Watching Excited State Dynamics with Optical and X-ray Probes: The Excited State Dynamics of Aquocobalamin and Hydroxocobalamin.

Femtosecond time-resolved X-ray absorption (XANES) at the Co K-edge, X-ray emission (XES) in the Co Kß and valence-to-core regions, and broadband UV-vis transient absorption are combined to probe the femtosecond to picosecond sequential atomic and electronic dynamics following photoexcitation of two vitamin B12 compounds, hydroxocobalamin and aquocobalamin. Polarized XANES difference spectra allow identification of sequential structural evolution involving first the equatorial and then the axial ligands, with the latter showing rapid coherent bond elongation to the outer turning point of the excited state potential followed by recoil to a relaxed excited state structure. Time-resolved XES, especially in the valence-to-core region, along with polarized optical transient absorption suggests that the recoil results in the formation of a metal-centered excited state with a lifetime of 2-5 ps. This combination of methods provides a uniquely powerful tool to probe the electronic and structural dynamics of photoactive transition-metal complexes and will be applicable to a wide variety of systems.

Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

Time-resolved spectroscopy: Advances in understanding the electronic structure and dynamics of cobalamins.

Time resolved spectroscopy provides unique insight into the structure and function of cobalamins. In these experiments, the cobalamin is initially excited by a short "pump" pulse in the UV-visible region and then characterized at some later time using a short "probe" pulse. The emphasis in this chapter is on both UV-visible and X-ray probe pulses, with a particular focus on the unique information provided by the latter. The principles of time-resolved spectroscopy are reviewed, with an emphasis on ultrafast measurements (time scales less than ~10ps) to characterize short-lived cobalamin excited states. Several practical considerations are discussed, with a focus on the technical details that are necessary to obtain high quality, interpretable data. These include sample delivery, polarization, and excitation power. Some of the theoretical approaches to interpreting data are discussed.

Using real-time PCR and Bayesian analysis to distinguish susceptible tubificid taxa important in the transmission of Myxobolus cerebralis, the cause of salmonid whirling disease.

Aquatic oligochaetes have long been appreciated for their value in assessing habitat quality because they are ubiquitous sediment-dwelling filter feeders. Many oligochaete taxa are also important in the transmission of fish diseases. Distinguishing resistant and susceptible taxa is important for managing fish disease, yet challenging in practice. Tubifex tubifex (Oligochaeta: Tubificidae) is the definitive host for the complex life-cycle parasite, Myxobolus cerebralis, the causative agent of salmonid whirling disease. We developed two hydrolysis probe-based qualitative real-time PCR (qPCR) multiplex assays that distinguish among tubificid taxa collected from the Madison River, Montana, USA. The first assay distinguishes T. tubifex from Rhyacodrilus spp.; while the second classifies T. tubifex identified by the first assay into two genetic lineages (I and III). Specificity and sensitivity were optimized for each assay; the two assays showed specificity of 94.3% and 98.6% for the target oligochaetes, respectively. DNA sequencing verified the results. The development of these assays allowed us to more fully describe tubificid community composition (the taxa and their abundance at a site) and estimate the relative abundances of host taxa. To relate tubificid relative abundance to fish disease risk, we determined M. cerebralis infection prevalence in samples identified as T. tubifex using similar molecular techniques. Given prior information (i.e., morphological identification of sexually mature worms), Bayesian analysis inferred that the first qPCR assay improved taxonomic identification. Bayesian inference of the relative abundance of T. tubifex, combined with infection assay results, identified sites with a high prevalence of infected T. tubifex. To our knowledge, this study represents both the first assessment of oligochaete community composition using a qPCR assay based on fluorescent probes and the first use of Bayesian analysis to fully characterize the dominant infected taxa in streams where whirling disease is observed.

Gamma-hydroxybutyrate serum levels and clinical syndrome after severe overdose.

STUDY OBJECTIVE: We discuss a prospective case series of patients who present with a severe gamma-hydroxybutyrate intoxication with confirmatory serum and urine gamma-hydroxybutyrate levels. METHODS: Patients with a clinical suspicion of gamma-hydroxybutyrate-like drug overdoses and a Glasgow Coma Scale score of 8 or lower were identified from July 1998 through January 1999. Serial serum specimens and a single urine specimen were collected. The levels of gamma-hydroxybutyrate were performed by gas chromatography-mass spectrometry. RESULTS: All 16 suspected severe gamma-hydroxybutyrate overdose patients had significant serum or urine levels of gamma-hydroxybutyrate. Serum levels ranged from 45 to 295 mg/L, with a median of 180 mg/L (interquartile range [IQR] 235 to 118 mg/L). Patients who developed a Glasgow Coma Scale score of 3 had serum levels that ranged from 72 to 300 mg/L, with a median of 193 mg/L (IQR 242 to 124 mg/L). The time of awakening ranged from 30 minutes to 190 minutes, with a median of 120 minutes (IQR 150 to 83 minutes). Quantitative serum gamma-hydroxybutyrate levels did not correlate with the degree of coma or the time to awakening. Urine levels ranged from 432 to 2,407 mg/L, with a median of 1,263 mg/L (IQR 1,550 to 796 mg/L). Mild transitory hypoventilation occurred in 5 of the 16 patients. CONCLUSION: All of our patients with clinically suspected severe gamma-hydroxybutyrate overdose were confirmed to have significant serum and urine levels of exogenous gamma-hydroxybutyrate. They presented with severe coma that lasted 1 to 2 hours. Transient hypoventilation occurred in one third of these patients.