Rapid detection of resistance to pyrazinamide in Mycobacterium tuberculosis using the resazurin microtitre assay

Objectives: The resazurin microtitre plate assay (REMA) was evaluated to determine the susceptibility of Mycobacterium tuberculosis to pyrazinamide, and was compared with the broth microdilution method (BMM), the absolute concentration method (ACM) and pyrazinamidase (PZase) determination. Methods: Thirty-four M. tuberculosis clinical isolates (26 susceptible and 8 resistant to pyrazinamide) and reference strains M. tuberculosis H37Rv ATCC 27294 and Mycobacterium bovis AN5 were tested. Results: REMA and BMM showed 100% specificity and sensitivity when compared with ACM; BMM, however, demanded more reading time. The PZase determination assay showed 87.50% and 100% sensitivity and specificity, respectively. Conclusions: All tested methods in this preliminary study showed excellent sensitivity and specificity for the determination of pyrazinamide susceptibility of M. tuberculosis, but REMA was faster, low-cost and easy to perform and interpret. Additional studies evaluating REMA for differentiating pyrazinamide-resistant and-susceptible M. tuberculosis should be conducted on an extended panel of clinical isolates.

Comparison of the Salmonella/Microsome Microsuspension Assay with the new Microplate Fluctuation Protocol for Testing the Mutagenicity of Environmental Samples

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multi-channel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity. Environ. Mol. Mutagen. 51:31-38, 2010. (C) 2009 Wiley-Liss, Inc.

Sterilization by pure oxygen plasma and by oxygen-hydrogen peroxide plasma: An efficacy study

Plasma is an innovative sterilization method characterized by a low toxicity to operators and patients, and also by its operation at temperatures close to room temperatures. The use of different parameters for this method of sterilization and the corresponding results were analyzed in this study. A low-pressure inductive discharge was used to study the plasma sterilization processes. Oxygen and a mixture of oxygen and hydrogen peroxide were used as plasma source gases. The efficacy of the processes using different combinations of parameters such as plasma-generation method, type of gas, pressure, gas flow rate, temperature, power, and exposure time was evaluated. Two phases were developed for the processes, one using pure oxygen and the other a mixture of gases. Bacillus subtilis var. niger ATCC 9372 (Bacillus atrophaeus) spores inoculated on glass coverslips were used as biological indicators to evaluate the efficacy of the processes. All cycles were carried out in triplicate for different sublethal exposure times to calculate the D value by the enumeration method. The pour-plate technique was used to quantify the spores. D values of between 8 and 3 min were obtained. Best results were achieved at high power levels (350 and 40oW) using pure oxygen, showing that plasma sterilization is a promising alternative to other sterilization methods. (c) 2007 Elsevier B.V. All rights reserved.

Neuropharmacological effects in mice of Lychnophora species (Vernonieae, Asteraceae) and anticonvulsant activity of 4,5-di-O-[E]-caffeoylquinic acid isolated from the stem of L-rupestris and L-staavioides

Aiming at contributing with the search for neuroactive substances from natural sources, we report for the first time antinociceptive and anticonvulsant effects of some Lychnophora species. We verify the protective effects of polar extracts (600 mg/kg, intraperitoneally), and methanolic fractions of L. staavioides and L. rupestris (100 mg/kg, intraperitoneally) in pentylenetetrazole-induced seizures on mice. Previously, a screening was accomplished, evaluating the antinociceptive central activity (hot plate test), with different extracts of L. rupestris, L. staavioides and L. diamantinana. It was possible to select the possible extracts of Lychnophora with central nervous system activity. Some of the active extracts were submitted to fractionation and purification process and the methanolic fractions of L. rupestris (stem) and L. staavioides (stem), with anticonvulsant properties (100 mg/kg, intraperitoneally), yielded 4,5-di-O-[E]-caffeoylquinic acid. This substance was injected intraperitoneally in mice and showed anticonvulsant effect against pentylenetetrazole-induced seizures at doses of 25 and 50 mg/kg. It has often been shown that seizures induced by pentylenetetrazole are involved in inhibition and/or attenuation of GABAergic neurotransmission. However, other systems of the central nervous system such as adenosinergic and glutamatergic could be involved in the caffeoylquinic acid effects. Further studies should be conducted to verify that the target receptor could be participating in this anticonvulsant property. Although other investigations have reported a series of biological activities from Lychnophora species, this is the first report of central analgesic and anticonvulsant activity in species of this genus.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties. The aim of this study was to evaluate the in vitro effect of the essential oil of Thymus vulgaris (thyme) pure or incorporate in a alcohol-free pharmaceutical mouthwash formulation, against Streptococcus mutans (ATCC 25175), being determined the Minimal Inhibitory Concentration (MIC) and the effect in the bacterial plate formation. The MIC value obtained for the essential oil was 100 mu g/mL (1 %). The mouthwash pharmaceutical formulation containing commercial essential oil of T. vulgaris was preparated. Microbiological and macroscopic analysis as well as analyses for MEV confirmed the effectiveness of this new alcohol-free mouthwash formulation containing essential oil of T. vulgaris as agent with plaque-inhibiting properties and possible application in the preventive dentistry. The chemical characterization of the bioactive essential oil was accomplished by CG-MS, being verified the presence of carvacrol, p-cimene and alpha-pinene as major constituents.

Bed shear stress measurements in dam break and swash flows

A novel shear plate was used to make direct bed shear stress measurements in laboratory dam break and swash flows on smooth, fixed, impermeable beds. The pressure gradient due to the slope of the fluid free-surface across the plate was measured using pressure transducers. Surface elevation was measured at five locations using acoustic displacement sensors. Flow velocity was measured using an Acoustic-Doppler Velocimeter and calculated using the ANUGA inundation model. The measured bed shear stress at the dam break fluid tip for an initially dry, horizontal bed was close to twice that estimated using steady flow theory. The temporal variation of swash bed shear stress showed a large peak in landward directed stress at the uprush tip, followed by a rapid decay throughout the uprush flow interior. The peak seaward directed stress during the backwash phase was less than half that measured in the uprush. Close to the still water line, in the region of bore collapse and at the time of initial uprush, favourable pressure gradients were measured. In the lower swash region predominately weak adverse pressure gradients were measured.

Enhanced flow visualisation using near-resonant holographic interferometry

Holographic interferometry measurements have been performed on high-speed, high-temperature gas flows with a laser output tuned near a resonant sodium transition. The technique allows the detection and quantification of the sodium concentration in the flow. By controlling the laser detuning and seeded sodium concentration, we performed flow visualization in low-density flows that are not normally detectable with standard interferometry. The technique was also successfully used to estimate the temperature in the boundary layer of the flow over a flat plate.

Effects of viscous dissipation and boundary conditions on forced convection in a channel occupied by a saturated porous medium

Forced convection with viscous dissipation in a parallel plate channel filled by a saturated porous medium is investigated numerically. Three different viscous dissipation models are examined. Two different sets of wall conditions are considered: isothermal and isoflux. Analytical expressions are also presented for the asymptotic temperature profile and the asymptotic Nusselt number. With isothermal walls, the Brinkman number significantly influences the developing Nusselt number but not the asymptotic one. At constant wall heat flux, both the developing and the asymptotic Nusselt numbers are affected by the value of the Brinkman number. The Nusselt number is sensitive to the porous medium shape factor under all conditions considered.

Effects of viscous dissipation and boundary conditions on forced convection in a channel occupied by a saturated porous medium

Forced convection with viscous dissipation in a parallel plate channel filled by a saturated porous medium is investigated numerically. Three different viscous dissipation models are examined. Two different sets of wall conditions are considered: isothermal and isoflux. Analytical expressions are also presented for the asymptotic temperature profile and the asymptotic Nusselt number. With isothermal walls, the Brinkman number significantly influences the developing Nusselt number but not the asymptotic one. At constant wall heat flux, both the developing and the asymptotic Nusselt numbers are affected by the value of the Brinkman number. The Nusselt number is sensitive to the porous medium shape factor under all conditions considered.

Non-Equilibrium Reaction Rates in the Macroscopic Chemistry Method for DSMC Calculations

The Direct Simulation Monte Carlo (DSMC) method is used to simulate the flow of rarefied gases. In the Macroscopic Chemistry Method (MCM) for DSMC, chemical reaction rates calculated from local macroscopic flow properties are enforced in each cell. Unlike the standard total collision energy (TCE) chemistry model for DSMC, the new method is not restricted to an Arrhenius form of the reaction rate coefficient, nor is it restricted to a collision cross-section which yields a simple power-law viscosity. For reaction rates of interest in aerospace applications, chemically reacting collisions are generally infrequent events and, as such, local equilibrium conditions are established before a significant number of chemical reactions occur. Hence, the reaction rates which have been used in MCM have been calculated from the reaction rate data which are expected to be correct only for conditions of thermal equilibrium. Here we consider artificially high reaction rates so that the fraction of reacting collisions is not small and propose a simple method of estimating the rates of chemical reactions which can be used in the Macroscopic Chemistry Method in both equilibrium and non-equilibrium conditions. Two tests are presented: (1) The dissociation rates under conditions of thermal non-equilibrium are determined from a zero-dimensional Monte-Carlo sampling procedure which simulates ‘intra-modal’ non-equilibrium; that is, equilibrium distributions in each of the translational, rotational and vibrational modes but with different temperatures for each mode; (2) The 2-D hypersonic flow of molecular oxygen over a vertical plate at Mach 30 is calculated. In both cases the new method produces results in close agreement with those given by the standard TCE model in the same highly nonequilibrium conditions. We conclude that the general method of estimating the non-equilibrium reaction rate is a simple means by which information contained within non-equilibrium distribution functions predicted by the DSMC method can be included in the Macroscopic Chemistry Method.

Apparent insensitivity of the hotplate latency test for detection of antinociception following intraperitoneal, intravenous or intracerebroventricular M6G administration to rats

Although morphine-6-glucuronide (M6G) has been shown to be analgesically active, the relative involvement of spinal and supraspinal structures in mediating M6G's pain-relieving effects following central and systemic administration to rats is unclear. As the tail flick and hotplate latency tests are reported to quantify antinociception mediated primarily by spinal and supraspinal mechanisms respectively, these methods were used to determine the comparative apparent levels of antinociception (expressed as percentage maximum possible effect, % MPE) achieved after M6G or morphine administration. Following i.v. or i.p. M6G (1.9-5.4 mu mol) dosing or i.p. morphine (10 mu mol) dosing, high levels of antinociception (>50% MPE) were achieved using the tail flick test whereas base-line levels of antinociception were observed 30 sec later in the same rats using the hotplate test. By contrast, antinociception evoked by i.v. morphine (10 mu mol) exceeded 50% MPE using both the hotplate and tail flick tests although the apparent potency was approximately 2.5 times greater using the tail flick test. After i.c.v. dosing, M6G (0.22-3.3 nmol) was significantly (P < .05) more potent when assessed using the tail flick compared with the hotplate test. Taken together, these data strongly indicate that following central and systemic administration, M6G's antinociceptive effects are mediated primarily by spinal structures whereas both spinal and supraspinal mechanisms contribute to systemic morphine's antinociceptive effects.

The Fornax spectroscopic survey I. Survey strategy and preliminary results on the redshift distribution of a complete sample of stars and galaxies

The Fornax Spectroscopic Survey will use the Two degree Field spectrograph (2dF) of the Angle-Australian Telescope to obtain spectra for a complete sample of all 14000 objects with 16.5 less than or equal to b(j) less than or equal to 19.7 in a 12 square degree area centred on the Fornax Cluster. The aims of this project include the study of dwarf galaxies in the cluster (both known low surface brightness objects and putative normal surface brightness dwarfs) and a comparison sample of background field galaxies. We will also measure quasars and other active galaxies, any previously unrecognised compact galaxies and a large sample of Galactic stars. By selecting all objects-both stars and galaxies-independent of morphology, we cover a much larger range of surface brightness and scale size than previous surveys. In this paper we first describe the design of the survey. Our targets are selected from UK Schmidt Telescope sky survey plates digitised by the Automated Plate Measuring (APM) facility. We then describe the photometric and astrometric calibration of these data and show that the APM astrometry is accurate enough for use with the 2dF. We also describe a general approach to object identification using cross-correlations which allows us to identify and classify both stellar and galaxy spectra. We present results from the first 2dF field. Redshift distributions and velocity structures are shown for all observed objects in the direction of Fornax, including Galactic stars? galaxies in and around the Fornax Cluster, and for the background galaxy population. The velocity data for the stars show the contributions from the different Galactic components, plus a small tail to high velocities. We find no galaxies in the foreground to the cluster in our 2dF field. The Fornax Cluster is clearly defined kinematically. The mean velocity from the 26 cluster members having reliable redshifts is 1560 +/- 80 km s(-1). They show a velocity dispersion of 380 +/- 50 km s(-1). Large-scale structure can be traced behind the cluster to a redshift beyond z = 0.3. Background compact galaxies and low surface brightness galaxies are found to follow the general galaxy distribution.

Allelic loss of cyclophilin 40, an estrogen receptor-associated immunophilin, in breast carcinomas

Purpose: Cyclophilin 40 (CyP40) is an estrogen receptor-associated protein which appears to modify receptor function. The aim of this study was to determine the extent of allelic loss at the CyP40 locus in a panel of breast carcinomas using a newly characterized microsatellite marker located upstream of the CyP40 gene and then to correlate this with losses at chromosomal sites for cancer-associated genes. Methods: Allelic loss at CyP40 was determined from patients' matched tumor and normal breast tissue using Genescan 672 software analysis of fluorescently labeled, PAGE-separated PCR products incorporating the marker. For each patient, allelic loss at CyP40 was then assessed and compared with losses at markers for various cancer-associated genes. Results: Allelic loss was detected in 30% of breast carcinomas from patients heterozygous for the CyP40 marker. All carcinomas demonstrating allelic loss were grade II or III invasive ductal carcinomas and generally showed multiple losses at other sites near known cancer-associated genes. Conclusions: The polymorphic marker which we characterized was useful in determining allelic loss at the CyP40 locus in breast cancer patients and when applied in these studies in conjunction with various cancer-associated gene markers, suggests that deletions in the region of the CyP40 gene might be a late event in breast tumor progression.

Quasars from a complete spectroscopic survey

With the advent of multi-fibre spectrographs such as the 'Two-Degree Field' (2dF) instrument at the Angle-Australian Telescope, quasar surveys that are free of any preselection of candidates and any biases this implies have become possible for the first time. The first of these is that which is being undertaken as part of the Fornax Spectroscopic Survey, a survey of the area around the Fornax Cluster of galaxies, and aims to obtain the spectra of all objects in the magnitude range 16.5 < b(j) < 19.7. To date, 3679 objects in the central pi -deg(2) area have been successfully identified from their spectral characteristics. Of these, 71 are found to be quasars, 61 with redshifts 0.3 < z < 2.2 and 10 with redshifts z > 2.2. Using this complete quasar sample, a new determination of quasar number counts is made, enabling an independent check of existing quasars surveys. Cumulative counts per square degree at a magnitude limit of b(j) < 19.5 are found to be 11.5 +/- 2.2 for 0.3 < z < 2.2, 2.22 +/- 0.93 for z > 2.2 and 13.7 +/- 3.1 for z > 0.3. Given the likely detection of extra quasars in the Fornax survey, we make a more detailed examination of existing quasar selection techniques. First, looking at the use of a stellar criterion, four of the 71 quasars are 'non-stellar' on the basis of the automated plate measuring facility (APM) b(j) classification, however inspection shows all are consistent with stellar, but misclassified due to image confusion. Examining the ultraviolet excess and multicolour selection techniques, for the selection criteria investigated, ultraviolet excess would find 69 +/- 6 per cent of our 0.3 < z < 2.2 quasars and only 50(-18)(+14), per cent of our z > 2.2 quasars, while the completeness level for multicolour selection is found to be 90(-4)(+3) per cent for 0.3 < z < 2.2 quasars and 80(-12)(+14) per cent for z > 2.2 quasars. The extra quasars detected by our all-object survey thus have unusually red star-like colours, and this appears to be a result of the continuum shape rather than any emission features. An intrinsic dust extinction model may, at least partly, account for the red colours.