodicals, Inc.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

PubMed

Vasilchenko, Liliya G; Ludwig, Roland; Yershevich, Olga P; Haltrich, Dietmar; Rabinovich, Mikhail L

2012-07-01

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³⁺ (pathway 1) and reduction of ferric ions to Fe²⁺ reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antioxidant and antimicrobial capacities of ethanolic extract of Pergularia daemia leaves: a possible substitute in diabetic management.

PubMed

Sarkodie, Joseph Adusei; Squire, Sylvia Afriyie; Oppong Bekoe, Emelia; Fosu Domozoro, Charles Yaw; Kretchy, Irene Awo; Ahiagbe, Mariesta Kurukulasuriya Jayaroshini; Frimpong-Manso, Samuel; Oboba Kwakyi, Nana Akua; Edoh, Dominic Adotei; Sakyiama, Maxwel; Lamptey, Vida Korkor; Affedzi-Obresi, Seigfried; Duncan, John Lee; Debrah, Philip; N'guessa, Benoit Banga; Asiedu-Gyekye, Julius Isaac; Kwadwo Nyarko, Alexander

2016-09-01

The leaves of Pergularia daemia Forsk (family Asclepidaceae) provide alternative plant-based treatments for the management of diabetes mellitus and diarrhoea in both humans and indigenous poultry species like the Guinea fowls (Numida meleagris). However, no scientific investigations to validate its usefulness in Ghana have been established. This study therefore sought to investigate the anti-hyperglycaemic activity of the 70 % ethanolic extract of P. daemia using streptozotocin (STZ)-induced diabetic male Sprague-Dawley rats. Additionally, the antioxidant and antimicrobial properties of the extract were investigated. The total phenolic content, total flavonoids content, radical scavenging activity and reducing power assays were estimated using Folin-Ciocalteu method, aluminium chloride colorimetric assay, Fe3+ reduction assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, respectively. The results showed that P. daemia extract caused anti-hyperglycaemic activity in the STZ-induced rats at doses of 30, 60 and 90 mg/kg body weight with significant reduction in blood glucose levels. The phytosterols, saponins, phenols, alkaloids, tannins and triterpenes found in the extract may be responsible for the observed anti-hyperglycaemia and antioxidant activities. The extract also showed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. These findings justify the folkloric use of P. daemia as an anti-diabetic and antibacterial agents for susceptible species.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation 1

PubMed Central

Hardy, R. W. F.; Holsten, R. D.; Jackson, E. K.; Burns, R. C.

1968-01-01

The methodology, characteristics and application of the sensitive C2H2-C2H4 assay for N2 fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N2ase-catalyzed reduction of C2H2 to C2H4, gas chromatographic isolation of C2H2 and C2H4, and quantitative measurement with a H2-flame analyzer. As little as 1 μμmole C2H4 can be detected, providing a sensitivity 103-fold greater than is possible with 15N analysis. A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C2H2-reducing activity in the field. Applications to field samples included an evaluation of N2 fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N2 fixation rate of 30 to 33 kg N2 fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N2 fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N2 fixing activity of soybeans. The validity of measuring N2 fixation in terms of C2H2 reduction was established through extensive comparisons of these activities using defined systems, including purified N2ase preparations and pure cultures of N2-fixing bacteria. With this assay it now becomes possible and practicable to conduct comprehensive surveys of N2 fixation, to make detailed comparisons among different N2-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N2 fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N2 fixation reaction. PMID:16656902

Efficacy of individualised diets in patients with irritable bowel syndrome: a randomised controlled trial

PubMed Central

Ali, Ather; Weiss, Theresa R; McKee, Douglas; Scherban, Alisa; Khan, Sumiya; Fields, Maxine R; Apollo, Damian; Mehal, Wajahat Z

2017-01-01

Background Patients with irritable bowel syndrome (IBS) are often placed on diets guided by food intolerance assays, although these have not been validated. We assessed the effects of individualised diets in patients with IBS guided by a leucocyte activation test. Methods This is a parallel-group, double-blind, randomised controlled trial of 58 adults with IBS seen at an academic health centre in Northeast USA. Peripheral venous blood was analysed using a leucocyte activation test; individual foods were reported to produce positive or negative results. Participants were randomised to a 4-week diet with either individualised guidance to eliminate foods with positive assay results and allow foods with negative assay results (intervention), or with individualised guidance, matched in rigour and complexity, to eliminate foods with negative assay results and allow foods with positive assay results (comparison). The primary outcome was between-group differences in the IBS Global Improvement Scale (GIS). Secondary outcomes included reductions in IBS Symptom Severity Scale (SSS) scores and increases in IBS Adequate Relief (AR) and Quality of Life (QOL) scores. An aptamer-based proteomic analysis was conducted in strong responders. Results The intervention group had significantly greater increases in mean GIS score after 4 weeks (0.86 vs comparison; 95% CI 0.05 to 1.67; p=0.04) and 8 weeks (1.22 vs comparison; 95% CI 0.22 to 2.22; p=0.02). The intervention group also had significantly greater reductions in mean SSS score at 4 weeks (–61.78 vs comparison; 95% CI –4.43 to –119.14; p=0.04) and 8 weeks (–66.42 vs comparison; 95% CI –5.75 to –127.09; p=0.03). There were no significant differences between intervention and comparison groups in mean AR or QOL scores. A reduction in neutrophil elastase concentration was associated with reduced symptoms. Conclusions Elimination diets guided by leucocyte activation tests reduced symptoms. These findings could lead to insights into the pathophysiology of IBS. Trial registration number NCT02186743. PMID:29018540

Characterization of a sensitive mouse Aβ40 PD biomarker assay for Alzheimer's disease drug development in wild-type mice.

PubMed

Lu, Yanmei; Hoyte, Kwame; Montgomery, William H; Luk, Wilman; He, Dongping; Meilandt, William J; Zuchero, Y Joy Yu; Atwal, Jasvinder K; Scearce-Levie, Kimberly; Watts, Ryan J; DeForge, Laura E

2016-05-01

Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aβ40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aβ reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

Methanogenesis in the sediment of the acidic Lake Caviahue in Argentina

NASA Astrophysics Data System (ADS)

Koschorreck, Matthias; Wendt-Potthoff, Katrin; Scharf, Burkhard; Richnow, Hans H.

2008-12-01

The biogeochemistry of methane in the sediments of Lake Caviahue was examined by geochemical analysis, microbial activity assays and isotopic analysis. The pH in the water column was 2.6 and increased up to a pH of 6 in the deeper sediment pore waters. The carbon isotope composition of CH 4 was between - 65 and - 70‰ which is indicative for the biological origin of the methane. The enrichment factor ɛ increased from - 46‰ in the upper sediment column to more than - 80 in the deeper sediment section suggesting a transition from acetoclastic methanogenesis to CO 2 reduction with depth. In the most acidic surface layer of the sediment (pH < 4) methanogenesis is inhibited as suggested by a linear CH 4 concentration profile, activity assays and MPN analysis. The CH 4 activity assays and the CH 4 profile indicate that methanogenesis in the sediment of Lake Caviahue was active below 40 cm depth. At that depth the pH was above 4 and sulfate reduction was sulfate limited. Methane was diffusing with a flux of 0.9 mmol m - 2 d - 1 to the sediment surface where it was probably oxidized. Methanogenesis contributed little to the sediments carbon budget and had no significant impact on lake water quality. The high biomass content of the sediment, which was probably caused by the last eruption of Copahue Volcano, supported high rates of sulfate reduction which probably raised the pH and created favorable conditions for methanogens in deeper sediment layers.

Inactivation of tannins in milled sorghum grain through steeping in dilute NaOH solution.

PubMed

Adetunji, Adeoluwa I; Duodu, Kwaku G; Taylor, John R N

2015-05-15

Steeping milled sorghum in up to 0.4% NaOH was investigated as a method of tannin inactivation. NaOH steeping substantially reduced assayable total phenols and tannins in both Type III and Type II sorghums and with Type III sorghum caused a 60-80% reduction in α-amylase inhibition compared to a 20% reduction by water steeping. NaOH treatment also reduced starch liquefaction time and increased free amino nitrogen. Type II tannin sorghum did not inhibit α-amylase and consequently the NaOH treatment had no effect. HPLC and LC-MS of the tannin extracts indicated a general trend of increasing proanthocyanidin/procyanidin size with increasing NaOH concentration and steeping time, coupled with a reduction in total area of peaks resolved. These show that the NaOH treatment forms highly polymerised tannin compounds, too large to assay and to interact with the α-amylase. NaOH pre-treatment of Type III sorghums could enable their utilisation in bioethanol production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biosorption of landfill leachate by Phanerochaete sp. ISTL01: isotherms, kinetics and toxicological assessment.

PubMed

Ghosh, Pooja; Thakur, Indu Shekhar

2017-07-01

The study investigates the ability of fungus Phanerochaete sp. ISTL01 for biosorption of color from landfill leachate. Batch mode experiments were conducted to study the effects of pH, temperature, adsorbent dose, contact time and initial leachate concentration on biosorption. Maximum biosorption capacity was determined as 17.73 mg g -1 of biomass. Equilibrium isotherms and kinetics were further studied. The biosorption data were found to fit well to the Freundlich isotherm and pseudo-second-order kinetic model. The value of activation energy suggested that chemisorption mechanism was involved. Biosorption efficiency was also evaluated by the Methyltetrazolium (MTT) assay for cytotoxicity and alkaline comet assay in HepG2 human hepato-carcinoma cells. The fungus reduced toxicity as shown by 1.3-fold increase in MTT EC 50 and 1.5- and 1.1-fold reduction in Tail moment and Olive tail moment, respectively, after 12 h biosorption. The fungus showed good biosorption characteristics in terms of contaminant-level reduction per unit mass of adsorbent, process kinetics and toxicity reduction, envisaging its application in leachate treatment.

Milk fat globule membrane isolate induces apoptosis in HT-29 human colon cancer cells.

PubMed

Zanabria, Romina; Tellez, Angela M; Griffiths, Mansel; Corredig, Milena

2013-02-01

A native milk fat globule membrane (MFGM) isolate obtained from raw milk was assessed for its anticarcinogenic capacity using a colon cancer cell line (HT-29). To prevent microbial contamination and eliminate the presence of lipopolysaccharide (LPS) in the milk used for MFGM isolation, the milk was obtained from the mammary glands of cows using a catheter. Cell proliferation assays demonstrated a reduction of exponentially growing cancer cells of up to 53%, expressed as DNA synthesis (BrdU test), after 72 h stimulation with 100 μg of MFGM protein per mL. Using a similar MFGM concentration, the sulforhodamine B assay resulted in 57% reduction of cell density after 48 h incubation. This bioactivity was comparable to that of known anticancer drugs, 0.1 mM melphalan and 20 μM C2-ceramide, which achieved a cell division reduction of 25 and 40%, respectively, under the same experimental conditions. The toxic effect of the MFGM extracts on HT-29 cells was confirmed by the significant reduction in lactate dehydrogenase enzyme (LDH) by the residual viable cells. An increase of caspase-3 activity (up to 26%) led to the conclusion that MFGM has an apoptotic effect on HT-29 cancer cells.

In vitro free radical scavenging activity of ethanolic extract of the whole plant of Evolvulus alsinoides (L.) L.

PubMed

Gomathi, Duraisamy; Ravikumar, Ganesan; Kalaiselvi, Manokaran; Vidya, Balasubramaniam; Uma, Chandrasekar

2015-06-01

To identify the free radical scavenging activity of ethanolic extract of Evolvulus alsinoides. The free radical scavenging activity was evaluated by in vitro methods like reducing power assay, total antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction, superoxide radical scavenging activity, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(+)) scavenging activity, hydroxyl radical scavenging assay, and nitric oxide radical scavenging assay, which were studied by using ascorbic acid as standard. The extract showed significant activities in all antioxidant assays compared with the reference antioxidant ascorbic acid. The total antioxidant activity as well as the reducing power was also found to increase in a dose-dependent manner. Evolvulus alsinoides may act as a chemopreventive agent, providing antioxidant properties and offering effective protection from free radicals.

Response to Vicriviroc in Treatment-Experienced Subjects Using an Enhanced Sensitivity Co-receptor Tropism Assay: Reanalysis of AIDS Clinical Trials Group A5211

PubMed Central

Su, Zhaohui; Gulick, Roy M.; Krambrink, Amy; Coakley, Eoin; Hughes, Michael D.; Han, Dong; Flexner, Charles; Wilkin, Timothy J.; Skolnik, Paul R.; Greaves, Wayne L.; Kuritzkes, Daniel R.; Reeves, Jacqueline D.

2009-01-01

The enhanced sensitivity Trofile assay was used to re-test co-receptor usage at study screening and entry for the 118 ACTG A5211 treatment-experienced subjects who had CCR5-tropic (R5) virus by the original Trofile assay at study screening. Among 90 vicriviroc recipients, a significantly (P<0.001) greater mean reduction in HIV-1 RNA was observed in 72 subjects with R5 virus versus 15 subjects reclassified with dual/mixed-tropic viruses at screening: −1.11 vs. −0.09 (day 14), −1.91 vs. −0.57 (week 24) log10 copies/mL, respectively. Results suggest that the enhanced sensitivity assay is a better screening tool for determining patient eligibility for CCR5 antagonist therapy. PMID:19874179

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

PubMed Central

McSharry, James J.; McDonough, Ann C.; Olson, Betty A.; Drusano, George L.

2004-01-01

A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses. PMID:14715540

Nitrate reduction in sediments of lowland tropical streams draining swamp forest in Costa Rica: An ecosystem perspective

USGS Publications Warehouse

Duff, J.H.; Pringle, C.M.; Triska, F.J.

1996-01-01

Nitrate reduction and denitrification were measured in swamp forest streams draining lowland rain forest on Costa Rica's Atlantic slope foothills using the C2H2-block assay and sediment-water nutrient fluxes. Denitrification assays using the C2H2-block technique indicated that the full suite of denitrifying enzymes were present in the sediment but that only a small fraction of the functional activity could be expressed without adding NO3/-. Under optimal conditions, denitrification enzyme activity averaged 15 nmoles cm-3 sediment h-1. Areal NO3/- reduction rates measured from NO3/- loss in the overlying water of sediment- water flux chambers ranged from 65 to 470 umoles m-2 h-1. Oxygen loss rates accompanying NO3/-depletion averaged 750 umoles m-2 h-1. Corrected for denitrification of NO3/- oxidized from NH4/+ in the sediment, gross NO3/- reduction rates increase by 130 umoles m-2 h-1, indicating nitrification may be the predominant source of NO3/- for NO3/- reduction in swamp forest stream sediments. Under field conditions approximately 80% of the increase in inorganic N mass along a 1250-m reach of the Salto River was in the form of NO3/- with the balance NH4/+. Scrutiny of potential inorganic N sources suggested that mineralized N released from the streambed was a major source of the inorganic N increase. Despite significant NO3/- reduction potential, swamp forest stream sediments appear to be a source of inorganic N to downstream communities.

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses.

PubMed

Shoshan, Maria C; Havelka, Associate Professor Principal Investigator Aleksandra Mandic; Neumann, Frank; Linder, Stig

2006-11-01

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.

One-week 96-well soft agar growth assay for cancer target validation.

PubMed

Ke, Ning; Albers, Aaron; Claassen, Gisela; Yu, De-hua; Chatterton, Jon E; Hu, Xiuyuan; Meyhack, Bernd; Wong-Staal, Flossie; Li, Qi-Xiang

2004-05-01

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Apolar Bioactive Fraction of Melipona scutellaris Geopropolis on Streptococcus mutans Biofilm.

PubMed

da Cunha, Marcos Guilherme; Franchin, Marcelo; Galvão, Lívia Câmara de Carvalho; Bueno-Silva, Bruno; Ikegaki, Masaharu; de Alencar, Severino Matias; Rosalen, Pedro Luiz

2013-01-01

The aim of this study was to evaluate the influence of the bioactive nonpolar fraction of geopropolis on Streptococcus mutans biofilm. The ethanolic extract of Melipona scutellaris geopropolis was subjected to a liquid-liquid partition, thus obtaining the bioactive hexane fraction (HF) possessing antimicrobial activity. The effects of HF on S. mutans UA159 biofilms generated on saliva-coated hydroxyapatite discs were analyzed by inhibition of formation, killing assay, and glycolytic pH-drop assays. Furthermore, biofilms treated with vehicle control and HF were analyzed by scanning electron microscopy (SEM). HF at 250  μ g/mL and 400  μ g/mL caused 38% and 53% reduction in the biomass of biofilm, respectively, when compared to vehicle control (P < 0.05) subsequently observed at SEM images, and this reduction was noticed in the amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides, and proteins. In addition, the S. mutans viability (killing assay) and acid production by glycolytic pH drop were not affected (P > 0.05). In conclusion, the bioactive HF of geopropolis was promising to control the S. mutans biofilm formation, without affecting the microbial population but interfering with its structure by reducing the biochemical content of biofilm matrix.

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

PubMed Central

Townson, Simon; Tagboto, Senyo; McGarry, Helen F; Egerton, Gillian L; Taylor, Mark J

2006-01-01

Background The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. Methods Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. Results Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 × 10-5M) was the most effective anti-mycobacterial agent; clofazimine (1.25 × 10-5M and 3.13 × 10-6M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 × 10-5M) was filaricidal, but minocycline was more effective and at a lower concentration (5 × 10-5M and 1.25 × 10-5M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. Conclusion The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode. PMID:16563157

Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro.

PubMed

Marin-Kuan, Maricel; Fussell, Karma C; Riederer, Nicolas; Latado, Helia; Serrant, Patrick; Mollergues, Julie; Coulet, Myriam; Schilter, Benoit

2017-12-01

In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance. Copyright © 2017 Elsevier Ltd. All rights reserved.

A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)

PubMed Central

Deshpande, Kanchanmala; Mishra, Rupesh K.; Bhand, Sunil

2010-01-01

A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg·mL−1 with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%. PMID:22163555

Effect of ADAMTS13 activity turnaround time on plasma utilization for suspected thrombotic thrombocytopenic purpura.

PubMed

Connell, Nathan T; Cheves, Tracey; Sweeney, Joseph D

2016-02-01

Thrombotic thrombocytopenic purpura (TTP) due to deficiency of the von Willebrand-cleaving protease ADAMTS13 is a hematologic emergency that requires prompt initiation of therapeutic plasma exchange (TPE). Long turnaround times (TATs) have precluded the use of pre-TPE measurement of ADAMTS13 activity for the initial diagnosis in most institutions. An in-house rapid TAT (r-TAT) assay for ADAMTS13 activity was implemented after 18 months of validation. In a quasi-experimental design using interrupted time series analysis, patterns of plasma utilization in patients with suspected TTP were assessed after implementation of this assay for ADAMTS13 activity and compared to utilization patterns for patients who received plasma exchange before r-TAT assay implementation designated the standard TAT period. In the 18 months after implementation of the r-TAT ADAMTS13 assay, there was a significant reduction in plasma utilization per patient suspected of having TTP (mean, 144.5 units vs. 63.3 units of plasma per patients suspected of having TTP; p = 0.002). The mean number of exchanges per patient and mean number of exchanges after achieving a platelet count of at least 150 × 10(9) /L were lower in the r-TAT cohort (p < 0.001 for both). There was no significant difference in 30-day mortality. Implementation of a rapid turnaround assay for ADAMTS13 resulted in a significant reduction in plasma utilization for patients with suspected TTP, without an increase in mortality. This study demonstrates that these data, provided in a timely fashion, can avoid unnecessary plasma exchange in patients who do not have TTP. © 2015 AABB.

Impact of the New Abbott mPLUS Feature on Clinical Laboratory Efficiencies of Abbott RealTime Assays for Detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae

PubMed Central

Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin

2013-01-01

Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents. PMID:24088850

Impact of the New Abbott mPLUS feature on clinical laboratory efficiencies of abbott RealTime assays for detection of HIV-1, Hepatitis C Virus, Hepatitis B Virus, Chlamydia trachomatis, and Neisseria gonorrhoeae.

PubMed

Lucic, Danijela; Jones, Sara; Wiesneth, Russ; Barry, Cathy; Webb, Erika; Belova, Larissa; Dolan, Peggy; Ho, Shiaolan; Abravaya, Klara; Cloherty, Gavin

2013-12-01

Diagnostic laboratories are under increasing pressure to improve and expand their services. Greater flexibility in sample processing is a critical factor that can improve the time to results while reducing reagent waste, making laboratories more efficient and cost-effective. The introduction of the Abbott mPLUS feature, with the capacity for extended use of amplification reagents, significantly increases the flexibility of the m2000 platform and enables laboratories to customize their workflows based on sample arrival patterns. The flexibility in sample batch size offered by mPLUS enables significant reductions in processing times. For hepatitis B virus tests, a reduction in sample turnaround times of up to 30% (105 min) was observed for batches of 12 samples compared with those for batches of 24 samples; for Chlamydia trachomatis/Neisseria gonorrhoeae tests, the ability to run batches of 24 samples reduced the turnaround time by 83% (54 min) compared with that for batches of 48 samples. Excellent correlations between mPLUS and m2000 standard condition results were observed for all RealTime viral load assays evaluated in this study, with correlation r values of 0.998 for all assays tested. For the qualitative RealTime C. trachomatis/N. gonorrhoeae assay, the overall agreements between the two conditions tested were >98% for C. trachomatis and 100% for N. gonorrhoeae. Comparable precision results were observed for the two conditions tested for all RealTime assays. The enhanced mPLUS capability provides clinical laboratories with increased efficiencies to meet increasingly stringent turnaround time requirements without increased costs associated with discarding partially used amplification reagents.

Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma

PubMed Central

Mok, Wei Chuen; Wasser, Shanthi; Tan, Theresa; Lim, Seng Gee

2012-01-01

AIM: To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC). METHODS: PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls. RESULTS: RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls. CONCLUSION: Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway. PMID:22826617

The effect of the plasma needle on the human keratinocytes related to the wound healing process

NASA Astrophysics Data System (ADS)

Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

2016-01-01

In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

Reduction in lipophilicity improved the solubility, plasma–protein binding, and permeability of tertiary sulfonamide RORc inverse agonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fauber, Benjamin P.; René, Olivier; de Leon Boenig, Gladys

2014-08-01

Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma–protein unbound fraction and improvements in cellular permeability and aqueous solubility.

Detection of measles, mumps and rubella viruses by immuno-colorimetric assay and its application in focus reduction neutralization tests.

PubMed

Vaidya, Sunil R; Kumbhar, Neelakshi S; Bhide, Vandana S

2014-12-01

Measles, mumps and rubella are vaccine-preventable diseases; however limited epidemiological data are available from low-income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture-based rapid and reliable immuno-colorimetric assay (ICA) was established and its utility studied. Twenty-three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT-PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post-infection in Vero or Vero-human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post-infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero-epidemiological, cross-neutralization and pre/post-vaccine studies. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Response to vicriviroc in treatment-experienced subjects, as determined by an enhanced-sensitivity coreceptor tropism assay: reanalysis of AIDS clinical trials group A5211.

PubMed

Su, Zhaohui; Gulick, Roy M; Krambrink, Amy; Coakley, Eoin; Hughes, Michael D; Han, Dong; Flexner, Charles; Wilkin, Timothy J; Skolnik, Paul R; Greaves, Wayne L; Kuritzkes, Daniel R; Reeves, Jacqueline D

2009-12-01

The enhanced-sensitivity Trofile assay (Monogram Biosciences) was used to retest coreceptor use at both study screening and study entry for 118 treatment-experienced subjects in AIDS Clinical Trials Group A5211 who had CCR5-tropic (R5) virus detected by the original Trofile assay at study screening. Among 90 recipients of vicriviroc, a significantly (P< .001) greater mean reduction in HIV-1 RNA was observed in 72 subjects with R5 virus versus 15 subjects reclassified as having dual/mixed-tropic viruses at screening: -1.11 versus -0.09 log(10) copies/mL at day 14 and -1.91 versus -0.57 log(10) copies/mL at week 24, respectively. Results suggest that the enhanced-sensitivity assay is a better screening tool for determining patient eligibility for CCR5 antagonist therapy.

Quantitative and Cost Comparison of Ultrasensitive Human Immunodeficiency Virus Type 1 RNA Viral Load Assays: Bayer bDNA Quantiplex Versions 3.0 and 2.0 and Roche PCR Amplicor Monitor Version 1.5

PubMed Central

Elbeik, Tarek; Charlebois, Edwin; Nassos, Patricia; Kahn, James; Hecht, Frederick M.; Yajko, David; Ng, Valerie; Hadley, Keith

2000-01-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay. PMID:10699005

Quantitative and cost comparison of ultrasensitive human immunodeficiency virus type 1 RNA viral load assays: Bayer bDNA quantiplex versions 3.0 and 2.0 and Roche PCR Amplicor monitor version 1.5.

PubMed

Elbeik, T; Charlebois, E; Nassos, P; Kahn, J; Hecht, F M; Yajko, D; Ng, V; Hadley, K

2000-03-01

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.

Comparing an in vivo egg reduction test and in vitro egg hatching assay for different anthelmintics against Fasciola species, in cattle.

PubMed

Arafa, Waleed M; Shokeir, Khalid M; Khateib, Abdelrahman M

2015-11-30

This study aimed to compare between the efficiency of in vivo fecal egg reduction test (FERT) and in vitro egg hatching assay (EHA) in evaluating of the anti-Fasciola activity of albendazole, triclabendazole, oxyclozanide and praziquantel. A field trial was carried out on fifty naturally Fasciola infected cattle that were divided equally into 5 groups (A-E). On day zero; groups A-D were drenched with albendazole, triclabendazole, oxyclozanide or praziquantel, respectively, while the remaining one, group E, was kept as untreated control. Fecal egg counts of the different groups were conducted weekly over a period of one month post-treatment. In vitro, commercial albendazole and oxyclozanide were diluted to 0.0002, 0.002, 0.02, 0.2 and 2.0 μg/ml, while commercial triclabendazole and praziquantel were diluted to concentrations of 25, 50, 75 and 100 μg/ml with dimethyl sulfoxide (DMSO). In vivo, at the 2nd week post-treatment, triclabendazole and oxyclozanide showed 100% fecal egg reduction (FER), and albendazole had a maximum of 73.7% reduction (P < 0.0001), however, praziquantel did not record any reduction of Fasciola egg counts. In vitro, triclabendazole treated Fasciola gigantica eggs showed early embryonic lysis with zero% hatching at the different concentrations (P < 0.01). In albendazole, the hatching varied according to the drug concentration. At the highest two concentrations; 0.2 and 2.0 μg/ml, the hatching percentages were 7.4 ± 1.6 and 5.6 ± 1.5 (P < 0.01) respectively. On the contrary, there were no significant differences in egg development and hatching percentage of oxyclozanide or praziquantel treated groups. In conclusion, the efficacy of triclabendazole and albendazole as fasciolicdes could be predicted by Egg Hatching Assay (EHA). Meanwhile fasciolicide activity of oxyclozanide could not be assessed with EHA. Based on in vivo and in vitro findings, paraziquantel did not show any fasciolicide effect. Copyright © 2015 Elsevier B.V. All rights reserved.

The non-nucleoside antiviral, BAY 38-4766, protects against cytomegalovirus (CMV) disease and mortality in immunocompromised guinea pigs

PubMed Central

Schleiss, Mark R.; Bernstein, David I.; McVoy, Michael A.; Stroup, Greg; Bravo, Fernando; Creasy, Blaine; McGregor, Alistair; Henninger, Kristin; Hallenberger, Sabine

2008-01-01

New antiviral drugs are needed for the treatment of cytomegalovirus (CMV) infections, particularly in immunocompromised patients. These studies evaluated the in vitro and in vivo activity of the non-nucleosidic CMV inhibitor, BAY 38-4766, against guinea pig cytomegalovirus (GPCMV). Plaque reduction assays indicated that BAY 38-4766 was active against GPCMV, with an IC50 of 0.5 μM. Yield reduction assays demonstrated an ED90 and ED99 of 0.4 and 0.6 μM, respectively, of BAY 38-4766 against GPCMV. Guinea pigs tolerated oral administration of 50 mg/kg/day of BAY 38-4766 without evidence of biochemical or hematologic toxicity. Plasma concentrations of BAY 38-4766 were high following oral dosing, with a mean peak level at 1-h post-dose of 26.7 mg/ml (n = 6; range, 17.8-35.4). Treatment with BAY 38-4766 reduced both viremia and DNAemia, as determined by a real-time PCR assay, following GPCMV infection of cyclophosphamide-immunosuppressed strain 2 guinea pigs (p < 0.05, Mann-Whitney test). BAY 38-4766 also reduced mortality following lethal GPCMV challenge in immunosuppressed Hartley guinea pigs, from 83% (20/24) in placebo-treated guinea pigs, to 17% (4/24) in BAY 38-4766-treated animals (p < 0.0001, Fisher’s exact test). Mortality differences were accompanied by reduction in DNAemia in Hartley guinea pigs. Based upon its favorable safety, pharmacokinetic, and therapeutic profiles, BAY 38-4766 warrants further investigation in the GPCMV model. PMID:15652969

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

PubMed

Boriollo, Marcelo Fabiano Gomes; Souza, Luiz Silva; Resende, Marielly Reis; Silva, Thaísla Andrielle da; Oliveira, Nelma de Mello Silva; Resck, Maria Cristina Costa; Dias, Carlos Tadeu dos Santos; Fiorini, João Evangelista

2014-04-02

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

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

PubMed Central

2014-01-01

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

Development of a voltammetric assay, using screen-printed electrodes, for clonazepam and its application to beverage and serum samples.

PubMed

Honeychurch, Kevin C; Brooks, Joshua; Hart, John P

2016-01-15

This paper describes the development of an electrochemical assay based on screen-printed carbon sensors for the determination of clonazepam in serum and in wine. The cyclic voltammetric behaviour of the drug was investigated and the effects of pH and scan rate on the peak current and peak potential determined. Two reduction peaks were recorded on the initial negative going scan, which were considered to result from the 2e(-), 2 H(+) reduction of the 4,5-azomethine and from the 4e(-), 4 H(+) reduction of the 7-NO2 to a hydroxylamine. On the return positive going scan an oxidation peak was seen, which was considered to result from the 2e(-), 2 H(+) oxidation (O1) of the hydroxylamine to the corresponding nitroso species. At pH 11 the solution of clonazepam was found to turn from clear to yellow in colour and the voltammetric signal of the O1 oxidation process was found to be adsorptive in nature, this was exploited in the development of an adsorptive stripping voltammetric assay. Experimental conditions were then optimised for the differential pulse adsorptive voltammetric measurement of clonazepam in wine and serum samples. It was shown that these analyses could be performed on only 100µL of sample which was deposited on the sensor surface. Mean recoveries of 79.53% (%CV=9.88%) and 88.22% (%CV=14.1%) were calculated for wine fortified with 3.16µg/mL and serum fortified with 12.6µg/mL. Copyright © 2015 Elsevier B.V. All rights reserved.

Towards a More Complete Picture: Dissimilatory Metal Reduction by Anaeromyxobacter Species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loeffler, Frank E.

2004-06-01

We investigate the physiological requirements of available Anaeromyxobacter isolates, and assess their distribution and abundance in the environment, including DOE sites. The performers on this project include Frank Loeffler (PI), Robert Sanford (Co-PI), Qingzhong Wu (postdoc), Sara Henry (graduate student) and Cornell Gayle (undergraduate student). Year-1 efforts focused on method and tool development to address the research objectives. First, we compared different analytical assays (based on fluorescent light emission and calorimetric methods) to quantify U(VI) in cultures of Anaeromyxobacter dehalogenans strain 2CP-C. The assays were optimized to reflect specific culture conditions, and we found that a laser-excited spectrofluorescence assay providedmore » reproducible and accurate information on the amount of U(VI) reduced in bacterial cultures. To demonstrate the ability of Anaeromyxobacter dehalogenans strain 2CP-C to reduce U(VI), washed suspensions of fumarate-grown cells were prepared. These experiments confirmed that the rapid reduction of U(VI) to U(IV) depended on the presence of live cells, and no U(VI) reduction occurred in cell-free controls. Additional experiments explored the ability of three different Anaeromyxobacter strains to grow with the mineral hematite, an insoluble form of ferric iron, as electron acceptor. All strain grew equally well with soluble ferric iron (provided as ferric citrate) but distinct differences were observed between strains when grown with hematite. All strains tested shared a 16S rRNA gene similarity of >99.5%, suggesting that closely related strains may differ in their ability to access insoluble forms of ferric iron.« less

A comparison of antibacterial and antibiofilm efficacy of phenothiazinium dyes between Gram positive and Gram negative bacterial biofilm.

PubMed

Misba, Lama; Zaidi, Sahar; Khan, Asad U

2017-06-01

Antimicrobial photodynamic therapy (APDT) is a process that generates reactive oxygen species (ROS) in presence of photosensitizer, visible light and oxygen which destroys the bacterial cells. We investigated the photoinactivation efficiency of phenothiazinium dyes and the effect of ROS generation on Gram positive and Gram negative bacterial cell as well as on biofilm. Enterococcus faecalis and Klebsiella pneumonia were incubated with all the three phenothiazinium dyes and exposed to 630nm of light. After PDT, colony forming unit (CFU) were performed to estimate the cell survival fraction. Intracellular reactive oxygen species (ROS) was detected by DCFH-DA. Crystal violet (CV) assay and extracellular polysaccharides (EPS) reduction assay were performed to analyze antibiofilm effect. Confocal laser electron microscope (CLSM) scanning electron microscope (SEM) was performed to assess the disruption of biofilm. 8log 10 reduction in bacterial count was observed in Enterococcus faecalis while 3log 10 in Klebsiella pneumoniae. CV and EPS reduction assay revealed that photodynamic inhibition was more pronounced in Enterococcus faecalis. In addition to this CLSM and SEM study showed an increase in cell permeability of propidium iodide and leakage of cellular constituents in treated preformed biofilm which reflects the antibiofilm action of photodynamic therapy. We conclude that Gram-positive bacteria (Enterococcus faecalis) are more susceptible to APDT due to increased level of ROS generation inside the cell, higher photosensitizer binding efficiency and DNA degradation. Phenothiazinium dyes are proved to be highly efficient against both planktonic and biofilm state of cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment.

PubMed

Grant, W F; Lee, H G; Logan, D M; Salamone, M F

1992-11-01

Tests have shown plant bioassays to be excellent for mutagenicity studies. Most studies with plant bioassays, however, have been carried out either in the laboratory, or if, in situ, as monitors of atmospheric contaminants. The primary purpose of this study was to assess the utility of in situ plant mutagenicity bioassays in monitoring water contaminants. The assay systems tested were the Tradescantia stamen hair and micronucleus assays for the detection of gene mutations and chromosomal aberrations respectively, and the Vicia faba bioassay system which detects chromosomal aberrations in root tips. The assays were used to test the effluent from a pulp and paper mill located on the north shore of Lake Superior. Assays were performed in a creek containing raw effluent and in the bay of Lake Superior into which the creek emptied. All in situ treatments were carried out for 24 h. The effluent from the creek was heavy with pulp and debris which coated the plant cuttings and the Vicia faba seedlings and may have restricted the uptake from the effluent. In the creek, at test sites 11.5 km from the source, the effluent was toxic to the Vicia faba roots as evidenced by a reduction in the mitotic index. The data for the Tradescantia stamen hair assay in the creek were equivocal. The cuttings from the creek test sites and the air and water control sites appeared to have undergone a physiological delay. Within a day or two after the return to the laboratory, that is 6-8 days after testing, flowering almost ceased and did not fully resume until about day 35. This reduction in flowering was particularly severe with the cuttings from the effluent and air control sites, making it very difficult to interpret the results. In contrast, the Tradescantia micronucleus and Vicia faba chromosomal aberration data were unequivocal; each produced positive responses at both test sites relative to the air and water controls. The results obtained for the bay sites with all 3 assays were in agreement. In that section of the bay visibly contaminated by the creek effluent, increases in stamen hair mutants, micronuclei, and chromosome aberrations were measured. In general, there was a considerable reduction in the number of mutant events observed for the water samples brought back from the test sites and tested in the laboratory.(ABSTRACT TRUNCATED AT 400 WORDS)

Microbiological cleanliness of the Mars Exploration Rover spacecraft

NASA Technical Reports Server (NTRS)

Newlin, L.; Barengoltz, J.; Chung, S.; Kirschner, L.; Koukol, R.; Morales, F.

2002-01-01

Planetary protection for Mars missions is described, and the approach being taken by the Mars Exploration Rover Project is discussed. Specific topics include alcohol wiping, dry heat microbial reduction, microbiological assays, and the Kennedy Space center's PHSF clean room.

Glyphosate-resistant goosegrass from Mississippi

USDA-ARS?s Scientific Manuscript database

A glyphosate resistant population of goosegrass (Eleusine indica (L.) Gaertn.) was documented near Stoneville, Mississippi, USA, in an area which had received multiple applications of glyphosate each year for the previous eleven years. Resistance ratios based on dose response growth reduction assays...

The Use of Bioluminescence in Detecting Biohazardous Substances in Water.

ERIC Educational Resources Information Center

Thomulka, Kenneth William; And Others

1993-01-01

Describes an inexpensive, reproducible alternative assay that requires minimal preparation and equipment for water testing. It provides students with a direct method of detecting potentially biohazardous material in water by observing the reduction in bacterial luminescence. (PR)

Propidium monoazide RTqPCR assays for the assessment of hepatitis A inactivation and for a better estimation of the health risk of contaminated waters.

PubMed

Fuster, Noemí; Pintó, Rosa M; Fuentes, Cristina; Beguiristain, Nerea; Bosch, Albert; Guix, Susana

2016-09-15

The waterborne transmission of hepatitis A virus (HAV), the main cause of acute hepatitis, is well documented. Recently, two ISO proposals for sensitive determination of this pathogen by RTqPCR in water and food have been published (ISO/TS 15216-1 and ISO/TS 15216-2), and could enable the formulation of regulatory standards for viruses in the near future. However, since detected viral genomes do not always correlate with virus infectivity, molecular approaches need to be optimized to better predict infectivity of contaminated samples. Two methods involving the use of propidium monoazide (PMA), with or without Triton X-100, prior to RTqPCR amplification were optimized and adapted to infer the performance of infectious viral inactivation upon two different water treatments: free chlorine and high temperature. Significant correlations between the decrease of genome copies and infectivity were found for both inactivation procedures. The best procedure to infer chlorine inactivation was the PMA-RTqPCR assay, in which 1, 2 or 3-log genome copies reductions corresponded to reductions of infectious viruses of 2.61 ± 0.55, 3.76 ± 0.53 and 4.92 ± 0.76 logs, respectively. For heat-inactivated viruses, the best method was the PMA/Triton-RTqPCR assay, with a 1, 2 or 3-log genome reduction corresponding to reductions of infectious viruses of 2.15 ± 1.31, 2.99 ± 0.79 and 3.83 ± 0.70 logs, respectively. Finally, the level of damaged virions was evaluated in distinct types of water naturally contaminated with HAV. While most HAV genomes quantified in sewage corresponded to undamaged capsids, the analysis of a river water sample indicated that more than 98% of viruses were not infectious. Although the PMA/Triton-RTqPCR assay may still overestimate infectivity, it is more reliable than the RTqPCR alone and it seems to be a rapid and cost-effective method that can be applied on different types of water, and that it undeniably provides a more accurate measure of the health risk associated to contaminated waters. Copyright © 2016. Published by Elsevier Ltd.

Identification of the inactivating factors and mechanisms exerted on MS2 coliphage in concentrated synthetic urine.

PubMed

Oishi, Wakana; Sano, Daisuke; Decrey, Loic; Kadoya, Syunsuke; Kohn, Tamar; Funamizu, Naoyuki

2017-11-15

Volume reduction (condensation) is a key for the practical usage of human urine as a fertilizer because it enables the saving of storage space and the reduction of transportation cost. However, concentrated urine may carry infectious disease risks resulting from human pathogens frequently present in excreta, though the survival of pathogens in concentrated urine is not well understood. In this study, the inactivation of MS2 coliphage, a surrogate for single-stranded RNA human enteric viruses, in concentrated synthetic urine was investigated. The infectious titer reduction of MS2 coliphage in synthetic urine samples was measured by plaque assay, and the reduction of genome copy number was monitored by reverse transcription-quantitative PCR (RTqPCR). Among chemical-physical conditions such as pH and osmotic pressure, uncharged ammonia was shown to be the predominant factor responsible for MS2 inactivation, independently of urine concentration level. The reduction rate of the viral genome number varied among genome regions, but the comprehensive reduction rate of six genome regions was well correlated with that of the infectious titer of MS2 coliphage. This indicates that genome degradation is the main mechanism driving loss of infectivity, and that RT-qPCR targeting the six genome regions can be used as a culture-independent assay for monitoring infectivity loss of the coliphage in urine. MS2 inactivation rate constants were well predicted by a model using ion composition and speciation in synthetic urine samples, which suggests that MS2 infectivity loss can be estimated solely based on the solution composition, temperature and pH, without explicitly accounting for effects of osmotic pressure. Copyright © 2017 Elsevier B.V. All rights reserved.

A Molecular Approach Designed to Limit the Replication of Mature DENV2 in Host Cells.

PubMed

Raheel, Ummar; Jamal, Muhsin; Zaidi, Najam Us Sahar Sadaf

2015-09-01

Dengue virus (DENV) is an arthropod-borne virus, which belongs to the Flaviviridae family, and completes its life cycle in two hosts: humans and mosquitoes. For DENV maturation, the surface pre-membrane (prM) protein is cleaved to form a mature membrane protein (M) by furin, which is a cellular enzyme subsequently releasing the mature virus from the host dendritic cell. The objective of the current study was to inhibit mature DENV isotype 2 (DENV2) by RNA-interference in a Vero-81 cell line. Mature DENV2 was propagated in and isolated from U937 cells expressing dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin. Maturation of DENV2 was confirmed by Western blot analysis, where virus stock lacking prM was considered mature. Inhibition studies were carried out by transfection of Vero-81 cells with six synthetic siRNAs along with a control siRNA. Reduction in cellular DENV2 was observed also by focus-reduction assay, immunofluorescence assay (IFA), and real-time quantitative polymerase chain reaction (RT-qPCR). Cells transfected with DENV2SsiRNA2, which was targeting the structural region M of mature DENV2, was able to reduce DENV2 titer by up to 85% in focus reduction assays. A significant reduction in mature DENV2 RNA load was observed by RT-qPCR, confirming the previous findings. IFA also revealed reduced levels of cellular DENV2. These results demonstrated that mature DENV2 can be effectively inhibited by synthetic siRNA targeting the structural region of the genome. Mature DENV2 can be successfully inhibited by siRNAs, and specifically high knock-down efficiency is observed by siRNAs against M region of mature DENV2. This study shows that M represents a potential target for RNAi based inhibitory approaches.

Inactivation of Norovirus by Lemongrass Essential Oil Using a Norovirus Surrogate System.

PubMed

Kim, Ye Won; You, Hyun Ju; Lee, Soyoung; Kim, Bomi; Kim, Do Kyung; Choi, Joo-Bong; Kim, Ji-Ah; Lee, Hee Jung; Joo, In Sun; Lee, Jeong Su; Kang, Dong Hyun; Lee, Giljae; Ko, Gwang Pyo; Lee, Sung-Joon

2017-08-01

This study investigated the effect of lemongrass essential oil (LGEO) on the infectivity and viral replication of norovirus. Murine norovirus 1 (MNV-1), a surrogate of human norovirus, was preincubated with LGEO and then used to infect RAW 264.7 cells in a plaque reduction assay. LGEO exhibited a significant reduction in MNV-1 plaque formation in both time- and dose-dependent manners. The quantification of viral genome by quantitative real-time PCR showed similar results in line with those of the plaque reduction assay. It was revealed that citral, a single compound in LGEO, showed dramatic reduction in MNV-1 infectivity (-73.09% when using a treatment of 0.02%, v/v). The inhibitory activity of LGEO on viral replication was further investigated in HG23 cells that harbored a human norovirus replicon. LGEO treatment significantly reduced viral replication in HG23 cells, which suggests that LGEO may have dual inhibitory activities that inactivate viral coat proteins required for viral infection and suppress norovirus genome replication in host cells. In animal experiments, oral administration of murine norovirus preincubated with LGEO significantly suppressed virus infectivity in vivo. Collectively, these results suggest that LGEO, in particular the LGEO component citral, inactivates the norovirus and its subsequent replication in host cells. Thus, LGEO shows promise as a method of inhibiting norovirus within the food industry.

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

PubMed

Fornalé, Silvia; Capellades, Montserrat; Encina, Antonio; Wang, Kan; Irar, Sami; Lapierre, Catherine; Ruel, Katia; Joseleau, Jean-Paul; Berenguer, Jordi; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2012-07-01

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Blasted copper slag as fine aggregate in Portland cement concrete.

PubMed

Dos Anjos, M A G; Sales, A T C; Andrade, N

2017-07-01

The present work focuses on assessing the viability of applying blasted copper slag, produced during abrasive blasting, as fine aggregate for Portland cement concrete manufacturing, resulting in an alternative and safe disposal method. Leaching assays showed no toxicity for this material. Concrete mixtures were produced, with high aggregate replacement ratios, varying from 0% to 100%. Axial compressive strength, diametrical compressive strength, elastic modulus, physical indexes and durability were evaluated. Assays showed a significant improvement in workability, with the increase in substitution of fine aggregate. With 80% of replacement, the concrete presented lower levels of water absorption capacity. Axial compressive strength and diametrical compressive strength decreased, with the increase of residue replacement content. The greatest reductions of compressive strength were found when the replacement was over 40%. For tensile strength by diametrical compression, the greatest reduction occurred for the concrete with 80% of replacement. After the accelerated aging, results of mechanic properties showed a small reduction of the concrete with blasted copper slag performance, when compared with the reference mixture. Results indicated that the blasted copper slag is a technically viable material for application as fine aggregate for concrete mixtures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro.

PubMed

Ocazionez, Raquel Elvira; Meneses, Rocio; Torres, Flor Angela; Stashenko, Elena

2010-05-01

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

CMV viral load measurements in whole blood and plasma--which is best following renal transplantation?

PubMed

Barrett-Muir, W; Breuer, J; Millar, C; Thomas, J; Jeffries, D; Yaqoob, M; Aitken, C

2000-07-15

Quantitative commercial assays for cytomegalovirus (CMV) detection have recently been developed. Their role in the management of patients after transplantation needs to be evaluated. Widespread use of these assays will allow for comparison of results between centers and meaningful interpretation of the significance of viral load measurements. Sequential samples from 52 patients after renal transplantation were tested in the murex hybrid capture assay (HCA) and the Roche Amplicor CMV DNA assay (QPCR) and correlated with the development of CMV disease. A comparison of viral loads in plasma and whole blood was also made. Both assays were sensitive and detected all cases of CMV disease. The specificity and positive predictive value increased from 0.34 and 0.36 to 0.85 and 0.96 for the HCA and 0.37, 0.37 to 0.72 and 0.63 for the QPCR following a receiver operator curve analysis. Higher viral loads were measured using the HCA compared to the QPCR. Response to ganciclovir was associated with a greater than 80% reduction in viral load by HCA or greater than 70% using the QPCR. Both assays were highly sensitive. By using a receiver operator curve analysis a cutoff viral load can be determined which maximizes the clinical utility of these assays.

A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

PubMed Central

Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

2014-01-01

The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

Advantages and limitations of common testing methods for antioxidants.

PubMed

Amorati, R; Valgimigli, L

2015-05-01

Owing to the importance of antioxidants in the protection of both natural and man-made materials, a large variety of testing methods have been proposed and applied. These include methods based on inhibited autoxidation studies, which are better followed by monitoring the kinetics of oxygen consumption or of the formation of hydroperoxides, the primary oxidation products. Analytical determination of secondary oxidation products (e.g. carbonyl compounds) has also been used. The majority of testing methods, however, do not involve substrate autoxidation. They are based on the competitive bleaching of a probe (e.g. ORAC assay, β-carotene, crocin bleaching assays, and luminol assay), on reaction with a different probe (e.g. spin-trapping and TOSC assay), or they are indirect methods based on the reduction of persistent radicals (e.g. galvinoxyl, DPPH and TEAC assays), or of inorganic oxidizing species (e.g. FRAP, CUPRAC and Folin-Ciocalteu assays). Yet other methods are specific for preventive antioxidants. The relevance, advantages, and limitations of these methods are critically discussed, with respect to their chemistry and the mechanisms of antioxidant activity. A variety of cell-based assays have also been proposed, to investigate the biological activity of antioxidants. Their importance and critical aspects are discussed, along with arguments for the selection of the appropriate testing methods according to the different needs.

Alternatives to animal testing: research, trends, validation, regulatory acceptance.

PubMed

Huggins, Jane

2003-01-01

Current trends and issues in the development of alternatives to the use of animals in biomedical experimentation are discussed in this position paper. Eight topics are considered and include refinement of acute toxicity assays; eye corrosion/irritation alternatives; skin corrosion/irritation alternatives; contact sensitization alternatives; developmental/reproductive testing alternatives; genetic engineering (transgenic) assays; toxicogenomics; and validation of alternative methods. The discussion of refinement of acute toxicity assays is focused primarily on developments with regard to reduction of the number of animals used in the LD(50) assay. However, the substitution of humane endpoints such as clinical signs of toxicity for lethality in these assays is also evaluated. Alternative assays for eye corrosion/irritation as well as those for skin corrosion/irritation are described with particular attention paid to the outcomes, both successful and unsuccessful, of several validation efforts. Alternative assays for contact sensitization and developmental/reproductive toxicity are presented as examples of methods designed for the examination of interactions between toxins and somewhat more complex physiological systems. Moreover, genetic engineering and toxicogenomics are discussed with an eye toward the future of biological experimentation in general. The implications of gene manipulation for research animals, specifically, are also examined. Finally, validation methods are investigated as to their effectiveness, or lack thereof, and suggestions for their standardization and improvement, as well as implementation are reviewed.

Screening of bacterial antagonists for biological control of Phytophthora blight of pepper.

PubMed

Rajkumar, M; Lee, Wang Hyu; Lee, Kui Jae

2005-01-01

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici. ((c) 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12

PubMed Central

2012-01-01

Background When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo′ = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays. Results Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT. Conclusions The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex. PMID:22769583

Assays for estimating HIV incidence: updated global market assessment and estimated economic value.

PubMed

Morrison, Charles S; Homan, Rick; Mack, Natasha; Seepolmuang, Pairin; Averill, Megan; Taylor, Jamilah; Osborn, Jennifer; Dailey, Peter; Parkin, Neil; Ongarello, Stefano; Mastro, Timothy D

2017-11-01

Accurate incidence estimates are needed to characterize the HIV epidemic and guide prevention efforts. HIV Incidence assays are cost-effective laboratory assays that provide incidence estimates from cross-sectional surveys. We conducted a global market assessment of HIV incidence assays under three market scenarios and estimated the economic value of improved incidence assays. We interviewed 27 stakeholders, and reviewed journal articles, working group proceedings, and manufacturers' sales figures. We determined HIV incidence assay use in 2014, and estimated use in 2015 to 2017 and in 5 to 10-years under three market scenarios, as well as the cost of conducting national and key population surveys using an HIV incidence assay with improved performance. Global 2014 HIV incidence assay use was 308,900 tests, highest in Asia and mostly for case- and population-based surveillance. Estimated 2015 to 2017 use was 94,475 annually, with declines due to China and the United States discontinuing incidence assay use for domestic surveillance. Annual projected 5 to 10 year use under scenario 1 - no change in technology - was 94,475. For scenario 2 - a moderately improved incidence assay - projected annual use was 286,031. Projected annual use for scenario 3 - game-changing technologies with an HIV incidence assay part of (a) standard confirmatory testing, and (b) standard rapid testing, were 500,000 and 180 million, respectively. As HIV incidence assay precision increases, decreased sample sizes required for incidence estimation resulted in $5 to 23 million annual reductions in survey costs and easily offset the approximately $3 million required to develop a new assay. Improved HIV incidence assays could substantially reduce HIV incidence estimation costs. Continued development of HIV incidence assays with improved performance is required to realize these cost benefits. © 2017 The Authors. Journal of the International AIDS Society published by John Wiley & sons Ltd on behalf of the International AIDS Society.

Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays.

PubMed

Kintzios, Spiridon; Papageorgiou, Katerina; Yiakoumettis, Iakovos; Baricevic, Dea; Kusar, Anita

2010-11-02

We investigated the antioxidant activity of methanolic and water extracts of Slovene accessions of four medicinal plant species (Salvia officinalis, Achillea millefolium, Origanum vulgare subsp. vulgare and Gentiana lutea). Their free radical-scavenging activity against the DPPH. free radical was studied with a spectrophotometric assay, while their biological activity with the help of a laboratory-made biosensor based on immobilized fibroblast cells (assay duration: 3 min). The observed antioxidant activity of the extracts from the four investigated medicinal plant species was dependent on both the solvent used for extraction and the assay method (conventional or biosensor-based). Independently from the assay method and the solvent used for extraction, the lowest scavenging activity was observed in root extracts of G. lutea. Treatment of the immobilized cells with the plant extracts resulted in an increase of the cell membrane potential (membrane hyperpolarization), possibly due to the reduction of membrane damage due to oxidation. The novel cell biosensor could be utilized as a rapid, high throughput tool for screening the antioxidant properties of plant-derived compounds. Copyright (c) 2010 Elsevier B.V. All rights reserved.

High performance liquid chromatography with two simultaneous on-line antioxidant assays: Evaluation and comparison of espresso coffees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnett, Neil W; Gritti, Fabrice; Guiochon, Georges A

The antioxidant profiles of various espresso coffees were established using HPLC with UV-absorbance detection and two rapid, simultaneous, on-line chemical assays that enabled the relative reactivity of sample components to be screened. The assays were based on (i) the colour change associated with reduction of the 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH{sm_bullet}); and (ii) the emission of light (chemiluminescence) upon reaction with acidic potassium permanganate. Results from the two approaches were similar and reflected the complex array of antioxidant species present in the samples. However, some differences in selectivity were observed. Chromatograms generated with the chemiluminescence assay contained more peaks, which was ascribedmore » to the greater sensitivity of the reagent towards minor, readily oxidisable sample components. The three coffee samples produced closely related profiles, signifying their fundamentally similar chemical compositions and origin. Nevertheless, the overall intensity and complexity of the samples in both UV absorption and antioxidant assay chromatograms were aligned with the manufacturers description of flavour intensity and character.« less

A novel drug-free strategy of nano-pulse stimulation sequence (NPSS) in oral cancer therapy: In vitro and in vivo study.

PubMed

Guo, Jinsong; Dong, Feihong; Ding, Lian; Wang, Kaile; Zhang, Jue; Fang, Jing

2018-04-19

Nano-pulse stimulation (NPS) is a novel technology to induce cancer apoptosis. In this study, based on the energy-dose effect of NPS, we designed a special NPS sequence (NPSS) with low field intensity. The effectiveness and mechanisms of NPSS on oral cancer therapy were evaluated by cell proliferation assays, microscopic investigation, JC-1 mitochondrial membrane potential assay, tumor inhibition assays, immunohistochemistry (IHC) assay, Ca 2+ , NOS and ROS detection assays, respectively. The results demonstrated that NPSS treatment significantly inhibited oral cancer growth in vitro and in vivo. Furthermore, we found that NPSS treatment induced an obviously apoptosis and mitochondrial membrane potential (ΔΨm) reduction in Cal-27 cells. Notably, further experiments revealed that the mechanisms of crosstalk signaling between NO, ROS and Ca 2+ involvement in NPSS treatment. In conclusion, this is a proof-of-concept study that provides a potential alternative strategy for developing and applying NPSS in oral cancer therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Does Zika virus infection affect mosquito response to repellents?

PubMed Central

Leal, Walter S.; Barbosa, Rosângela M. R.; Zeng, Fangfang; Faierstein, Gabriel B.; Tan, Kaiming; Paiva, Marcelo H. S.; Guedes, Duschinka R. D.; Crespo, Mônica M.; Ayres, Constância F. J.

2017-01-01

The World Health Organization (WHO) recommends that people travelling to or living in areas with Zika virus (ZIKV) outbreaks or epidemics adopt prophylactic measures to reduce or eliminate mosquito bites, including the use of insect repellents. It is, however, unknown whether repellents are effective against ZIKV-infected mosquitoes, in part because of the ethical concerns related to exposing a human subject’s arm to infected mosquitoes in the standard arm-in-cage assay. We used a previously developed, human subject-free behavioural assay, which mimics a human subject to evaluate the top two recommended insect repellents. Our measurements showed that DEET provided significantly higher protection than picaridin provided against noninfected, host-seeking females of the southern house mosquito, Culex quinquefasciatus, and the yellow fever mosquito, Aedes aegypti. When tested at lower doses, we observed a significant reduction in DEET-elicited protection against ZIKV-infected yellow fever mosquitoes from old and recent laboratory colonies. The reduction in protection is more likely associated with aging than the virus infection and could be compensated by applying a 5x higher dose of DEET. A substantial protection against ZIKV-infected and old noninfected mosquitoes was achieved with 5% DEET, which corresponds approximately to a 30% dose in the conventional arm-in-cage assays. PMID:28205633

Apolar Bioactive Fraction of Melipona scutellaris Geopropolis on Streptococcus mutans Biofilm

PubMed Central

da Cunha, Marcos Guilherme; Galvão, Lívia Câmara de Carvalho; de Alencar, Severino Matias; Rosalen, Pedro Luiz

2013-01-01

The aim of this study was to evaluate the influence of the bioactive nonpolar fraction of geopropolis on Streptococcus mutans biofilm. The ethanolic extract of Melipona scutellaris geopropolis was subjected to a liquid-liquid partition, thus obtaining the bioactive hexane fraction (HF) possessing antimicrobial activity. The effects of HF on S. mutans UA159 biofilms generated on saliva-coated hydroxyapatite discs were analyzed by inhibition of formation, killing assay, and glycolytic pH-drop assays. Furthermore, biofilms treated with vehicle control and HF were analyzed by scanning electron microscopy (SEM). HF at 250 μg/mL and 400 μg/mL caused 38% and 53% reduction in the biomass of biofilm, respectively, when compared to vehicle control (P < 0.05) subsequently observed at SEM images, and this reduction was noticed in the amounts of extracellular alkali-soluble glucans, intracellular iodophilic polysaccharides, and proteins. In addition, the S. mutans viability (killing assay) and acid production by glycolytic pH drop were not affected (P > 0.05). In conclusion, the bioactive HF of geopropolis was promising to control the S. mutans biofilm formation, without affecting the microbial population but interfering with its structure by reducing the biochemical content of biofilm matrix. PMID:23843868

A novel quantitative immunomagnetic reduction assay for Nervous necrosis virus.

PubMed

Yang, Shieh Yueh; Wu, Jen Leih; Tso, Chun Hsi; Ngou, Fang Huar; Chou, Hsin Yiu; Nan, Fan Hua; Horng, Herng Er; Lu, Ming Wei

2012-09-01

Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper (Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay's limit of detection was approximately 2 × 10(1) TCID(50)/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 10(7) TCID(50)/ml for Infectious pancreatic necrosis virus and 1 × 10(6.5) TCID(50)/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

2013-12-01

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

PubMed

Huber, Alexander; France, Richard M; Riccalton-Banks, Lisa; McLaren, Jane; Cox, Helen; Quirk, Robin A; Shakesheff, Kevin M; Thompson, David; Panjwani, Naveed; Shipley, Sarah; Pickett, Andy

2008-05-01

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

Bimetallic Effect of Single Nanocatalysts Visualized by Super-Resolution Catalysis Imaging

DOE PAGES

Chen, Guanqun; Zou, Ningmu; Chen, Bo; ...

2017-11-01

Compared with their monometallic counterparts, bimetallic nanoparticles often show enhanced catalytic activity associated with the bimetallic interface. Direct quantitation of catalytic activity at the bimetallic interface is important for understanding the enhancement mechanism, but challenging experimentally. Here using single-molecule super-resolution catalysis imaging in correlation with electron microscopy, we report the first quantitative visualization of enhanced bimetallic activity within single bimetallic nanoparticles. We focus on heteronuclear bimetallic PdAu nanoparticles that present a well-defined Pd–Au bimetallic interface in catalyzing a photodriven fluorogenic disproportionation reaction. Our approach also enables a direct comparison between the bimetallic and monometallic regions within the same nanoparticle. Theoreticalmore » calculations further provide insights into the electronic nature of N–O bond activation of the reactant (resazurin) adsorbed on bimetallic sites. Subparticle activity correlation between bimetallic enhancement and monometallic activity suggests that the favorable locations to construct bimetallic sites are those monometallic sites with higher activity, leading to a strategy for making effective bimetallic nanocatalysts. Furthermore, the results highlight the power of super-resolution catalysis imaging in gaining insights that could help improve nanocatalysts.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Guanqun; Zou, Ningmu; Chen, Bo

Compared with their monometallic counterparts, bimetallic nanoparticles often show enhanced catalytic activity associated with the bimetallic interface. Direct quantitation of catalytic activity at the bimetallic interface is important for understanding the enhancement mechanism, but challenging experimentally. Here using single-molecule super-resolution catalysis imaging in correlation with electron microscopy, we report the first quantitative visualization of enhanced bimetallic activity within single bimetallic nanoparticles. We focus on heteronuclear bimetallic PdAu nanoparticles that present a well-defined Pd–Au bimetallic interface in catalyzing a photodriven fluorogenic disproportionation reaction. Our approach also enables a direct comparison between the bimetallic and monometallic regions within the same nanoparticle. Theoreticalmore » calculations further provide insights into the electronic nature of N–O bond activation of the reactant (resazurin) adsorbed on bimetallic sites. Subparticle activity correlation between bimetallic enhancement and monometallic activity suggests that the favorable locations to construct bimetallic sites are those monometallic sites with higher activity, leading to a strategy for making effective bimetallic nanocatalysts. Furthermore, the results highlight the power of super-resolution catalysis imaging in gaining insights that could help improve nanocatalysts.« less

Imaging the photoinduced charge injection in CdS/TiO2 nanoparticles by the sequential fluorescence mapping method

NASA Astrophysics Data System (ADS)

Frederice, Rafael; Lencione, Diego; Gehlen, Marcelo H.

2017-03-01

The combination of a sensitizer and TiO2 nanoparticles forming a photocatalytic material is a central issue in many fields of applied photochemistry. The charge injection of emissive sensitizers into the conduction band of the semiconductor TiO2 may form a photoactive region that becomes dark, or it has a very low emission signal due to the generation of sensitizer radicals. However, by sequential coupling of a selected photoredox dye, such as resazurin, the dark region may become fluorescent at the interfaces where the charge injection has taken place due to the concomitant formation of fluorescent resorufin by cascade electron transfer. Using this strategy and a total internal reflection fluorescence microscopy (TIRFM) image, the charge injection in TiO2/CdS and SiO2/TiO2/CdS nanoparticles is investigated The method allows the charge injection efficiency of the excited CdS into TiO2 to be evaluated qualitatively, explaining the differences observed for these photocatalytic materials in H2 generation.

Imaging the photoinduced charge injection in CdS/TiO2 nanoparticles by the sequential fluorescence mapping method.

PubMed

Frederice, Rafael; Lencione, Diego; Gehlen, Marcelo H

2017-02-10

The combination of a sensitizer and TiO 2 nanoparticles forming a photocatalytic material is a central issue in many fields of applied photochemistry. The charge injection of emissive sensitizers into the conduction band of the semiconductor TiO 2 may form a photoactive region that becomes dark, or it has a very low emission signal due to the generation of sensitizer radicals. However, by sequential coupling of a selected photoredox dye, such as resazurin, the dark region may become fluorescent at the interfaces where the charge injection has taken place due to the concomitant formation of fluorescent resorufin by cascade electron transfer. Using this strategy and a total internal reflection fluorescence microscopy (TIRFM) image, the charge injection in TiO 2 /CdS and SiO 2 /TiO 2 /CdS nanoparticles is investigated The method allows the charge injection efficiency of the excited CdS into TiO 2 to be evaluated qualitatively, explaining the differences observed for these photocatalytic materials in H 2 generation.

Influence of antibody formation on reduction of globotriaosylceramide (GL-3) in urine from Fabry patients during agalsidase beta therapy.

PubMed

Ohashi, Toya; Sakuma, Mio; Kitagawa, Teruo; Suzuki, Ken; Ishige, Nobuyuki; Eto, Yoshikatsu

2007-11-01

Two recombinant human agalsidase preparations are available for treatment of Fabry disease. We assayed urinary GL-3 (uGL-3) concentration in seronegative and seropositive patients receiving agalsidase beta (1mg/kg). Antibody formation and residual enzyme activity were strongly correlated. Normalization of uGL-3 was achieved more efficiently in seronegatives. But different from previous reports, reduction of uGL-3 level was observed in some seropositive patients.

Comparison of cardiac TnI outliers using a contemporary and a high-sensitivity assay on the Abbott Architect platform.

PubMed

Ryan, J B; Southby, S J; Stuart, L A; Mackay, R; Florkowski, C M; George, P M

2014-07-01

Assays for cardiac troponin (cTn) have undergone improvements in sensitivity and precision in recent years. Increased rates of outliers, however, have been reported on various cTn platforms, typically giving irreproducible, falsely higher results. We aimed to evaluate the outlier rate occurring in patients with elevated cTnI using a contemporary and high-sensitivity assay. All patients with elevated cTnI (up to 300 ng/L) performed over a 21-month period were assayed in duplicate. A contemporary assay (Abbott STAT Troponin-I) was used for the first part of the study and subsequently a high-sensitivity assay (Abbott STAT High-Sensitive Troponin-I) was used. Outliers exceeded a calculated critical difference (CD) (CD = z × √2 × SDAnalytical) where z = 3.5 (for probability of 0.0005) and critical outliers also were on a different side of the decision level. The respective outlier and critical outlier rates were 0.22% and 0.10% for the contemporary assay (n = 4009) and 0.18% and 0.13% for the high-sensitivity assay (n = 3878). There was no significant reduction in outlier rate between the two assays (χ(2) = 0.034, P = 0.854). Fifty-six percent of outliers occurred in samples where cTn was an 'add-on' test (and was stored and refrigerated prior to assay). Despite recent improvements in cTn methods, outliers (including critical outliers) still occur at a low rate in both a contemporary and high-sensitivity cTnI assay. Laboratory and clinical staff should be aware of this potential analytical error, particularly in samples with suboptimal sample handling such as add-on tests. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Differential sensitivity of von Willebrand factor (VWF) 'activity' assays to large and small VWF molecular weight forms: a cross-laboratory study comparing ristocetin cofactor, collagen-binding and mAb-based assays.

PubMed

Favaloro, E J; Bonar, R; Chapman, K; Meiring, M; Funk Adcock, D

2012-06-01

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura. © 2012 International Society on Thrombosis and Haemostasis.

Nitrotriazole- and imidazole-based amides and sulfonamides as antitubercular agents.

PubMed

Papadopoulou, Maria V; Bloomer, William D; Rosenzweig, Howard S; Arena, Alexander; Arrieta, Francisco; Rebolledo, Joseph C J; Smith, Diane K

2014-11-01

Twenty-three 3-nitrotriazole-based and 2-nitroimidazole-based amides and sulfonamides were screened for antitubercular (anti-TB) activity in aerobic Mycobacterium tuberculosis H37Rv by using the BacTiter-Glo (BTG) microbial cell viability assay. In general, 3-nitrotriazole-based sulfonamides demonstrated anti-TB activity, whereas 3-nitrotriazole-based amides and 2-nitroimidazole-based amides and sulfonamides were inactive. Three 3-nitrotriazole-based sulfonamides (compounds 4, 2, and 7) demonstrated 50% inhibitory concentration (IC50), IC90, and MIC values of 0.38, 0.43, and 1.56 μM (compound 4), 0.57, 0.98, and 3.13 μM (compound 2), and 0.79, 0.87, and 3.13 μM (compound 7), respectively. For 3-nitrotriazole-based sulfonamides, anti-TB activity increased with lipophilicity, whereas the one-electron reduction potential (E1/2) did not play a role. 2-Nitroimidazole-based analogs, which were inactive in the BTG assay, were significantly more active in the low-oxygen assay and more active than the 3-nitrotriazoles. All active nitrotriazoles in the BTG assay were similarly active or more potent (lower MIC values) against resistant strains, with the exception of compounds 2, 3, 4, and 8, which demonstrated greater MIC values against isoniazid-resistant strains. Five 3-nitrotriazole-based sulfonamides demonstrated activity in infected murine J774 macrophages, causing log reductions similar to those seen with rifampin. However, some compounds caused toxicity in uninfected macrophages. In conclusion, the classes of 3-nitrotriazole-based amides and sulfonamides merit further investigation as potential antitubercular agents. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Nitrotriazole- and Imidazole-Based Amides and Sulfonamides as Antitubercular Agents

PubMed Central

Bloomer, William D.; Rosenzweig, Howard S.; Arena, Alexander; Arrieta, Francisco; Rebolledo, Joseph C. J.; Smith, Diane K.

2014-01-01

Twenty-three 3-nitrotriazole-based and 2-nitroimidazole-based amides and sulfonamides were screened for antitubercular (anti-TB) activity in aerobic Mycobacterium tuberculosis H37Rv by using the BacTiter-Glo (BTG) microbial cell viability assay. In general, 3-nitrotriazole-based sulfonamides demonstrated anti-TB activity, whereas 3-nitrotriazole-based amides and 2-nitroimidazole-based amides and sulfonamides were inactive. Three 3-nitrotriazole-based sulfonamides (compounds 4, 2, and 7) demonstrated 50% inhibitory concentration (IC50), IC90, and MIC values of 0.38, 0.43, and 1.56 μM (compound 4), 0.57, 0.98, and 3.13 μM (compound 2), and 0.79, 0.87, and 3.13 μM (compound 7), respectively. For 3-nitrotriazole-based sulfonamides, anti-TB activity increased with lipophilicity, whereas the one-electron reduction potential (E1/2) did not play a role. 2-Nitroimidazole-based analogs, which were inactive in the BTG assay, were significantly more active in the low-oxygen assay and more active than the 3-nitrotriazoles. All active nitrotriazoles in the BTG assay were similarly active or more potent (lower MIC values) against resistant strains, with the exception of compounds 2, 3, 4, and 8, which demonstrated greater MIC values against isoniazid-resistant strains. Five 3-nitrotriazole-based sulfonamides demonstrated activity in infected murine J774 macrophages, causing log reductions similar to those seen with rifampin. However, some compounds caused toxicity in uninfected macrophages. In conclusion, the classes of 3-nitrotriazole-based amides and sulfonamides merit further investigation as potential antitubercular agents. PMID:25182645

Polymer nanoassemblies with hydrophobic pendant groups in the core induce false positive siRNA transfection in luciferase reporter assays.

PubMed

Rheiner, Steven; Reichel, Derek; Rychahou, Piotr; Izumi, Tadahide; Yang, Hsin-Sheng; Bae, Younsoo

2017-08-07

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(l-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P' caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimizing Escherichia coli's metabolism for fuel cell applications

NASA Astrophysics Data System (ADS)

Nieves, Ismael U.

In the last few years there have been many publications about applications that center on the generation of electrons from bacterial cells. These applications take advantage of the catabolic diversity of microbes to generate electrical power. The practicality of these applications depends on the microorganism's ability to effectively donate electrons, either directly to the electrode or indirectly through the use of a mediator. After establishing the limitations of electrical output in microbial fuel cells (MFCs) imposed by the bacterial cells, a spectrophotometric assay measuring the indirect reduction of the electronophore neutral red via iron reduction was used to measure electron production from Escherichia coli resting cells. Using this assay I identified NADH dehydrogenase I as a likely site of neutral red reduction. The only previously reported site of interaction between E. coli cells and NR is at the hydrogenases. Although we cannot rule out the possibility that NR is reduced by soluble hydrogenases in the cytoplasm, this previous report indicated that hydrogenase activity does not account for all of the NR reduction activity. Supporting this, data in this thesis suggest that the hydrogenases play a small role in NR reduction. It seems that NR reduction is largely taking place within the cytoplasmic membrane of the bacterial cells, serving as a substrate of enzymes that typically reduce quinones. Furthermore, it seems that under the experimental conditions used here, E. coli's catabolism of glucose is rather inefficient. Instead of using the complete TCA cycle, the bacterial cells are carrying out fermentation, leading to incomplete oxidation of the fuel and low yields of electrons. The results obtained from the TC31 strain suggest that eliminating fermentation pathways to improve NR reduction was the correct approach. Following up on this a new strain was created, KN02, which, in addition to the mutations on strain TC31, lacks acetate kinase activity.

Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

PubMed Central

Tøndervik, Anne; Sletta, Håvard; Klinkenberg, Geir; Emanuel, Charlotte; Powell, Lydia C.; Pritchard, Manon F.; Khan, Saira; Craine, Kieron M.; Onsøyen, Edvar; Rye, Phil D.; Wright, Chris; Thomas, David W.; Hill, Katja E.

2014-01-01

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity. PMID:25409186

Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

PubMed Central

Gilling, Damian H.; Kitajima, Masaaki; Torrey, Jason R.

2014-01-01

Numerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P ≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log10 to 1.88 log10). After 24 h, the reductions were 2.74, 3.00, and 3.41 log10 for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses. PMID:24907316

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

PubMed

Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

2015-06-01

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inhibitory Effect of a Nucleotide Analog on Infectious Salmon Anemia Virus Infection ▿

PubMed Central

Rivas-Aravena, Andrea; Vallejos-Vidal, Eva; Cortez-San Martin, Marcelo; Reyes-Lopez, Felipe; Tello, Mario; Mora, Patricia; Sandino, Ana María; Spencer, Eugenio

2011-01-01

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-β-d-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC50) of 0.02 μg/ml and an IC90 of 0.4 μg/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo. PMID:21653663

Cost of gentamicin assays carried out by microbiology laboratories.

PubMed Central

Vacani, P F; Malek, M M; Davey, P G

1993-01-01

AIMS--To assess the current range of prices charged for gentamicin assays in United Kingdom laboratories; and to examine the laboratories' likely response to increases or decreases in the demand for the service. METHODS--A postal survey of the 420 members of the Association of Medical Microbiologists was used to establish the range of prices charged for aminoglycoside assays. Additionally, eight private institutions were contacted to determine what the private sector was charging for aminoglycoside assays. Reagent costs in the NHS laboratories were calculated by dividing the total cost of all aminoglycoside assay kits by the number of samples analysed. RESULTS--The NHS and the private institutions both showed a wide price variation. Prices charged to an in-hospital requester for a peak and trough assay ranged from 5.00 pounds to 68.20 pounds (n = 44), and to an external private hospital, under a bulk service contract, from 5.00 pounds to 96.00 pounds (n = 47). Prices charged by private laboratories ranged from 49.00 pounds to 84.00 pounds (n = 8). There was a log linear correlation in the NHS laboratories between the reagent costs per assay and the number of assays performed per year, and most laboratories thought that their price per assay would be sensitive to increases or decreases in demand. Laboratories which had purchased their assay machines had lower reagent costs per assay but higher repair and maintenance costs. Overall, number of assays performed and method of payment for assay machinery only accounted for 44.8% of the observed variation in assay kit costs. CONCLUSIONS--The price range for gentamicin assays in the United Kingdom is wide and is only partially explained by the number of assays performed. Most laboratories believe that they would experience a reduction in unit cost as output increases. The currently offered range of prices is, in part, due to variation in the laboratories' approach to costing the service provided and some laboratories charge prices which do not even cover the cost of assay kits. Overall, we believe that prices charged should be as close as possible to the marginal cost of the tests performed. PMID:8227402

Application of firefly luciferase assay for adenosine triphosphate (ATP) to antimicrobial drug sensitivity testing

NASA Technical Reports Server (NTRS)

Picciolo, G. L.; Tuttle, S. A.; Schrock, C. G.; Deming, J. W.; Barza, M. J.; Wienstein, L.; Chappelle, E. W.

1977-01-01

The development of a rapid method for determining microbial susceptibilities to antibiotics using the firefly luciferase assay for adenosine triphosphate (ATP) is documented. The reduction of bacterial ATP by an antimicrobial agent was determined to be a valid measure of drug effect in most cases. The effect of 12 antibiotics on 8 different bacterial species gave a 94 percent correlation with the standard Kirby-Buer-Agar disc diffusion method. A 93 percent correlation was obtained when the ATP assay method was applied directly to 50 urine specimens from patients with urinary tract infections. Urine samples were centrifuged first to that bacterial pellets could be suspended in broth. No primary isolation or subculturing was required. Mixed cultures in which one species was predominant gave accurate results for the most abundant organism. Since the method is based on an increase in bacterial ATP with time, the presence of leukocytes did not interfere with the interpretation of results. Both the incubation procedure and the ATP assays are compatible with automation.

DNA-protective activities of hyperforin and aristoforin.

PubMed

Ševčovičová, A; Šemeláková, M; Plšíková, J; Loderer, D; Imreová, P; Gálová, E; Kožurková, M; Miadoková, E; Fedoročko, P

2015-04-01

The aim of this study was to explain the molecular mechanisms of action of hyperforin, a phluoroglucinol derivative found in Hypericum perforatum L. and its more stable derivative aristoforin. DNA-topology assay revealed partial DNA-protective activities of hyperforin and aristoforin against Fe(2+)-induced DNA breaks. In order to assess molecular mechanisms underlying DNA-protective activity, the potential antioxidant activity of hyperforin and aristoforin was investigated using DPPH and OH scavenging assays, reducing power assay and Fe(2+)-chelating assay. We also studied interaction of hyperforin and aristoforin with DNA using established protocols for fluorescence titration. The ability of the studied compounds to relax topoisomerase I with electrophoretic techniques was investigated. The reduction in the fluorescence of hyperforin indicated an interaction between hyperforin and DNA with a binding constant of 0.2×10(8)M(-1). We suggest that a mechanism of hyperforin/aristoforin DNA-protective abilities is based on free radicals (mainly OH) scavenging activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

PubMed Central

Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

2012-01-01

A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

Assessing cellulolysis in passive treatment systems for mine drainage: a modified enzyme assay.

PubMed

McDonald, Corina M; Gould, W Douglas; Lindsay, Matthew B J; Blowes, David W; Ptacek, Carol J; Condon, Peter D

2013-01-01

A modified cellulase enzyme assay was developed to monitor organic matter degradation in passive treatment systems for mine drainage. This fluorogenic substrate method facilitates assessment of exo-(1,4)-β-D-glucanase, endo-(1,4)-β-D-glucanase, and β-glucosidase, which compose an important cellulase enzyme system. The modified method was developed and refined using samples of organic carbon-amended mine tailings from field experiments where sulfate reduction was induced as a strategy for managing water quality. Sample masses (3 g) and the number of replicates ( ≥ 3) were optimized. Matrix interferences within these metal-rich samples were found to be insignificant. Application of this modified cellulase assay method provided insight into the availability and degradation of organic carbon within the amended tailings. Results of this study indicate that cellulase enzyme assays can be applied to passive treatment systems for mine drainage, which commonly contain elevated concentrations of metals. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR

PubMed Central

Witte, Anna Kristina; Mester, Patrick; Fister, Susanne; Witte, Matthias; Schoder, Dagmar; Rossmanith, Peter

2016-01-01

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as “rain” in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay. PMID:27992475

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

PubMed

Witte, Anna Kristina; Mester, Patrick; Fister, Susanne; Witte, Matthias; Schoder, Dagmar; Rossmanith, Peter

2016-01-01

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

Duddingtonia flagrans formulated in rice bran in the control of Oesophagostomum spp. intestinal parasite of swine.

PubMed

Facchini Rodrigues, João Victor; Braga, Fabio Ribeiro; Campos, Artur Kanadani; de Carvalho, Lorendane Millena; Araujo, Juliana Milani; Aguiar, Anderson Rocha; Ferraz, Carolina Magri; da Silveira, Wendeo Ferreira; Valadão, Marisa Caixeta; de Oliveira, Thais; de Freitas, Samuel Galvão; de Araújo, Jackson Victor

2018-01-01

Three experimental assays with Duddingtonia flagrans (isolated AC001) were carried out. The growth of the genus Duddingtonia present in formulation of rice bran, its predatory capability on Oesophagostomum spp. infective larvae (L 3 ) in petri dishes (assay 1), its action in faecal cultures with eggs of that parasite (assay 2) and isolate's capability of predation after passing through gastrointestinal tract of swine (assay 3) was evaluated. At assay 3, feces were collected at time intervals of 12, 24, 36, 48, and 60 h after feed animals with the formulation. Assays 1 and 2 showed a statistical difference (p < 0.01) by the F test when comparing the treated group with the control group. At the both assays, was observed in the treated group a reduction percentage of 74.18% and 88.38%, respectively. In assay 3, there was a statistical difference between the treated group and the control group at all collection times (p < 0.01). Regarding the collection periods, there was no statistical difference over time in the treatment group (p > 0.05). The results demonstrate that the fungal isolate AC001 formulated in rice bran can prey on L 3 of Oesophagostomum spp., in vitro and after passing through the gastrointestinal tract, without loss of viability. This isolate may be an alternative in the control of Oesophagostomum spp. in swine. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids.

PubMed

Chanput, Wasaporn; Krueyos, Narumol; Ritthiruangdej, Pitiporn

2016-11-01

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids. Copyright © 2016 Elsevier B.V. All rights reserved.

Radiorespirometric assays for the detection of biogenic sulfides from sulfate-reducing bacteria.

PubMed

de Queiroz, J C; de Melo Ferreira, A C; da Costa, A C A

2013-04-01

The detection of trace concentrations of biogenic sulfides can be carried out through radiorespirometric assays. The objective of this work was to improve the methodology for detection of H2 S in trace concentrations, to correlate with sulfate-reducing bacterial activity. Serial dilutions of synthetic sea water with a pure culture of Desulfovibrio alaskensis, a mixed anaerobic microbial culture and a natural saline sample from a petroleum offshore platform indicated that dilutions were followed, accordingly, by sulfate reduction. Tests performed indicated that increasing the time of incubation of a mixed anaerobic microbial culture contributed to an increase in the sulfate reduction rates, as well as the amount of carbon source and inoculum. The technique here developed proved to be a rapid test for the detection of biogenic sulfides, particularly those associated with corrosion products, being an useful tool for monitoring and controlling oil/water storage tanks, petroleum continental platforms and several types of reservoirs. © 2013 The Society for Applied Microbiology.

The pentose phosphate pathway leads to enhanced succinic acid flux in biofilms of wild-type Actinobacillus succinogenes.

PubMed

Bradfield, Michael F A; Nicol, Willie

2016-11-01

Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady state. The results of the assays were coupled to a kinetic model that revealed an increase in oxidative pentose phosphate pathway (OPPP) flux relative to total substrate flux with increasing SA titre, for both substrates. Furthermore, applying metabolite concentration data to metabolic flux models that include the OPPP revealed similar flux relationships to those observed in the experimental kinetic analysis. A relative increase in OPPP flux produces additional reduction power that enables increased flux through the reductive branch of the TCA cycle, leading to increased SA yields, reduced by-product formation and complete closure of the overall redox balance.

Reduction-responsive multifunctional hyperbranched polyaminoglycosides with excellent antibacterial activity, biocompatibility and gene transfection capability.

PubMed

Huang, Yajun; Ding, Xiaokang; Qi, Yu; Yu, Bingran; Xu, Fu-Jian

2016-11-01

There is an increasing demand in developing of multifunctional materials with good antibacterial activity, biocompatibility and drug/gene delivery capability for next-generation biomedical applications. To achieve this purpose, in this work series of hydroxyl-rich hyperbranched polyaminoglycosides of gentamicin, tobramycin, and neomycin (HP and SS-HP with redox-responsive disulfide bonds) were readily synthesized via ring-opening reactions in a one-pot manner. Both HP and SS-HP exhibit high antibacterial activity toward Escherichia coli and Staphylococcus aureus. Meanwhile, the hemolysis assay of the above materials shows good biocompatibility. Moreover, SS-HPs show excellent gene transfection efficiency in vitro due to the breakdown of reduction-responsive disulfide bonds. For an in vivo anti-tumor assay, the SS-HP/p53 complexes exhibit potent inhibition capability to the growth of tumors. This study provides a promising approach for the design of next-generation multifunctional biomedical materials. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

PubMed

O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

2017-01-27

Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

The Effects of HSP27 on Gemcitabine-Resistant Pancreatic Cancer Cell Line Through Snail.

PubMed

Zhang, Song; Zhang, Xiao-qi; Huang, Shu-ling; Chen, Min; Shen, Shan-shan; Ding, Xi-wei; Lv, Ying; Zou, Xiao-ping

2015-10-01

To evaluate the regulation mechanism of heat shock protein 27 (HSP27) on gemcitabine (GEM) resistance of pancreatic cancer cell. The expression vectors pEGFP-C1-HSP27 and the vectors of MicroRNA targeting Snail were introduced into GEM-sensitive pancreatic cancer SW1990 cells, and the vectors of small hairpin RNA targeting HSP27 were transfected into SW1990 and GEM-resistant SW1990/GEM cells. The expressions of HSP27, p-HSP27 (Ser82), Snail, ERCC1, and E-cadherin were evaluated by Western blotting. The sensitivity of transfected cells to GEM was detected by CCK-8 assay and Annexin V-FITC apoptosis assay. As compared to SW1990, SW1990/GEM showed significantly increased expressions of HSP27, p-HSP27, Snail and ERCC1 with decreased expression of E-cadherin. By increasing HSP27 expression, we found increase of Snail and ERCC1 with reduction of E-cadherin expressions, while reduction of HSP27 expression caused reduction of Snail and ERCC1 but increase of E-cadherin expressions. Downregulation of Snail resulted in the reduction of ERCC1 expression and increase of E-cadherin. Furthermore, downregulation of HSP27 or snail caused increased GEM sensitivity of pancreatic cancer cells, and upregulation of HSP27 showed the opposite results. There is an inverse correlation between HSP27 expression and GEM sensitivity of SW1990 cells, which might be realized by regulating E-cadherin and ERCC1 expressions through Snail.

Steroid sulfatase inhibition success and limitation in breast cancer clinical assays: an underlying mechanism.

PubMed

Sang, Xiaoye; Han, Hui; Poirier, Donald; Lin, Sheng Xiang

2018-05-24

Steroid sulfatase is detectable in most hormone-dependent breast cancers. STX64, an STS inhibitor, induced tumor reduction in animal assay. Despite success in phase І clinical trial, the results of phase II trial were not that significant. Breast Cancer epithelial cells (MCF-7 and T47D) were treated with two STS inhibitors (STX64 and EM1913). Cell proliferation, cell cycle, and the concentrations of estradiol and 5α-dihydrotestosterone were measured to determine the endocrinological mechanism of sulfatase inhibition. Comparisons were made with inhibitions of reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs). Proliferation studies showed that DNA synthesis in cancer cells was modestly decreased (approximately 20%), accompanied by an up to 6.5% in cells in the G0/G1 phase and cyclin D1 expression reduction. The concentrations of estradiol and 5α-dihydrotestosterone were decreased by 26% and 3% respectively. However, supplementation of 5α-dihydrotestosterone produced a significant increase (approximately 35.6%) in the anti-proliferative effect of sulfatase inhibition. This study has clarified sex-hormone control by sulfatase in BC, suggesting that the different roles of estradiol and 5α-dihydrotestosterone can lead to a reduction in the effect of sulfatase inhibition when compared with 17β-HSD7 inhibition. This suggests that combined treatment of sulfatase inhibitors with 17β-HSD inhibitors such as the type7 inhibitor could hold promise for hormone-dependent breast cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biofilms and Antifungal Susceptibility Testing.

PubMed

Simitsopoulou, Maria; Chatzimoschou, Athanasios; Roilides, Emmanuel

2016-01-01

Yeasts and filamentous fungi both exist as single cells and hyphal forms, two morphologies used by most fungal organisms to create a complex multilayered biofilm structure. In this chapter we describe the most widely used assays for the determination of biofilm production and assessment of susceptibility of biofilms to antifungal agents or host phagocytes as various methods, the most frequent of which are staining, confocal laser scanning microscopy, quantification of extracellular DNA and protein associated with extracellular matrix and XTT metabolic reduction assay. Pathway-focused biofilm gene expression profiling is assessed by real-time reverse transcriptase polymerase chain reaction.

Evaluation of the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction assay and the INFINITI® Respiratory Viral Panel Plus assay for the detection of human metapneumovirus in Kuwait.

PubMed

Al-Turab, Mariam; Chehadeh, Wassim; Al-Mulla, Fahd; Al-Nakib, Widad

2012-04-01

Human metapneumovirus (hMPV) is a respiratory pathogen that was discovered in 2001 and is considered a major cause of both upper and lower respiratory tract infections. A sensitive, fast, and high-throughput diagnostic test is needed for the detection of hMPV that may assist in the clinical management as well as in the reduction of inappropriate therapy. Therefore, a comparison assessment was performed in this study between the PrimerDesign™ genesig real-time reverse transcription-polymerase chain reaction (RT-PCR) Assay and the INFINITI(®) Respiratory Viral Panel Plus Assay (RVP-Plus) for the detection of hMPV infection in patients with respiratory tract infections. A total of 200 respiratory samples were collected from 185 hospitalized patients, during the winter season in Kuwait. Of 185 patients, 10 (5.4%) were positive for hMPV RNA by the in-house RT-PCR assay, while 7 (4%) were positive for hMPV RNA by the real-time RT-PCR assay and 9 (5%) were positive for hMPV RNA by the INFINITI(®) RVP-Plus assay. The high incidence rate (60%) of hMPV infection was in January 2011. The sensitivity of the real-time RT-PCR and INFINITI(®) RVP-Plus assays was 70% and 90%, respectively, with specificity of 100% for both assays. hMPV types A and B could be identified in this study; however, discordant genotyping results were found between the direct sequencing method and the INFINITI(®) RVP-Plus assay in 33% of hMPV-positive patients. Copyright © 2012 Elsevier Inc. All rights reserved.

Comparison of four different cardiac troponin assays in patients with end-stage renal disease on chronic haemodialysis.

PubMed

Helleskov Madsen, Lene; Ladefoged, Søren; Hildebrandt, Per; Atar, Dan

2008-01-01

Several studies have documented the importance of troponin elevation as a prognostic marker in end-stage renal disease (ESRD). The reason for the elevated concentrations is not clarified. We do not know whether the different assays recognize the same patients within ESRD populations. The aim of this study was to compare concentrations of troponin measured by four different assays in a cohort of patients with ESRD, to investigate whether haemodialysis affects troponin concentrations, and to compare the prognostic potential of the different assays. We included 109 patients on chronic haemodialysis. Serum cardiac troponin T (cTnT) was measured pre- and postdialysis using Elecsys 2010 and troponin I (cTnI) using Access AccuTnI, Dimension RxL and AIA-600II. The cTnT assay had the highest percentage of elevated concentrations for all chosen cut-offs with a reduction in percentage of patients with elevated concentrations during haemodialysis. Elecsys 2010 and AIA-600II demonstrated a significant increased mortality with raised concentrations of troponin. The diverging results in previous studies are most likely based on substantial differences in the analytical performance of the assays. The prognostic value of cTnT appears superior to cTnI, which amplifies the prognostic significance of this cardiovascular marker in patients with ESRD.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

PubMed Central

Dreier, Jens; Störmer, Melanie; Mäde, Dietrich; Burkhardt, Sabine; Kleesiek, Knut

2006-01-01

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities. PMID:16891482

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

PubMed

Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

2012-03-01

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

Development of a high-throughput assay for rapid screening of butanologenic strains.

PubMed

Agu, Chidozie Victor; Lai, Stella M; Ujor, Victor; Biswas, Pradip K; Jones, Andy; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2018-02-21

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP + to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

PubMed

Shalaby, Mohamed A; El-Deeb, Ayman; El-Tholoth, Mohamed; Hoffmann, Donata; Czerny, Claus-Peter; Hufert, Frank T; Weidmann, Manfred; Abd El Wahed, Ahmed

2016-11-02

Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Assessment of antioxidant activity by using different in vitro methods.

PubMed

Schlesier, K; Harwat, M; Böhm, V; Bitsch, R

2002-02-01

In this study, six common tests for measuring antioxidant activity were evaluated by comparing four antioxidants and applying them to beverages (tea and juices): Trolox equivalent antioxidant capacity assay (TEAC I-III assay), Total radical-trapping antioxidant parameter assay (TRAP assay), 2,2-diphenyl-l-picrylhydrazyl assay (DPPH assay), N,N-dimethyl-p-phenylendiamine assay (DMPD assay), Photochemiluminescence assay (PCL assay) and Ferric reducing ability of plasma assay (FRAP assay). The antioxidants included gallic acid representing the group of polyphenols, uric acid as the main antioxidant in human plasma, ascorbic acid as a vitamin widely spread in fruits and Trolox as water soluble vitamin E analogue. The six methods presented can be divided into two groups depending on the oxidising reagent. Five methods use organic radical producers (TEAC I-III, TRAP, DPPH, DMPD, PCL) and one method works with metal ions for oxidation (FRAP). Another difference between these tests is the reaction procedure. Three assays use the delay in oxidation and determine the lag phase as parameter for the antioxidant activity (TEAC I, TRAP, PCL). They determine the delay of radical generation as well as the ability to scavenge the radical. In contrast, the assays TEAC II and III, DPPH, DMPD and FRAP analyse the ability to reduce the radical cation (TEAC II and III, DPPH, DMPD) or the ferric ion (FRAP). The three tests acting by radical reduction use preformed radicals and determine the decrease in absorbance while the FRAP assay measures the formed ferrous ions by increased absorbance. Gallic acid was the strongest antioxidant in all tests with exception of the DMPD assay. In contrast, uric acid and ascorbic acid showed low activity in some assays. Most of the assays determine the antioxidant activity in the micromolar range needing minutes to hours. Only one assay (PCL) is able to analyse the antioxidant activity in the nanomolar range. Black currant juice showed highest antioxidant activity in all tests compared to tea, apple juice and tomato juice. Despite these differences, results of these in vitro assays give an idea of the protective efficacy of secondary plant products. It is strongly recommended to use at least two methods due to the differences between the test systems investigated.

Cell oxidation-reduction imbalance after modulated radiofrequency radiation.

PubMed

Marjanovic, Ana Marija; Pavicic, Ivan; Trosic, Ivancica

2015-01-01

Aim of this study was to evaluate an influence of modulated radiofrequency field (RF) of 1800 MHz, strength of 30 V/m on oxidation-reduction processes within the cell. The assigned RF field was generated within Gigahertz Transversal Electromagnetic Mode cell equipped by signal generator, modulator, and amplifier. Cell line V79, was irradiated for 10, 30, and 60 min, specific absorption rate was calculated to be 1.6 W/kg. Cell metabolic activity and viability was determined by MTT assay. In order to define total protein content, colorimetric method was used. Concentration of oxidised proteins was evaluated by enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) marked with fluorescent probe 2',7'-dichlorofluorescin diacetate were measured by means of plate reader device. In comparison with control cell samples, metabolic activity and total protein content in exposed cells did not differ significantly. Concentrations of carbonyl derivates, a product of protein oxidation, insignificantly but continuously increase with duration of exposure. In exposed samples, ROS level significantly (p < 0.05) increased after 10 min of exposure. Decrease in ROS level was observed after 30-min treatment indicating antioxidant defence mechanism activation. In conclusion, under the given laboratory conditions, modulated RF radiation might cause impairment in cell oxidation-reduction equilibrium within the growing cells.

Impact of Introducing the Line Probe Assay on Time to Treatment Initiation of MDR-TB in Delhi, India

PubMed Central

Singla, Neeta; Satyanarayana, Srinath; Sachdeva, Kuldeep Singh; Van den Bergh, Rafael; Reid, Tony; Tayler-Smith, Katherine; Myneedu, V. P.; Ali, Engy; Enarson, Donald A.; Behera, Digamber; Sarin, Rohit

2014-01-01

Setting National Institute of Tuberculosis and Respiratory Diseases (erstwhile Lala Ram Sarup Institute) in Delhi, India. Objectives To evaluate before and after the introduction of the line Probe Assay (LPA) a) the overall time to MDR-TB diagnosis and treatment initiation; b) the step-by-step time lapse at each stage of patient management; and c) the lost to follow-up rates. Methods A retrospective cohort analysis was done using data on MDR-TB patients diagnosed during 2009–2012 under Revised National Tuberculosis Control Programme at the institute. Results Following the introduction of the LPA in 2011, the overall median time from identification of patients suspected for MDR-TB to the initiation of treatment was reduced from 157 days (IQR 127–200) to 38 days (IQR 30–79). This reduction was attributed mainly to a lower diagnosis time at the laboratory. Lost to follow-up rates were also significantly reduced after introduction of the LPA (12% versus 39% pre-PLA). Conclusion Introduction of the LPA was associated with a major reduction in the delay between identification of patients suspected for MDR-TB and initiation of treatment, attributed mainly to a reduction in diagnostic time in the laboratory. PMID:25058124

Resistance of faecal coliforms and enterococci populations in sludge and biosolids to different hygienisation treatments.

PubMed

Bonjoch, X; Blanch, A R

2009-04-01

The composition of the most abundant facultative anaerobic bacteria populations [faecal coliforms (FC) and enterococci (ENT)] in sludge can be modified after different treatments. These involve the disposal or reuse of sludge and include: anaerobic digesters, incineration, composting, pasteurization and lime treatments. In this study, three treatment types (mesophilic anaerobic digestion, composting and pasteurization) were compared in terms of their ability to reduce both bacterial populations. The diversity and any changes in composition of main phenotypic groups for both populations were also analyzed. Mesophilic anaerobic digestion (MAD) was carried out at 35 degrees C for 20 days. Digested sludge was then dehydrated by centrifugation at 2,500 rpm. Composting (COM) was performed at 55 degrees C with windrow phases. Pasteurization was assayed at 60 degrees C for 90 min (P60), at 80 degrees C for 60 min (P80). A 1-1.5 log unit reduction was observed for FC, and 1 log unit reduction was noted for ENT by MAD treatment. In composting, this reduction proved higher for FC than for ENT (6 log and 3-4 log units, respectively). Optimal pasteurization was obtained at 80 degrees C for 60 min, resulting in a 5 log unit reduction for FC and a 2 log unit reduction for ENT. High diversity indices (Di) for both bacterial populations were detected both before and after implementation of the different treatments. Analyses of the population's similarity provided that FC were diverse both before and after COM, P60 and P80 treatments. However, no differences were observed on the composition of ENT populations after the different treatments assayed.

Effects of X-shaped reduction-sensitive amphiphilic block copolymer on drug delivery.

PubMed

Xiao, Haijun; Wang, Lu

2015-01-01

To study the effects of X-shaped amphiphilic block copolymers on delivery of docetaxel (DTX) and the reduction-sensitive property on drug release, a novel reduction-sensitive amphiphilic copolymer, (PLGA)2-SS-4-arm-PEG2000 with a Gemini-like X-shape, was successfully synthesized. The formation of nanomicelles was proved with respect to the blue shift of the emission fluorescence as well as the fluorescent intensity increase of coumarin 6-loaded particles. The X-shaped polymers exhibited a smaller critical micelle concentration value and possessed higher micellar stability in comparison with those of linear ones. The size of X-shaped (PLGA)2-SS-4-arm-PEG2000 polymer nanomicelles (XNMs) was much smaller than that of nanomicelles prepared with linear polymers. The reduction sensitivity of polymers was confirmed by the increase of micellar sizes as well as the in vitro drug release profile of DTX-loaded XNMs (DTX/XNMs). Cytotoxicity assays in vitro revealed that the blank XNMs were nontoxic against A2780 cells up to a concentration of 50 µg/mL, displaying good biocompatibility. DTX/XNMs were more toxic against A2780 cells than other formulations in both dose- and time-dependent manners. Cellular uptake assay displayed a higher intracellular drug delivery efficiency of XNMs than that of nanomicelles prepared with linear polymers. Besides, the promotion of tubulin polymerization induced by DTX was visualized by immunofluorescence analysis, and the acceleration of apoptotic process against A2780 cells was also imaged using a fluorescent staining method. Therefore, this X-shaped reduction-sensitive (PLGA)2-SS-4-arm-PEG2000 copolymer could effectively improve the micellar stability and significantly enhance the therapeutic efficacy of DTX by increasing the cellular uptake and selectively accelerating the drug release inside cancer cells.

Fisetin inhibits the proliferation of gastric cancer cells and induces apoptosis through suppression of ERK 1/2 activation

PubMed Central

Yan, Weixin; Chen, Shouhui; Zhao, Yiyang; Ye, Xiaoyu

2018-01-01

The present study aimed to investigate the effect of fisetin on proliferation and apoptosis of gastric cancer cells, as well as the underlying mechanism. Proliferation in SGC7901 cancer and GES-1 normal cells was analyzed using a CCK-8 assay. Apoptosis was analyzed using an Annexin V/Propidium Iodide apoptosis kit and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was analyzed by western blot assay. Treatment of SGC7901 cells with various concentrations (1, 5, 10, 15 and 20 µM) of fisetin for 48 h resulted in a concentration dependent reduction in proliferation. Flow cytometry revealed a marked increase in apoptosis from 5 µM concentration of fisetin after 48 h. The percentage of apoptotic cells increased to 87% following treatment with 15 µM fisetin for 48 h, compared with 2% in control. Treatment of SGC7901 cells with fisetin for 48 h resulted in a reduction in the activation of ERK 1/2 in a concentration-dependent manner. The reduction in activation of ERK 1/2 was significant following treatment with 15 µM fisetin for 48 h. The inhibitory effect of fisetin on activation of ERK 1/2 was further demonstrated using the ERK 1/2 inhibitor, PD98059. The results indicated a significant reduction in the proliferation of SGC7901 cells following treatment with PD98059 (P<0.002). The reduction by PD98059 administration was comparable to that observed following fisetin treatment for 48 h. In conclusion, the current study demonstrates that fisetin inhibits the proliferation of gastric cancer cells and induces apoptosis through suppression of ERK 1/2 activation. Thus, fisetin may have therapeutic applications in the treatment of gastric cancer. PMID:29805580

Fisetin inhibits the proliferation of gastric cancer cells and induces apoptosis through suppression of ERK 1/2 activation.

PubMed

Yan, Weixin; Chen, Shouhui; Zhao, Yiyang; Ye, Xiaoyu

2018-06-01

The present study aimed to investigate the effect of fisetin on proliferation and apoptosis of gastric cancer cells, as well as the underlying mechanism. Proliferation in SGC7901 cancer and GES-1 normal cells was analyzed using a CCK-8 assay. Apoptosis was analyzed using an Annexin V/Propidium Iodide apoptosis kit and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was analyzed by western blot assay. Treatment of SGC7901 cells with various concentrations (1, 5, 10, 15 and 20 µM) of fisetin for 48 h resulted in a concentration dependent reduction in proliferation. Flow cytometry revealed a marked increase in apoptosis from 5 µM concentration of fisetin after 48 h. The percentage of apoptotic cells increased to 87% following treatment with 15 µM fisetin for 48 h, compared with 2% in control. Treatment of SGC7901 cells with fisetin for 48 h resulted in a reduction in the activation of ERK 1/2 in a concentration-dependent manner. The reduction in activation of ERK 1/2 was significant following treatment with 15 µM fisetin for 48 h. The inhibitory effect of fisetin on activation of ERK 1/2 was further demonstrated using the ERK 1/2 inhibitor, PD98059. The results indicated a significant reduction in the proliferation of SGC7901 cells following treatment with PD98059 (P<0.002). The reduction by PD98059 administration was comparable to that observed following fisetin treatment for 48 h. In conclusion, the current study demonstrates that fisetin inhibits the proliferation of gastric cancer cells and induces apoptosis through suppression of ERK 1/2 activation. Thus, fisetin may have therapeutic applications in the treatment of gastric cancer.

Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts.

PubMed

Crawford, N A; Droux, M; Kosower, N S; Buchanan, B B

1989-05-15

Results obtained with isolated intact chloroplasts maintained aerobically under light and dark conditions confirm earlier findings with reconstituted enzyme assays and indicate that the ferredoxin/thioredoxin system functions as a light-mediated regulatory thiol chain. The results were obtained by application of a newly devised procedure in which a membrane-permeable thiol labeling reagent, monobromobimane (mBBr), reacts with sulfhydryl groups and renders the derivatized protein fluorescent. The mBBr-labeled protein in question is isolated individually from chloroplasts by immunoprecipitation and its thiol redox status is determined quantitatively by combining sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorescence measurements. The findings indicate that each member of the ferredoxin/thioredoxin system containing a catalytically active thiol group is reduced in isolated intact chloroplasts after a 2-min illumination. The extents of reduction were FTR, 38%; thioredoxin m, 75% (11-kDa form) and 87% (13-kDa form); thioredoxin f, 95%. Reduction of each of these components was negligible both in the dark and when chloroplasts were transferred from light to dark conditions. The target enzyme, NADP-malate dehydrogenase, also underwent net reduction in illuminated intact chloroplasts. Fructose-1,6-bisphosphatase showed increased mBBr labeling under these conditions, but due to interfering gamma globulin proteins it was not possible to determine whether this was a result of net reduction as is known to take place in reconstituted assays. Related experiments demonstrated that mBBr, as well as N-ethylmaleimide, stabilized photoactivated NADP-malate dehydrogenase and fructose-1,6-bisphosphatase so that they remained active in the dark. By contrast, phosphoribulokinase, another thioredoxin-linked enzyme, was immediately deactivated following mBBr addition. These latter results provide new information on the relation between the regulatory and active sites of these enzymes.

Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng

Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor,more » we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.« less

Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during the production process of deep-frozen onions and spinach.

PubMed

Baert, Leen; Uyttendaele, Mieke; Vermeersch, Mattias; Van Coillie, Els; Debevere, Johan

2008-08-01

The reduction of murine norovirus 1 (MNV-1) on onions and spinach by washing was investigated as was the risk of contamination during the washing procedure. To decontaminate wash water, the industrial sanitizer peracetic acid (PAA) was added to the water, and the survival of MNV-1 was determined. In contrast to onions, spinach undergoes a heat treatment before freezing. Therefore, the resistance of MNV-1 to blanching of spinach was examined. MNV-1 genomic copies were detected with a real-time reverse transcription PCR assay in PAA-treated water and blanched spinach, and PFUs (representing infectious MNV-1 units) were determined with a plaque assay. A < or = 1-log reduction in MNV-1 PFUs was achieved by washing onion bulbs and spinach leaves. More than 3 log PFU of MNV-1 was transmitted to onion bulbs and spinach leaves when these vegetables were washed in water containing approximately 5 log PFU/ml. No decline of MNV-1 occurred in used industrial spinach wash water after 6 days at room temperature. A concentration of 20 ppm of PAA in demineralized water (pH 4.13) and in potable water (pH 7.70) resulted in reductions of 2.88 +/- 0.25 and 2.41 +/- 0.18 log PFU, respectively, after 5 min of exposure, but no decrease in number of genomic copies was observed. No reduction of MNV-1 PFUs was observed on frozen onions or spinach during storage for 6 months. Blanching spinach (80 degrees C for 1 min) resulted in at least 2.44-log reductions of infectious MNV-1, but many genomic copies were still present.

Recovery of palladium(II) by methanogenic granular sludge.

PubMed

Pat-Espadas, Aurora M; Field, James A; Otero-Gonzalez, Lila; Razo-Flores, Elías; Cervantes, Francisco J; Sierra-Alvarez, Reyes

2016-02-01

This is the first report that demonstrates the ability of anaerobic methanogenic granular sludge to reduce Pd(II) to Pd(0). Different electron donors were evaluated for their effectiveness in promoting Pd reduction. Formate and H2 fostered both chemically and biologically mediated Pd reduction. Ethanol only promoted the reduction of Pd(II) under biotic conditions and the reduction was likely mediated by H2 released from ethanol fermentation. No reduction was observed in biotic or abiotic assays with all other substrates tested (acetate, lactate and pyruvate) although a large fraction of the total Pd was removed from the liquid medium likely due to biosorption. Pd(II) displayed severe inhibition towards acetoclastic and hydrogenotrophic methanogens, as indicated by 50% inhibiting concentrations as low as 0.96 and 2.7 mg/L, respectively. The results obtained indicate the potential of utilizing anaerobic granular sludge bioreactor technology as a practical and promising option for Pd(II) reduction and recovery offering advantages over pure cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Milk Oligosaccharides Inhibit Human Rotavirus Infectivity in MA104 Cells.

PubMed

Laucirica, Daniel R; Triantis, Vassilis; Schoemaker, Ruud; Estes, Mary K; Ramani, Sasirekha

2017-09-01

Background: Oligosaccharides in milk act as soluble decoy receptors and prevent pathogen adhesion to the infant gut. Milk oligosaccharides reduce infectivity of a porcine rotavirus strain; however, the effects on human rotaviruses are less well understood. Objective: In this study, we determined the effect of specific and abundant milk oligosaccharides on the infectivity of 2 globally dominant human rotavirus strains. Methods: Four milk oligosaccharides-2'-fucosyllactose (2'FL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and galacto-oligosaccharides-were tested for their effects on the infectivity of human rotaviruses G1P[8] and G2P[4] through fluorescent focus assays on African green monkey kidney epithelial cells (MA104 cells). Oligosaccharides were added at different time points in the infectivity assays. Infections in the absence of oligosaccharides served as controls. Results: When compared with infections in the absence of glycans, all oligosaccharides substantially reduced the infectivity of both human rotavirus strains in vitro; however, virus strain-specific differences in effects were observed. Compared with control infections, the maximum reduction in G1P[8] infectivity was seen with 2'FL when added after the onset of infection (62% reduction, P < 0.01), whereas the maximum reduction in G2P[4] infectivity was seen with the mixture of 3'SL + 6'SL when added during infection (73% reduction, P < 0.01). The mixture of 3'SL + 6'SL at the same ratio as is present in breast milk was more potent in reducing G2P[4] infectivity (73% reduction, P < 0.01) than when compared with 3'SL (47% reduction) or 6'SL (40% reduction) individually. For all oligosaccharides the reduction in infectivity was mediated by an effect on the virus and not on the cells. Conclusions: Milk oligosaccharides reduce the infectivity of human rotaviruses in MA104 cells, primarily through an effect on the virus. Although breastfed infants are directly protected, the addition of specific oligosaccharides to infant formula may confer these benefits to formula-fed infants. © 2017 American Society for Nutrition.

The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

ERIC Educational Resources Information Center

Splittgerber, A. G.; And Others

1975-01-01

Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)

Measuring potential denitrification enzyme activity rates using the membrane inlet mass spectrometer

EPA Science Inventory

The denitrification enzyme activity (DEA) assay, provides a quantitative assessment of the multi enzyme, biological process of reactive nitrogen removal via the reduction of N03 to N2. Measured in soil, usually under non limiting carbon and nitrate concentrations, this short ter...

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

PubMed

Khan, Arshad; Sarkar, Dhiman

2008-04-01

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.

PubMed

First, Eric A

2015-08-15

A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors.

PubMed

Doughan, Samer; Uddayasankar, Uvaraj; Krull, Ulrich J

2015-06-09

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min. Copyright © 2015 Elsevier B.V. All rights reserved.

Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

NASA Astrophysics Data System (ADS)

Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

2017-04-01

Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P < 0.05), suggesting that a sperm-related enzymatic reaction is involved in sperm motility. Under this protocol, MTT reduction was coupled with catalysis of GAPDH and was promoted by electron transfer from NADH. Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

A modified method for determining tannin-protein precipitation capacity using accelerated solvent extraction (ASE) and microplate gel filtration.

PubMed

McArt, Scott H; Spalinger, Donald E; Kennish, John M; Collins, William B

2006-06-01

The protein precipitation assay used by Robbins et al., (1987) Ecology 68:98-107 has been shown to predict successfully the reduction in protein availability to some ruminants due to tannins. The procedure, however, is expensive and laborious, which limits its utility, especially for quantitative ecological or nutritional applications where large numbers of assays may be required. We have modified the method to decrease its cost and increase laboratory efficiency by: (1) automating the extraction by using Accelerated Solvent Extraction (ASE); and (2) by scaling and automating the precipitation reaction, chromatography, and spectrometry with microplate gel filtration and an automated UV-VIS microplate spectrometer. ASE extraction is shown to be as effective at extracting tannins as the hot methanol technique. Additionally, the microplate assay is sensitive and precise. We show that the results from the new technique correspond in a nearly 1:1 relationship to the results of the previous technique. Hence, this method could reliably replace the older method with no loss in relevance to herbivore protein digestion. Moreover, the ASE extraction technique should be applicable to other tannin-protein precipitation assays and possibly other phenolic assays.

Determination of estrogenic potential in waste water without sample extraction.

PubMed

Avberšek, Miha; Žegura, Bojana; Filipič, Metka; Uranjek-Ževart, Nataša; Heath, Ester

2013-09-15

This study describes the modification of the ER-Calux assay for testing water samples without sample extraction (NE-(ER-Calux) assay). The results are compared to those obtained with ER-Calux assay and a theoretical estrogenic potential obtained by GC-MSD. For spiked tap and waste water samples there was no statistical difference between estrogenic potentials obtained by the three methods. Application of NE-(ER-Calux) to "real" influent and effluents from municipal waste water treatment plants and receiving surface waters found that the NE-(ER-Calux) assay gave higher values compared to ER-Calux assay and GC-MSD. This is explained by the presence of water soluble endocrine agonists that are usually removed during extraction. Intraday dynamics of the estrogenic potential of a WWTP influent and effluent revealed an increase in the estrogenic potential of the influent from 12.9 ng(EEQ)/L in the morning to a peak value of 40.0 ng(EEQ)/L in the afternoon. The estrogenic potential of the effluent was

Moricandia arvensis extracts protect against DNA damage, mutagenesis in bacteria system and scavenge the superoxide anion.

PubMed

Skandrani, Ines; Bouhlel, Ines; Limem, Ilef; Boubaker, Jihed; Bhouri, Wissem; Neffati, Aicha; Ben Sghaier, Mohamed; Kilani, Soumaya; Ghedira, Kamel; Ghedira-Chekir, Leila

2009-02-01

The mutagenic potential of total aqueous, total oligomers flavonoids (TOF), ethyl acetate (EA), chloroform (Chl), petroleum ether (PE) and methanol (MeOH) extracts from aerial parts of Moricandia arvensis was assessed using Ames Salmonella tester strains TA100 and TA1535 with and without metabolic activation (S9), and using plasmid pBluescript DNA assay. None of the different extracts produced a mutagenic effect, except aqueous extract when incubated with Salmonella typhimurium TA100 after metabolic activation. Likewise, the antimutagenicity of the same extracts was tested using the "Ames test". Our results showed that M. arvensis extracts possess antimutagenic effects against sodium azide (SA) in the two tested Salmonella assay systems, except metabolized aqueous and PE extracts when tested with S. typhimurium TA100 assay system. Different extracts were also found to be effective in protecting plasmid DNA against the strand breakage induced by hydroxyl radicals, except PE and aqueous extracts. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non enzymatic (NBT/Riboflavine assay) systems. TOF extract was the more effective one in inhibiting both xanthine oxidase activity and NBT reduction.

Ex Vivo Oxidation in Tissue and Plasma Assays of Hydroxyoctadecadienoates: (Z,E/E,E)-Stereoisomer Ratios

PubMed Central

Liu, Wei; Yin, Huiyong; Akazawa, Yoko Ogawa; Yoshida, Yasukazu; Niki, Etsuo; Porter, Ned A.

2010-01-01

The primary products from peroxidation of linoleate in biological tissues and fluids are the hydroperoxy octadecadienoates and the products normally assayed, after reduction of the hydroperoxides, are the corresponding hydroxy octadecadienoates (HODEs). The HODEs are found in tissues and fluids as a mixture of Z,E and E,E stereoisomers. Two regioisomeric sets of Z,E and E,E stereoisomers are normally observed with substitution at the 9 and 13 position of the 18-carbon chain. The Z,E/E,E product ratio has proved to be a useful means for assessing the reducing capacity of the medium undergoing peroxidation. The HODE Z,E/E,E product ratios previously reported for tissues such as liver and brain vary from 0.5 to 2.0 and plasma ratios are somewhat higher, between 2.0 and 3.0. The reported literature protocols for HODE assay in tissues involve homogenization, reduction with sodium borohydride in the presence of BHT, and ester hydrolysis with KOH to give the free HODEs. This is followed by either reverse-phase HPLC of the free acid HODEs or by conversion to TMS derivatives and GC/MS. When sodium borohydride is replaced in the protocol by triphenylphosphine, a gentler reducing agent, HODE Z,E/E,E product ratios are much higher and lower total HODEs levels of are found. It is proposed that inclusion of sodium borohydride in the isolation procedures leads to ex vivo reactions that are avoided if triphenylphosphine is used as the reducing agent. Modified protocols for HODE analyses (Tissue and Plasma Methods #2) are described that should be used for assays of tissues and fluids. PMID:20423158

Positive allosteric modulators of the human sweet taste receptor enhance sweet taste

PubMed Central

Servant, Guy; Tachdjian, Catherine; Tang, Xiao-Qing; Werner, Sara; Zhang, Feng; Li, Xiaodong; Kamdar, Poonit; Petrovic, Goran; Ditschun, Tanya; Java, Antoniette; Brust, Paul; Brune, Nicole; DuBois, Grant E.; Zoller, Mark; Karanewsky, Donald S.

2010-01-01

To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste. PMID:20173092

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals.

PubMed

Ebiloma, Godwin Unekwuojo; Igoli, John Ogbaji; Katsoulis, Evangelos; Donachie, Anne-Marie; Eze, Anthonius; Gray, Alexander Ian; de Koning, Harry P

2017-04-18

Leaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. To investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. Plants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC 50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. Seven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC 50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC 50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. We have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

New Isoflavonoids from the extract of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC. and their antimycobacterial activity.

PubMed

Coronado-Aceves, Enrique Wenceslao; Gigliarelli, Giulia; Garibay-Escobar, Adriana; Zepeda, Ramón Enrique Robles; Curini, Massimo; López Cervantes, Jaime; Inés Espitia-Pinzón, Clara Inés; Superchi, Stefano; Vergura, Stefania; Marcotullio, Maria Carla

2017-07-12

The evaluation of the antimycobacterial activity of extracts of medicinal plants used by Mayos against tuberculosis and respiratory problems, allowed the identification of Rhynchosia precatoria (Humb. & Bonpl. ex Willd.) DC (Fabaceae) as the best candidate to find new antimycobacterial compounds. To isolate and characterize the compounds of R. precatoria responsible for the inhibitory and bactericidal activity against Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis ATCC 700084. To determine antimycobacterial synergistic effect of pure compounds and their selectivity index towards Vero cells. A total of six flavonoids were purified by silica gel column chromatography. Structural elucidation of the isolated compounds was achieved by using 1D and 2D NMR spectroscopy techniques. The configuration at the C-3 chiral center was established by quantum mechanical calculation of the electronic circular dichroism (ECD) spectrum. In vitro inhibitory and bactericidal activity against M. tuberculosis and M. smegmatis were determined with the redox indicator Alamar Blue (resazurin). Synergy was determined by X/Y quotient. Cytotoxicity was measured by MTT assay. The isolated compounds were identified as precatorin A (1), precatorin B (2), precatorin C (3), lupinifolin (4), cajanone (5) and lupinifolinol (6). Compounds 1-3 are new. Compounds 1 to 5 inhibited the growth of M. tuberculosis (MIC ≥31.25µg/mL); compounds 1, 2, 4 and 5 killed the bacteria (MBC ≥31.25µg/mL) and also inhibited M. smegmatis (MIC ≥125µg/mL), while 1 and 4 also resulted bactericidal (MBC ≥125µg/mL). Compounds 4 and 5 presented synergistic effect (X/Y quotient value <0.5) at a concentration of 1/2 MIC of each compound in the combination. Cytotoxicity in murine macrophages (RAW 264.7 cells) gave IC 50 values of 13.3-46.98µM, for compounds 1-5. In this work we isolated two new isoflavanones (1 and 2), and one new isoflavone (3) with a weak antimycobacterial activity. The (3R) absolute configuration was assigned to 1 by computational analysis of its ECD spectrum and to 2 and 5 by similarity of their ECD spectra with that of 1. We are also reporting by first time, activity against virulent strain of M. tuberculosis for compounds 4 and 5 and their antimycobacterial synergistic effect. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

A Simple Method for Measuring Ground-Level Ozone in the Atmosphere

ERIC Educational Resources Information Center

Seeley, John V.; Seeley, Stacy K.; Bull, Arthur W.; Fehir, Richard J., Jr.; Cornwall, Susan; Knudsen, Gabriel A.

2005-01-01

An iodometric assay that allows the ground-level ozone concentration to be determined with an inexpensive sampling apparatus and a homemade photometer is described. This laboratory experiment applies a variety of different fundamental concepts including oxidation-reduction chemistry, the ideal gas law, and spectroscopic analysis and also provides…

FACTORS INFLUENCING IN VITRO KILLING OF BACTERIA BY HEMOCYTES OF THE EASTERN OYSTER (CRASSOSTREA VIRGINICA)

EPA Science Inventory

A tetrazolium dye reduction assay was used to study factors governing killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains...

Mechanistic Approach to Understanding the Toxicity of the Azole Fungicide Triadimefon to a Nontarget Aquatic Insect and Implications for Exposure Assessment

EPA Science Inventory

We utilized mechanistic and stereoselective based in vitro metabolism assays and sublethal exposures of triadimefon to gain insight into the extent of carbonyl reduction and the toxic mode of action of triadimefon with black fly (Diptera: Simuliidae) larvae.

Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors

PubMed Central

Ziuzina, Dana; Boehm, Daniela; Patil, Sonal; Cullen, P. J.; Bourke, Paula

2015-01-01

The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP) against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin, elastase (Las B) and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated ‘inpack’ using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence for the inhibition of QS and mechanisms by which this may occur. PMID:26390435

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

PubMed

Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Expeditious neutralization assay for human metapneumovirus based on a recombinant virus expressing Renilla luciferase.

PubMed

Zhou, Min; Kitagawa, Yoshinori; Yamaguchi, Mayu; Uchiyama, Chika; Itoh, Masae; Gotoh, Bin

2013-01-01

Human metapneumovirus (HMPV) is a common cause of respiratory diseases in persons of all ages. Because of its slow replication and weak cytopathic effect in cultured cells, conventional neutralization assays for HMPV require around one week for completion. The purpose of this study is to establish a rapid neutralization assay based on a recombinant virus expressing Renilla luciferase (Rluc). A recombinant HMPV expressing both Rluc and green fluorescent protein (GFP) was created by reverse genetics method. Two-fold serial dilutions of human 23 sera were made in a 96-well plate and incubated with 50 pfu/well of the recombinant virus at 4°C for 1 h. The mixtures were then transferred to LLC-MK2 cells in a 96-well plate, incubated for 2 h, and replaced with trypsin-free fresh media. After incubation at 32°C for 24 h, the cells were lysed and measured for Rluc activity. The neutralization titer was defined as the reciprocal of the highest serum dilution that resulted in 50% reduction of Rluc activity. The novel assay could be completed within 24 h and eliminated the requirement of trypsin supporting multistep replication in cultured cells, as well as laborious processes including the plaque assay with immunostaining. Neutralization titers correlated well with those determined by a GFP-based assay previously developed. The neutralization assay based on Rluc activity is the fastest and the most straightforward of all previous assays, and may be available for high throughput screening of neutralizing antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster.

PubMed

Bauer, Johannes H; Goupil, Stephan; Garber, Graham B; Helfand, Stephen L

2004-08-31

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. Copyright 2004 The National Academy of Sciencs of the USA

Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection.

PubMed

Paris, Daniel H; Blacksell, Stuart D; Nawtaisong, Pruksa; Jenjaroen, Kemajittra; Teeraratkul, Achara; Chierakul, Wirongrong; Wuthiekanun, Vanaporn; Kantipong, Pacharee; Day, Nicholas P J

2011-09-01

There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

An accelerated assay for the identification of lifespan-extending interventions in Drosophila melanogaster

PubMed Central

Bauer, Johannes H.; Goupil, Stephan; Garber, Graham B.; Helfand, Stephen L.

2004-01-01

Recent advances in aging research have uncovered genes and genetic pathways that influence lifespan in such diverse organisms as yeast, nematodes, flies, and mice. The discovery of genes and drugs that affect lifespan has been delayed by the absence of a phenotype other than survivorship, which depends on the measurement of age at death of individuals in a population. The use of survivorship to identify genetic and pharmacological interventions that prolong life is time-consuming and requires a large number of homogeneous animals. Here, we report the development of an assay in Drosophila melanogaster using the expression of molecular biomarkers that accelerates the ability to evaluate potential lifespan-altering interventions. Coupling the expression of an age-dependent molecular biomarker to a lethal toxin reduces the time needed to perform lifespan studies by 80%. The assay recapitulates the effect of the three best known environmental life-span-extending interventions in the fly: ambient temperature, reproductive status, and calorie reduction. Single gene mutations known to extend lifespan in the fly such as Indy and rpd3 also extend lifespan in this assay. We used this assay as a screen to identify drugs that extend lifespan in flies. Lipoic acid and resveratrol were identified as being beneficial in our assay and shown to extend lifespan under normal laboratory conditions. We propose that this assay can be used to screen pharmacological as well as genetic interventions more rapidly for positive effects on lifespan. PMID:15328413

A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

NASA Astrophysics Data System (ADS)

Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

2011-05-01

Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate this potential, on-chip adhesion islands are fabricated to immobilize MCF-7 human breast cancer cells. Viability studies are performed to assess the functionalization efficiency.

Evaluation of a novel delayed-type hypersensitivity assay to Candida albicans in adult and neonatal rats.

PubMed

Thorn, Mitchell; Hudson, Adam W; Kreeger, John; Kawabe, Thomas T; Bowman, Christopher J; Collinge, Mark

2015-01-01

Delayed-type hypersensitivity (DTH) is a T-cell-mediated immune response that may be used for immunotoxicity testing in non-clinical species. However, in some cases DTH assays using T-dependent antigens may be confounded by the production of antibodies to the antigen. The authors have previously modified a DTH assay, initially validated in the mouse, for use in juvenile rats to assess the effect of immunosuppressive drugs on the developing rat immune system. The assay measures footpad swelling induced by subcutaneous footpad injection of Candida albicans (C. albicans) derived-chitosan in rats previously sensitized with C. albicans. Antibodies to chitosan are not produced in this model. However, considerable inter-animal variability inherent in the footpad swelling assay can make it difficult to precisely quantify the magnitude of the immune response and inhibition by immunosuppressants, particularly if complete suppression is not observed. This report describes the development of an ex vivo assay to assess DTH in rats using interferon (IFN)-γ production by splenocytes, obtained from rats sensitized with C. albicans, as the quantifiable measure of the DTH response. Adult and neonatal rats administered dexamethasone (DEX), a known immunosuppressant, exhibited immunosuppression as evidenced by a reduction in ex vivo IFNγ production from splenocytes challenged with C. albicans-derived chitosan. Current data indicate that the ex vivo based DTH assay is more sensitive than the conventional footpad swelling assay due to a lower background response and the ability to detect a response as early as post-natal day (PND) 12. The ex vivo based rat DTH assay offers a highly sensitive and quantitative alternative to the footpad swelling assay for the assessment of the immunotoxic potential of drugs. The increased sensitivity of the ex vivo DTH assay may be useful for identifying smaller changes in response to immunotoxic drugs, as well as detecting responses earlier in animal development.

Non-invasive assessment of adrenocortical activity as a measure of stress in giraffe (Giraffa camelopardalis).

PubMed

Bashaw, Meredith J; Sicks, Florian; Palme, Rupert; Schwarzenberger, Franz; Tordiffe, Adrian S W; Ganswindt, Andre

2016-10-18

Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected. Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3β,11β-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3-7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day. We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5β-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5β-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes.

Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy

NASA Astrophysics Data System (ADS)

Han, Haijie; Wang, Haibo; Chen, Yangjun; Li, Zuhong; Wang, Yin; Jin, Qiao; Ji, Jian

2015-12-01

A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy.A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06734k

Thermal modification of activated carbon surface chemistry improves its capacity as redox mediator for azo dye reduction.

PubMed

Pereira, L; Pereira, R; Pereira, M F R; van der Zee, F P; Cervantes, F J; Alves, M M

2010-11-15

The surface chemistry of a commercial AC (AC(0)) was selectively modified, without changing significantly its textural properties, by chemical oxidation with HNO(3) (AC(HNO3)) and O(2) (AC(O2)), and thermal treatments under H(2) (AC(H2)) or N(2) (AC(N2)) flow. The effect of modified AC on anaerobic chemical dye reduction was assayed with sulphide at different pH values 5, 7 and 9. Four dyes were tested: Acid Orange 7, Reactive Red 2, Mordant Yellow 10 and Direct Blue 71. Batch experiments with low amounts of AC (0.1 g L(-1)) demonstrated an increase of the first-order reduction rate constants, up to 9-fold, as compared with assays without AC. Optimum rates were obtained at pH 5 except for MY10, higher at pH 7. In general, rates increased with increasing the pH of point zero charge (pH(pzc)), following the trend AC(HNO3) < AC(O2) < AC(0) < AC(N2) < AC(H2). The highest reduction rate was obtained for MY10 with AC(H2) at pH 7, which corresponded to the double, as compared with non-modified AC. In a biological system using granular biomass, AC(H2) also duplicated and increase 4.5-fold the decolourisation rates of MY10 and RR2, respectively. In this last experiment, reaction rate was independent of AC concentration in the tested range 0.1-0.6 g L(-1). Copyright © 2010 Elsevier B.V. All rights reserved.

A screening dye-uptake assay to evaluate in vitro susceptibility of herpes simplex virus isolates to acyclovir.

PubMed

Danve, Christelle; Morfin, Florence; Thouvenot, Danielle; Aymard, Michèle

2002-09-01

The widespread use of acyclovir (ACV) could increase the prevalence of herpes simplex virus (HSV) ACV-resistant isolates, and a screening assay are thus important for routine surveillance of the ACV susceptibility of HSV. A screening dye-uptake assay was developed, based on the conventional dye-uptake assay [J. Biol. Stand. 14 (1986) 201]. The susceptibility of HSV was measured by testing two virus dilutions (10(-1) and 10(-2)) against two ACV concentrations (5 and 10 microM) on Vero cells and expressed as a reduced percentage of viral replication. The reproducibility was evaluated with HSV1 and HSV2 ACV-sensitive and ACV-resistant reference strains introduced as controls in successive series. The dye-uptake by Vero cells, the growth capacity of the HSV strains and the reduction of the viral replication in the presence of acyclovir varied by less than 14, 20 and 30%, respectively. This assay allowed the detection of a heterogenous population containing as few as 20% of ACV-resistant strain. The screening test was applied to 500 HSV isolates in a prospective study, and over 95% of the HSV isolates tested were characterised using a single test. This test appeared to be half the cost and much easier to carry out than the conventional dye-uptake assay, and consequently is well suited for large scale surveillance.

Anti-angiogenic activity and phytochemical screening of fruit fractions from Vitex agnus castus.

PubMed

Certo, Giovanna; Costa, Rosaria; D'Angelo, Valeria; Russo, Marina; Albergamo, Ambrogina; Dugo, Giacomo; Germanò, Maria Paola

2017-12-01

Although the antitumour activity of Vitex agnus castus fruits has been already addressed, no work has yet assessed their anti-angiogenic potential. To this purpose, several extractive fractions of such fruits were tested on zebrafish embrios by EAP assay, so that only the bioactive fractions could be subsequently tested on the chick chorioallantoic membrane by CAM assay. Bioactive fractions were also phytochemically screened to identify those bioactive compounds responsible for anti-angiogenic activity. A marked inhibition of vessel formation was detected only in zebrafish embryos treated with chloroform or ethyl acetate fractions. Considering CAM assay, chloroform fraction induced a strong reduction of microvasculature and haemoglobin content; while lower anti-angiogenic effects of the ethyl acetate fraction were determined. Phytochemical analyses confirmed the presence of several bioactive anti-angiogenic compounds. Overall, obtained preliminary results highlighted a potential anti-angiogenic activity of V. agnus castus fruits.

Development of a new class of chemical and biological ultrasensors: Ribonuclease contamination and control

NASA Technical Reports Server (NTRS)

1984-01-01

In order to define ribonuclease contamination, an assay for ribonuclease having picogram level sensitivity was established. In this assay, polycytidylic acid is digested by ribonuclease leading to smaller fragments of poly C that remain soluble after treatment of the sample with perchloric acid and lanthanum acetate. An absorbance measurement at 260 nm of the supernatant from the centrifuged sample measures the ribonuclease. A standard curve is shown. Using this assay procedure, ribonuclease contamination was found to be significant in routine laboratory proteins, in particular, bovine serum albumin, lysozyme, catalase, and cytochrome C. This was confirmed by demonstrating a considerable reduction in this activity in the presence of phosphate buffer since phosphate inhibits ribonuclease. Ribonuclease contamination was not significantly encountered in routine laboratory glassware, plasticware, column surfaces, chromatographic particles, and buffer reagents, including airborne contamination. Some contamination could be introduced by fingerprints, however.

Surface reactivity measurements as required for grouping and read-across: An advanced FRAS protocol

NASA Astrophysics Data System (ADS)

Gandon, Arnaud; Werle, Kai; Neubauer, Nicole; Wohlleben, Wendel

2017-06-01

Oxidative stress is a widely accepted paradigm associated with different adverse outcomes of particulate matter, including nanomaterials. It has frequently been identified in in vitro and in vivo studies and different assays have been developed for this purpose. Here we describe a newly developed multi-dose protocol of the FRAS assay (Ferric Reduction Ability of Serum). The purpose of this SOP is the measurement of the surface reactivity of nanomaterials under physiological conditions. Antioxidative components as present in human blood serum (HBS) serve as reporter molecules. The assay separates the oxidative damage from the read-out of the reporter molecules. The results show significantly enhanced repeatability with better sensitivity towards low reactivity, enabling application of FRAS both to a rough grouping by reactive vs. passive nanomaterials and further to substantiation of read-across by enhanced resolution of the similarity between different nanoforms of the same substance.

Nanoengineered capsules for selective SERS analysis of biological samples

NASA Astrophysics Data System (ADS)

You, Yil-Hwan; Schechinger, Monika; Locke, Andrea; Coté, Gerard; McShane, Mike

2018-02-01

Metal nanoparticles conjugated with DNA oligomers have been intensively studied for a variety of applications, including optical diagnostics. Assays based on aggregation of DNA-coated particles in proportion to the concentration of target analyte have not been widely adopted for clinical analysis, however, largely due to the nonspecific responses observed in complex biofluids. While sample pre-preparation such as dialysis is helpful to enable selective sensing, here we sought to prove that assay encapsulation in hollow microcapsules could remove this requirement and thereby facilitate more rapid analysis on complex samples. Gold nanoparticle-based assays were incorporated into capsules comprising polyelectrolyte multilayer (PEMs), and the response to small molecule targets and larger proteins were compared. Gold nanoparticles were able to selectively sense small Raman dyes (Rhodamine 6G) in the presence of large protein molecules (BSA) when encapsulated. A ratiometric based microRNA-17 sensing assay exhibited drastic reduction in response after encapsulation, with statistically-significant relative Raman intensity changes only at a microRNA-17 concentration of 10 nM compared to a range of 0-500 nM for the corresponding solution-phase response.

High-Throughput Biosensor Discriminates Between Different Algal H 2-Photoproducing Strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wecker, Matt S. A.; Maria L. Ghirardi

2014-02-27

A number of species of microalgae and cyanobacteria photosynthetically produce H 2 gas by coupling water oxidation with the reduction of protons to molecular hydrogen, generating renewable energy from sunlight and water. Photosynthetic H 2 production, however, is transitory, and there is considerable interest in increasing and extending it for commercial applications. Here we report a Petri-plate version of our previous, microplate-based assay that detects photosynthetic H 2 production by algae. The assay consists of an agar overlay of H 2-sensing Rhodobacter capsulatus bacteria carrying a green fluorescent protein that responds to H 2 produced by single algal colonies inmore » the bottom agar layer. The assay distinguishes between algal strains that photoproduce H 2 at different levels under high light intensities, and it does so in a simple, inexpensive, and high-throughput manner. The assay will be useful for screening both natural populations and mutant libraries for strains having increased H 2 production, and useful for identifying various genetic factors that physiologically or genetically alter algal hydrogen production.« less

Precision-cut tissue chips as an in vitro toxicology system

PubMed Central

Catania, J. M.; Pershing, A. M.; Gandolfi, A. J.

2007-01-01

Precision-cut tissue slices mimic specific organ toxicity because normal cellular heterogeneity and organ architecture are retained. To optimize the use of the smaller tissues of the mouse and to establish easy assays for tissue viability, a tissue chip based system was used to generate large numbers of samples from a single organ. Iodoacetamide (IAM), was used as a model toxicant, and assays for intracellular potassium (normalized to DNA content) were used to establish viability and toxicant susceptibility. Thereafter, assays that were more rapid and specific were pursued. Lysates from tissues incubated in 6-carboxyfluorescein fluoresced proportionately to concentrations of IAM, indicating disruption of cellular membranes. Similarly, FURA-2, a probe applied to lysates to measure calcium levels, fluoresced proportionately to IAM dosage. Monobromobimane, a fluorescent sulfhydryl probe, displayed a decrease in fluorescent intensity at higher IAM challenge; a finding confirmed with an absorbance assay with Ellman’s reagent. Importantly, the number of samples per organ/mouse was increased at least 3-fold and a significant time reduction per analysis was realized. PMID:17376647

Tangeretin from Citrus reticulate Inhibits Respiratory Syncytial Virus Replication and Associated Inflammation in Vivo.

PubMed

Xu, Jiao-Jiao; Liu, Zhong; Tang, Wei; Wang, Guo-Cai; Chung, Hau Yin; Liu, Qiu-Ying; Zhuang, Ling; Li, Man-Mei; Li, Yao-Lan

2015-11-04

Human respiratory syncytial virus (RSV) is a common pathogen that causes pneumonia and bronchiolitis in infants and young children. Our previous study showed that tangeretin from Citrus reticulate possessed potent in vitro anti-RSV effects comparable to that of ribavirin. Therefore, in this study, we investigated the in vivo anti-RSV activity of tangeretin in 3-week-old male BALB/c mice. A plaque reduction assay and fluorescence quantitative polymerase chain reaction (FQ-PCR) showed that tangeretin inhibited RSV replication in the lung of mice. Moreover, a luminex assay indicated tangeretin relieved RSV-induced lung inflammation by attenuating interleukin (IL)-1β secretion. Possible anti-inflammatory mechanisms of tangeretin were preliminarily explored using a RSV-infected macrophage model. A FQ-PCR, enzyme-linked immunosorbent assay (ELISA), and luciferase assay revealed that tangeretin inhibited RSV-induced inflammation by suppressing nuclear factor-κB (NF-κB) activation. This study demonstrates that tangeretin inhibited RSV replication and RSV-induced lung inflammation in vivo and may be useful in preventing and treating RSV infections and inflammation.

Evaluation of in vitro antioxidant properties of methanol and aqueous extracts of Parkinsonia aculeata L. leaves.

PubMed

Sharma, Sonia; Vig, Adarsh Pal

2013-01-01

In the present study, methanol and aqueous extracts of Parkinsonia aculeata L. leaves were prepared and analyzed for phytochemical analysis and antioxidant potential in different in vitro assays. Antioxidant activity was studied using DPPH, CUPRAC, reducing power assay, deoxyribose degradation (site and nonsite specific), ferric reducing antioxidant potential (FRAP), ferric thiocyanate (FTC), thiobarbituric acid (TBA), and molybdate ion reduction, respectively. The total phenolic contents of the methanol and aqueous leaf extract were 39 mg GAE/g and 38 mg GAE/g, whereas flavonoid contents of these extracts were found to be 0.013 mg RE/g and 0.006 mg RE/g, respectively. From the two extracts, the methanol extract shows maximum inhibition (%) of 57.82%, 71.23%, 48.26%, 69.85%, and 52.78% in DPPH, nonsite- and site-specific, FTC, and TBA assays and absorbance of 0.669 and 0.241 in reducing power and CUPRAC assays at the highest concentration tested. UPLC analysis was done to determine the presence of various types of polyphenols present in plant extracts.

Evaluation of In Vitro Antioxidant Properties of Methanol and Aqueous Extracts of Parkinsonia aculeata L. Leaves

PubMed Central

Vig, Adarsh Pal

2013-01-01

In the present study, methanol and aqueous extracts of Parkinsonia aculeata L. leaves were prepared and analyzed for phytochemical analysis and antioxidant potential in different in vitro assays. Antioxidant activity was studied using DPPH, CUPRAC, reducing power assay, deoxyribose degradation (site and nonsite specific), ferric reducing antioxidant potential (FRAP), ferric thiocyanate (FTC), thiobarbituric acid (TBA), and molybdate ion reduction, respectively. The total phenolic contents of the methanol and aqueous leaf extract were 39 mg GAE/g and 38 mg GAE/g, whereas flavonoid contents of these extracts were found to be 0.013 mg RE/g and 0.006 mg RE/g, respectively. From the two extracts, the methanol extract shows maximum inhibition (%) of 57.82%, 71.23%, 48.26%, 69.85%, and 52.78% in DPPH, nonsite- and site-specific, FTC, and TBA assays and absorbance of 0.669 and 0.241 in reducing power and CUPRAC assays at the highest concentration tested. UPLC analysis was done to determine the presence of various types of polyphenols present in plant extracts. PMID:24348173

Decreased number of steroid receptors of circulating lymphocytes in Crohn's disease and ulcerative colitis.

PubMed

Krasznai, A; Krajcsi, P; Arányi, P; Mészáros, K; Horváth, I

1986-01-01

The number of steroid receptors of circulating lymphocytes was determined in 13 patients with inflammatory bowel disease and in controls. Marked reduction of the number of receptors was observed both in Crohn's disease and in ulcerative colitis; no receptors were detected by radioactive hormone binding assay in 4 cases.

Co-Contamination of DON and NIV in Domestic Flour in Japan: Survey, Intake, Reduction and Rapid Assay

USDA-ARS?s Scientific Manuscript database

Nivalenol (NIV) and Deoxynivalenol (DON) are trichothecene mycotoxins produced by Fusarium spp. that contaminate mainly cereal crops, such as wheat, barley, and maize. These mycotoxins are serious health hazards to humans and domestic animals. In Japan, there have been many reports of DON and NIV ...

ASCORBIC ACID REDUCTION OF ACTIVE CHLORINE PRIOR TO DETERMINING AMES MUTAGENICITY OF CHLORINATED NATURAL ORGANIC MATTER (NOM)

EPA Science Inventory

Many potable water disinfection byproducts (DBPs) that result from the reaction of natural organic matter (NOM) with oxidizing chlorine are known or suspected to be carcinogenic and mutagenic. The Ames assay is routinely used to assess an overall level of mutagenicity for all com...

Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

EPA Science Inventory

Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

PubMed Central

Hyakutake, Manami; Tomizawa, Satoshi; Mizuno, Kouhei; Abe, Hideki

2014-01-01

Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRCYB-4) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRCYB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRCYB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRCBm) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature. PMID:24334666

In vitro and in vivo antibiofilm potential of 2,4-Di-tert-butylphenol from seaweed surface associated bacterium Bacillus subtilis against group A streptococcus.

PubMed

Viszwapriya, Dharmaprakash; Prithika, Udayakumar; Deebika, Sundaresan; Balamurugan, Krishnaswamy; Pandian, Shunmugiah Karutha

2016-10-01

Biofilm formation of Group A Streptococcus (GAS) is recognized as an important virulent determinant. The present study reports the antibiofilm potential of seaweed (Gracilaria gracilis) surface associated Bacillus subtilis against GAS. Purification revealed 2,4-Di-tert-butyl-phenol (DTBP) as the active principle. DTBP exhibited a dose dependent antibiofilm activity against GAS (SF370 & six different clinical M serotypes). Microscopic analysis revealed changes in cell surface architecture and reduced thickness upon DTBP treatment. Results of extracellular polymeric substance quantification, microbial adhesion to hydrocarbon assay and fourier transform infrared spectroscopic analysis suggested that DTBP probably interferes with the initial adhesion stage of biofilm formation cascade. Reduction in hyaluronic acid synthesis goes in unison with blood survival assay wherein, increased susceptibility to phagocytosis was observed. In vivo studies using Caenorhabditis elegans manifested the reduction in adherence and virulence, which prompts further investigation of the potential of DTBP for the treatment of GAS infections. Copyright © 2016 Elsevier GmbH. All rights reserved.

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bavari, Sina; Kaplan, Gerardo

2018-04-01

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials. Published by Elsevier B.V.

Optimisation of entrapped activated carbon conditions to remove coloured compounds from winery wastewaters.

PubMed

Devesa-Rey, R; Bustos, G; Cruz, J M; Moldes, A B

2011-06-01

The objective of this work was to study the entrapped conditions of activated carbon in calcium-alginate beads for the clarification of winery wastewaters. An incomplete 3(3) factorial design was carried out to study the efficiency of activated carbon (0.5-2%); sodium alginate (1-5%); and calcium chloride (0.050-0.900 M), on the following dependent variables: colour reduction at 280, 465, 530 and 665 nm. The activated carbon and calcium chloride were the most influential variables in the colour reduction. Nearly 100% colour reductions were found for the wavelengths assayed when employing 2% of activated carbon, 5% of sodium alginate and intermediate concentrations of calcium chloride (0.475 M). Instead, other conditions like, 2% of activated carbon, 4% of sodium alginate and 0.580 M of calcium chloride can also give absorbance reductions close to 100%. Copyright © 2011 Elsevier Ltd. All rights reserved.

Photocatalytic self-cleaning transparent 2Bi2O3-B2O3 glass ceramics

NASA Astrophysics Data System (ADS)

Sharma, Sumeet Kumar; Singh, V. P.; Chauhan, Vishal S.; Kushwaha, H. S.; Vaish, Rahul

2017-09-01

Photocatalytic response of as-quenched and heat-treated 2Bi2O3-B2O3 glasses was studied. X ray diffraction reveals that the controlled heat treatment of glasses at 380 °C for 1 h, 2 h, and 3 h shows the formation of Bi4B2O9 crystals embedded in 2Bi2O3-B2O3 the host glass matrix. Scanning electron microscopic images reveal the presence of nanocrystallization in as-quenched glass. Significant photocatalytic activities were observed in as-quenched transparent glass. Photocatalytic activities were studied using the degradation of Resazurin as well as pharmaceutical 17 β-Estradiol under UV irradiation. Measurement of contact angle shows enhanced hydrophilicity with the increase in crystallization of the samples. Further, for as quenched 2Bi2O3-B2O3 glass ceramic, under UV irradiation, the water contact angle decreased from 92.7° to 39.5° and the sample surface transformed from hydrophobic to hydrophilic. Effective photocatalytic performance along with photoinduced hydrophilicity promotes 2Bi2O3-B2O3 glass ceramics in self-cleaning applications.

Synthesis and In Vitro Evaluation of Thiolated Carrageenan.

PubMed

Suchaoin, Wongsakorn; Bonengel, Sonja; Hussain, Shah; Huck, Christian W; Ma, Benjamin N; Bernkop-Schnürch, Andreas

2015-08-01

The aim of this study was to generate and characterize a thiolated carrageenan. Thiolated carrageenan (carrageenan-SH) was synthesized from kappa (κ)- and iota (ι)-carrageenan by bromine replacement of the hydroxyl moieties followed by substitution to thiol groups using thiourea. Thiolated κ- and ι-carrageenan exhibited 176.57 ± 20.11 and 109.51 ± 18.26 μmol thiol groups per gram polymer, respectively. The resazurin test in Caco-2 cells revealed no toxic effect of both thiolated carrageenans at a concentration below 0.1% (w/v). Regarding efflux pump inhibitory effect, cellular accumulation of multidrug-resistance protein 2 substrate, sulforhodamine 101, was 1.38- and 1.35-fold increased in cells treated with thiolated κ- and ι-carrageenan, respectively. Modification of κ- and ι-carrageenan led to 3.9- and 2.0-fold increase in dynamic viscosity of mucus-thiolated carrageenan mixture within 4 h. Furthermore, residence time of κ- and ι-carrageenan-SH on porcine intestinal mucosa was 6.4- and 1.8-fold prolonged, respectively, as demonstrated by rotating cylinder method, indicating improved mucoadhesive properties. Hence, thiolation of carrageenans led to novel pharmaceutical excipients for various applications. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Interactions of protamine with the marine bacterium, Pseudoalteromonas sp. NCIMB 2021.

PubMed

Pustam, A; Smith, C; Deering, C; Grosicki, K M T; Leng, T Y; Lin, S; Yang, J; Pink, D; Gill, T; Graham, L; Derksen, D; Bishop, C; Demont, M E; Wyeth, R C; Smith-Palmer, T

2014-03-01

Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021) was grown in synthetic seawater (SSW) containing pyruvate, in the presence (SSW(++) ) and absence (SSW(-) ) of divalent cations. Cultures contained single cells. Addition of the cationic antibacterial peptide (CAP), protamine, did not inhibit, but rather increased, the growth of NCIMB 2021 in SSW(++) and caused the bacteria to grow in chains. Bacterial growth was assessed using turbidity, cell counts and the sodium salt of resazurin. In SSW(-) , NCIMB 2021 was no longer resistant to protamine. The minimum inhibitory concentration (MIC) was 5 mg ml(-1) . Protamine is a cationic antimicrobial peptide (CAP), which is active against a variety of bacteria. This is the first in-depth study of the interaction of protamine with a marine bacterium, Pseudoalteromonas sp. NCIMB 2021. Our results show that protamine is only active in seawater in the absence of divalent cations. In the presence of the divalent cations, Mg(2+) and Ca(2+) , protamine enhances the growth of Pseudoalteromonas sp. NCIMB 2021 and produces chains rather than individual cells. These are important considerations when deciding on applications for protamine and in terms of understanding its mechanism of action. © 2013 The Society for Applied Microbiology.

Prospective Clinical Utility Study of the Use of the 21-Gene Assay in Adjuvant Clinical Decision Making in Women With Estrogen Receptor-Positive Early Invasive Breast Cancer: Results From the SWITCH Study

PubMed Central

Pivot, Xavier B.; Jacot, William; Naman, Hervé L.; Spaeth, Dominique; Misset, Jean-Louis; Largillier, Rémy; Sautiere, Jean-Loup; de Roquancourt, Anne; Pomel, Christophe; Rouanet, Philippe; Rouzier, Roman; Penault-Llorca, Frederique M.

2015-01-01

Background. The 21-gene Oncotype DX Recurrence Score assay is a validated assay to help decide the appropriate treatment for estrogen receptor-positive (ER+), early-stage breast cancer (EBC) in the adjuvant setting. The choice of adjuvant treatments might vary considerably in different countries according to various treatment guidelines. This prospective multicenter study is the first to assess the impact of the Oncotype DX assay in the French clinical setting. Methods. A total of 100 patients with ER+, human epidermal growth factor receptor 2-negative EBC, and node-negative (pN0) disease or micrometastases in up to 3 lymph nodes (pN1mi) were enrolled. Treatment recommendations, physicians’ confidence before and after knowing the Recurrence Score value, and physicians’ perception of the assay were recorded. Results. Of the 100 patients, 95 were evaluable (83 pN0, 12 pN1mi). Treatment recommendations changed in 37% of patients, predominantly from chemoendocrine to endocrine treatment alone. The proportion of patients recommended chemotherapy decreased from 52% pretest to 25% post-test. Of patients originally recommended chemotherapy, 61% were recommended endocrine treatment alone after receiving the Recurrence Score result. For both pN0 and pN1mi patients, post-test recommendations appeared to follow the Recurrence Score result for low and high values. Physicians’ confidence improved significantly. Conclusion. These are the first prospective data on the impact of the Oncotype DX assay on adjuvant treatment decisions in France. Using the assay was associated with a significant change in treatment decisions and an overall reduction in chemotherapy use. These data are consistent with those presented from European and non-European studies. Implications for Practice: This study shows that in estrogen receptor-positive, human epidermal growth factor receptor 2-negative early breast cancer (either node-negative or with micrometastases in up to 3 lymph nodes), Oncotype DX testing is associated with a treatment recommendation change in more than a third of patients (primarily from chemoendocrine treatment to endocrine treatment alone but also in the opposite direction) and an overall reduction in chemotherapy use. These results are consistent with those from other decision impact studies worldwide and further emphasize the role of Oncotype DX testing in management of early breast cancer, as reflected in international treatment guidelines. PMID:26112003

Mitoxantrone is More Toxic than Doxorubicin in SH-SY5Y Human Cells: A 'Chemobrain' In Vitro Study.

PubMed

Almeida, Daniela; Pinho, Rita; Correia, Verónica; Soares, Jorge; Bastos, Maria de Lourdes; Carvalho, Félix; Capela, João Paulo; Costa, Vera Marisa

2018-05-05

The potential neurotoxic effects of anticancer drugs, like doxorubicin (DOX) and mitoxantrone (MTX; also used in multiple sclerosis), are presently important reasons for concern, following epidemiological data indicating that cancer survivors submitted to chemotherapy may suffer cognitive deficits. We evaluated the in vitro neurotoxicity of two commonly used chemotherapeutic drugs, DOX and MTX, and study their underlying mechanisms in the SH-SY5Y human neuronal cell model. Undifferentiated human SH-SY5Y cells were exposed to DOX or MTX (0.13, 0.2 and 0.5 μM) for 48 h and two cytotoxicity assays were performed, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and the neutral red (NR) incorporation assays. Phase contrast microphotographs, Hoechst, and acridine orange/ethidium bromide stains were performed. Mitochondrial membrane potential was also assessed. Moreover, putative protective drugs, namely the antioxidants N -acetyl-l-cysteine (NAC; 1 mM) and 100 μM tiron, the inhibitor of caspase-3/7, Ac-DEVD-CHO (100 μM), and a protein synthesis inhibitor, cycloheximide (CHX; 10 nM), were tested to prevent DOX- or MTX-induced toxicity. The MTT reduction assay was also done in differentiated SH-SY5Y cells following exposure to 0.2 μM DOX or MTX. MTX was more toxic than DOX in both cytotoxicity assays and according to the morphological analyses. MTX also evoked a higher number of apoptotic nuclei than DOX. Both drugs, at the 0.13 μM concentration, caused mitochondrial membrane potential depolarization after a 48-h exposure. Regarding the putative neuroprotectors, 1 mM NAC was not able to prevent the cytotoxicity caused by either drug. Notwithstanding, 100 μM tiron was capable of partially reverting MTX-induced cytotoxicity in the NR uptake assay. One hundred μM Ac-DEVD-CHO and 10 nM cycloheximide (CHX) also partially prevented the toxicity induced by DOX in the NR uptake assay. MTX was more toxic than DOX in differentiated SH-SY5Y cells, while MTX had similar toxicity in differentiated and undifferentiated SH-SY5Y cells. In fact, MTX was the most neurotoxic drug tested and the mechanisms involved seem dissimilar among drugs. Thus, its toxicity mechanisms need to be further investigated as to determine the putative neurotoxicity for multiple sclerosis and cancer patients.

Systematic review of the clinical and economic value of gene expression profiles for invasive early breast cancer available in Europe.

PubMed

Blok, E J; Bastiaannet, E; van den Hout, W B; Liefers, G J; Smit, V T H B M; Kroep, J R; van de Velde, C J H

2018-01-01

Gene expression profiles with prognostic capacities have shown good performance in multiple clinical trials. However, with multiple assays available and numerous types of validation studies performed, the added value for daily clinical practice is still unclear. In Europe, the MammaPrint, OncotypeDX, PAM50/Prosigna and Endopredict assays are commercially available. In this systematic review, we aim to assess these assays on four important criteria: Assay development and methodology, clinical validation, clinical utility and economic value. We performed a literature search covering PubMed, Embase, Web of Science and Cochrane, for studies related to one or more of the four selected assays. We identified 147 papers for inclusion in this review. MammaPrint and OncotypeDX both have evidence available, including level IA clinical trial results for both assays. Both assays provide prognostic information. Predictive value has only been shown for OncotypeDX. In the clinical utility studies, a higher reduction in chemotherapy was achieved by OncotypeDX, although the number of available studies differ considerably between tests. On average, economic evaluations estimate that genomic testing results in a moderate increase in total costs, but that these costs are acceptable in relation to the expected improved patient outcome. PAM50/prosigna and EndoPredict showed comparable prognostic capacities, but with less economical and clinical utility studies. Furthermore, for these assays no level IA trial data are available yet. In summary, all assays have shown excellent prognostic capacities. The differences in the quantity and quality of evidence are discussed. Future studies shall focus on the selection of appropriate subgroups for testing and long-term outcome of validation trials, in order to determine the place of these assays in daily clinical practice. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Initial experience with GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.

PubMed

Patil, Naveen; Saba, Hamida; Marco, Asween; Samant, Rohan; Mukasa, Leonard

2014-01-01

Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay. To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance. Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program.Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas.Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas.Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation. Between June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Löwenstein-Jensen (LJ) slants. Surveillance data for January 2009-June 2013 was analysed to characterise sputum smear-negative but culture-positive cases. Seventy-one TB cases were reported from June 2012- June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports. GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one hand, and quickly ruling out TB whenever suspected.

Effect of the fungus Pochonia chlamydosporia on Echinostoma paraensei (Trematoda: Echinostomatidae).

PubMed

Lelis, Rosane Teixeira; Braga, Fabio Ribeiro; de Carvalho, Lorendane Millena; de Paula, Alessandra Teixeira; Araujo, Juliana Milani; Fausto, Mariana Costa; Junior, Arnaldo Maldonado; Rodrigues, João Victor Facchini; de Freitas Soares, Filippe Elias; Garcia, Juberlan Silva; de Araújo, Jackson Victor

2014-11-01

Echinostoma paraensei is a trematode of the genus Echinostoma that causes echinostomiasis in humans. The objectives of this study were to: evaluate the ovicidal activity of the nematophagous fungus Pochonia chlamydosporia (VC1 and VC4) on a solid medium 2% water-agar (2% WA) against E. paraensei eggs (assay A); evaluate ovicidal effect (destruction of eggs) of the isolate VC4 in supplemented culture media (assay B); and evaluate the ovicidal ability of the crude extract (VC4) on E. paraensei eggs (assay C). Eggs of E. paraensei (assay A) were placed in Petri dishes containing 2% WA with an isolate of the fungus P. chlamydosporia (VC1 and VC4) grown for 10 days, and without fungus as a control and evaluated regarding their destruction. In assay B, eggs of E. paraensei were placed in Petri dishes with different supplemented culture media and with VC4 isolate and the destruction of eggs was examined at the end of 25 days of interaction. In assay C, effects of the crude extract of P. chlamydosporia (VC4) on eggs were evaluated at the end of 7 days. In assay A, there was no difference (p>0.05) in ovicidal activity among the tested isolates (VC1 and VC4); however, the highest percentage for ovicidal activity (type 3 effect) was demonstrated by the isolate VC4. In assay B, the culture medium starch-agar showed the best results for the destruction of the eggs, with a percentage of 46.6% at the end of the assay. In assay C, the crude extract of VC4 was effective in the destruction of E. paraensei eggs, with a percentage reduction of 53%. The results of this study demonstrate that a rich culture medium with a greater availability of carbon and nitrogen may interfere directly in the predatory characteristics of ovicidal fungi. Copyright © 2014 Elsevier B.V. All rights reserved.

Availability of highly sensitive troponin assays and acute coronary syndrome care: insights from the SNAPSHOT registry.

PubMed

Cullen, Louise; French, John K; Briffa, Tom G; Redfern, Julie; Hammett, Christopher J K; Brieger, David B; Parsonage, William A; Lefkovits, Jeffrey; Ellis, Chris; Astley, Carolyn; Howell, Tegwen Eleanor; Elliott, John M; Chew, Derek P B

2015-01-19

To examine differences in care and inhospital course of patients with possible acute coronary syndrome (ACS) in Australia and New Zealand based on whether a highly sensitive (hs) troponin assay was used at the hospital to which they presented. A snapshot study of consecutive patients presenting to hospitals in Australia and New Zealand from 14 to 27 May 2012 with possible ACS. Rates of major adverse cardiac events (inhospital death, new or recurrent myocardial infarction, stroke, cardiac arrest or worsening heart failure); association between assay type and outcome (via propensity score matching and a generalised estimating equation [GEE]; averages of the predicted outcomes among patients who were treated with and without the availability of an hs assay (via inverse probability-weighting [IPW] with regression-adjusted estimators). 4371 patients with possible ACS were admitted to 283 hospitals. Over half of the hospitals (156 [55%]) reported using the hs assay and most patients (2624 [60%]) had hs tests (P = 0.004). Use of the hs assay was independent of hospital coronary revascularisation capability. Patients tested with the hs assay had more non-invasive investigations (exercise tests, stress echocardiography, stress nuclear scans, and computed tomography coronary angiography) than those tested with the sensitive assay. However, there were no differences between the groups in rates of angiography or revascularisation. All adjusted analyses showed a consistently lower rate of inhospital events, including recurrent heart failure in patients for whom the hs assay was used (GEE odds ratio, 0.75; 95% CI, 0.60-0.94; P = 0.014); IPW analysis showed a 2.3% absolute reduction in these events with the use of the hs assay (P = 0.018). Use of hs troponin testing of patients hospitalised with possible ACS was associated with an increased rate of non-invasive cardiac investigations and fewer inhospital adverse events.

A rapid method for infectivity titration of Andes hantavirus using flow cytometry.

PubMed

Barriga, Gonzalo P; Martínez-Valdebenito, Constanza; Galeno, Héctor; Ferrés, Marcela; Lozach, Pierre-Yves; Tischler, Nicole D

2013-11-01

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Multigene assays and molecular markers in breast cancer: systematic review of health economic analyses.

PubMed

Rouzier, Roman; Pronzato, Paolo; Chéreau, Elisabeth; Carlson, Josh; Hunt, Barnaby; Valentine, William J

2013-06-01

Breast cancer is the most common female cancer and is associated with a significant clinical and economic burden. Multigene assays and molecular markers represent an opportunity to direct chemotherapy only to patients likely to have significant benefit. This systematic review examines published health economic analyses to assess the support for adjuvant therapy decision making. Literature searches of PubMed, the Cochrane Library, and congress databases were carried out to identify economic evaluations of multigene assays and molecular markers published between 2002 and 2012. After screening and data extraction, study quality was assessed using the Quality of Health Economic Studies instrument. The review identified 29 publications that reported evaluations of two assays: Oncotype DX(®) and MammaPrint. Studies of both tests provided evidence that their routine use was cost saving or cost-effective versus conventional approaches. Benefits were driven by optimal allocation of adjuvant chemotherapy and reduction in chemotherapy utilization. Findings were sensitive to variation in the frequency of chemotherapy prescription, chemotherapy costs, and patients' risk profiles. Evidence suggests that multigene assays are likely cost saving or cost-effective relative to current approaches to adjuvant therapy. They should benefit decision making in early-stage breast cancer in a variety of settings worldwide.

A new assay system for guinea pig interferon biological activity.

PubMed

Yamamoto, Toshiko; Jeevan, Amminikutty; Ohishi, Kazue; Nojima, Yasuhiro; Umemori, Kiyoko; Yamamoto, Saburo; McMurray, David N

2002-07-01

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.

PubMed

Raudonis, Raimondas; Raudone, Lina; Jakstas, Valdas; Janulis, Valdimaras

2012-04-13

ABTS and FRAP post-column techniques evaluate the antioxidant characteristics of HPLC separated compounds with specific reagents. ABTS characterize their ability to scavenge free radicals by electron-donating antioxidants, resulting in the absorbance decrease of the chromophoric radical. FRAP - is based on the reduction of Fe(III)-tripyridyltriazine complex to Fe(II)-tripyridyltriazine at low pH by electron-donating antioxidants, resulting in an absorbance increase. Both post-column assays were evaluated and compared according to the following validation parameters: specificity, precision, limit of detection (LoD), limit of quantitation (LoQ) and linearity. ABTS and FRAP post-column assays were specific, repeatable and sensitive and thus can be used for the evaluation of antioxidant active compounds. Antioxidant active compounds were quantified according to TEAC for each assay and ABTS/FRAP ratio was derived. No previous records of antioxidative activity of leaves and fruits of strawberries (Fragaria viridis, Fragaria moschata) research have been found. The research results confirm the reliability of ABTS and FRAP post-column assays for screening of antioxidants in complex mixtures and the determination of radical scavenging and ferric reducing ability by their TEAC values. Copyright © 2012 Elsevier B.V. All rights reserved.

Natural Variation of Drug Susceptibility in Wild-Type Human Immunodeficiency Virus Type 1

PubMed Central

Parkin, N. T.; Hellmann, N. S.; Whitcomb, J. M.; Kiss, L.; Chappey, C.; Petropoulos, C. J.

2004-01-01

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or “biological cutoffs,” for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs. PMID:14742192

HCV-specific immune responses induced by CIGB-230 in combination with IFN-α plus ribavirin

PubMed Central

Amador-Cañizares, Yalena; Martínez-Donato, Gillian; Álvarez-Lajonchere, Liz; Vasallo, Claudia; Dausá, Mariacarla; Aguilar-Noriega, Daylen; Valenzuela, Carmen; Raíces, Ivette; Dubuisson, Jean; Wychowski, Czeslaw; Cinza-Estévez, Zurina; Castellanos, Marlén; Núñez, Magdalys; Armas, Anny; González, Yaimé; Revé, Ismariley; Guerra, Ivis; Pérez Aguiar, Ángel; Dueñas-Carrera, Santiago

2014-01-01

AIM: To analyze hepatitis C virus (HCV)-specific immune responses in chronically infected patients under triple therapy with interferon-α (IFN-α) plus ribavirin and CIGB-230. METHODS: CIGB-230 was administered in different schedules with respect to IFN-α plus ribavirin therapy. Paired serum and peripheral blood mononuclear cells (PBMC) samples from baseline and end of treatment were analyzed. The HCV-specific humoral response was tested by enzyme-linked immunosorbent assay, neutralizing antibodies were evaluated by cell culture HCV neutralization assays, PBMC proliferation was assayed by carboxyfluorescein succinimidyl ester staining and IFN-γ secretion was assessed by enzyme-linked immunospot. Data on virological and histological response and their association with immune variables are also provided. RESULTS: From week 12 to week 48, all groups of patients showed a significant reduction in mean leukocyte counts. Statistically significant reductions in antibody titers were frequent, but only individuals immunized with CIGB-230 as early add-on treatment sustained the core-IgG response, and the neutralizing antibody response was enhanced only in patients receiving CIGB-230. Cell-mediated immune responses also tended to decline, but significant reductions in IFN-γ secretion and total absence of core-specific lymphoproliferation were exclusive of the control group. Only CIGB-230-immunized individuals showed de novo induced lymphoproliferative responses against the structural antigens. Importantly, it was demonstrated that the quality of the CIGB-230-induced immune response depended on the number of doses and timing of administration in relation to the antiviral therapy. Specifically, the administration of 6 doses of CIGB-230 as late add-on to therapy increased the neutralizing antibody activity and the de novo core-specific IFN-γ secretion, both of which were associated with the sustained virological response. CONCLUSION: CIGB-230, combined with IFN-α-based therapy, modifies the immune response in chronic patients. The study provides evidence for the design of more effective therapeutic vaccine interventions against HCV. PMID:24415868

Use of viscoelastic haemostatic assay in emergency and elective surgery.

PubMed

Yeung, Maximus C F; Tong, Steven Y T; Tong, Paul Y W; Cheung, Billy H H; Ng, Joanne Y W; Leung, Gilberto K K

2015-02-01

To review the current evidence for the use of viscoelastic haemostatic assays in different surgical settings including trauma, cardiac surgery, liver transplantation, as well as the monitoring of antiplatelet agents and anticoagulants prior to surgery. PubMed database. Key words for the literature search were "thromboelastography" or "ROTEM" in combination with "trauma", "antiplatelet", "cardiac surgery", "liver transplantation" or "anticoagulants". Original and major review articles related to the use of viscoelastic haemostatic assays. Haemostatic function is a critical factor determining patient outcomes in emergency or elective surgery. The increasing use of antiplatelet agents and anticoagulants has potentially increased the risks of haemorrhages and the need for transfusion. Conventional coagulation tests have limitations in detecting haemostatic dysfunctions in subgroups of patients and are largely ineffective in diagnosing hyperfibrinolysis. The viscoelastic haemostatic assays are potentially useful point-of-care tools that provide information on clot formation, clot strength, and fibrinolysis, as well as to guide goal-directed transfusion and antifibrinolytic therapy. They may also be used to monitor antiplatelet and anticoagulant therapy. However, standardisation of techniques and reference ranges is required before these tests can be widely used in different clinical settings. Viscoelastic haemostatic assays, as compared with conventional coagulation tests, are better for detecting coagulopathy and are the only tests that can provide rapid diagnosis of hyperfibrinolysis. Goal-directed administration of blood products based on the results of viscoelastic haemostatic assays was associated with reduction in allogeneic blood product transfusions in trauma, cardiac surgery, and liver transplantation cases. However, there is currently no evidence to support the routine use of viscoelastic haemostatic assays for monitoring platelet function prior to surgery.

Applicability of tetrazolium salts for the measurement of respiratory activity and viability of groundwater bacteria

USGS Publications Warehouse

Hatzinger, P.B.; Palmer, P.; Smith, R.L.; Penarrieta, C.T.; Yoshinari, T.

2003-01-01

A study was undertaken to measure aerobic respiration by indigenous bacteria in a sand and gravel aquifer on western Cape Cod, MA using tetrazolium salts and by direct oxygen consumption using gas chromatography (GC). In groundwater and aquifer slurries, the rate of aerobic respiration calculated from the direct GC assay was more than 600 times greater than that using the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT). To explain this discrepancy, the toxicity of INT and two additional tetrazolium salts, sodium 3???-[1-(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), to bacterial isolates from the aquifer was investigated. Each of the three tetrazolium salts was observed to be toxic to some of the groundwater isolates at concentrations normally used in electron transport system (ETS) and viability assays. For example, incubation of cells with XTT (3 mM) caused the density of four of the five groundwater strains tested to decline by more than four orders of magnitude. A reasonable percentage (>57%) of cells killed by CTC and INT contained visible formazan crystals (the insoluble, reduced form of the salts) after 4 h of incubation. Thus, many of the cells reduced enough CTC or INT prior to dying to be considered viable by microscopic evaluation. However, one bacterium (Pseudomonas fluorescens) that remained viable and culturable in the presence of INT and CTC, did not incorporate formazan crystals into more than a few percent of cells, even after 24 h of incubation. This strain would be considered nonviable based on traditional tetrazolium salt reduction assays. The data show that tetrazolium salt assays are likely to dramatically underestimate total ETS activity in groundwater and, although they may provide a reasonable overall estimate of viable cell numbers in a community of groundwater bacteria, some specific strains may be falsely considered nonviable by this assay due to poor uptake or reduction of the salts. ?? 2003 Elsevier Science B.V. All rights reserved.

Benzimidazole-Based Antibacterial Agents Against F. tularensis

PubMed Central

Kumar, Kunal; Awasthi, Divya; Lee, Seung-Yub; Cummings, Jason E.; Knudson, Susan E.; Slayden, Richard A.; Ojima, Iwao

2013-01-01

Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23 of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater than 90 % growth inhibition at 1 µg/mL. Among those hits, 21 compounds showed MIC90 values in the range of 0.35–48.6 µg/mL after accurate MIC determination. In ex-vivo efficacy assays, four of these compounds exhibited 2–3 Log reduction in colony forming units (CFU) per mL at concentrations of 10 and 50 µg/mL. PMID:23623254

Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.

PubMed

Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H

2015-10-01

To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.

Quantum dot-linked immunosorbent assay (QLISA) using orientation-directed antibodies.

PubMed

Suzuki, Miho; Udaka, Hikari; Fukuda, Takeshi

2017-09-05

An approach similar to the enzyme-linked immunosorbent assay (ELISA), with the advantage of saving time and effort but exhibiting high performance, was developed using orientation-directed half-part antibodies immobilized on CdSe/ZnS quantum dots. ELISA is a widely accepted assay used to detect the presence of a target substance. However, it takes time to quantify the target with specificity and sensitivity owing to signal amplification. In this study, CdSe/ZnS quantum dots are introduced as bright and photobleaching-tolerant fluorescent materials. Since hydrophilic surface coating of quantum dots rendered biocompatibility and functional groups for chemical reactions, the quantum dots were modified with half-sized antibodies after partial reduction. The half-sized antibody could be bound to a quantum dot through a unique thiol site to properly display the recognition domain for the core process of ELISA, which is an antigen-antibody interaction. The reducing conditions were investigated to generate efficient conjugates of quantum dots and half-sized antibodies. This was applied to IL-6 detection, as the quantification of IL-6 is significant owing to its close relationships with various biomedical phenomena that cause different diseases. An ELISA-like assay with CdSe/ZnS quantum dot institution (QLISA; Quantum dot-linked immunosorbent assay) was developed to detect 0.05ng/mL IL-6, which makes it sufficiently sensitive as an immunosorbent assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Hypoosmotic swelling modifies glutamate-glutamine cycle in the cerebral cortex and in astrocyte cultures

PubMed Central

Hyzinski-García, María C.; Vincent, Melanie Y.; Haskew-Layton, Renée E.; Dohare, Preeti; Keller, Richard W.; Mongin, Alexander A.

2011-01-01

In our previous work, we found that perfusion of the rat cerebral cortex with hypoosmotic medium triggers massive release of the excitatory amino acid L-glutamate but decreases extracellular levels of L-glutamine (R.E. Haskew-Layton et al., PLoS ONE, 3: e3543). The release of glutamate was linked to activation of volume-regulated anion channels (VRAC), while mechanism(s) responsible for alterations in extracellular glutamine remained unclear. When mannitol was added to the hypoosmotic medium in order to reverse reductions in osmolarity, changes in microdialysate levels of glutamine were prevented, indicating an involvement of cellular swelling. Since the main source of brain glutamine is astrocytic synthesis and export, we explored the impact of hypoosmotic medium on glutamine synthesis and transport in rat primary astrocyte cultures. In astrocytes, a 40% reduction in medium osmolarity moderately stimulated the release of L-[3H]glutamine by ~2-fold and produced no changes in L-[3H]glutamine uptake. In comparison, hypoosmotic medium stimulated the release of glutamate (traced with D[3H]aspartate) by more than 20-fold. In whole-cell enzymatic assays, we discovered that hypoosmotic medium caused a 20% inhibition of astrocytic conversion of L[3H]glutamate into L-[3H]glutamine by glutamine synthetase. Using an HPLC assay we further found a 35% reduction in intracellular levels of endogenous glutamine. Overall, our findings suggest that cellular swelling (1) inhibits astrocytic glutamine synthetase activity, and (2) reduces substrate availability for this enzyme due to the activation of VRAC. These combined effects likely lead to reductions in astrocytic glutamine export in vivo and may partially explain occurrence of hyperexcitability and seizures in human hyponatremia. PMID:21517854

Extensin network formation in Vitis vinifera callus cells is an essential and causal event in rapid and H2O2-induced reduction in primary cell wall hydration

PubMed Central

2011-01-01

Background Extensin deposition is considered important for the correct assembly and biophysical properties of primary cell walls, with consequences to plant resistance to pathogens, tissue morphology, cell adhesion and extension growth. However, evidence for a direct and causal role for the extensin network formation in changes to cell wall properties has been lacking. Results Hydrogen peroxide treatment of grapevine (Vitis vinifera cv. Touriga) callus cell walls was seen to induce a marked reduction in their hydration and thickness. An analysis of matrix proteins demonstrated this occurs with the insolubilisation of an abundant protein, GvP1, which displays a primary structure and post-translational modifications typical of dicotyledon extensins. The hydration of callus cell walls free from saline-soluble proteins did not change in response to H2O2, but fully regained this capacity after addition of extensin-rich saline extracts. To assay the specific contribution of GvP1 cross-linking and other wall matrix proteins to the reduction in hydration, GvP1 levels in cell walls were manipulated in vitro by binding selected fractions of extracellular proteins and their effect on wall hydration during H2O2 incubation assayed. Conclusions This approach allowed us to conclude that a peroxidase-mediated formation of a covalently linked network of GvP1 is essential and causal in the reduction of grapevine callus wall hydration in response to H2O2. Importantly, this approach also indicated that extensin network effects on hydration was only partially irreversible and remained sensitive to changes in matrix charge. We discuss this mechanism and the importance of these changes to primary wall properties in the light of extensin distribution in dicotyledons. PMID:21672244

The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation*

PubMed Central

Rishavy, Mark A.; Hallgren, Kevin W.; Wilson, Lee A.; Usubalieva, Aisulu; Runge, Kurt W.; Berkner, Kathleen L.

2013-01-01

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously. PMID:23918929

Mental Retardation Genes in Drosophila: New Approaches to Understanding and Treating Developmental Brain Disorders

ERIC Educational Resources Information Center

Restifo, Linda L.

2005-01-01

"Drosophila melanogaster" is emerging as a valuable genetic model system for the study of mental retardation (MR). MR genes are remarkably similar between humans and fruit flies. Cognitive behavioral assays can detect reductions in learning and memory in flies with mutations in MR genes. Neuroanatomical methods, including some at single-neuron…

Inactivation and UV Disinfection of Murine Norovirus with TiO2 under Various Environmental Conditions▿

PubMed Central

Lee, JungEun; Zoh, KyungDuk; Ko, GwangPyo

2008-01-01

We studied inactivation and UV disinfection of murine norovirus (MNV) as a surrogate for human norovirus. We investigated the effects of different surface characteristics, temperatures, and NaCl concentrations on MNV survival using both a plaque assay and a real-time TaqMan reverse transcription (RT)-PCR assay. MNV survived more than 40 days on diaper material, on gauze, and in a stool suspension. Compared to inactivation at lower temperatures (−20 and 4°C), inactivation of MNV was greater at higher temperatures (18 and 30°C). On the surface of both gauze and diaper material, there was a <2-log10 reduction in the amount of infectious MNV in 40 days after incubation at both −20 and 4°C, compared to a >5-log10 reduction after incubation at 30°C in 24 days. MNV survived better in a stool suspension than on the surface of gauze or diaper material. A higher salt concentration increased the rate of inactivation of MNV. In 72 h, <0.3-, 1.5-, and 2.5-log10 reductions in the amount of infectious MNV occurred in distilled water and 0.5 and 1 M NaCl, respectively. We observed only minor reductions in the numbers of viral RNA copies as quantified by real-time TaqMan RT-PCR regardless of the temperature, the salt concentration, or the suspending medium. We also evaluated UV disinfection of infectious MNV with and without TiO2. The amount of MNV was significantly reduced by 254-nm UV with and without TiO2. When 25 mJ/cm2 UV was used, 3.3- and 3.6-log10 reductions in the amounts of infectious MNV occurred with and without TiO2, respectively. Our results demonstrate that MNV can persist in various environmental conditions and can be efficiently controlled by UV disinfection. PMID:18245239

Graphene Synthesis & Graphene/Polymer Nanocomposites

NASA Astrophysics Data System (ADS)

Liao, Ken-Hsuan

We successfully developed a novel, fast, hydrazine-free, high-yield method for producing single-layered graphene. Graphene sheets were formed from graphite oxide by reduction with de-ionized water at 130 ºC. Over 65% of the sheets are single graphene layers. A dehydration reaction of exfoliated graphene oxide was utilized to reduce oxygen and transform C-C bonds from sp3 to sp2. The reduction appears to occur in large uniform interconnected oxygen-free patches so that despite the presence of residual oxygen the sp2 carbon bonds formed on the sheets are sufficient to provide electronic properties comparable to reduced graphene sheets obtained using other methods. Cytotoxicity of aqueous graphene was investigated with Dr. Yu-Shen Lin by measuring mitochondrial activity in adherent human skin fibroblasts using two assays. The methyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) assay, a typical nanotoxicity assay, fails to predict the toxicity of graphene oxide and graphene toxicity because of the spontaneous reduction of MTT by graphene and graphene oxide, resulting in a false positive signal. An appropriate alternate assessment, using the water soluble tetrazolium salt (WST-8) assay, reveals that the compacted graphene sheets are more damaging to mammalian fibroblasts than the less densely packed graphene oxide. Clearly, the toxicity of graphene and graphene oxide depends on the exposure environment (i.e. whether or not aggregation occurs) and mode of interaction with cells (i.e. suspension versus adherent cell types). Ultralow percolation concentration of 0.15 wt% graphene, as determined by surface resistance and modulus, was observed from in situ polymerized thermally reduced graphene (TRG)/ poly-urethane-acrylate (PUA) nanocomposite. A homogeneous dispersion of TRG in PUA was revealed by TEM images. The aspect ratio of dispersed TRG, calculated from percolation concentration and modulus, was found to be equivalent to the reported aspect ratio of single-layered free standing TRG. This indicates TRG is mono-layer-dispersed in the matrix polymer. How graphene/polymer nanocomposite glass transition temperatures ( Tg) vary was investigated in this study. We measured Tg in PMMA. We used isotactic PMMA (i-PMMA) and syndiotactic-rich atactic PMMA (a-PMMA) to make TRG/PMMA nanocomposites using solvent blending and in situ polymerization in order to investigate the stereo-regularity and processing effects on the Tg. A T g increase was found in i-PMMA and in situ PMMA but not in a-PMMA. The results can be explained by the thin film confinement effect of polymer. We attribute the Tg increase to both a higher interaction density and a stronger hydrogen bonding at the interfaces. We have studied the elastic modulus of graphene oxide with various oxygen content. We used in situ AFM nano-indentation to measure the influence of oxygen on the elastic modulus of graphene oxide with various carbon/oxygen (C/O) ratios. The results show that chemical reduction (lower oxygen contents) decreases the elastic modulus of graphene oxide. We speculate that chemical reduction of oxygen atoms of epoxy groups on graphene oxide surface removes the bridging effect between carbon atoms, which leads to more flexible sheets. (Abstract shortened by UMI.).

Tracking Human Adenovirus Inactivation by Gamma Radiation under Different Environmental Conditions

PubMed Central

Pimenta, Andreia I.; Guerreiro, Duarte; Madureira, Joana; Margaça, Fernanda M. A.

2016-01-01

ABSTRACT Adenovirus is the most prevalent enteric virus in waters worldwide due to its environmental stability, which leads to public health concerns. Mitigation strategies are therefore required. The aim of this study was to assess the inactivation of human adenovirus type 5 (HAdV-5) by gamma radiation in aqueous environments. Various substrates with different organic loads, including domestic wastewater, were inoculated with HAdV-5 either individually or in a viral pool (with murine norovirus type 1 [MNV-1]) and were irradiated in a Cobalt-60 irradiator at several gamma radiation doses (0.9 to 10.8 kGy). The infectivity of viral particles, before and after irradiation, was tested by plaque assay using A549 cells. D10 values (dose required to inactivate 90% of a population or the dose of irradiation needed to produce a 1 log10 reduction in the population) were estimated for each substrate based on virus infectivity inactivation exponential kinetics. The capability of two detection methods, nested PCR and enzyme-linked immunosorbent assay (ELISA), to track inactivated viral particles was also assessed. After irradiation at 3.5 kGy, a reduction of the HAdV-5 titer of 4 log PFU/ml on substrates with lower organic loads was obtained, but in highly organic matrixes, the virus titer reduction was only 1 log PFU/ml. The D10 values of HAdV-5 in high organic substrates were significantly higher than in water suspensions. The obtained results point out some discrepancies between nested PCR, ELISA, and plaque assay on the assessments of HAdV-5 inactivation. These results suggest that the inactivation of HAdV-5 by gamma radiation, in aqueous environments, is significantly affected by substrate composition. This study highlights the virucidal potential of gamma radiation that may be used as a disinfection treatment for sustainable water supplies. IMPORTANCE Human adenovirus (HAdV) is the most prevalent of the enteric viruses in environmental waters worldwide. The purposes of this study are to provide new insights on the inactivation of enteric virus by gamma irradiation and to introduce new concepts and reinforce the benefits and utility of radiation technologies as disinfection processes. This may be an effective tool to guarantee the reduction of viral pathogens and to contribute to public health and sustainable water supplies. PMID:27316961

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

PubMed

Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

2014-08-18

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together with cryopreserved trout hepatocytes for screening estrogenic chemicals, resulting in a reduction of the time required to perform the assay and enabling greater access to the model system through the approach of cryopreservation.

Drosophila Courtship Conditioning As a Measure of Learning and Memory.

PubMed

Koemans, Tom S; Oppitz, Cornelia; Donders, Rogier A T; van Bokhoven, Hans; Schenck, Annette; Keleman, Krystyna; Kramer, Jamie M

2017-06-05

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male's copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to naïve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats.

PubMed

Oliveira, Bruna Vasconcelos; Barros Silva, Paulo Goberlânio; Nojosa, Jacqueline de Santiago; Brizeno, Luiz André Cavalcante; Ferreira, Jamile Magalhães; Sousa, Fabrício Bitú; Mota, Mário Rogério Lima; Alves, Ana Paula Negreiros Nunes

2016-01-01

to evaluate the influence of Tumor Necrosis Factor alpha (TNF-α) and apoptosis in rats with DM treated with chamomile extract or triamcinolone. Wistar male rats (210.0±4.2 g) were divided into five groups: negative control group (NCG) without diabetes; positive control group (PCG) with DM (alloxan, 45 mg/kg); and groups treated with chamomile extract (normoglycemic= NCG group and diabetic= DCG group) and with triamcinolone (TG). Traumatic ulcers were performed on all animals that received topical triamcinolone, chamomile extract or saline 12/12 hours for ten days. On days five and ten the animals were euthanized and the ulcers were analyzed by light microscopy, TUNEL assay, and immunohistochemically (TNF-α). The NCG (p=0.0062), PCG (p=0.0285), NCG (p=0.0041), and DCG (p<0.0001) groups were completely healed on the 10th day, however, there was no healing on the TG (p=0.5127) group. The TNF-α expression showed a significant reduction from the 5th to the 10th day in NCG (p=0.0266) and DCG (p=0.0062). In connective tissue, the TUNEL assay showed a significant reduction in the number of positive cells in NCG (p=0.0273) and CNG (p=0.0469) and in the epithelium only in CDG (p=0.0320). Chamomile extract can optimize the healing of traumatic oral ulcers in diabetic rats through the reduction of apoptosis in the epithelium and TNF-α expression.

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

PubMed

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Clinical utility of the ARCHITECT HCV Ag assay for early treatment monitoring in patients with chronic hepatitis C genotype 1 infection.

PubMed

Vermehren, Johannes; Susser, Simone; Berger, Annemarie; Perner, Dany; Peiffer, Kai-Henrik; Allwinn, Regina; Zeuzem, Stefan; Sarrazin, Christoph

2012-09-01

Virologic response-monitoring is essential for determining therapy duration in patients with chronic hepatitis C virus (HCV) infection. This is usually performed using highly sensitive HCV-RNA assays. However, HCV-RNA assays are time-consuming, expensive and require highly trained personnel. Quantitative determination of HCV core-antigen (HCVAg) levels may be used to supplement treatment monitoring. The clinical utility of the ARCHITECT HCV Ag assay (Abbott Diagnostics) for response-guided therapy was investigated. We analyzed serum from 160 patients with HCV genotype 1 infection who had been treated with peg-interferon alfa-2b/ribavirin. HCVAg levels were determined at baseline, weeks 1, 2, 4 and 12. HCVAg levels were compared to those obtained with HCV-RNA assays: VERSANT HCV Quantitative 3.0 (bDNA) and Qualitative (TMA, both Siemens Healthcare) assay and the Abbott RealTime HCV assay (ART; Abbott Diagnostics). Baseline HCVAg levels correlated well with HCV-RNA as assessed by bDNA (r=0.91; p<0.0001) and ART (r=0.92; p<0.0001), respectively. Patients with undetectable HCVAg levels at week 1 had a 90.9% probability (positive predictive value) to achieve a rapid virologic response (HCV-RNA undetectable at week 4) based on TMA and 86.4% based on ART, respectively. Patients with less than 1 log(10) reduction in HCVAg between baseline and week 12 had a 90% probability (negative predictive value) to achieve a nonresponse (<2 log(10) decline in HCV-RNA between baseline and week 12) based on bDNA and 100% based on ART, respectively. Determination of HCVAg may be useful for antiviral response-monitoring in patients with HCV genotype 1 infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Prospective Clinical Utility Study of the Use of the 21-Gene Assay in Adjuvant Clinical Decision Making in Women With Estrogen Receptor-Positive Early Invasive Breast Cancer: Results From the SWITCH Study.

PubMed

Gligorov, Joseph; Pivot, Xavier B; Jacot, William; Naman, Hervé L; Spaeth, Dominique; Misset, Jean-Louis; Largillier, Rémy; Sautiere, Jean-Loup; de Roquancourt, Anne; Pomel, Christophe; Rouanet, Philippe; Rouzier, Roman; Penault-Llorca, Frederique M

2015-08-01

The 21-gene Oncotype DX Recurrence Score assay is a validated assay to help decide the appropriate treatment for estrogen receptor-positive (ER+), early-stage breast cancer (EBC) in the adjuvant setting. The choice of adjuvant treatments might vary considerably in different countries according to various treatment guidelines. This prospective multicenter study is the first to assess the impact of the Oncotype DX assay in the French clinical setting. A total of 100 patients with ER+, human epidermal growth factor receptor 2-negative EBC, and node-negative (pN0) disease or micrometastases in up to 3 lymph nodes (pN1mi) were enrolled. Treatment recommendations, physicians' confidence before and after knowing the Recurrence Score value, and physicians' perception of the assay were recorded. Of the 100 patients, 95 were evaluable (83 pN0, 12 pN1mi). Treatment recommendations changed in 37% of patients, predominantly from chemoendocrine to endocrine treatment alone. The proportion of patients recommended chemotherapy decreased from 52% pretest to 25% post-test. Of patients originally recommended chemotherapy, 61% were recommended endocrine treatment alone after receiving the Recurrence Score result. For both pN0 and pN1mi patients, post-test recommendations appeared to follow the Recurrence Score result for low and high values. Physicians' confidence improved significantly. These are the first prospective data on the impact of the Oncotype DX assay on adjuvant treatment decisions in France. Using the assay was associated with a significant change in treatment decisions and an overall reduction in chemotherapy use. These data are consistent with those presented from European and non-European studies. ©AlphaMed Press.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

PubMed

Zhao, Yue; Liu, Guowen; Angeles, Aida; Hamuro, Lora L; Trouba, Kevin J; Wang, Bonnie; Pillutla, Renuka C; DeSilva, Binodh S; Arnold, Mark E; Shen, Jim X

2015-04-15

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 μm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 μg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessing Bacillus subtilis biosurfactant effects on the biodegradation of petroleum products.

PubMed

Montagnolli, Renato Nallin; Lopes, Paulo Renato Matos; Bidoia, Ederio Dino

2015-01-01

Microbial pollutant removal capabilities can be determined and exploited to accomplish bioremediation of hydrocarbon-polluted environments. Thus, increasing knowledge on environmental behavior of different petroleum products can lead to better bioremediation strategies. Biodegradation can be enhanced by adding biosurfactants to hydrocarbon-degrading microorganism consortia. This work aimed to improve petroleum products biodegradation by using a biosurfactant produced by Bacillus subtilis. The produced biosurfactant was added to biodegradation assays containing crude oil, diesel, and kerosene. Biodegradation was monitored by a respirometric technique capable of evaluating CO₂ production in an aerobic simulated wastewater environment. The biosurfactant yielded optimal surface tension reduction (30.9 mN m(-1)) and emulsification results (46.90% with kerosene). Biodegradation successfully occurred and different profiles were observed for each substance. Precise mathematical modeling of biosurfactant effects on petroleum degradation profile was designed, hence allowing long-term kinetics prediction. Assays containing biosurfactant yielded a higher overall CO₂ output. Higher emulsification and an enhanced CO2 production dataset on assays containing biosurfactants was observed, especially in crude oil and kerosene.

Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions

PubMed Central

Marques, Sara S.; Magalhães, Luís M.; Tóth, Ildikó V.; Segundo, Marcela A.

2014-01-01

Total antioxidant capacity assays are recognized as instrumental to establish antioxidant status of biological samples, however the varying experimental conditions result in conclusions that may not be transposable to other settings. After selection of the complexing agent, reagent addition order, buffer type and concentration, copper reducing assays were adapted to a high-throughput scheme and validated using model biological antioxidant compounds of ascorbic acid, Trolox (a soluble analogue of vitamin E), uric acid and glutathione. A critical comparison was made based on real samples including NIST-909c human serum certified sample, and five study samples. The validated method provided linear range up to 100 µM Trolox, (limit of detection 2.3 µM; limit of quantification 7.7 µM) with recovery results above 85% and precision <5%. The validated developed method with an increased sensitivity is a sound choice for assessment of TAC in serum samples. PMID:24968275

Chemical sensing of Benzo[a]pyrene using Corchorus depressus fluorescent flavonoids.

PubMed

Ahmad, Wajiha; Rana, Nosheen Fatima; Riaz, Sundus; Ahmad, Nasir Mehmood; Hameed, Maryam; Naeem, Ayesha; Tahir, Rabbiya

2018-04-01

Plant phytochemicals, such as flavonoids are in use for the development of optical biosensor. Benzo[a]pyrene (B[a]P), is a pervasive environmental and dietary carcinogen. A fluorescent assay is developed using plant isolated flavonoid for the detection of B[a]P. High content saponins are excluded from the flavonoid-containing methanolic extract of Corchorus depressus by implying reduction of silver ions by saponins resulting in formation of silver nanoparticles. Isolated plant flavonoids are used to develop a spectrofluorometric assay for the detection of B[a]P. Decrease in the flavonoid fluorescence intensity by B[a]P is found to be based on both static and dynamic quenching. Specificity of the assay for B[a]P was tested for other carcinogens belonging to different classes of compounds. Flavonoids-mediated sensing can be implied for the development of new generation of nanoparticle-based biosensors that can be more sensitive and less susceptible to external factors, such as temperature and humidity.

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

PubMed Central

Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

Application of an in vitro DNA protection assay to visualize stress mediation properties of the Dps protein.

PubMed

Karas, Vlad O; Westerlaken, Ilja; Meyer, Anne S

2013-05-31

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

PubMed

Kang, Chung Hyo; Yun, Jeong In; Lee, Kwangho; Lee, Chong Ock; Lee, Heung Kyoung; Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho; Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un; Jeong, Hye Gwang; Kim, Hyoung Rae; Park, Chi Hoon

2015-08-28

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

Two continuous coupled assays for ornithine-δ-aminotransferase.

PubMed

Juncosa, Jose I; Lee, Hyunbeom; Silverman, Richard B

2013-09-15

We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme. Copyright © 2013 Elsevier Inc. All rights reserved.

Establishing a safe, rapid, convenient and low-cost antiviral assay of interferon bioactivity based on recombinant VSV expressing GFP.

PubMed

Chen, Weiye; Wen, Zhiyuan; Zhang, Jialin; Li, Cuicui; Huang, Kehe; Bu, Zhigao

2018-02-01

The methods of the quantitative assay of the antiviral activity of interferons (IFNs) (type I, II or III) are very important during carrying out of the research of them, since they were found. Here a recombinant vesicular stomatitis virus expressing green fluorescent protein (GFP) (VSV/GFP) and MDBK cells were used to develop an antiviral assay (AVA) for IFNs. This method was carried out on a 96-well cell culture plate, and the half reduction of virus replication was quantified by assaying GFP. To quantify GFP, cell lysis buffer was directly added to the wells infected with VSV/GFP to lyse cells, the VSV/GFP was then inactivated, and relative fluorescence unit (RFU) of GFP was measured and used to calculate the antiviral activity. This method needed only one step instead of three steps in the staining method with naphthol blue black, medium with phenol red can be used, and it had good reproducibility. The GFP-containing samples could be stored at 4°C in a wet box for at least 1 week without affecting the assay results. In addition, the results obtained with this method were similar to those obtained with the staining method. In conclusion, a safe, rapid, convenient and low-cost AVA of IFN based on recombinant VSV/GFP was established. Copyright © 2017. Published by Elsevier B.V.

Detection of Zaire ebolavirus in swine: Assay development and optimization.

PubMed

Pickering, B S; Collignon, B; Smith, G; Marszal, P; Kobinger, G; Weingartl, H M

2018-02-01

Ebolaviruses (family Filoviridae, order Mononegavirales) cause often fatal, haemorrhagic fever in primates including humans. Pigs have been identified as a species susceptible to Reston ebolavirus (RESTV) infection, with indicated transmission to humans in the Philippines; however, their role during Ebola outbreaks in Africa needs to be clarified. To perform surveillance studies, detection of ebolavirus requires a prerequisite validation of viral RNA and antibody detection methods in swine samples. These diagnostic tests also need to be suitable for deployment to low-level containment laboratories. In this study, we developed a set of tests for detection of antibodies against Zaire ebolavirus (EBOV) in swine. Recombinant EBOV nucleoprotein was produced using a baculovirus expression system for indirect ELISA development. Evaluation of this assay was performed using laboratory and field samples, achieving a diagnostic specificity of 99%. Importantly, the indirect ELISA was able to detect antibodies to EBOV at 7 dpi, 3 days earlier than virus neutralization tests (VNT). The format of the VNT in this work was modified to a microtitre plaque reduction neutralization assay (miPRNT) complemented with immunostaining to provide a more rapid and highly specific assay. Finally, a confirmatory immunoblot assay was generated to supplement the indirect ELISA results. © 2017 Her Majesty the Queen in Right of Canada Reproduced with the permission of the Minister of Health and Agriculture, Canadian Food Inspection Agency.

In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes.

PubMed

Kubiça, Thaís F; Alves, Sydney H; Weiblen, Rudi; Lovato, Luciane T

2014-01-01

The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 μg mL(-1)) and 1,8-cineole (CC50 = 2996.10 μg mL(-1)) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.

Antioxidant and neuroprotector effect of Lepidium meyenii (maca) methanol leaf extract against 6-hydroxy dopamine (6-OHDA)-induced toxicity in PC12 cells.

PubMed

Rodríguez-Huamán, Ángel; Casimiro-Gonzales, Sandra; Chávez-Pérez, Jorge Antonio; Gonzales-Arimborgo, Carla; Cisneros-Fernández, Richard; Aguilar-Mendoza, Luis Ángel; Gonzales, Gustavo F

2017-05-01

Reactive oxygen species (ROS) are normally produced during cell metabolism, there is strong evidence to suggest that ROS produced in excess impair the cell and may be etiologically related to various neurodegenerative diseases. This study was undertaken to examine the effects of Lepidium meyenii (MACA) methanol leaf extract on neurotoxicity in PC12 cell exposed to 6-hydroxydopamine (6-OHDA). Fresh samples of "maca" leaves were processed in order to obtain foliar extracts and to evaluate the neurobiological activity on PC12 cells, subjected to the cytotoxic effect of 6-OHDA through the determination of the capacity antioxidant, cell viability and cytotoxicity assays on PC12 cells. The results of the tests of antioxidant activity, showed maximum values of 2262.37 and 1305.36 expressed in Trolox equivalents (TEAC), for the methanolic and aqueous fractions respectively. Cell viability assays at a dose of 10 μg extract showed an increase of 31% and 60% at 6 and 12 h of pretreatment, respectively. Cytotoxicity assays at the same dose and exposure time showed a 31.4% and 47.8% reduction in lactate dehydrogenase (LDH) activity and an increase in superoxide dismutase (SOD) activity. The results allow us to affirm that the methanolic foliar extract of "maca" presents in vitro neurobiological activity of antioxidant protection, increase in cell viability and reduction of cytotoxicity against oxidative stress generated by 6-OHDA. In conclusion, the present study shows a protective role for Lepidium meyenii leaf extract on 6-OHDA-induced toxicity by an antioxidant effect.

Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis.

PubMed

Alegre, Isabel; Viñas, Inmaculada; Usall, Josep; Teixidó, Neus; Figge, Marian J; Abadias, Maribel

2013-06-01

The consumption of fresh-cut fruit has substantially risen over the last few years, leading to an increase in the number of outbreaks associated with fruit. Moreover, consumers are currently demanding wholesome, fresh-like, safe foods without added chemicals. As a response, the aim of this study was to determine if the naturally occurring microorganisms on fruit are "competitive with" or "antagonistic to" potentially encountered pathogens. Of the 97 and 107 isolates tested by co-inoculation with Escherichia coli O157:H7, Salmonella and Listeria innocua on fresh-cut apple and peach, respectively, and stored at 20 °C, seven showed a strong antagonistic capacity (more than 1-log unit reduction). One of the isolates, CPA-7, achieved the best reduction values (from 2.8 to 5.9-log units) and was the only isolate able to inhibit E. coli O157:H7 at refrigeration temperatures on both fruits. Therefore, CPA-7 was selected for further assays. Dose-response assays showed that CPA-7 should be present in at least the same amount as the pathogen to adequately reduce the numbers of the pathogen. From the results obtained in in vitro assays, competition seemed to be CPA-7's mode of action against E. coli O157:H7. The CPA-7 strain was identified as Pseudomonas graminis. Thus, the results support the potential use of CPA-7 as a bioprotective agent against foodborne pathogens in minimally processed fruit. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cost-benefit and effectiveness analysis of rapid testing for MRSA carriage in a hospital setting.

PubMed

Henson, Gay; Ghonim, Elham; Swiatlo, Andrea; King, Shelia; Moore, Kimberly S; King, S Travis; Sullivan, Donna

2014-01-01

A cost-effectiveness analysis was conducted comparing the polymerase chain reaction assay and traditional microbiological culture as screening tools for the identification of methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted to the pediatric and surgical intensive care units (PICU and SICU) at a 722 bed academic medical center. In addition, the cost benefits of identification of colonized MRSA patients were determined. The cost-effectiveness analysis employed actual hospital and laboratory costs, not patient costs. The actual cost of the PCR assay was higher than the microbiological culture identification of MRSA ($602.95 versus $364.30 per positive carrier identified). However, this did not include the decreased turn-around time of PCR assays compared to traditional culture techniques. Patient costs were determined indirectly in the cost-benefit analysis of clinical outcome. There was a reduction in MRSA hospital-acquired infection (3.5 MRSA HAI/month without screening versus 0.6/month with screening by PCR). A cost-benefit analysis based on differences in length of stay suggests an associated savings in hospitalization costs: MRSA HAI with 29.5 day median LOS at $63,810 versus MRSA identified on admission with 6 day median LOS at $14,561, a difference of $49,249 per hospitalization. Although this pilot study was small and it is not possible to directly relate the cost-effectiveness and cost-benefit analysis due to confounding factors such as patient underlying morbidity and mortality, a reduction of 2.9 MRSA HAI/month associated with PCR screening suggests potential savings in hospitalization costs of $142,822 per month.

Cytosine arabinoside, vinblastine, 5-fluorouracil and 2-aminoanthracene testing in the in vitro micronucleus assay with L5178Y mouse lymphoma cells at Sanofi Aventis, with different cytotoxicity measurements, in support of the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test.

PubMed

Cariou, Olivier; Laroche-Prigent, Nathalie; Ledieu, Sandrine; Guizon, Isabelle; Paillard, Françoise; Thybaud, Véronique

2010-10-29

Cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair), vinblastine (an aneugen that inhibits tubulin polymerisation), 5-fluorouracil (a nucleoside analogue with a steep response profile), and 2-aminoanthracene (a metabolism-dependent reference genotoxin) were tested in the in vitro micronucleus assay with L5178Y mouse lymphoma cells, without cytokinesis block. The four chemicals were independently evaluated in two Sanofi Aventis laboratories, one of which used an image analyser to score micronuclei, while the other scored micronucleated cells manually. Very similar results were obtained in the two laboratories, highlighting the robustness of the assay. The four test chemicals induced significant increases in the incidence of micronucleated cells at concentrations that produced no more than a 55±5% reduction in survival growth, as measured with the three parameters recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) for chemical testing, namely the relative increase in cell counts, relative population doubling, and the relative cell count. These results support the premise that the relative increase in cell counts and relative population doubling, that take into account both cell death and cytostasis, are appropriate measures of survival growth reduction in the in vitro micronucleus test conducted in the absence of cytokinesis block, as recommended in MNvit. Copyright © 2010 Elsevier B.V. All rights reserved.

Susceptibility of Candida albicans biofilms to caspofungin and anidulafungin is not affected by metabolic activity or biomass production.

PubMed

Marcos-Zambrano, Laura Judith; Escribano, Pilar; Bouza, Emilio; Guinea, Jesús

2016-02-01

Micafungin is more active against biofilms with high metabolic activity; however, it is unknown whether this observation applies to caspofungin and anidulafungin and whether it is also dependent on the biomass production. We compare the antifungal activity of anidulafungin, caspofungin, and micafungin against preformed Candida albicans biofilms with different degrees of metabolic activity and biomass production from 301 isolates causing fungemia in patients admitted to Gregorio Marañon Hospital (January 2007 to September 2014). Biofilms were classified as having low, moderate, or high metabolic activity according XTT reduction assay or having low, moderate, or high biomass according to crystal violet assay. Echinocandin MICs for planktonic and sessile cells were measured using the EUCAST E.Def 7.2 procedure and XTT reduction assay, respectively. Micafungin showed the highest activity against biofilms classified according to the metabolic activity and biomass production (P < .001). The activity of caspofungin and anidulafungin was not dependent on the metabolic activity of the biofilm or the biomass production. These observations were confirmed by scanning electron microscopy. None of the echinocandins produced major changes in the structure of biofilms with low metabolic activity and biomass production when compared with the untreated biofilms. However, biofilm with high metabolic activity or high biomass production was considerably more susceptible to micafungin; this effect was not shown by caspofungin or anidulafungin. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Pummelo Protects Doxorubicin-Induced Cardiac Cell Death by Reducing Oxidative Stress, Modifying Glutathione Transferase Expression, and Preventing Cellular Senescence

PubMed Central

Chularojmontri, L.; Gerdprasert, O.; Wattanapitayakul, S. K.

2013-01-01

Citrus flavonoids have been shown to reduce cardiovascular disease (CVD) risks prominently due to their antioxidant effects. Here we investigated the protective effect of pummelo (Citrus maxima, CM) fruit juice in rat cardiac H9c2 cells against doxorubicin (DOX-) induced cytotoxicity. Four antioxidant compositions (ascorbic acid, hesperidin, naringin, and gallic acid) were determined by HPLC. CM significantly increased cardiac cell survival from DOX toxicity as evaluated by MTT assay. Reduction of cellular oxidative stress was monitored by the formation of DCF fluorescent product and total glutathione (GSH) levels. The changes in glutathione-S-transferase (GST) activity and expression were determined by enzyme activity assay and Western blot analysis, respectively. Influence of CM on senescence-associated β-galactosidase activity (SA-β-gal) was also determined. The mechanisms of cytoprotection involved reduction of intracellular oxidative stress, maintaining GSH availability, and enhanced GST enzyme activity and expression. DOX-induced cellular senescence was also attenuated by long-term CM treatment. Thus, CM fruit juice can be promoted as functional fruit to protect cells from oxidative cell death, enhance the phase II GSTP enzyme activity, and decrease senescence phenotype population induced by cardiotoxic agent such as DOX. PMID:23401708

Toxicity and biocompatibility profile of 3D bone scaffold developed by Universitas Indonesia: A preliminary study

NASA Astrophysics Data System (ADS)

Rahyussalim A., J.; Kurniawati, T.; Aprilya, D.; Anggraini, R.; Ramahdita, Ghiska; Whulanza, Yudan

2017-02-01

Scaffold as a biomaterial must fulfill some requirements to be safely implanted to the human body. Toxicity and biocompatibility test are needed to evaluate scaffold material in mediating cell proliferation and differentiation, secreting extracelullar matrix and carrying biomolecular signals for cell communication. An in vitro study with mesenchymal stem cells consisted of direct contact test and indirect contact test using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was conducted on 4 scaffolds made of poly-L-lactic acid (PLA), polyvinyl alcohol (PVA), and hydroxyapatite-poly (vinyl alcohol) composite. There were cells-substrate adhesion impairment, morphological changes, cell death and reduction in cell proliferation seen at 2nd and 6th day in most tested scaffold. Cell count result at day-6 showed proliferation inhibition of more than 50% cell death (inhibition value >50) in all tested scaffold. In MTT assay, two scaffolds were proven non-toxic. In conclusion, various scaffold materials showed different toxicity effect. The toxicity and biocompatibility profile in this study is a preliminary data for further research aiming to use those local-made scaffolds to fill human bone defect in various needs.

Performance evaluation of PRIDE UNDA system with pyroprocessing feed material.

PubMed

An, Su Jung; Seo, Hee; Lee, Chaehun; Ahn, Seong-Kyu; Park, Se-Hwan; Ku, Jeong-Hoe

2017-04-01

The PRIDE (PyRoprocessing Integrated inactive DEmonstration) is an engineering-scale pyroprocessing test-bed facility that utilizes depleted uranium (DU) instead of spent fuel as a process material. As part of the ongoing effort to enhance pyroprocessing safeguardability, UNDA (Unified Non-Destructive Assay), a system integrating three different non-destructive assay techniques, namely, neutron, gamma-ray, and mass measurement, for nuclear material accountancy (NMA) was developed. In the present study, UNDA's NMA capability was evaluated by measurement of the weight, 238 U mass, and U enrichment of oxide-reduction-process feed material (i.e., porous pellets). In the 238 U mass determination, the total neutron counts for porous pellets of six different weights were measured. The U enrichment of the porous pellets, meanwhile, was determined according to the gamma spectrums acquired using UNDA's NaI-based enrichment measurement system. The results demonstrated that the UNDA system, after appropriate corrections, could be used in PRIDE NMA applications with reasonable uncertainty. It is expected that in the near future, the UNDA system will be tested with next-step materials such as the products of the oxide-reduction and electro-refining processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficacy of subantimicrobial-dose doxycycline against nitrosative stress in chronic periodontitis.

PubMed

Pârvu, Alina Elena; Alb, Sandu Florin; Crăciun, Alexandra; Taulescu, Marian Aurel

2013-02-01

To evaluate the effectiveness of subantimicrobial-dose doxycycline (SDD) as an adjunct to scaling and root planing (SRP) treatment against the nitrosative stress of moderate to advanced chronic periodontitis. Adults with untreated chronic periodontitis (n=174) were randomly administered SRP+SDD (n=87) (20 mg of doxycycline twice daily) or SRP+placebo (n=87) treatment for 3 months. At baseline and after 3 months, the probing depths (PD), bleeding on probing (BOP) and clinical attachment level (CAL) were measured, and a gingivomucosal biopsy was collected to assay the induction of nitric oxide synthase (iNOS) and 3-nitrotyrosine (3NT), and blood was collected to assay for total nitrites and nitrates (NO(x)) and 3NT. Compared to baseline, at the completion of treatment, significant decreases in the levels of tissue iNOS and 3NT and serum NO(x) and 3NT were observed in both groups. SRP+SDD yielded a greater reduction in the gingivomucosal and serum nitrosative stress markers than did SRP+placebo. PD, BOP, and CAL reduction were correlated with the nitrosative stress parameters. On a short-term basis, SDD therapy may be used as an adjunct to SRP treatment against nitrosative stress in moderate to advanced chronic periodontitis.

Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.

PubMed

Crowston, Jonathan G; Wang, Xiao Y; Khaw, Peng T; Zoellner, Hans; Healey, Paul R

2006-03-01

To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.

Identification and environmental distribution of dcpA encoding the 1,2-dichloropropane-to-propene reductive dehalogenase in organohalide-respiring Chloroflexi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Padilla-Crespo, Elizabeth; Yan, Jun; Swift, Cynthia M

2014-01-01

Dehalococcoides mccartyi (Dhc) strains KS and RC grow with 1,2-dichloropropane (1,2-D) as an electron acceptor in enrichment cultures derived from hydrocarbon-contaminated and pristine river sediments, respectively. Transcription, expression, enzymatic and PCR analyses implicated the reductive dehalogenase gene dcpA in 1,2-D dichloroelimination to propene and inorganic chloride. Quantitative real-time PCR (qPCR) analyses demonstrated Dhc cell increase during growth with 1,2-D and suggested that both Dhc strains carried a single dcpA gene copy per genome. Dhc strain RC and strain KS produced 1.8 0.1 x 107 and 1.4 0.5 x 107 cells per mole of propene formed, respectively. The dcpA gene wasmore » identified in 1,2-D-to-propene-dechlorinating microcosms established with sediment samples collected from different geographical locations in Europe and North and South America. Clone library analysis revealed two distinct dcpA phylogenetic clusters, both of which the dcpA gene-targeted qPCR assay captured, suggesting the qPCR assay is useful for site assessment and bioremediation monitoring at 1,2-D-contaminated sites.« less

Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones.

PubMed

Quezada, M A; Carballeira, J D; Sinisterra, J V

2012-05-01

Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog's rule. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of temperature and benzalkonium chloride on nitrate reduction.

PubMed

Hajaya, Malek G; Tezel, Ulas; Pavlostathis, Spyros G

2011-04-01

The effect of temperature and benzalkonium chloride (BAC) on nitrate reduction was investigated in batch assays using a mixed nitrate reducing culture. Nitrate was transformed completely, mainly through denitrification, to dinitrogen at 5, 10, 15 and 22 °C. In the absence of BAC, reduction of individual nitrogen oxides had different susceptibility to temperature and transient nitrite accumulation was observed at low temperatures. When the effect of BAC was tested up to 100 mg/L from 5 to 22 °C, denitrification was inhibited at and above 50mg BAC/L with transient nitrite accumulation at all temperatures. The effect of BAC was described by a competitive inhibition model. Nitrite reduction was the denitrification step most susceptible to BAC, especially at low temperatures. BAC was not degraded during the batch incubation and was mostly biomass-adsorbed. Overall, this study shows that low temperatures exacerbate the BAC inhibitory effect, which in turn is controlled by adsorption to biomass. Copyright © 2011 Elsevier Ltd. All rights reserved.

A digital enzyme-linked immunosorbent assay for ultrasensitive measurement of amyloid-β 1-42 peptide in human plasma with utility for studies of Alzheimer's disease therapeutics.

PubMed

Song, Linan; Lachno, D Richard; Hanlon, David; Shepro, Adam; Jeromin, Andreas; Gemani, Dipika; Talbot, Jayne A; Racke, Margaret M; Dage, Jeffrey L; Dean, Robert A

2016-12-15

Amyloid-β 1-42 peptide (Aβ 1-42 ) is associated with plaque formation in the brain of patients with Alzheimer's disease (AD). Pharmacodynamic studies of AD therapeutics that lower the concentrations of Aβ 1-42 in peripheral blood require highly sensitive assays for its measurement. A digital enzyme-linked immunosorbent assay (ELISA) using single molecule array (Simoa) technology has been developed that provides improved sensitivity compared with conventional ELISA methods using the same antibody reagents. A sensitive digital ELISA for measurement of Aβ 1-42 using antibodies 3D6 and 21F12 was developed. Assay performance was evaluated by repeated testing of pooled human plasma and buffer diluent quality control samples to determine relative accuracy, intra- and inter-assay precision, limit of detection (LOD), lower limit of quantification (LLOQ), dilutional linearity, and spike recovery. The optimized assay was used to quantify Aβ 1-42 in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721. The prototype assay measured Aβ 1-42 with an LOD of 0.3 pg/ml and an LLOQ of 2.8 pg/ml in plasma, calibrated using an Aβ 1-42 peptide standard from Fujirebio. Assay precision was acceptable with intra- and inter-assay coefficients of variation both being ≤10%. Dilutional linearity was demonstrated in sample diluent and immunodepleted human plasma. Analyte spike recovery ranged from 51% to 93% with a mean of 80%. This assay was able to quantify Aβ 1-42 in all of the 84 clinical samples tested. A rapid reduction in levels of Aβ 1-42 was detected within 1 h after drug treatment, and a dose-dependent decrease of Aβ 1-42 levels was also observed over the time course of sample collection. This digital ELISA has potential utility in clinical applications for quantification of Aβ 1-42 in plasma where high sensitivity and precision are required.

Rapid Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) by the Vitek MS Saramis system.

PubMed

Shan, Weiguang; Li, Jiaping; Fang, Ying; Wang, Xuan; Gu, Danxia; Zhang, Rong

2016-01-01

A rapid, sensitive, and accurate Vitek MS assay was developed to distinguish clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) from clinical isolates of methicillin-sensitive Staphylococcus aureus (MSSA) by developing an in-house knowledgebase of SuperSpectra. Three unique peaks, including peaks at 2305.6 and 3007.3 Da specific to MRSA, and 6816.7 Da specific to MSSA, were selected for differentiating MRSA and MSSA. This assay accurately identified 84 and 91% of clinical MRSA and MSSA strains out of the total 142 clinically acquired S. aureus strains that were tested. This method will greatly improve the efficiency of single clinical sample identification of MRSA, thereby facilitating a reduction in the transmission of MRSA in clinical settings.

Inhibition of intra-Golgi transport in vitro by mitotic kinase.

PubMed

Stuart, R A; Mackay, D; Adamczewski, J; Warren, G

1993-02-25

It has previously been shown that exocytic and endocytic membrane traffic are inhibited in mitotic mammalian cells. Here we have used a cell-free intra-Golgi transport assay supplemented with heterologous cytosols to mimic this effect in vitro. Cytosols with high histone kinase activity, made either from mitotic cells or by cyclin A treatment of interphase cells, inhibited intra-Golgi transport by up to 75%. Inhibition of transport was reversed by the kinase inhibitor staurosporine or by reduction in ATP levels leading to inactivation of histone kinase. The data indicate that cell cycle control of intra-Golgi transport is due to a reversible modification of cytosol, and this assay system may be used to study the molecular mechanism of mitotic transport inhibition in mammalian cells.

Iron Reduction and Carbonate Precipitation by Shewanella oneidensis

NASA Astrophysics Data System (ADS)

Zeng, Z.; Tice, M. M.

2011-12-01

This study is to contribute to better understanding of how Archean microbes induced carbonate diagenesis in mats and stromatolites. Previous studies showed sulfate reduction, a common promoter of carbonate precipitation in modern mats[1], is likely to have been less effective in Archean mats in marine fluids lower in sulfate[2]. Alternatively, iron reduction produces far more alkalinity per unit carbon respired than sulfate reduction. Therefore, we hypothesize iron reduction can promote much more carbonate precipitation than sulfate reduction. Our study might also have some relevance to banded iron formation on which microbial iron reduction played a potential role[3]. To test our hypothesis, Shewanella oneidensis MR-1, a dissimilatory iron reducing bacterium will be cultured anaerobically (79%N2, 20%CO2 and 1%H2) in basal medium to trigger iron reduction. Lactate will be used as electron donor, and the electron acceptor will be fresh ferrihydrite. Culture medium will be added with various metal ions, such as Ca2+ and Mg2+, to obtain potential carbonate precipitate. Escherichia coli (with fumarate added as an electron acceptor) will be used to provide a comparison to live but non-iron- reduction cells. After 20 days incubation, precipitate will be collected, washed and identified by X-ray diffraction (XRD). Besides, iron reduction rate (ferrozine assay)[4], PH and amount of precipitate (carbonate and oxidize fractions)[5] will be measured over time to well understand how S. oneidensis drives carbonate precipitation.

Transformation of serum-susceptible Escherichia coli O111 with p16Slux plasmid to allow for real-time monitoring of complement-based inactivation of bacterial growth in bovine milk.

PubMed

Maye, S; Stanton, C; Fitzgerald, G F; Kelly, P M

2016-01-01

Complement activity has only recently been characterized in raw bovine milk. However, the activity of this component of the innate immune system was found to diminish as milk was subjected to heat or partitioning during cream separation. Detection of complement in milk relies on a bactericidal assay. This assay exploits the specific growth susceptibility of Escherichia coli O111 to the presence of complement. Practical application of the assay was demonstrated when a reduction in complement activity was recorded in the case of pasteurized and reduced-fat milks. This presented an opportunity to improve the functionality of the bactericidal assay by incorporating bioluminescence capability into the target organism. Following some adaptation, the strain was transformed by correctly integrating the p16Slux plasmid. Growth properties of the transformed strain of E. coli O111 were unaffected by the modification. The efficacy of the strain adaptation was correlated using the LINEST function analysis [r=0.966; standard error of prediction (SEy)=0.957] bioluminescence with that of bactericidal assay total plate counts within the range of 7.5 to 9.2 log cfu/mL using a combination of raw and processed milk samples. Importantly, the transformed E. coli O111 p16Slux strain could be identified in milk and broth samples using bioluminescence measurement, thus enabling the bactericidal assay-viability test to be monitored in real time throughout incubation. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Immunoreactive LH in long-term frozen human urine samples.

PubMed

Singh, Gurmeet Kaur Surindar; Jimenez, Mark; Newman, Ron; Handelsman, David J

2014-04-01

Urine provides a convenient non-invasive alternative to blood sampling for measurement of certain hormones. Urinary luteinizing hormone (LH) measurements have been used for endocrinology research and anti-doping testing. However, the commercially available LH immunoassays are developed and validated for human blood samples but not urine so that LH assays intended for use with urine samples need thorough validation. Therefore, the present study evaluated the measurement of urinary LH immunoreactivity using previously validated immunofluorometric (IF) and immunochemiluminometric (ICL) LH assays after prolonged frozen storage. LH was measured in serial urine samples following administration of a single injection of one of two doses of recombinant human chorionic hormone (rhCG) with assays run at the end of study (2008) and again after four years of frozen (-20 °C) storage where samples were stored without adding preservatives. The ICL assay showed quantitatively reproducible LH measurements after prolonged -20 °C storage. However, the IF immunoassay gave consistently lower LH levels relative to ICL (2008) with a further proportionate reduction after four years of sample storage (2012). Yet, both the assays displayed similar patterns of the time-course of urine LH measurement both before and after four years of frozen storage. In conclusion, we found that both immunoassays are suitable for urinary LH measurements with ICL assay being more robust for quantitative urinary LH measurement such as for anti-doping purposes, whereas the IF could be applicable for research studies where urine LH levels are compared within-study but not in absolute terms. Copyright © 2013 John Wiley & Sons, Ltd.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

PubMed

Kulkarni, Rohan M; Dakoulas, Emily W; Miller, Ken E; Terse, Pramod S

2017-09-01

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Effect of time since exposure to Chlamydia trachomatis on chlamydia antibody detection in women: a cross-sectional study.

PubMed

Horner, Patrick J; Wills, Gillian S; Reynolds, Rosy; Johnson, Anne M; Muir, David A; Winston, Alan; Broadbent, Andrew J; Parker, David; McClure, Myra O

2013-08-01

To investigate what factors influence the detection of Chlamydia trachomatis antibody following genital tract infection. One hundred and sixty-four women with a previous history of C trachomatis infection contributed to an earlier report on the performance of chlamydia antibody ELISA assays. We undertook further analysis to explore how chlamydia antibody assay sensitivity changes with time since infection. Chlamydia antibody was detected in more women soon after the last detection of chlamydia at the lower genital tract than at later times. This holds true for all tests, but the Anilabsystems IgG EIA, Medac pELISA plus ELISA and the Savyon SeroCT-IgG ELISA were less sensitive than the pgp3 ELISA and the Anilabsystems microimmunofluorescence (MIF) assay at all time points except during current infection. Fall in seropositivity in women generally occurred in the early weeks and months following the last episode of chlamydia infection. There was no clear pattern of further reduction in seropositivity after 6 months. Multiple previous episodes were associated with increased seropositivity in the pgp3 assay (two or more vs one, OR 19, p<0.001) and other tests, but the effect was significantly smaller for the Anilabs, Medac and SeroCT MOMP peptide ELISAs, but not for the MIF assay. Chlamydia antibody detection decreases with time since infection and this is most apparent in the first 6 months. In women who have had more than one infection, antibody remained detectable longer for all tests, but this was more marked for the pgp3 ELISA and MIF assay.

Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

PubMed

Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

2015-08-01

Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

Clinical and economic impact of the introduction of a nucleic acid amplification assay for Clostridium difficile.

PubMed

Guinta, Margaret M; Bunnell, Kristen; Harrington, Amanda; Bleasdale, Susan; Danziger, Larry; Wenzler, Eric

2017-12-04

The clinical outcomes and cost implications of a diagnostic shift from an EIA- to PCR-based assay for Clostridium difficile infection (CDI) have not been completely described in the literature. The impact of the PCR-based assay on the incidence and duration of CDI therapy was compared to the EIA assay for patients with a negative CDI diagnostic result. Secondary clinical and economic outcomes were also evaluated. Independent predictors of receipt of antibiotic therapy were assessed via logistic regression. 141 EIA and 140 PCR patients were included. Significantly more patients were started or continued on anti-CDI antibiotic therapy after a known negative assay result in the EIA group (26 patients vs. 8 patients, P = 0.002). Duration of antibiotic therapy after a known negative result was significantly shorter in the PCR group (1 vs. 4 days, P = 0.029) and a 23% reduction in the number of tests obtained per patient was observed (1.41 ± 0.86 vs. 1.82 ± 1.35, P = 0.007). The over fourfold difference in per-test cost of the EIA assay ($8.33 vs. $42.86, P < 0.0001) was offset by the overall medication costs required for the increased treatment in the EIA group ($546.60 vs. $188.96, P = 0.191). Utilization of the EIA-based CDI assay was associated with increased odds of CDI treatment after a negative test (aOR 4.71, 95% CI 1.93-11.46, P = 0.001). The transition from an EIA to PCR-based assay for diagnosing CDI resulted in a significant decrease in the number of patients treated and the duration of treatment in response to a negative test result. This significant decrease in treatment resulted in decreased costs offsetting the utilization of a more expensive molecular test for patients with a negative CDI diagnostic result.

Oligonucleotide Based Magnetic Bead Capture of Onchocerca volvulus DNA for PCR Pool Screening of Vector Black Flies

PubMed Central

Gopal, Hemavathi; Hassan, Hassan K.; Rodríguez-Pérez, Mario A.; Toé, Laurent D.; Lustigman, Sara; Unnasch, Thomas R.

2012-01-01

Background Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). Methodology/Principal Findings We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. Conclusions/Significance The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. PMID:22724041

Metabolic adaptation and in situ attenuation of chlorinated ethenes by naturally occurring microorganisms in a fractured dolomite aquifer near Niagara Falls, New York

USGS Publications Warehouse

Yager, R.M.; Bilotta, S.E.; Mann, C.L.; Madsen, E.L.

1997-01-01

A combination of hydrogeological, geochemical, and microbiological methods was used to document the biotransformation of trichloroethene (TCE) to ethene, a completely dechlorinated and environmentally benign compound, by naturally occurring microorganisms within a fractured dolomite aquifer. Analyses of groundwater samples showed that three microbially produced TCE breakdown products (cis-1,2-dichloroethene, vinyl chloride, and ethene) were present in the contaminant plume. Hydrogen (H2) concentrations in groundwater indicated that iron reduction was the predominant terminal electron-accepting process in the most contaminated geologic zone of the site. Laboratory microcosms prepared with groundwater demonstrated complete sequential dechlorination of TCE to ethene. Microcosm assays also revealed that reductive dechlorination activity was present in waters from the center but not from the periphery of the contaminant plume. This dechlorination activity indicated that naturally occurring microorganisms have adapted to utilize chlorinated ethenes and suggested that dehalorespiring rather than cometabolic, microbial processes were the cause of the dechlorination. The addition of pulverized dolomite to microcosms enhanced the rate of reductive dechlorination, suggesting that hydrocarbons in the dolomite aquifer may serve as electron donors to drive microbially mediated reductive dechlorination reactions. Biodegradation of the chlorinated ethenes appears to contribute significantly to decontamination of the site.A combination of hydrogeological, geochemical, and microbiological methods was used to document the biotransformation of trichloroethene (TCE) to ethene, a completely dechlorinated and environmentally benign compound, by naturally occurring microorganisms within a fractured dolomite aquifer. Analyses of groundwater samples showed that three microbially produced TCE breakdown products (cis-1,2-dichloroethene, vinyl chloride, and ethene) were present in the contaminant plume. Hydrogen (H2) concentrations in groundwater indicated that iron reduction was the predominant terminal electron-accepting process in the most contaminated geologic zone of the site. Laboratory microcosms prepared with groundwater demonstrated complete sequential dechlorination of TCE to ethene. Microcosm assays also revealed that reductive dechlorination activity was present in waters from the center but not from the periphery of the contaminant plume. This dechlorination activity indicated that naturally occurring microorganisms have adapted to utilize chlorinated ethenes and suggested that dehalorespiring rather than cometabolic, microbial processes were the cause of the dechlorination. The addition of pulverized dolomite to microcosms enhanced the rate of reductive dechlorination, suggesting that hydrocarbons in the dolomite aquifer may serve as electron donors to drive microbially mediated reductive dechlorination reactions. Biodegradation of the chlorinated ethenes appears to contribute significantly to decontamination of the site.

Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

PubMed Central

Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

2007-01-01

Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores. Conclusion The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput. PMID:17697325

Can we estimate biogeochemical uptake rates in sediments from reach-scale data or vice versa?

NASA Astrophysics Data System (ADS)

Gonzalez-Pinzon, R.; Garayburu-Caruso, V. A.

2017-12-01

Hydrologists and stream ecologists want to understand how reactive transport processes from sub-meter to reach scales aggregate to determine nutrient and carbon export across watersheds. Mesocosm (sub-meter) scale experiments offer the advantage of being tractable and affordable but may be spatially and temporally irrelevant for describing watershed-scale processes. While reach scale experiments sample larger heterogeneities, they provide aggregated information that does not allow for easy detection of hot-spots and hot-moments, and might still be irrelevant for describing watershed processes if they are not conducted under varying flow conditions. We conducted mesocosm (column) and reach-scale experiments along a first-to-eight stream order continuum using nutrient and resazurin tracers to investigate how information collected at the sub-meter scale (mesocosom experiments) compares to that collected at the reach scale, and vice versa. Our work highlights the difficulty of finding useful patterns not only across stream orders (i.e., for the same type of experiment) but also across experiments. Our results offer quantitative perspective on why hydrologists and stream ecologists must depart from the status quo of conducting solute-specific (e.g., only N), site-specific (primarily headwaters) and single-season (mainly summer) experiments to understand controls on nutrient retention.

On the influence of DC electric fields on the aerosol assisted chemical vapor deposition growth of photoactive titanium dioxide thin films.

PubMed

Romero, Luz; Binions, Russell

2013-11-05

Titanium dioxide thin films were deposited on fluorine doped tin oxide glass substrate from the electric field assisted aerosol chemical vapor deposition (EACVD) reaction of titanium isopropoxide (TTIP, Ti(OC3H7)4) in toluene on glass substrates at a temperature of 450 °C. DC electric fields were generated by applying a potential difference between the electrodes of the transparent coated oxide coated glass substrates during the deposition. The deposited films were characterized using scanning electron microscopy, X-ray diffraction, atomic force microscopy, Raman spectroscopy, and UV-vis spectroscopy. The photoactivity and hydrophilicity of the deposited films were also analyzed using a dye-ink test and water-contact angle measurements. The characterization work revealed that the incorporation of DC electric fields produced significant reproducible changes in the film microstructure, preferred crystallographic orientation, roughness, and film thickness. Photocatalytic activity was calculated from the half-time (t1/2) or time taken to degrade 50% of the initial resazurin dye concentration. A large improvement in photocatalytic activity was observed for films deposited using an electric field with a strong orientation in the (004) direction (t1/2 17 min) as compared to a film deposited with no electric field (t1/2 40 min).

Assessment of the sensitivity and specificity of Xpert MTB/RIF assay as an early sputum biomarker of response to tuberculosis treatment.

PubMed

Friedrich, Sven O; Rachow, Andrea; Saathoff, Elmar; Singh, Kasha; Mangu, Chacha D; Dawson, Rodney; Phillips, Patrick Pj; Venter, Amour; Bateson, Anna; Boehme, Catharina C; Heinrich, Norbert; Hunt, Robert D; Boeree, Martin J; Zumla, Alimuddin; McHugh, Timothy D; Gillespie, Stephen H; Diacon, Andreas H; Hoelscher, Michael

2013-08-01

An accurate biomarker is urgently needed to monitor the response to treatment in patients with pulmonary tuberculosis. The Xpert MTB/RIF assay is a commercially available real-time PCR that can be used to detect Mycobacterium-tuberculosis-specific DNA sequences in sputum samples. We therefore evaluated this assay with serial sputum samples obtained over 26 weeks from patients undergoing treatment for tuberculosis. We analysed sputum samples from 221 patients with smear-positive tuberculosis enrolled at two sites (Cape Town, South Africa, and Mbeya, Tanzania) of a multicentre randomised clinical trial REMoxTB of antituberculosis treatment on a weekly basis (weeks 0 to 8), then at weeks 12, 17, 22, and 26 after treatment initiation. The Xpert MTB/RIF results over time were compared with the results of standard smear microscopy and culture methods. We obtained and analysed 2741 sputum samples from 221 patients. The reduction in positivity rates with Xpert MTB/RIF were slower than those with the standard methods. At week 8, positive results were obtained for 62 (29%) of 212 sputum samples with smear microscopy, 46 (26%) of 175 with solid culture (Löwenstein-Jensen medium), 77 (42%) of 183 with liquid culture (Bactec MGIT960 system), and 174 (84%) of 207 with Xpert MTB/RIF; at 26 weeks, positive results were obtained for ten (5%) of 199, four (3%) of 157, seven (4%) of 169, and 22 (27%) of 83 sputum samples, respectively. The reduction in detection of quantitative M tuberculosis DNA with Xpert MTB/RIF correlated with smear grades (ρ=-0·74; p<0·0001), solid culture grades (ρ=-0·73; p<0·0001), and time to liquid culture positivity (ρ=0·73; p<0·0001). Compared with the combined binary smear and culture results as a reference standard, the Xpert MTB/RIF assay had high sensitivity (97·0%, 95% CI 95·8-97·9), but poor specificity (48·6%, 45·0-52·2). The poor specificity precludes the use of the Xpert MTB/RIF assay as a biomarker for monitoring tuberculosis treatment, and should not replace standard smear microscopy and culture. Global Alliance for TB Drug Development, Bill & Melinda Gates Foundation, UK Medical Research Council, German Ministry of Science and Technology. Copyright © 2013 Elsevier Ltd. All rights reserved.

STUDIES ON ORGANIC ACID METABOLISM,

DTIC Science & Technology

Lipoic acid metabolism: The acetyl and succinyl thio esters of civinyl dimercapto were prepared by chemical and enzymatic means. The oxidation...reduction reactions of the disulfide-dimercapto groups in pyrimidine nucleotide-linked reactions were explored in the initial lipoic acid assay organiam...disulfide couple. The studies appeared to indicate a bound form of lipoic acid to be the coenzyme, and suggested that an amide or possibly another

THE ANTIMUTAGENIC EFFECT OF VANILLIN AND CINNAMALDEHYDE ON SPONTANEOUS MUTATION IN SALMONELLA TA104 IS DUE TO A REDUCTION IN MUTATIONS AT GC BUT NOT AT SITES

EPA Science Inventory

Abstract
Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that, when added to assay plates, reduced the spontaneous mutant frequency in Salmonella typhimurium strain TA104 (hisG428, rfa, uvrB, pKM101) by 50%. To date, no study has demonstrated whether or not...

Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

PubMed Central

Egland, Paul G.; Gibson, Jane; Harwood, Caroline S.

2001-01-01

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation. PMID:11229940

The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase.

PubMed

Misuri, Livia; Cappiello, Mario; Balestri, Francesco; Moschini, Roberta; Barracco, Vito; Mura, Umberto; Del-Corso, Antonella

2017-12-01

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition 1

PubMed Central

Riens, Burgi; Heldt, Hans Walter

1992-01-01

In leaves of spinach plants (Spinacia oleracea L.) performing CO2 and NO3− assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells. PMID:16668679

Minimal residual disease in acute lymphoblastic leukemia: optimal methods and clinical relevance, pitfalls and recent approaches.

PubMed

Salari, Fatemeh; Shahjahani, Mohammad; Shahrabi, Saeid; Saki, Najmaldin

2014-11-01

After advances in experimental and clinical testing, minimal residual disease (MRD) assay results are considered a determining factor in treatment of acute lymphoblastic leukemia patients. According to MRD assay results, bone marrow (BM) leukemic burden and the rate of its decline after treatment can be directly evaluated. Detailed knowledge of the leukemic burden in BM can minimize toxicity and treatment complications in patients by tailoring the therapeutic dose based on patients' conditions. In addition, reduction of MRD before allo-HSCT is an important prerequisite for reception of transplant by the patient. In direct examination of MRD by morphological methods (even by a professional hematologist), leukemic cells can be under- or over-estimated due to similarity with hematopoietic precursor cells. As a result, considering the importance of MRD, it is necessary to use other methods including flow cytometry, polymerase chain reaction (PCR) amplification and RQ-PCR to detect MRD. Each of these methods has its own advantages and disadvantages in terms of accuracy and sensitivity. In this review article, different MRD assay methods and their sensitivity, correlation of MRD assay results with clinical symptoms of the patient as well as pitfalls in results of these methods are evaluated. In the final section, recent advances in MRD have been addressed.

The combination of two novel tobacco blends and filter technologies to reduce the in vitro genotoxicity and cytotoxicity of prototype cigarettes.

PubMed

Crooks, Ian; Scott, Ken; Dalrymple, Annette; Dillon, Debbie; Meredith, Clive

2015-04-01

Tobacco smoke from a combustible cigarette contains more than 6000 constituents; approximately 150 of these are identified as toxicants. Technologies that modify the tobacco blend to reduce toxicant emissions have been developed. These include tobacco sheet substitute to dilute toxicants in smoke and blend treated tobacco to reduce the levels of nitrogenous precursors and some polyphenols. Filter additives to reduce gas (vapour) phase constituents have also been developed. In this study, both tobacco blend and filter technologies were combined into an experimental cigarette and smoked to International Organisation on Standardisation and Health Canada puffing parameters. The resulting particulate matter was subjected to a battery of in vitro genotoxicity and cytotoxicity assays - the Ames test, mouse lymphoma assay, the in vitro micronucleus test and the Neutral Red Uptake assay. The results indicate that cigarettes containing toxicant reducing technologies may be developed without observing new additional genotoxic hazards as assessed by the assays specified. In addition, reductions in bacterial mutagenicity and mammalian genotoxicity of the experimental cigarette were observed relative to the control cigarettes. There were no significant differences in cytotoxicity relative to the control cigarettes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Anti-inflammatory and antinociceptive properties of blueberry extract (Vaccinium corymbosum).

PubMed

Torri, Eliane; Lemos, Marivane; Caliari, Vinícius; Kassuya, Cândida A L; Bastos, Jairo K; Andrade, Sérgio F

2007-04-01

Blueberries are among the edible fruits that are recognized best for their potential health benefits. The crude extract from Vaccinium corymbosum was assessed in anti-inflammatory and antinociceptive models. The crude hydroalcoholic extract was administered orally at doses of 100, 200 or 300 mg kg (-1) for all the assays. In the carrageenan test, the crude extract reduced rat paw oedema by 9.8, 28.5 and 65.9%, respectively. For the histamine assay, the reductions of oedema were 70.1, 71.7 and 81.9%, respectively. In the myeloperoxidase (MPO) assay, 300 mg kg (-1) crude extract produced a significant inhibition of the MPO activity, at 6 h and 24 h after injection of carrageenan, by 42.8 and 46.2%, respectively. With the granulomatous tissue assay dexamethasone displayed significant activity, whereas the blueberry extract was inactive. For the abdominal constriction test, inhibitions of 49.0, 54.5, 53.5%, respectively, were observed for the crude extract, and 61.4% for indometacin. In the formalin test, the crude extract (200 and 300 mg kg (-1)) and indometacin inhibited only the second phase by 36.2, 35.3 and 45.8%, respectively. Considering that the crude extract of blueberry displayed antinociceptive and anti-inflammatory activity, its consumption may be helpful for the treatment of inflammatory disorders.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

PubMed

Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

2016-05-01

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Alcohols which have been in contact with any plastics may interfere in radioimmunoassays of progesterone.

PubMed

Ocvirk, Rok; Bisson, Jennifer M; Murphy, Beverley E Pearson

2009-01-01

In recent years there has been increasing use of plastic rather than glass containers for many liquids, including wine. However we have found that residue from commercially obtained 'pure' ethanol dispensed in plastic bottles interferes in some biochemical assays. We have observed a volume-dependent decrease in maximally bound ligand in radioimmunoassays of progesterone. The resulting shift in the standard curve leads to an underestimation of the analyte concentrations and to altered estimation of cross reactivity by competing ligands. These effects became apparent in assays with high sensitivity (500 pg or less). All sources of ethanol obtainable in Quebec contained impurities. A similar effect was also produced by 'pure' methanol. The reduction in maximally bound ligand was amplified when the alcohol was aliquoted using plastic pipette tips. We conclude that alcohols which have had any contact with plastics are not safe to use in immunoassays of progesterone (or its metabolites as estimated according to cross-reactivity after HPLC) and may affect other assays. If the use of alcohol and plastic tips cannot be avoided, the amount of alcohol used should be reduced to 1% or less. This can be accomplished by preparing steroid standards in assay buffers containing albumin or gelatin, which enhance the solubility of steroids in aqueous media.

Competitive Binding to Cuprous Ions of Protein and BCA in the Bicinchoninic Acid Protein Assay

PubMed Central

Huang, Tao; Long, Mian; Huo, Bo

2010-01-01

Although Bicinchoninic acid (BCA) has been widely used to determine protein concentration, the mechanism of interaction between protein, copper ion and BCA in this assay is still not well known. Using the Micro BCA protein assay kit (Pierce Company), we measured the absorbance at 562 nm of BSA solutions with different concentrations of protein, and also varied the BCA concentration. When the concentration of protein was increased, the absorbance exhibited the known linear and nonlinear increase, and then reached an unexpected plateau followed by a gradual decrease. We introduced a model in which peptide chains competed with BCA for binding to cuprous ions. Formation of the well-known chromogenic complex of BCA-Cu1+-BCA was competed with the binding of two peptide bonds (NTPB) to cuprous ion, and there is the possibility of the existence of two new complexes. A simple equilibrium equation was established to describe the correlations between the substances in solution at equilibrium, and an empirical exponential function was introduced to describe the reduction reaction. Theoretical predictions of absorbance from the model were in good agreement with the measurements, which not only validated the competitive binding model, but also predicted a new complex of BCA-Cu1+-NTPB that might exist in the final solution. This work provides a new insight into understanding the chemical bases of the BCA protein assay and might extend the assay to higher protein concentration. PMID:21625379

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

PubMed

Cabillic, F; Bouet-Toussaint, F; Toutirais, O; Rioux-Leclercq, N; Fergelot, P; de la Pintière, C Thomas; Genetet, N; Patard, J-J; Catros-Quemener, V

2006-12-01

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

PubMed

Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole

2016-10-15

Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples. Copyright © 2016 Elsevier B.V. All rights reserved.

The prognostic value of histidine-rich glycoprotein RNA in breast tissue using unmodified gold nanoparticles assay.

PubMed

Eissa, Sanaa; Azzazy, Hassan M E; Matboli, Marwa; Shawky, Sherif M; Said, Hebatallah; Anous, Fatin A

2014-09-01

The aim of is this study is to explore the role of tissue histidine-rich glycoprotein (HRG) RNA as a promising clinically useful biomarker for breast cancer patients prognosis using nanogold assay. Expression of the HRG RNA was assessed by gold nanoparticles and conventional RT-PCR after purification by magnetic nanoparticles in breast tissue samples. The study included 120 patients, 60 of which were histologically proven breast carcinoma cases, 30 had benign breast lesions and 30 were healthy individuals who had undergone reductive plastic surgery. ER, PR and HER2 status were also investigated. The prognostic significance of tissue HRG RNA expression in breast cancer was explored. The magnetic nanoparticles coated with specific thiol modified oligonucleotide probe were used successfully in purification of HRG RNA from breast tissue total RNAs with satisfactory yield. The developed HRG AuNPs assay had a sensitivity and a specificity of 90 %, and a detection limit of 1.5 nmol/l. The concordance rate between the HRG AuNPs assay with RT-PCR after RNA purification using magnetic nanoparticles was 93.3 %. The median follow-up period was 60 months. Among traditional prognostic biomarkers, HRG was a significant independent prognostic marker in relapse-free survival (RFS). HRG RNA is an independent prognostic marker for breast cancer and can be detected using gold NPs assay, which is rapid, sensitive, specific, inexpensive to extend the value for breast cancer prognosis.

Genotoxic effects of old landfill leachate on HepG2 cells after nitration/ultrafiltration/reverse osmosis membrane treatment process.

PubMed

Cheng, Rong; Zhao, Ling; Yin, Pinghe

2017-12-01

Toxicity assessment of nitration/ultrafiltration/reverse osmosis (nitration/UF/RO) project, which has recently been widely used as an efficient process with applications in practical leachate treatment, was very limited. In the present study, DNA damage of leachates was investigated before and after the nitration/UF/RO process by a battery of assays with human hepatoma cells. Methyletrazolium assay showed a high cytotoxicity of 97.1% after being exposed to the highest concentration of raw leachate for 24 h, and a cytotoxicity of 26% in effluent at a concentration of 30% (v/v). Both comet assay (24 h) and γH2AX flow cytometer assay (3 h) showed increased levels of DNA damage in cells exposed to raw leachate and after nitration/UF-treated leachate followed by a significant increase of 7-ethoxyresorufin-O-deethylase activity. However, the effluent after nitration/UF/RO treatment showed no significant difference compared to negative control for γH2AX flow cytometer assay but slight DNA damage at concentrations of 20% and 30% (v/v) as well as increase of 7-ethoxyresorufin-O-deethylase. Analysis showed that nitration/UF/RO process exhibited high removal of physicochemical indexes and significant reduction of toxic and genotoxic effects of leachate, but still demands an improvement to reduce all possible negative risks to the environment and humans. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

A new DPYD genotyping assay for improving the safety of 5-fluorouracil therapy.

PubMed

Sistonen, Johanna; Smith, Chingying; Fu, Yung-Kang; Largiadèr, Carlo R

2012-12-24

Chemotherapeutic use of 5-fluorouracil (5FU) is compromised by 10-20% of patients developing severe toxicity. Recently described genetic variation in dihydropyrimidine dehydrogenase (DPYD) has been shown to be a major predictor of 5FU toxicity. Here, we describe a new genotyping assay for routine clinical use that covers all the major DPYD risk variants. Genomic regions targeting DPYD risk variants (c.1129-5923C>G, c.1679T>G/A, c.1905+1G>A, c.2846A>T) and additional markers (c.234-123G>C, c.496A>G, c.775A>G) were amplified in a multiplex PCR reaction. The subsequent steps including allele-specific primer extension, hybridization of the primers to a microarray, scanning of the array, and data analysis were automated within the INFINITI® Analyzer (AutoGenomics). The assay was validated by analyzing 107 blood samples obtained from patients previously re-sequenced for the DPYD. The genotypes obtained with the developed assay were 100% concordant with the re-sequencing. The procedure is suitable for routine clinical use since the results are obtained within one day. For heterozygous risk variant carriers (~7% of Europeans), the treatment can be adjusted by 5FU dose reduction, whereas carriers of two risk alleles should be treated with an alternative therapy. The developed assay provides a novel tool to improve the safety of commonly used 5FU-based chemotherapies. Copyright © 2012 Elsevier B.V. All rights reserved.