Achieving Health Security and Threat Reduction through Sharing Sequence Data.

With the rapid development and broad applications of next-generation sequencing platforms and bioinformatic analytical tools, genomics has become a popular area for biosurveillance and international scientific collaboration. Governments from countries including the United States (US), Canada, Germany, and the United Kingdom have leveraged these advancements to support international cooperative programs that aim to reduce biological threats and build scientific capacity worldwide. A recent conference panel addressed the impacts of the enhancement of genomic sequencing capabilities through three major US bioengagement programs on international scientific engagement and biosecurity risk reduction. The panel contrasted the risks and benefits of supporting the enhancement of genomic sequencing capabilities through international scientific engagement to achieve biological threat reduction and global health security. The lower costs and new bioinformatic tools available have led to the greater application of sequencing to biosurveillance. Strengthening sequencing capabilities globally for the diagnosis and detection of infectious diseases through mutual collaborations has a high return on investment for increasing global health security. International collaborations based on genomics and shared sequence data can build and leverage scientific networks and improve the timeliness and accuracy of disease surveillance reporting needed to identify and mitigate infectious disease outbreaks and comply with international norms. Further efforts to promote scientific transparency within international collaboration will improve trust, reduce threats, and promote global health security.

Simple and rapid extraction, separation, and detection of alkaloids in beverages.

Implementation of an uncomplicated SPE process for the rapid extraction and preconcentration of the alkaloids, colchicine, strychnine, aconitine, and nicotine, from water, apple juice, and nonfat milk samples is presented. When coupled to analysis via micellar EKC (MEKC), the total analysis time per sample was less than 15 min for the water and juice samples and less than 20 min for the milk. The SPE process allowed for anywhere from a three to a fourteen-fold improvement in the LOD for each alkaloid when compared to detecting the alkaloids in a nontreated water sample matrix. Following SPE, the LODs for colchicine, strychnine, and nicotine were sufficient to meet levels from 150 to 5000 times more dilute than the LD(50) for a 50 kg individual drinking 12 oz of a contaminated beverage. Aconitine, on the other hand, was detected at approximately the LD(50) level. The percent recoveries for the SPE ranged from 37% to as high as 99%. Nicotine attained the highest recovery efficiencies, followed by colchicine, and finally, aconitine and strychnine, which were nearly identical. The greatest recovery efficiencies were achieved from apple juice and water, whereas nonfat milk yielded the lowest.

Microchip micellar electrokinetic chromatography separation of alkaloids with UV-absorbance spectral detection.

A microchip device is demonstrated for the electrophoretic separation and UV-absorbance spectral detection of four toxic alkaloids: colchicine, aconitine, strychnine, and nicotine. A fused-silica (quartz) microchip containing a simple cross geometry is utilized to perform the separations, and a miniature, fiber-optic CCD spectrometer is coupled to the microchip for detection. Sensitive UV-absorbance detection is achieved via the application of online preconcentration techniques in combination with the quartz microchip substrate which contains an etched bubble-cell for increased pathlength. The miniature CCD spectrometer is configured to detect light between 190 and 645 nm and LabView programming written in-house enables absorbance spectra as well as separations to be monitored from 210 to 400 nm. Consequently, the configuration of this microchip device facilitates qualitative and quantitative separations via simultaneous spatial and spectral resolution of solutes. UV-absorbance limits of quantification for colchicine, 20 microM (8 mg/L); strychnine, 50 microM (17 mg/L); aconitine, 50 microM (32 mg/L); and nicotine, 100 microM (16 mg/L) are demonstrated on the microchip. With the exception of aconitine, these concentrations are > or =20-times more sensitive than lethal dose monitoring requirements. Finally, this device is demonstrated to successfully detect each toxin in water, skim milk, and apple juice samples spiked at sublethal dose concentrations after a simple, SPE procedure.

Advances in CE for kinetic studies.

CE is a promising technique for the investigation of molecular interactions because it affords evaluation of multiple interaction modes, does not require immobilization of molecules, and has no dead time. In order to perform these investigations, numerous methods have been developed for determining binding constants and other thermodynamic parameters. These methods have been reviewed extensively in recent years. However, methods for determining the rates of reaction are less prolific. Nonetheless, numerous theoretical and experimental advances have been made in recent years to address this discrepancy. Some of these methods employ computer simulations to determine first-order or second-order rate constants numerically, whereas other methods calculate rate constants directly as solutions to analytical equations. It is the object of this review to provide descriptions of these methods in terms of their underlying assumptions, experimental methodology, calculation of rate constants, and inherent limitations.

Micelle stacking in micellar electrokinetic chromatography.

In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.

Capillary electrophoresis for thermodynamic and kinetic studies of peptidyl-proline isomerization by the theoretical plate height model.

The theoretical plate height model, extended to include reactive CE, is used to calculate equilibrium constants and rate constants for the reversible, first-order isomerization of proline dipeptides. This model is consistent with chromatographic theory and enables calculation of equilibrium constants from velocity and calculation of rate constants from plate height. Thermodynamic and kinetic parameters for isomerization of Ala-Pro and Phe-Pro are calculated by using the plate height model, and are shown to be in good agreement with literature values. Additionally, the efficacy of the plate height model is compared to ChromWin, an existing simulation method for calculating rate constants from zone profiles. It is shown that ChromWin and the plate height model are complementary methods. ChromWin is best used for calculating rate constants for reactions that are far from steady state, where the zone profiles exhibit plateau formation. On the other hand, the plate height model is best used for calculating rate constants for reactions that are at or near steady state, where the zone profiles exhibit a single zone containing both reacting species.

Effects of nonequilibrium on velocity and plate height in reactive capillary electrophoresis.

Models for velocity and plate height for reactive CE are developed under the formalism of generalized nonequilibrium theory, as described by Giddings. The resultant equations are consistent with chromatographic theory and validated with an independent stochastic simulation. Moreover, unlike prior methods for CE, this model allows calculation of thermodynamic equilibrium constants and kinetic rate constants from a single, undistorted peak. The theoretical development shows that velocity is directly dependent on the equilibrium constant and is independent of the rate constant. On the other hand, plate height varies little with equilibrium constant and is inversely proportional to rate constant. The ability to evaluate equilibrium constants from velocity and rate constants from plate height is most greatly influenced by electric field strength and mobility difference. The accuracy in calculated equilibrium constants is limited by mobility difference; however, the accuracy in rate constants is limited by plate height and equilibrium constant.

Stochastic simulation of reactive separations in capillary electrophoresis.

A stochastic (Monte Carlo) simulation is used to investigate thermodynamic and kinetic contributions from the reversible A <--> B reaction in capillary electrophoresis (CE). The effects of equilibrium constant, rate constant, and electrophoretic mobility on the molecular zone profiles and the corresponding statistical moments are evaluated. As the reaction approaches steady state, the velocity of the zone is governed by the equilibrium constant and the electrophoretic mobilities of the reacting molecules. When the equilibrium constant is less than unity, the mean zone velocity is more similar to that of the reactant A. Conversely, when the equilibrium constant is greater than unity, the velocity is more similar to that of the product B. The extent of zone-broadening and asymmetry at steady state is dependent upon the equilibrium constant, the characteristic reaction lifetime, and the electrophoretic mobility difference between reacting molecules. If all other parameters are held constant, the plate height is greatest and skew is least when the equilibrium constant is unity. The plate height increases linearly with the characteristic reaction lifetime and electrophoretic mobility difference, whereas the skew is independent of these parameters. These conclusions have important implications for the elucidation of thermodynamic and kinetic information from experimental data.