Rapid Identification of Emerging Pathogens: Coronavirus

New surveillance approach can analyze >900 polymerase chain reactions per day

Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

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.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species 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.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

COMPARISON OF THE SYMMETRIZED CARTESIAN AND ANGULAR MOMENTUM BASIS SETS TO DESCRIBE VIBRATIONALLY EXCITED STATES IN SPHERICAL TOP MOLECULES

Author Institution: Chemistry Department, University of Rochester River StationAnalysis of the $3\nu_{3}$ band in two spherical tops, $SF_{6}{^{1}}$ and $SiF_{4}{^{2}}$, has shown that the cartesian basis is 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 $F_{1u}/F_{2}$ subblocks of the vibrational Hamiltonian matrices of the bands of four vibrational quanta and one band of five quanta, $2\nu_{2}3\nu_{3}$, are diagonalized in each basis set for both $SF_{6}$ and $CF_{4}$ to extend the comparison to more general vibrational levels. Both bases fail to adequately describe $CF_{4}$. Examination of the relative quality of the various angular momentum and cartesian quantum numbers suggests a hybrid basis as a better representation of the tetrahedral case. $SF_{6}$ is poorly described by the angular momentum basis. Within a vibrational band of $SF_{6}$, only a component whose vibrational motion lies along a single cartesian axis is well described by the cartesian basis. It is the lowest energy component, well separated in energy from the remaining components because of the single bond anharmonicity. Overtone spectroscopy was performed with an optical parametric oscillator in the region $2800 - 4200 cm^{-1}$. Only components well described by the cartesian basis have measureable oscillator strength. $^{1}$C. W. Patterson, B. J. Krohn and A. S. Pine, J. Mol. Spectrose. 88(1) 133-166 (1981). $^{2}$C. W. Patterson and A. S. Pine, J. Mol. Spectrose. 96(2) 404-421 (1982)

ON THE NATURE OF HIGH CH4CH_{4} VIBRATIONAL LEVELS EXCITED BY PROTON ENERGY LOSS SPECTROSCOPY

Address of Levene: Department of Chemistry, University of California, Irvine, CA 92717 Address of Perry: Department of Chemistry, University of Rochester, Rochester, NY 14627Author Institution:The forced harmonic oscillator model, which has been applied to proton energy loss spectroscopy (PELS), is extended here to probe the nature of $CH_{4}$ vibrational levels excited by the proton beam. At each overtone of the triply degenerate $\nu_{3}$ vibration, there is a collection of vibrational sublevels which differ in the way amplitude is distributed 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 $SF_{6}$ and with single photon overtone absorption which selects the most localized states. Peak positions in a simulated spectrum match the experiment up to $8\nu_{3}$ 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. It is suggested that high resolution PELS spectra could provide spectroscopic information not accessible by other methods

Ultraviolet photodissociation dynamics of H2S and D2S

Nascent SH(X2 Πi, v = 0,1) and SD(X2 Πi, v = 0,1) rotational state population distributions, spin-orbit state population ratios, and A-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(X2 Πi, v = 0) rotational state distributions following 193 nm photodissociation of cold H2 S in a free jet expansion vs 300 K H2 S 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(X2 Πi, v = 1) and SD (X2 Πi, v = 1) have been recorded for the first time. Rotational state distributions for SH(X2 Πi, v ) and SD(X2 Πi, v ) are independent of v . A-doublet population ratios of the nascent photofragments are essentially unity for all the cases measured. The nascent rotational state distributions are consistent with an impact parameter model for the dissociating triatomic molecule

High Spatial Resolution Observation of Single-Molecule Dynamics in Living Cell Membranes

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an ∼100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface

Identification of conserved regulatory RNA structures in prokaryotic metabolic pathway genes

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