In situ-synthesized virulence and marker gene biochip for detection of bacterial pathogens in water.

Pathogen detection tools with high reliability are needed for various applications, including food and water safety and clinical diagnostics. In this study, we designed and validated an in situ-synthesized biochip for detection of 12 microbial pathogens, including a suite of pathogens relevant to water safety. To enhance the reliability of presence/absence calls, probes were designed for multiple virulence and marker genes (VMGs) of each pathogen, and each VMG was targeted by an average of 17 probes. Hybridization of the biochip with amplicon mixtures demonstrated that 95% of the initially designed probes behaved as predicted in terms of positive/negative signals. The probes were further validated using DNA obtained from three different types of water samples and spiked with pathogen genomic DNA at decreasing relative abundance. Excellent specificity for making presence/absence calls was observed by using a cutoff of 0.5 for the positive fraction (i.e., the fraction of probes yielding a positive signal for a given VMG). A split multiplex PCR design for simultaneous amplification of the VMGs resulted in a detection limit of between 0.1 and 0.01% relative abundance, depending on the type of pathogen and the VMG. Thermodynamic analysis of the hybridization patterns obtained with DNA from the different water samples demonstrated that probes with a hybridization Gibbs free energy of approximately -19.3 kcal/mol provided the best trade-off between sensitivity and specificity. The developed biochip may be used to detect the described bacterial pathogens in water samples when parallel and specific detection is required.

Development and experimental validation of a predictive threshold cycle equation for quantification of virulence and marker genes by high-throughput nanoliter-volume PCR on the OpenArray platform.

Development of quantitative PCR (QPCR) assays typically requires extensive screening within and across a given species to ensure specific detection and lucid identification among various pathogenic and nonpathogenic strains and to generate standard curves. To minimize screening requirements, multiple virulence and marker genes (VMGs) were targeted simultaneously to enhance reliability, and a predictive threshold cycle (C(T)) equation was developed to calculate the number of starting copies based on an experimental C(T). The empirical equation was developed with Sybr green detection in nanoliter-volume QPCR chambers (OpenArray) and tested with 220 previously unvalidated primer pairs targeting 200 VMGs from 30 pathogens. A high correlation (R(2) = 0.816) was observed between the predicted and experimental C(T)s based on the organism's genome size, guanine and cytosine (GC) content, amplicon length, and stability of the primer's 3' end. The performance of the predictive C(T) equation was tested using 36 validation samples consisting of pathogenic organisms spiked into genomic DNA extracted from three environmental waters. In addition, the primer success rate was dependent on the GC content of the target organisms and primer sequences. Targeting multiple assays per organism and using the predictive C(T) equation are expected to reduce the extent of the validation necessary when developing QPCR arrays for a large number of pathogens or other targets.

Diagnostic microarray for 14 water and foodborne pathogens using a flatbed scanner.

Parallel detection approaches are of interest to many researchers interested in identifying multiple water and foodborne pathogens simultaneously. Availability and cost-effectiveness are two key factors determining the usefulness of such approaches for laboratories with limited resources. In this study, we developed and validated a high-density microarray for simultaneous screening of 14 bacterial pathogens using an approach that employs gold labeling with silver enhancement (GLS) protocol. In total, 8887 probes (50-mer) were designed using an in-house database of virulence and marker genes (VMGs), and synthesized in quadruplicate on glass slides using an in-situ synthesis technology. Target VMG amplicons were obtained using multiplex polymerase chain reaction (PCR), labeled with biotin, and hybridized to the microarray. The signals generated after gold deposition and silver enhancement, were quantified using a flatbed scanner having 2-µm resolution. Data analysis indicated that reliable presence/absence calls could be made, if: i) over four probes were used per gene, ii) the signal-to-noise ratio (SNR) cutoff was greater than or equal to two, and iii) the positive fraction (PF), i.e., number of probes with SNR≥2 for a given VMG was greater than 0.75. Hybridization of the array with blind samples resulted in 100% correct calls, and no false positive. Because amplicons were obtained by multiplex PCR, sensitivity of this method is similar to PCR. This assay is an inexpensive and reliable technique for high throughput screening of multiple pathogens.

Personality, Cognitive Style, Motivation, and Aptitude Predict Systematic Trends in Analytic Forecasting Behavior.

The decision sciences are increasingly challenged to advance methods for modeling analysts, accounting for both analytic strengths and weaknesses, to improve inferences taken from increasingly large and complex sources of data. We examine whether psychometric measures-personality, cognitive style, motivated cognition-predict analytic performance and whether psychometric measures are competitive with aptitude measures (i.e., SAT scores) as analyst sample selection criteria. A heterogeneous, national sample of 927 participants completed an extensive battery of psychometric measures and aptitude tests and was asked 129 geopolitical forecasting questions over the course of 1 year. Factor analysis reveals four dimensions among psychometric measures; dimensions characterized by differently motivated "top-down" cognitive styles predicted distinctive patterns in aptitude and forecasting behavior. These dimensions were not better predictors of forecasting accuracy than aptitude measures. However, multiple regression and mediation analysis reveals that these dimensions influenced forecasting accuracy primarily through bias in forecasting confidence. We also found that these facets were competitive with aptitude tests as forecast sampling criteria designed to mitigate biases in forecasting confidence while maximizing accuracy. These findings inform the understanding of individual difference dimensions at the intersection of analytic aptitude and demonstrate that they wield predictive power in applied, analytic domains.

Enhanced arsenic removal using mixed metal oxide impregnated chitosan beads.

Mixed metal oxide impregnated chitosan beads (MICB) containing nanocrystalline Al2O3 and nanocrystalline TiO2 were successfully developed. This adsorbent exploits the high capacity of Al2O3 for arsenate and the photocatalytic activity of TiO2 to oxidize arsenite to arsenate, resulting in a removal capacity higher than that of either metal oxide alone. The composition of the beads was optimized for maximum arsenite removal in the presence of UV light. The mechanism of removal was investigated and a mode of action was proposed wherein TiO2 oxidizes arsenite to arsenate which is then removed from solution by Al2O3. Pseudo-second order kinetics were used to validate the proposed mechanism. MICB is a more efficient and effective adsorbent for arsenic than TiO2-impregnated chitosan beads (TICB), previously reported on, yet maintains a desirable life cycle, free of complex synthesis processes, toxic materials, and energy inputs.

Optimization of capacity and kinetics for a novel bio-based arsenic sorbent, TiO2-impregnated chitosan bead.

The optimization of TiO(2)-impregnated chitosan beads (TICB) as an arsenic adsorbent is investigated to maximize the capacity and kinetics of arsenic removal. It has been previously reported that TICB can 1) remove arsenite, 2) remove arsenate, and 3) oxidize arsenite to arsenate in the presence of UV light and oxygen. Herein, it is reported that adsorption capacity for TICB is controlled by solution pH and TiO(2) loading within the bead and enhanced with exposure to UV light. Solution pH is found to be a critical parameter, whereby arsenate is effectively removed below pH 7.25 and arsenite is effectively removed below pH 9.2. A model to predict TICB capacity, based on TiO(2) loading and solution pH, is presented for arsenite, arsenate, and total arsenic in the presence of UV light. The rate of removal is increased with reductions in bead size and with exposure to UV light. Phosphate is found to be a direct competitor with arsenate for adsorption sites on TICB, but other relevant common background groundwater ions do not compete with arsenate for adsorption sites. TICB can be regenerated with weak NaOH and maintain full adsorption capacity for at least three adsorption/desorption cycles.

Novel, bio-based, photoactive arsenic sorbent: TiO2-impregnated chitosan bead.

A novel sorbent for arsenic, TiO(2)-impregnated chitosan bead (TICB), has been synthesized and successfully tested. Kinetic plots, pH dependence, isotherm data, and bead morphology are reported. Equilibrium is achieved after 185 h in batch experiments with exposure to UV light. The TICB system performs similarly to the mass equivalent of neat TiO(2) nanopowder. The point of zero charge (pzc) for TICB was determined to be 7.25, and as with other TiO(2)-based arsenic removal technologies, the optimal pH range for sorption is below this pH(pzc). Without exposure to UV light, TICB removes 2198 µg As(III)/g TICB and 2050 µg As(V)/g TICB. With exposure to UV light, TICB achieves photo-oxidation of As(III) to As(V), the less toxic and more easily sequestered arsenic form. UV irradiation also results in enhanced arsenic removal, reaching sorption capacities of 6400 µg As/g TICB and 4925 µg As/g TICB, where arsenic is initially added as As(III) and As(V), respectively. Because the TICB system obviates filtration post-treatment, TICB is superior to TiO(2) nanopowder from the perspective of implementation for decentralized water treatment.

Toward understanding the efficacy and mechanism of Opuntia spp. as a natural coagulant for potential application in water treatment.

Historically, there is evidence to suggest that communities in the developing world have used plant-based materials as one strategy for purifying drinking water. In this study, the coagulant properties of Opuntia spp., a species of cactus, are quantitatively evaluated for the first time. Opuntia spp. was evaluated for turbidity removal from synthetic water samples, and steps were made toward elucidating the underlying coagulation mechanism. In model turbid water using kaolin clay particles at pH 10, Opuntia spp. reduced turbidity by 98% for a range of initial turbidities. This is similar to the observed coagulation activities previously described for Moringa oleifera, a widely studied natural coagulant. Although it has been reported that Moringa oleifera predominantly operates through charge neutralization, comparison of zeta potential measurements and transmission electron microscopy images of flocs formed by Opuntia spp. suggest that these natural coagulants operate through different mechanisms. It is suggested that Opuntia spp. operates predominantly through a bridging coagulation mechanism. Once optimized, application of these readily available plants as a part of point-of-use water treatment technology may offer a practical, inexpensive, and appropriate solution for producing potable water in some developing communities.

Stable colloidal dispersions of C60 fullerenes in water: evidence for genotoxicity.

Stable aqueous suspensions of colloidal C60 fullerenes free of toxic organic solvents were prepared by two methods: ethanol to water solvent exchange (EthOH/nC60 suspensions) and extended mixing in water (aqu/nC60 suspensions). The extended mixing method resulted in the formation of larger (dp approximately 178 nm) and less negatively charged (zeta approximately -13.5 mV) nC60 colloids than nC60 prepared by ethanol to water solvent exchange (dp approximately 122 nm, zeta approximately -31.6 mV). Genotoxicity of these suspensions was evaluated with respect to human lymphocytes using single-cell gel electrophoresis assay (Comet assay). The assay demonstrated genotoxicity for both types of suspensions with a strong correlation between the genotoxic response and nC60 concentration, and with genotoxicity observed at concentrations as low as 2.2 microg/L for aqu/nC60 and 4.2 microg/L for EtOH/nC60. The Olive tail moments (OTM) for these two concentrations were 1.54 +/- 0.24 and 1.34 +/- 0.07, respectively, which in comparison to the negative control OTM of 0.98 +/- 0.17 is statistically different with a p value of at least 0.05. Aqu/nC60 suspensions elicited higher genotoxic response than EthOH/nC60 for the same nC60 concentration. The results represent the first genotoxicity data for colloidal fullerenes produced by simple mixing in water.