Solution structure refinement comparing reference model NOE volumes

NMR is a powerful experimental technique which can be used to determine protein structures. Central to this process is conversion of peak volumes in the NOE data sets into distance constraints and then structural refinement against those constraints. In general, each solution structural analysis is initiated de novo. We are interested in the structural analysis of a series of hybrids derived from two parental proteins (rubredoxins from mesophilic bacterium Clostridium pasteurianum (Cp) and the hyperthermophilic archaeon Pyrococcus furiosus (Pf)). Near 1 A resolution X-ray structures of Cp and Pf rubredoxins are available to provide a basis for quantitative comparison of the NOE volumes for analogous 1H-1H pairs in a difference analysis. In this approach, the volume differences are used to drive the structure away from the initial parentally derived model

Faecal virome of the Australian grey-headed flying fox from urban/ suburban environments contains novel coronaviruses, retroviruses and sapoviruses

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of greyheaded flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalianinfecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution

Improvement to sample introduction in plasma spectrochemistry with biological and environmental applications and potential for arsenic speciation in waters by anodic stripping voltammetry

Methods for the determination of trace elements in environmental and biological samples were developed and optimized. Parameters affecting sample introduction efficiency, matrix effects, sample throughput and tolerance to interfering species were evaluated for methods based on plasma spectrochemistry and electrochemistry. A method was developed for the determination of copper in the presence of β-2-microglobulin. A mass balance of copper was performed at the end of in vitro incubations which required method development to quantify copper in the liquid and solid portions of the incubation solutions, along with copper adsorbed and mass transferred into the walls of the incubation vessel. A flow injection method was developed to accommodate the small sample volume and high dissolved solids content to allow for the determination of copper by ICP-MS. A commercial introduction system was evaluated for the determination of trace elements in drinking waters and wastewaters by ICP-MS and ICP-OES. Parameters including increased throughput, reduced memory effects, increased stability and lower reagent consumption were evaluated as the system was successfully applied to U.S. EPA Methods 200.8 and 200.7, respectively. Particular attention was paid to the retention of mercury and sodium and long term stability during the analysis of samples containing high total dissolved salts. Results obtained with the new introduction system were compliant with EPA Methods 200.8 and 200.7 while increasing sample throughput two- or three-fold and significantly reducing memory effects. A method was developed for the enhancement of signals for trace elemental analysis by DRC-ICP-MS. Signal enhancement was evaluated as instrumental and reaction gas parameters were optimized. Background noise levels remained relatively unchanged as signal intensities were improved, resulting in improved signal to noise ratios. The developed method was successfully applied to the determination of gold in synthetic geological samples. A method was developed for the preconcentration and speciation of arsenic by anodic stripping voltammetry using ion exchange resins. Ion exchange and instrument parameters were optimized. The developed method was successfully applied to the determination of arsenic(III) and arsenic(V) in waters without interference from problematic species such as Cu(II). Suitable detection limits were obtained and the method was validated using a certified reference material

Cu(II) organizes β-2-microglobulin oligomers but is released upon amyloid formation

β-2-Microglobulin (β2m) is deposited as amyloid fibrils in the bones and joints of patients undergoing long-term dialysis treatment as a result of kidney failure. Previous work has shown that biologically relevant amounts of Cu(II) can cause β2m to be converted to amyloid fibrils under physiological conditions in vitro. In this work, dynamic light scattering, mass spectrometry, and size-exclusion chromatography are used to characterize the role that Cu plays in the formation of oligomeric intermediates that precede fibril formation. Cu(II) is found to be necessary for the stability of the dimer and an initial form of the tetramer. The initially formed tetramer then undergoes a structural change to a state that no longer binds Cu(II) before progressing to a hexameric state. Based on these results, we propose that the lag phase associated with β2m fibril formation is partially accounted for by the structural transition of the tetramer that results in Cu(II) loss. Consistent with this observation is the determination that the mature β2m amyloid fibrils do not contain Cu. Thus, Cu(II) appears to play a catalytic role by enabling the organization of the necessary oligomeric intermediates that precede β2m amyloid formation