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We describe a new approach to the sensitive and specific identification of bacteria, viruses, fungi, and protozoa based on broad-range PCR and high-performance mass spectrometry. The Ibis T5000 is based on technology developed for the Department of Defense known as T.I.G.E.R. (Triangulation Identification for the Genetic Evaluation of Risks) for pathogen surveillance. The technology uses mass spectrometry—derived base composition signatures obtained from PCR amplification of broadly conserved regions of the pathogen genomes to identify most organisms present in a sample. The process of sample analysis has been automated using a combination of commercially available and custom instrumentation. A software system known as T-Track manages the sample flow, signal analysis, and data interpretation and provides simplified result reports to the user. No specialized expertise is required to use the instrumentation. In addition to pathogen surveillance, the Ibis T5000 is being applied to reducing health care—associated infections (HAIs), emerging and pandemic disease surveillance, human forensics analysis, and pharmaceutical product and food safety, and will be used eventually in human infectious disease diagnosis. In this review, we describe the automated Ibis T5000 instrument and provide examples of how it is used in HAI control.

A combination of algorithms to search RNA sequence for the potential for secondary structure formation, and search large numbers of sequences for structural similarity, were used to search the 5'UTRs of annotated genes in the Escherichia coli genome for regulatory RNA structures. Using this approach, similar RNA structures that regulate genes in the thiamin metabolic pathway were identified. In addition, several putative regulatory structures were discovered upstream of genes involved in other metabolic pathways including glycerol metabolism and ethanol fermentation. The results demonstrate that this computational approach is a powerful tool for discovery of important RNA structures within prokaryotic organisms.

In this work, we describe a strategy for the detection and characterization of microorganisms associated with a potential biological warfare attack or a natural outbreak of an emerging infectious disease. This approach, termed TIGER (Triangulation Identification for the Genetic Evaluation of Risks), relies on mass spectrometry-derived base composition signatures obtained from PCR amplification of broadly conserved regions of the microbial genome(s) in a sample. The sample can be derived from air filtration devices, clinical samples, or other sources. Core to this approach are “intelligent PCR primers” that target broadly conserved regions of microbial genomes that flank variable regions. This approach requires that high-performance mass measurements be made on PCR products in the 80–140 bp size range in a high-throughput, robust modality. As will be demonstrated, the concept is equally applicable to bacteria and viruses and could be further applied to fungi and protozoa. In addition to describing the fundamental strategy of this approach, several specific examples of TIGER are presented that illustrate the impact this approach could have on the way biological weapons attacks are detected and the way that the etiologies of infectious diseases are determined. The first example illustrates how any bacterial species might be identified, using Bacillus anthracis as the test agent. The second example demonstrates how DNA-genome viruses are identified using five members of Poxviridae family, whose members includes Variola virus, the agent responsible for smallpox. The third example demonstrates how RNA-genome viruses are identified using the Alphaviruses (VEE, WEE, and EEE) as representative examples. These examples illustrate how the TIGER technology can be applied to create a universal identification strategy for all pathogens, including those that infect humans, livestock, and plants.

A new algorithm called RNAMotif containing RNA structure and sequence constraints and a thermodynamic scoring system was used to search for intrinsic rho-independent terminators in the Escherichia coli K-12 genome. We identified all 135 reported terminators and 940 putative terminator sequences beginning no more than 60 nt away from the 3′-end of the annotated transcription units (TU). Putative and reported terminators with the scores above our chosen threshold were found for 37 of the 53 non-coding RNA TU and for almost 50% of the 2592 annotated protein-encoding TU, which correlates well with the number of TU expected to contain rho-independent terminators. We also identified 439 terminators that could function in a bi-directional fashion, servicing one gene on the positive strand and a different gene on the negative strand. Approximately 700 additional termination signals in non-coding regions (NCR) far away from the nearest annotated gene were predicted. This number correlates well with the excess number of predicted ‘orphan’ promoters in the NCR, and these promoters and terminators may be associated with as yet unidentified TU. The significant number of high scoring hits that occurred within the reading frame of annotated genes suggests that either an additional component of rho-independent terminators exists or that a suppressive mechanism to prevent unwanted termination remains to be discovered.

Quantitative measurements of second harmonic and first harmonic Doppler were carried out using two ultrasound contrast agents, Albunex ® and FS069. The RMS amplitudes of the Doppler shift spectra were measured as a function of the concentration of the agents, frequency and transmitted acoustic pressure. The results showed that, for a given lot of contrast agent investigated, FS069 was able to produce higher levels of first and, especially, second harmonic signals compared to Albunex. Under the same experimental conditions, the RMS Doppler amplitude (RDA) of FS069 was 3.8 ± 0.8 dB higher than Albunex at first harmonic and 12.6 ± 0.8 dB higher at second harmonic. The ratio of the second harmonic to first harmonic RDA, which we called R2R1, decreases at a rate of 7 dB/MHz for both agents with increasing frequency. The difference in the value of R2R1 between FS069 and Albunex at any frequency was approximately 4.5 dB. R2R1 was found to increase linearly as a function of the transmitted acoustic pressure for both agents. Simulations using the Rayleigh-Plesset equation show a decrease of R2R1 at a rate of 5 dB/MHz. Comparison of experimental results with theory indicates that the shell elasticity parameter may be an increasing function of the mean diameter of the bubbles.

Background. Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/ electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology. Methods and Principal Findings. Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide subspecies identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999–2006) showed correct identification of the viral species and subtypes with .97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005–2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution. Conclusion/Significance. Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance.

Quantitating regional myocardial perfusion has been the much sought-after but still elusive goal of many intensive investigations over the years. Videodensitometry of the variation of myocardial echogenicity in two-dimensional (2-D) echocardiograms as a function of time in conjunction with the injection of a bolus of an ultrasound contrast agent has been used clinically as a tool for a direct assessment of regional myocardial perfusion, despite that the precise relationship between tissue echogenicity observed on an image and the echoes detected by the ultrasonic probe is unknown. A study was undertaken to determine whether ultrasonic backscatter calculated from unprocessed radio frequency (RF) echoes returned from myocardium could be used to quantitate regional myocardium perfusion. A real-time ultrasonic scanner has been modified and interfaced to a microcomputer to acquire RF data at a rate up to 10 frames per second. Preliminary experimental data were obtained from four open-chest dogs following intracoronary injection of a bolus of Albunex® and two dogs following intravenous injection with this modified scanner. On one hand, these results indicate that the integrated backscatter measured from the region of myocardium perfused by the coronary artery where Albunex® is injected and selected for monitoring initially increases, reaches a peak, and then decreases as the contrast agent is washed out and that the magnitude of the peak is approximately linearly proportional to the volume concentration of Albunex® microspheres injected, clearly demonstrating the feasibility of this approach for quantitating region myocardial perfusion. On the other hand, intravenous injections did not result in any appreciable change in myocardial backscatter in the left ventricle although a response could be observed in the left ventricular blood pool.

AlbunexR is an ultrasound contrast agent prepared by sonicating 5% human serum albumin to produce stable, air‐filled, albumin‐coated microspheres. It is an effective ultrasound contrast agent for use during echocardiography and other ultrasound radiological procedures. Current research is directed toward understanding the physics of the interaction of ultrasound with AlbunexR in tissue. A theoretical model describing the dynamics of these bubbles has been developed by assuming a solid elastic shell. The shell (1) supports a strain that counters the effect of surface tension and thus stabilizes the bubble against dissolution; (2) increases the stiffness of the bubble and thus raises the resonance frequency of the bubble significantly; (3) increases the effective damping of the bubble and thus reduces the scattering cross section of the bubble markedly. Experimental measurements of speed of sound, attenuation coefficient, and backscatter coefficient have been made as functions of acoustic frequency and gas ...

Albunex® is an ultrasound contrast agent prepared by sonicating 5% human serum albumin to produce stable, air‐filled, albumin microspheres. It is an effective ultrasound contrast agent for use with echocardiography and other ultrasound radiology procedures. The characteristics of Albunex that are responsible for the clinically observed high echogenicity of Albunex are discussed. Albunex is composed of a high concentration of noncoalescing microspheres (400–500 μ/nl) with a mean diameter of ≈4 μm and a shell thickness of 30–50 nm, as determined by particle analysis, light microscopy, and electron microscopy. Attenuation and backscatter measurements as a function of ultrasonic frequency and amplitude are reported for varying concentration and size distributions of Albunex.

The present invention is a novel method for producing real-time colorized, contrast enhanced images from a sequence of grey-scale video images obtained during diagnostic ultrasound. The particular colorizing scheme varies according to which information parameter is desired to be displayed in real-time. The information parameters used to colorize a segment of video images include: time-to-arrival, duration of brightening, and absolute brightening. Time-to-arrival colorization depicts the time that a given pixel achieves a given intensity threshold. Duration of brightening depicts the time that a given pixel's intensity stays above a given threshold. Absolute brightening depicts various threshold values obtained by the region's pixels.

Recent analyses of the 3ν3 vibrational band in two spherical tops, SF6 and SiF4, have shown the Cartesian basis to be an appropriate description for this band in the octahedral case, while the tetrahedral case is adequately represented by the angular momentum basis. Using spectroscopic constants derived from force field analyses, the F1u/F2 subblocks of the vibrational Hamiltonian matrices of the bands of four vibrational quanta and one band of five quanta 2ν23ν3 are diagonalized in each bases set for SF6, CF4, and CH4 to extend the comparison to more general vibrational levels. Both bases fail to adequately describe CF4 and CH4. The validity of the various angular momentum designations as quantum numbers is strongly dependent on the particular set of spectroscopic parameters. SF6 is poorly described by the angular momentum basis. Within a vibrational band of SF6, only a component whose vibrational motion lies along a single Cartesian axis is well described by the Cartesian basis. It is the lowest energy ...

Nascent SH(X 2Πi, v‘=0,1) and SD(X 2Πi, v‘=0,1) rotational state population distributions, spin–orbit state population ratios, and Λ‐doublet state population ratios have been measured following the UV excimer laser photodissociation of H2S (λ=193, 222, and 248 nm) and D2S (λ=193 and 222 nm), respectively. Nascent SH(X 2Πi, v‘=0) rotational state distributions following 193 nm photodissociation of cold H2S in a free jet expansion vs 300 K H2S in a flowing gas cell were essentially the same, indicating that photofragment angular momentum must be originating predominantly in the dissociation event, and not from rotational energy contained in the parent triatom. Laser excitation spectra of SH(X 2Πi, v‘=1) and SD(X 2Πi, v‘=1) have been recorded for the first time. Rotational state distributions for SH(X 2Πi, v‘) and SD(X 2Πi, v‘) are independent of v‘. Λ‐doublet population ratios of the nascent photofragments are essentially unity for all the cases measured. The nascent rotational state distributions are consi...

A semiclassical calculation is presented that fully accounts for the angular momentum disposal in photodissociation of triatomic molecules. Rotational distributions are calculated for O2(3Σ−g) produced by the visible photolysis of ozone, O2(1Δg) by the UV photolysis of ozone, and OH by the 157 nm dissociation of water, to illustrate the effects of parent internal motion on fragment rotational distributions in the dissociation of C2v geometry molecules. A simple, but realistic, impulsive model of the energy release is used to describe the dissociation dynamics. The calculations are carried out for parent molecules at room temperature, as well as at the low temperatures characteristic of molecular beams. The contributions to the diatomic fragment rotational distribution from both parent triatom rotation and zero‐point bending vibration are computed. Comparison of the calculated distributions with experimentally measured distributions indicates that the spread in rotational and bending vibrational angular mo...

The photolysis of ozone at 300 K in the Chappuis band has been investigated through collision‐free coherent anti‐Stokes Raman scattering (CARS) spectroscopy of the molecular oxygen photofragment. We have obtained the nascent electronic, vibrational, rotational, and translational energy distributions for nine photolysis wavelengths over the range 560–638 nm. The O2 photofragments are always formed in the ground (3Σ−g) state, there is no evidence for production of the excited (1Δg) state. Only a narrow range of high rotational levels are populated. The rotational distribution shifts to higher rotational states as the available energy increases. The vibrational distribution, however, is independent of photolysis wavelength. Only states v=0 to v=4 are populated, and there is a population inversion between v=2 and v=3. The average partitioning of energy among the vibrational, rotational, and translational degrees of freedom is 10%, 24%, and 66%, respectively. These results are interpreted to imply vibrationall...

We report the first measurement of rotationally and vibrationally product-state-resolved absolute cross sections for H+H 2 and H+D 2 collisions. There is generally good agreement between these experimentally determined partial reaction cross sections and those that result from quasiclassical trajectory calculations.

We report the results of state‐to‐state dynamics experiments on the D+H2→HD+H reaction as well as D+H2→H°2+D energy transfer at relative energies of 0.67 and 0.79 eV. Both product state distributions and absolute partial cross sections have been determined, from coherent anti‐Stokes Raman scattering (CARS) spectra of the HD and H°2 products recorded under single‐collision conditions following pulsed‐laser photolysis of DI to generate the D atom reactant. At both energies and for both reactive and inelastic collisions there is a strong dynamical bias against rotational and vibrational excitation of the product. However, at 0.67 eV there is an enhancement of both the relative and absolute yield of HD (v’=1), and to a lesser extent H2(v’=1), the only energetically accessible vibrationally excited product states. This may be the result of a Feshbach resonance at ≈0.65 eV, just above the v’=1 threshold energy. Product quantum state distributions from quasiclassical trajectory calculations are in fairly good agreement with the experimental results, except that they do not show the v’=1 enhancement at 0.67 eV. However, the partial cross sections from the trajectory calculations are systematically larger than those measured.

The forced harmonic oscillator model, which has been applied to proton energy loss spectroscopy (PELS), is extended here to probe the nature of CF4 vibrational levels excited by the proton beam. At each overtone of the triply degenerate ν3 vibration, there is a collection of vibrational sublevels which differ in the way amplitude is distributed among the four C–F bonds. The model predicts that PELS populates a broad distribution of these sublevels with a propensity for the highest energy, most delocalized vibrations in each overtone band. Comparison is made with infrared multiphoton absorption which excites delocalized vibrations in SF6 and with single photon overtone absorption which selects the most localized states. Peak positions in a simulated spectrum match the experiment up to 8ν3 but for higher overtones the anharmonic shift is up to 0.05 eV less than observed. A demanding test of the model must wait for higher resolution experiments and better spectroscopic constants.

The forced harmonic oscillator model, which has been applied to proton energy loss spectroscopy (PELS), is extended here to probe the nature of CF4 vibrational levels excited by the proton beam. At each overtone of the triply degenerate ν3 vibration, there is a collection of vibrational sublevels which differ in the way amplitude is distributed among the four C–F bonds. The model predicts that PELS populates a broad distribution of these sublevels with a propensity for the highest energy, most delocalized vibrations in each overtone band. Comparison is made with infrared multiphoton absorption which excites delocalized vibrations in SF6 and with single photon overtone absorption which selects the most localized states. Peak positions in a simulated spectrum match the experiment up to 8ν3 but for higher overtones the anharmonic shift is up to 0.05 eV less than observed. A demanding test of the model must wait for higher resolution experiments and better spectroscopic constants.

The response to dietary deprivation in late pregnancy, as compared to the non-pregnant condition, is more rapid and profound in terms of mobilization of fuels frm peripheral tissues as well as hepatic ketogenesis and gluconeogenesis ("accelerated starvation"). We examined the potential role of hepatic insulin and glucagon receptors in mediating these changes by comparing 48-h fasted 18-day pregnant and age-matched nongravid rats. Molar ratios for insulin:glucagon in peripheral and portal blood were significantly higher in the pregnant rats. Insulin binding to purified liver plasma membrane receptors, when appropriately corrected for differences in insulin degradation by the membrane system, was marginally diminished in the pregnant animals. Glucagon binding and adenylate cyclase activation by glucagon was indistinguishable in the two groups of animals. On the basis of portal vein hormone concentrations and the values for receptor binding, liver insulinization relative to glucagonization appears to be unchanged or slightly increased in the fasted pregnant rat compared to the fasted nongravid rat. Thus, it seems unlikely that much of the "accelerated starvation" response in late pregnancy can be ascribed to diminished insulin and/or increased glucagon availability at the hepatocellular level. Instead, it is hypothesized that postreceptor events play the major role in sustaining the intrahepatic realignments of established fasting in late pregnancy.