Neutral Red Dye Research Articles

Use of Oxalic Acid-Modified Rice Husk for the Adsorption of Neutral Red from Aqueous Solutions

Rice husk modified with oxalic acid (MRH) was tested as a low-cost adsorbent for the removal of Neutral Red (NR) dye from aqueous solutions employing batch adsorption procedures. Such studies were conducted by varying various parameters such as the pH, the adsorbent dosage, the salt concentration, the contact time, the concentration of the adsorbate and the temperature. The kinetic experimental data were analyzed using three kinetic equations, viz. the pseudo-first-order equation, the pseudo-second-order equation and the intraparticle diffusion model equation, to examine the mechanism of adsorption and the potential rate-controlling step.The mechanism of the process was found to be complex, consisting of both surface adsorption and pore diffusion. The values of the effective diffusion parameter, Deff, were estimated to be of the order of 10-8 cm2/s, indicated that intra-particle diffusion was not the rate-controlling step. The equilibrium adsorption data obtained at various temperatures were analyzed usin...

Modulating effect of Leptadenia reticulata (Retz) Wight & arn against chromate (VI)-induced immunosuppression and oxidative stress on mouse splenic lymphocytes and bone marrow derived macrophages

Ethnopharmacological relevance Leptadenia reticulata (Retz) Wight & arn is mentioned in the ancient ayurvedic literature as an immune booster and rejuvenator. Aims of the study To investigate, the effects of different forms of the extract of Leptadenia reticulata [Aqueous extract (JAE), Padavashesha kashaya (JPK) and Tarpana kashaya (JTK)] to alleviate the experimental immunosuppression induced by the immunotoxicant chromate (VI) in vitro. Materials and methods Standard cell proliferation and cytotoxicity assays like MTT assay, trypan blue dye exclusion test, neutral red dye uptake test, NBT reduction test, determination of percentage cell survival and estimation of markers of oxidative stress were performed in the study. The study was conducted on primary cultures of mouse splenic lymphocytes and bone marrow derived macrophages. Results and Conclusions Treatment with all the three forms of the extract used in the study offered protection against chromate (VI)-induced immunosuppression and the overall protective effect was found to be superior in the case of the aqueous extract of Leptadenia reticulata (JAE). These results confirm that Leptadenia reticulata acts as a modulator and alleviates the immunosuppressive conditions induced by chromate (VI).

Adsorption and magnetic removal of neutral red dye from aqueous solution using Fe 3O 4 hollow nanospheres

Fe 3O 4 hollow nanospheres were prepared via a simple one-pot template-free hydrothermal method and were fully characterized. These magnetic spheres have been investigated for application as an adsorbant for the removal of dye contaminants from water. Because of the high specific surface area, nano-scale particle size, and hollow porous material, Fe 3O 4 hollow spheres showed favorable adsorption behavior for Neutral red. Factors affecting adsorption, such as, initial dye concentration, pH and contact time were evaluated. Langmuir and the Freundlich adsorption isotherms were selected to explicate the interaction of the dye and magnetic adsorbant. The characteristic parameters for each isotherm have been determined. The overall trend followed an increase of the sorption capacity with increasing dye concentration with a maximum of 90% dye removal. The monolayer adsorption capacity of magnetic hollow spheres (0.05 g) for NR in the concentration range studied, as calculated from the Langmuir isotherm model at 25 °C and pH 6, was found to be 105 mg g −1. Adsorption kinetic followed pseudo-second-order reaction kinetics. Thermodynamic study showed that the adsorption processes are spontaneous and endothermic. The combination of the superior adsorption and the magnetic properties of Fe 3O 4 nanospheres can be useful as a powerful separation tool to deal with environmental pollution.

Evaluation of cytotoxicity and degree of conversion of orthodontic adhesives over different time periods

As new orthodontic resin adhesives continue to be marketed, rapid and sensitive tests for examining their toxic effects at the ' cell and tissue level ' are needed because patient safety has been identifi ed as a legal concept. The objective of the present study was to evaluate the cytotoxicity and degree of monomer conversion of orthodontic adhesives over different time periods. Seven adhesives: Transbond® XT, Transbond® Color Change, Quick Cure, EagleBond, Orthobond®, Fill Magic® and Biofix® were evaluated for their cytotoxicity in L929 fibroblastic cells and for their degree of monomer conversion over different time periods. Three control groups were also analysed: Positive control (C+), consisting of Tween 80 cell detergent; Negative control (C-), consisting of PBS; and cell control (CC), consisting of cells exposed to any material. The dye-uptake technique that involves the absorption of a neutral red dye in viable cells was used for the cytotoxicity evaluation and the degree of conversion was evaluated using spectroscopy with infrared. The results showed the cytotoxicity of the adhesives at 24, 48, 72 and 168 hours. At these times, the viability values presented for these materials were statistically different from the groups CC and C- (p 0.05). In the monomer conversions there was a percentage increase of monomer conversion from 24 to 72 hours. A direct correlation could be observed between cytotoxicity and monomer conversions. From this work it can be concluded that all adhesives evaluated are cytotoxic at the times of 24, 48 and 72 hours. Monomers continued conversion even after photopolymerization had stopped.

The attachment of Fe 3O 4 nanoparticles to graphene oxide by covalent bonding

Hybrids of graphene oxide (GO) nanosheets and surface-modified Fe 3O 4 nanoparticles (NPs) were fabricated by a two-step process. First, Fe 3O 4 was modified by tetraethyl orthosilicate and (3-aminopropyl) triethoxysilane to introduce amino groups on its surface. Second, the amino groups of Fe 3O 4 were reacted with the carboxylic groups of GO with the aid of 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide and N-hydroxysuccinnimide to form a GO–Fe 3O 4 hybrid. The attachment of Fe 3O 4 NPs on the GO nanosheet surface was confirmed by transmission electron microscopy and Fourier-transform infrared spectroscopy. The adsorption capacity of GO–Fe 3O 4 for methylene blue and neutral red cationic dyes was as high as 190.14 and 140.79 mg/g, respectively. The GO–Fe 3O 4 hybrids could be reduced to form graphene–Fe 3O 4 hybrids by using NaBH 4 as reducing agent and be used to prepare magnetic GO films.

The role of small molecular weight compounds to increase vacuolation induced by VacA toxin in vitro

VacA is a vacuolation protein toxin secreted by Helicobacter pylori. Many compounds have been implicated in the regulation of VacA toxin activity. In this study, regulation of cell vacuolation induced by VacA was observed with the addition of glycine, glycine hydrochloride, xylitol, and taurine by neutral red dye uptake assay using gastric human epithelial cell cultures. Glycine, xylitol, and taurine increased cell vacuolation significantly after 48 h ( p < 0.05), with their effect apparent in a wide concentration range (0.2 mM to about 100 mM). Changes were sharp in respect of concentration and showed little dose–response characteristics. In contrast, upregulation of glycine hydrochloride on cell vacuolation in weak acidic extracellular pH was much retarded with VacA activity not initiated until 72 h. In addition, our results showed that cell vacuolation was highest when the pH was 6.8. The increase in vacuolation was gentle in weak acidic extracellular pH and the increase dose-dependent with a Pearson correlation coefficient ( r) of 0.986 from 0.2 to 6.25 mM. In this concentration range and at the same time point, the pH decrease was negatively correlated with vacuolating activity ( r = 0.922, p < 0.01). In conclusion, our study showed that three small molecular compounds can increase vacuolation induced by VacA toxin in vitro.

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food-grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02-2.5 microg x ml(-1)) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 microg x ml(-1) respectively, while values of nisin found were 0.50 and 1.04 microg x ml(-1). In the LDH assay, the EC50 values were 0.65 and 0.62 microg x ml(-1) for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.

Contrasting guest binding interaction of cucurbit[7-8]urils with neutral red dye: controlled exchange of multiple guests

Interactions among macrocyclic hosts and dyes/drugs have been explored extensively for their direct usage in controlled uptake and release of large number of potential drug molecules. In this paper we report the non-covalent interaction of cucurbit[8]uril macrocycle (CB8) with a biologically important dye, neutral red, by absorption and fluorescence spectroscopy. A comparative analysis with the complexation behaviour of the dye with CB7, the lower homologue of CB8, indicates contrasting guest binding behaviour with significant changes in the photophysical characteristics of the dye. While CB7 interaction leads to a 1 ratio 1 stoichiometry resulting in approximately 6 fold enhancement in the fluorescence emission of the dye, CB8 displays signatures for a 1 ratio 2 host-guest stoichiometry with drastic reduction in the fluorescence emission. Apart from the evaluation of approximately 2 unit shift in the protolytic equilibrium on complexation (pK(a) shift), the measurements with tryptophan established a selective guest exchange to favour a co-localized dimer inside the CB8 cavity. In a protein medium (BSA), the 1 ratio 2 complex was converted to a 1 ratio 1 ratio 1 CB8-NRH(+)-BSA complex. The finding that NRH(+) can be transferred from CB8 to BSA, even though the binding constant for NRH(+)-CB8 is much higher than NRH(+)-BSA, is projected for a controlled slow release of NRH(+) towards BSA. Since the release and activity of drugs can be controlled by regulating the protolytic equilibrium, the macromolecular encapsulation and release of NRH(+) demonstrated here provide information relevant to host-guest based drug delivery systems and its applications.

Immunomodulatory effects of betulinic acid from the bark of white birch on mice

The objective of this study was to explore the immunomodulatory effects of betulinic acid (BA) extracted from the bark of white birch on mice. Female mice were orally administered BA for 14 days in doses of 0, 0.25, 0.5, and 1 mg/kg body weight. We found that BA significantly enhanced the thymus and spleen indices, and stimulated lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide as shown by MTT assay. Flow cytometry revealed that BA increased the percentage of CD4+ cells in thymus as well as the percentage of CD19+ and the ratios of CD4+/CD8+ in spleen. BA increased the number of plaque-forming cell and macrophage phagocytic activity as indicated by a neutral red dye uptake assay, and the peritoneal macrophages levels of TNF-α were also increased. In contrast, serum levels of IgG and IgM and serum concentrations of IL-2 and IL-6 were significantly decreased in BA-treated mice compared to the control as assayed by haemagglutination tests and ELISA, respectively. Taken together, these results suggest that BA enhances mouse cellular immunity, humoral immunity, and activity of macrophages. Thus, BA is a potential immune stimulator and may strengthen the immune response of its host.

Study of interaction between kaempferol–Eu 3+ complex and DNA with the use of the Neutral Red dye as a fluorescence probe

The interaction between kaempferol–Eu 3+ complex and calf thymus DNA was investigated in physiological buffer (pH 7.4) using the Neutral Red (NR) dye as a spectral probe by UV–vis absorption and fluorescence spectroscopy, as well as viscosity measurements and DNA melting techniques. The results indicated that kaempferol–Eu 3+ complex can bind to DNA and the major binding mode is intercalative binding. The binding constants K and number of binding sites n of kaempferol–Eu 3+ complex with DNA were obtained by fluorescence quenching method. The thermodynamic parameters (Δ H θ , Δ S θ and Δ G θ ) were calculated from the fluorescence data measured at three different temperatures, which showed that the binding of kaempferol–Eu 3+ complex to DNA was driven mainly by hydrophobic interaction. Furthermore, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data, which provided simultaneously the concentration information for the three reaction components, NR, kaempferol–Eu 3+ and DNA–NR. This ALS analysis indicated that the intercalation of the kaempferol–Eu 3+ complex into the DNA proceeds by exchanging with the NR probe.

Single-walled carbon nanotubes dispersed in aqueous media via non-covalent functionalization: Effect of dispersant on the stability, cytotoxicity, and epigenetic toxicity of nanotube suspensions

As the range of applications for carbon nanotubes (CNTs) rapidly expands, understanding the effect of CNTs on prokaryotic and eukaryotic cell systems has become an important research priority, especially in light of recent reports of the facile dispersion of CNTs in a variety of aqueous systems including natural water. In this study, single-walled carbon nanotubes (SWCNTs) were dispersed in water using a range of natural (gum arabic, amylose, Suwannee River natural organic matter) and synthetic (polyvinyl pyrrolidone, Triton X-100) dispersing agents (dispersants) that attach to the CNT surface non-covalently via different physiosorption mechanisms. The charge and the average effective hydrodynamic diameter of suspended SWCNTs as well as the concentration of exfoliated SWCNTs in the dispersion were found to remain relatively stable over a period of 4 weeks. The cytotoxicity of suspended SWCNTs was assessed as a function of dispersant type and exposure time (up to 48 h) using general viability bioassay with Escherichia coli and using neutral red dye uptake (NDU) bioassay with WB-F344 rat liver epithelia cells. In the E. coli viability bioassays, three types of growth media with different organic loadings and salt contents were evaluated. When the dispersant itself was non-toxic, no losses of E. coli and WB-F344 viability were observed. The cell viability was affected only by SWCNTs dispersed using Triton X-100, which was cytotoxic in SWCNT-free (control) solution. The epigenetic toxicity of dispersed CNTs was evaluated using gap junction intercellular communication (GJIC) bioassay applied to WB-F344 rat liver epithelial cells. With all SWCNT suspensions except those where SWCNTs were dispersed using Triton X-100 (wherein GJIC could not be measured because the sample was cytotoxic), no inhibition of GJIC in the presence of SWCNTs was observed. These results suggest a strong dependence of the toxicity of SWCNT suspensions on the toxicity of the dispersant and point to the potential of non-covalent functionalization with non-toxic dispersants as a method for the preparation of stable aqueous suspensions of biocompatible CNTs.

Optimal Cytocompatibility of a Bioceramic Nanoparticulate Cement in Primary Human Mesenchymal Cells

IntroductionThe aim of this study was to evaluate and compare the cytotoxic effects of BioAggregate (Innovative BioCaramix Inc, Vancouver, BC, Canada), a novel bioceramic nanoparticulate cement, on human mesenchymal cells. White Pro-Root MTA (Dentsply, Tulsa Dental, Tulsa, OK) was used as a reference for comparison. MethodsFifty-six human maxillary incisor teeth were submitted to a step-back flaring technique and prepared for cytotoxicity assay in an in situ root-end filling experimental model. After retro filling, each root containing MTA, BioAggregate, or empty root canals (control) was exposed to culture media for 24, 48, or 72 hours, providing several extraction media. Mesenchymal cells were incubated with each extract medium for 24 hours, and toxicity was evaluated by three different parameters of cell survival and integrity on the same sample: XTT, neutral red, and crystal violet dye elution. ResultsNo statistically significant differences between MTA and BioAggregate were found in all the experimental periods (p > 0.05). ConclusionDiaRoot BioAggregate displayed in vitro compatibility similar to MTA.

Haemocyte lysosomal fragility facing an environmental reality: A toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case

The present study investigates the potential use of haemocyte lysosomal fragility as a reliable indicator of toxic injury in snails subjected to herbicide atrazine. Two cytochemical assays were used. The first one was based on microscopic observations of neutral red dye, and the second one on a fluorescence spectroscopy approach using acridine orange, which had the advantage of being more reliable in assessing minor cell damage. Two sets of experiments were carried out in controlled laboratory conditions (temperature, photoperiod) and in outdoor microcosms, which produce more realistic ecological conditions. An unequivocal relationship was shown, linking atrazine exposure to Lymnaea stagnalis haemocytes lysosomal permeability. High temperatures also had a negative effect on the lysosomal membrane. In the microcosms, the response was affected by seasonal factors, and led to an integrated cell response. It was concluded that lysosomal sensitivity reflects environmental changes. Moreover, the technique using acridine orange was shown to be sufficiently sensitive and reliable to serve as a lysosomal marker in a controlled environment.

Pulmonary toxicity induced by three forms of titanium dioxide nanoparticles via intra-tracheal instillation in rats

Titanium dioxide (TiO 2) nanoparticles are in wide commercial use worldwide. To evaluate if acute pulmonary toxicity can be induced by nano-TiO 2 particles, rats were intra-tracheally instilled with 0.5, 5, or 50 mg/kg of 5, 21, and 50 nm TiO 2 primary particles. Toxic effects were determined with the coefficients of lung tissues to body weight, histopathology, biochemical parameters of blood, activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and acid phosphatase (ACP) in tissues, and the phagocytotic ability of alveolar macrophages (AMs). All the indicators were observed in sacrificed rats one week post-exposure. There was a significant difference of coefficients of pulmonary tissues between the high-dose group and the low- or moderate-dose groups with an exposure of 5 nm TiO 2. At the same time, 5 nm TiO 2 primary particles increased the activity of LDH and ALP when exposure dose was >5 mg/kg. A significant difference in LDH and ALP activity was observed between the 50 mg/kg group and 0.5 or 5 mg/kg group with exposure of 5 nm TiO 2. Lung tissues showed increased ALP activity only if treated with 5 and 50 mg/kg of 21 nm TiO 2 particles. There was no significant difference in LDH and ALP activity in the 50 nm TiO 2 group and control group. Histopathologic examination of lung tissues indicated that the pulmonary response to exposure to TiO 2 particles in rats manifested as dose-dependent inflammatory lesions, which mainly consisted of infiltration of inflammatory cells and interstitial thickening. Analysis of uptake of neutral red dye showed that 50 nm TiO 2 particles significantly increased phagocytotic ability of AMs compared with controls ( P < 0.05), whereas exposure with 5 nm TiO 2 reduced the phagocytotic ability of AMs when the exposure dose was 50 mg/kg. These results suggest that particle size and exposure dose may have important roles in pulmonary toxicity. The toxic effect of TiO 2 nanoparticles in lung tissue exhibited a dose–response relationship. After exposure with TiO 2 particles of >5.0 mg/kg, 5 and 21 nm TiO 2 particles induced standing pulmonary lesions; and 5 nm TiO 2 particles may suppress the phagocytotic ability of AMs if exposure dose was ⩾50 mg/kg. Pulmonary toxicity caused by 5 nm TiO 2 particles was more severe than that caused by 21 and 50 nm TiO 2 particles.

Anti-influenza A virus effect of Hypericum perforatum L. extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin’s minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.

L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay

This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37 degrees C in a humidified atmosphere of 5% CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.

The use of established skeletal muscle cell lines to assess potential toxicity from embedded metal fragments

The use of novel materials on the modern battlefield, both in military munitions as well as in Improvised Explosive Devices, opens the possibility of wounds with embedded fragments whose health effects and toxicity characteristics have not been fully investigated, if at all. The costly and time-consuming nature of standard two-year lifespan studies prohibits the testing of many materials. In this report, we describe an in vitro system for rapidly assessing potential toxicity of metals and metal mixtures. Using rat L6 and mouse C2C12 skeletal muscle cells and tests for cellular viability, we have shown that two militarily relevant tungsten alloy mixtures (W/Ni/Co and W/Ni/Fe) significantly decreased the metabolic viability of rat L6 cells, whereas the viability of mouse C2C12 cells was not affected by W/Ni/Co and only slightly affected by W/Ni/Fe. In addition, viability assessed through lysosomal uptake of neutral red dye was not affected by either mixture in either cell line indicating that the mitochondria may be the target organelle of these unique metal mixtures. Development of this in vitro screening system may provide a procedure by which the potential toxicities of embedded metal fragments can be rapidly assessed.

Solvent effect on the spectral properties of Neutral Red

BackgroundThe study was aimed at investigating the effect of various solvents on the absorption spectra of Neutral Red, a dye belonging to the quinone-imine class of dyes. The solvents chosen for the study were water, ethanol, acetonitrile, acetone, propan-1-ol, chloroform, nitrobenzene, ethyleneglycol, acetic acid, DMSO and DMF.ResultsThe results have shown that the absorption maxima of dyes are dependent on solvent polarity. In non-hydrogen-bond donating solvents, solvation of dye molecules probably occurs via dipole-dipole interactions, whereas in hydrogen-bond donating solvents the phenomenon is more hydrogen bonding in nature. To estimate the contribution of the different variables on the wave number of the Neutral Red dye, regression analyses using the ECW model were compared with the π* scale model. This showed that the unified scale for estimating the solvent effect on the absorption of the Neutral Red dye is more adopted and more applicable than the π* scale model.ConclusionAbsorption maxima of dyes are dependent on solvent polarity. Solvation of dye molecules probably occurs via dipole-dipole interactions in non-hydrogen-bond donating solvents, whereas in hydrogen-bond donating solvents the phenomenon is more hydrogen bonding in nature. The unified scale for estimating the solvent effect on the absorption of Neutral Red dye is more adopted and more applicable than the π* scale model. This may be due to complications from both π-π* charge transfer interactions and incomplete complexation of the solute; these effects are averaged out in the derived β and π parameters and thus limit their applicability.

A comparison between the responses of neutral red and acridine orange: Acridine orange should be preferential and alternative to neutral red as a dye for the monitoring of contaminants by means of biological sensors

The acridine orange (AO) and neutral red (NR) dyes, commonly used as probes to measure the internal pH in acidic vesicles, are compared in this article. The comparison between the two dyes (arising from calculations taking into account their analytical constants) illustrated that the use of AO is preferential to that of NR because the AO response is sensitive over the whole pH range between 4.0 and 7.4, whereas the NR response is effective only between pHs 4.0 and 6.0. In addition, it became evident from the mitochondrial respiration response that NR, unlike AO, is a protonophore. When taken into consideration, these two properties suggest that AO is more suitable than NR as an indicator of toxicity measurements in water samples because the environmental toxic compounds induce pH changes in the acidic vesicles of biological structures that are used as environmental biosensors.

Development of robust antiviral assays for profiling compounds against a panel of positive-strand RNA viruses using ATP/luminescence readout

The development of antiviral assays using an ATP/luminescence-based readout to profile antiviral compounds against the positive-strand RNA viruses: yellow fever virus (YFV), West Nile virus (WNV), Sindbis virus, and Coxsackie B virus, representing three virus families, is described. This assay readout is based upon the bioluminescent measurement of ATP in metabolically active cells. Antiviral efficacy was determined by measuring the ATP level in cells that were protected from the viral cytopathic effect (CPE) by the presence of antiviral compounds. The antiviral assay parameters were optimized and the assays were validated using a panel of different reference compounds to determine the intra- and inter-assay reproducibility. The signal-to-noise ratios for the yellow fever virus and West Nile virus assays were 7.5 and 36, respectively, comparing favorably with a signal-to-noise ratio of only 1.5 in the yellow fever virus neutral red dye uptake assay, an alternative readout for CPE inhibition. For Coxsackie B and Sindbis viruses, the signal-to-noise ratios were 40 and 50, respectively. These assays are robust, high-throughput, reproducible, and give much improved signal-to-noise ratios than those of dye uptake assays.