A new method for quantifying 64Cu in nuclear debris samples.

Quantifying 64Cu in post-detonation nuclear debris samples can provide important diagnostic information regarding the structural materials used within a nuclear device. However, this task is challenging due to the weak gamma emissions associated with the decay of 64Cu, its short half-life (12.701 h), and the presence of interfering fission product radioisotopes. Large quantities of debris sample are generally needed to accurately quantify 64Cu, which can be problematic in sample-limited scenarios where other radiometric analyses are required. Herein, we present a new method for the separation of 64Cu from solutions of mixed fission products and demonstrate the quantification of its activity through use of gas-flow proportional beta counting. The new method was validated through a series of rigorous tests and was shown to improve the detection limit of 64Cu by over two orders of magnitude, from 2.5 × 106 to 1.3 × 104 atoms/sample for 100 min measurements.

Chemical separation and measurement of platinum activation products.

A method has been developed to purify and measure platinum radioisotopes in the presence of fission products and environmental constituents. The method uses a combination of cation exchange and anion exchange chromatography and selective precipitation steps to remove other radioisotopes from the sample. The addition of stable platinum carrier allows for a gravimetric determination of the chemical yield of the procedure. Overall, the method is fast, simple, and potentially applicable for rapid turnaround of unknown samples. Using this method, multiple platinum radioisotopes were measured in two different irradiation experiments. The measured ratios of the platinum radioisotopes clearly reflect the neutron spectrum of the irradiation, suggesting that platinum radioisotopes could be valuable signatures in nuclear forensic analyses.

Development of forensic assay signatures for ebolaviruses.

Ebolaviruses are a diverse group of RNA viruses comprising five different species, four of which cause fatal hemorrhagic fever in humans. Because of their high infectivity and lethality, ebolaviruses are considered major biothreat agents. Although detection assays exist, no forensic assays are currently available. Here, we report the development of forensic assays that differentiate ebolaviruses. We performed phylogenetic analyses and identified canonical SNPs for all species, major clades and isolates. TaqMan-MGB allelic discrimination assays based on these SNPs were designed, screened against synthetic RNA templates, and validated against ebolavirus genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the species and variants with additional resolution at the isolate level. These assays enabled accurate forensic analysis on 4 "unknown" ebolaviruses. Unknowns were correctly classified to species and variant. A goal of providing resolution below the isolate level was not successful. These high-resolution forensic assays allow rapid and accurate genotyping of ebolaviruses for forensic investigations.

Automated genomic context analysis and experimental validation platform for discovery of prokaryote transcriptional regulator functions.

BACKGROUND: The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity. RESULTS: A software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018. CONCLUSION: These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.

Forensic signatures for Marburgviruses.

Marburgvirus is one of the most important hemorrhagic fever viruses with extremely high infectivity and fatality rate (~90%). It is transmitted easily in human populations through a respiratory route and therefore considered as a major biothreat agent. Although detection assays have been developed, no assay is available for forensic analysis. Here we report development of forensic assays for Marburgvirus. We performed detailed phylogenetic analysis of strains and isolates from all known Marburg virus outbreaks as well as from several laboratory strains and identified canonical SNPs for all major clades (outbreaks) and strains. TaqMan-MGB allelic discrimination assays targeting these SNPs were designed and experimentally screened against synthetic RNA templates and genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the clades (outbreaks) and 91% at the strain level (21 out of the 23 targeted Marburgvirus strains) with built-in redundancy for increased robustness. Using these validated assays, we were able to provide accurate forensic analysis on 3 "unknown" Marburgviruses. These high-resolution forensic assays allow rapid and accurate genotyping of Marburgviruses for forensic investigations.