NMR of petrochemical-type chemical reactions.

A simple technique is presented for NMR of chemically reacting systems at conditions of high temperature and pressure. The method can follow reactions that are typical of refinery operations - hydrogenation, transfer dehydrogenation, methanol synthesis, and isomerization. All of the reacting materials are flame-sealed into a glass capillary. Gaseous agents such as O2 and CO are loaded into the capillary by condensation at liquid N2 temperature. H2 is provided by loading LiAlH4. The capillary holds the high pressure, up to 7 MPa, so the NMR probe can be a simple design with hot air flowing over the sample tube, up to 350 °C. Example reaction results are presented, including hydrogenation of benzene, hydrogenation/dehydrogenation of cyclohexene to benzene and cyclohexane (a disproportionation), and synthesis of methane, methanol and dimethyl ether from CO and H2. In this work we present a simple, inexpensive method with rapid temperature response for tracking chemical reactions in real-time at high temperature and high pressure.

Electric field driven flow in natural porous media.

Electric fields were applied to fluid-saturated packed sand beds (0.23+/-0.03 mm average pore diameter), and the effects on the mobility of the water molecules were monitored using stimulated echo (STE) and pulsed field gradient (PFG) experiments. The mean flow velocity, averaged over the entire sample, is expected to vanish in closed systems, but the PFG and time dependent signal decay was enhanced beyond the effects of thermal diffusion, due to velocity dispersion. The internal flow generated by the electric field was shown to be fully time-reversible upon inverting the electric field polarity (for total flow times of up to 0.4s), a strong indication that the NMR detected displacements were mainly due to electro-osmotic flow (EOF). However, a comparison of the velocity dispersion for different electrolyte concentrations showed that the measured effect scaled with the applied power VI (V = voltage, I = electric current), rather than with the voltage alone, contrary to the prediction of the basic model for EOF in a single capillary channel.

Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The assays may be extended to detect a large number of pathogens, are applicable to the evaluation of both environmental and clinical samples, and have the potential to be applied in military, public health, and clinical diagnostic settings.

A multiplexed microfluidic PCR assay for sensitive and specific point-of-care detection of Chlamydia trachomatis.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes. METHODOLOGY AND PRINCIPAL FINDINGS: Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively. CONCLUSIONS: This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.

Optically detected magnetic resonance in the photoexcited triplet states of Ti(IV) and Zr(IV) cylopentadienyl complexes.

The class of compounds (RCp)2MX2, where M is a group IV metal, Cp is cyclopentadienyl, R is an alkyl, and X is a halide, has been of continuing interest as precursors for olefin coordination polymerization catalysts. In this communication, we demonstrate that the technique of optically detected magnetic resonance (ODMR) reveals subtle differences in the composition of the frontier molecular orbitals associated with the nature of the alkyl substituents on the Cp rings.

Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate.

In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde. Soluble homodimeric E. coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE. During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E. coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate. The resulting thioester intermediate was trapped and detected by autoradiography. In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed. In the absence of NADPH, E. coli GluTR exhibited substrate esterase activity. The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E. coli GluTR. Eight 5-aminolevulinic acid auxotrophic E. coli hemA mutants were genetically selected, and the corresponding mutations were determined. Most of the recombinant purified mutant GluTR enzymes lacked detectable activity. Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F). Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.