Paired stable carbon and oxygen isotope analyses of human enamel for forensic human geolocation: An exploratory study.

Stable carbon isotope analysis has proven utility for reconstructing dietary information in humans in past populations. The usefulness of stable carbon for forensic geolocation has been little investigated, largely because of the globalization of the human diet seemingly rendering it inconsequential. This study queried this assumption at a country-wide level on a known sample group. Stable carbon isotope values were obtained from human enamel with known biographical and geographical information to determine whether stable carbon, when paired with stable oxygen isotope values, could differentiate Canadians from non-Canadians. Samples originating outside of Canada were separated into three regions, and a linear discriminant analysis was used to generate discriminant functions that best separate the regions according to the stable carbon and oxygen isotope values. The results revealed two functions, where the first function explained 92.1% and the second 7.9% of the variance. Although some overlap in stable carbon and oxygen values was observed for individuals from both the United States and Canada, differences were observed between those from Canada and other geographical regions. This study demonstrated that pairing the dietary isotope carbon with the geolocator stable oxygen isotope, produced an interesting separation geographically, one that might well be helpful when attempting a geolocation query on unknown human remains.

The application of a CART model for forensic human geolocation using stable hydrogen and oxygen isotopes.

The utility of stable hydrogen and oxygen isotope analysis of human tissues for geolocation is an important area of study within forensic science. This study aimed to first validate the latitudinal relationship between stable hydrogen and oxygen isotopes in drinking water and human keratinous tissues through the analysis of human samples with known geographical origin. And secondly, to explore the use of classification and regression tree (CART) models to geographically classify individuals based on the stable isotope values of tissues themselves. Human hair and toenails were collected from four distinct study sites across Canada. The comparison of stable isotope values in drinking water and human tissues produced low R2 values indicating that linear models may not fully explain the variation observed for both hydrogen and oxygen values. Additionally, large intrapopulation variations were observed for Canadian cities and highlights the importance of understanding the regional isotopic spread of human values. Further, this study demonstrated that a closed group of unknown individuals known to have originated from a limited number of geographically distinct regions may be classified into their respective groups through the use of CART models. The potential for the CART model approach for human geolocation presents a promising new tool.

Assessing the predictability of existing water-to-enamel geolocation models against known human teeth.

Stable isotope analysis of human tissues has become a valuable tool for mapping human geolocation. This study adds to the existing knowledge of the relationship between oxygen stable isotopes in human enamel and drinking water by presenting enamel oxygen values in clinic-extracted human dental enamel with known provenance. The results from this study indicate that the theoretical isotopic relationship between enamel and drinking water oxygen is weak at the city and country-level. Differences of up to 15‰ were observed between predicted drinking water oxygen values using existing models and observed values, highlighting the complexity of using water/enamel conversion equations. The lower isotopic boundary of enamel oxygen values is now understood for Metro Vancouver at δ18Oc(VPDB) = - 11.0‰ and presents the possibility of using stable isotope analysis as an exclusionary tool where individuals falling below threshold value can be identified as non-local. Overall, this study's results support the development of geographical reference maps for human enamel oxygen.

Histidine-Mediated Intramolecular Electrostatic Repulsion for Controlling pH-Dependent Protein-Protein Interaction.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on Streptococcal protein G, an affinity ligand for immunoglobulin G, and showed that even a single point mutation effectively improved the pH sensitivity of the binding interactions without adversely affecting its structural stability or its innate binding function. Depending on the pH of the environment, the protein-protein interaction was disrupted by the electrostatic repulsion between the substituted histidine and its neighboring positively charged residues. Structurally, the substituted histidine residue was located adjacent to a lysine residue that could form hydrogen bonds with immunoglobulin G. Thermodynamically, the introduced electrostatic repulsion was reflected in the significant loss of the exothermic heat of the binding under acidic conditions, whereas accompanying enthalpy-entropy compensation partly suppressed the improvement of the pH sensitivity. Thus, the engineered pH-sensitive protein G could enable antibody purification under mildly acidic conditions. This intramolecular design can be combined with conventional protein-protein interface design. Moreover, the method proposed here provides us with additional design criteria for optimization of pH-dependent molecular interactions.

Assessing dual hair sampling for isotopic studies of grizzly bears.

RATIONALE: The stable isotope ratios of carbon (δ13 C values), nitrogen (δ15 N values) and sulfur (δ34 S values) in bear hair can be used to obtain information on dietary history. Sample protocols often require hair sampling from multiple anatomical locations; however, there remains a question as to whether this is necessary for isotopic studies of hair. The purpose of this study was to determine whether significant differences can be observed for the δ13 C, δ15 N and δ34 S values between paired hair samples taken from the rump and shoulder of grizzly bears (Ursus arctos). METHODS: Paired hair samples were collected from the rump and the shoulder of 81 grizzly bears in the Yukon, Canada. Hair samples were analyzed using a thermal combustion elemental analyzer coupled with a continuous flow isotope ratio mass spectrometer. RESULTS: Statistical comparisons of paired hair samples for both males and females showed no meaningful differences in δ13 C, δ15 N and δ34 S values in hair taken from the rump and shoulder, and any observed differences fell within the instrumental error. CONCLUSIONS: Based on these results, hair may be safely sampled on either the rump or the shoulder without loss of isotopic information and thus this finding allows for refinement of sampling.

A City-wide Investigation of the Isotopic Distribution and Source of Tap Waters for Forensic Human Geolocation Ground-truthing.

Human geolocation is prefaced on the accuracy of the geographic precision of mapped isotopic values for drinking water. As most people live in cities, it becomes important to understand city water supplies and how the isotopic values uniquely reflect that city. This study investigated the isotopic distribution of δ2 H and δ18 O from sourced tap waters that were collected from across the Metro Vancouver (MV) area (n = 135). The results revealed that the isotopic values reflect their water sources with a range of 5.3‰ for δ18 Otap and 29.3‰ for δ2 Htap for MV. The results indicate that individual cities need higher resolution studies to determine their tap water isotopic ranges, and a good understanding of the water supply network itself for human geolocation work. With an extended high-resolution understanding of each city, human tissue may be compared with more certainty for geolocation.

Characterization of ergothionase from Burkholderia sp. HME13 and its application to enzymatic quantification of ergothioneine.

We identified ergothionase, which catalyzes conversion of ergothioneine to thiolurocanic acid and trimethylamine, in a newly isolated ergothioneine-utilizing strain, Burkholderia sp. HME13. The enzyme was purified and its N-terminal amino acid sequence was determined. Based on the amino acid sequence, the gene encoding the enzyme was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and characterized. The enzyme consisted of four identical 55-kDa subunits. The enzyme showed maximum activity at pH 8.0 and 65 °C and was stable between pH 7.0 and pH 10.0 and up to 60 °C. The enzyme acted on ergothioneine (K m: 19 µM, V max: 270 µmol/min/mg), but not D-histidine, L-histidine, D-tyrosine, L-tyrosine, D-phenylalanine, or L-phenylalanine. The enzyme was activated by BaCl2 and strongly inhibited by CuSO4, ZnSO4, and HgCl2. The amino acid sequence of ergothionase showed 23 % similarity to histidine ammonia-lyase (HAL) from Pseudomonas putida and 17 % similarity to phenylalanine ammonia-lyase (PAL) from parsley. However, the tripeptide sequence, Ala-Ser-Gly, which is important for catalysis in both HAL and PAL, was not conserved in ergothionase. The application of ergothionase for the quantification of ergothioneine contained in practical food and blood samples was investigated by performing a recovery test. Satisfactory recovery data (98.7-104 %) were obtained when ergothioneine was added to extract of tamogitake and hemolysis blood.