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The isotopic signatures of human tissues can provide valuable information on geographic origin for medicolegal investigations involving unidentified persons. It is important to understand the impact of diagenetic processes on isotopic signatures, as alterations could result in incorrect estimation of geographic origin. This study examines alterations in isotope signatures of different tissues of five human body donors studied throughout decomposition at the Forensic Anthropology Research Facility (FARF), San Marcos, TX. Two body donors were buried, two were placed in open pits, and one was first allowed to naturally mummify and then buried. Remains were recovered after a period of 7-34 months. The preplacement and post-recovery Sr-Pb isotope data of scalp hair, bone (iliac and tibia), and tooth enamel and dentine were compared. The hair samples record significant shifts in Sr-Pb isotope compositions, with hair keratin Pb isotope composition shifting towards the Pb signature of local soil samples. Hair keratin Sr isotope compositions were altered by the burial environment and possibly also by the lab sample cleaning method. The spongy iliac bone samples show inconsistencies in the recoverability of the preplacement Sr-Pb isotope signatures. The post-placement signatures of the buried donors show slight elevation over preplacement signatures. The post-placement signatures of donors placed in open pits are significantly elevated. The tibia and dental samples record the most consistent isotopic data with the least alteration. These more densely mineralised elements show good recoverability of the preplacement isotope signatures in burials and open pits and are thus deemed better targets for forensic investigative purposes.

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The combined use of multiple omics allows to study complex interrelated biological processes in their entirety. We applied a combination of metabolomics, lipidomics and proteomics to human bones to investigate their combined potential to estimate time elapsed since death (i.e., the postmortem interval [PMI]). This 'ForensOMICS' approach has the potential to improve accuracy and precision of PMI estimation of skeletonized human remains, thereby helping forensic investigators to establish the timeline of events surrounding death. Anterior midshaft tibial bone was collected from four female body donors before their placement at the Forensic Anthropology Research Facility owned by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PMIs (219-790-834-872days). Liquid chromatography mass spectrometry (LC-MS) was used to obtain untargeted metabolomic, lipidomic, and proteomic profiles from the pre- and post-placement bone samples. The three omics blocks were investigated independently by univariate and multivariate analyses, followed by Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies (DIABLO), to identify the reduced number of markers describing postmortem changes and discriminating the individuals based on their PMI. The resulting model showed that pre-placement metabolome, lipidome and proteome profiles were clearly distinguishable from post-placement ones. Metabolites in the pre-placement samples suggested an extinction of the energetic metabolism and a switch towards another source of fuelling (e.g., structural proteins). We were able to identify certain biomolecules with an excellent potential for PMI estimation, predominantly the biomolecules from the metabolomics block. Our findings suggest that, by targeting a combination of compounds with different postmortem stability, in the future we could be able to estimate both short PMIs, by using metabolites and lipids, and longer PMIs, by using proteins.

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In this study, we present an experiment design and assess the capability of multiple geophysical techniques to image buried human remains in mass and individual graves using human cadavers willingly donated for scientific research. The study is part of a novel, interdisciplinary mass grave experiment established in May 2021 which consists of a mass grave with 6 human remains, 3 individual graves and 2 empty control graves dug to the same size as the mass grave and individual graves. Prior to establishing the graves, we conducted background measurements of electrical resistivity tomography (ERT), electromagnetics (EM), and ground penetrating radar (GPR) while soil profiles were analyzed in situ after excavating the graves. All graves were also instrumented with soil sensors for monitoring temporal changes in soil moisture, temperature, and electrical conductivity in situ. Measurements of ERT, EM and GPR were repeated 3, 37, 71 and 185 days after burial with further repeated measurements planned for another twelve months. ERT results show an initial increase in resistivity in all graves including the control graves at 3 days after burial and a continuous decrease thereafter in the mass and individual graves with the strongest decrease in the mass grave. Conductivity distribution from the EM shows a similar trend to the ERT with an initial decrease in the first 3 days after burial. Distortion in linear reflectors, presence of small hyperbolas and isolated strong amplitude reflectors in the GPR profiles across the graves is associated with known locations of the graves. These initial results validate the capability of geoelectrical methods in detecting anomalies associated with disturbed ground and human decay while GPR though show some success is limited by the geology of the site.

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Human DNA samples can remain unaltered for years and preserve important genetic information for forensic investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which were stored frozen for 5-16 years. Results demonstrated that the microbiome can be co-extracted with human DNA using forensic kits designed to extract the human host's DNA from different tissues and fluids during decomposition. We compared the microbial communities identified in these samples with microbial DNA recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University (FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from "old" (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples. The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The results obtained from the human DNA extracts were compared with each other and with the microbial DNA from the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition stage, the type of tissue, and the depositional environment. We found no indications of contamination in the microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demonstrating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research purposes. It is therefore important to create common protocols on the storage of biological material collected at crime scenes. We review existing legislation and guidelines, and identify some important limitations for the further development and application of forensic microbiomics.

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Bone proteomic studies using animal proxies and skeletonized human remains have delivered encouraging results in the search for potential biomarkers for precise and accurate post-mortem interval (PMI) and the age-at-death (AAD) estimation in medico-legal investigations. The development of forensic proteomics for PMI and AAD estimation is in critical need of research on human remains throughout decomposition, as currently the effects of both inter-individual biological differences and taphonomic alteration on the survival of human bone protein profiles are unclear. This study investigated the human bone proteome in four human body donors studied throughout decomposition outdoors. The effects of ageing phenomena (in vivo and post-mortem) and intrinsic and extrinsic variables on the variety and abundancy of the bone proteome were assessed. Results indicate that taphonomic and biological variables play a significant role in the survival of proteins in bone. Our findings suggest that inter-individual and inter-skeletal differences in bone mineral density (BMD) are important variables affecting the survival of proteins. Specific proteins survive better within the mineral matrix due to their mineral-binding properties. The mineral matrix likely also protects these proteins by restricting the movement of decomposer microbes. New potential biomarkers for PMI estimation and AAD estimation were identified. Future development of forensic bone proteomics should include standard measurement of BMD and target a combination of different biomarkers.

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Multi-isotope analysis (e.g., Sr-Pb-O-H-C-N) of human scalp hair is routinely used in forensic investigations of human remains to constrain the geographic origin of unidentified bodies, and to investigate antemortem mobility patterns. However, while it is known that postmortem processes can affect the preservation of, or even overprint, the biogenic isotopic signatures in hair, the speed and nature of these processes have rarely been studied. This study investigates the effects of decomposition and environment on the H-Pb-Sr isotope compositions of human hair as well as the relationship between structural hair shaft degradation and isotopic signature change over time. Human scalp hair samples from four body donations were collected at different stages throughout gross body decomposition. The willed-donated bodies were placed to decompose outdoors at the Forensic Anthropology Research Facility (FARF) at Texas State University. Hair fibres from two of the donations were examined using scanning electron microscopy (SEM) and high-resolution light microscopy (HRLM). Chemical and microbiological degradation of hair fibres occurred rapidly after placement of the body outdoors. Measurements of scalp hair isotopic composition demonstrated that H-Pb-Sr isotope ratios were altered within days after environmental exposure, presumably by deposition, leaching and/or exchange with the local bioavailable soil, and vapour. The degree of physical hair degradation and changes in H-Pb-Sr isotope composition were not correlated. We conclude that antemortem isotopic H-Pb-Sr isotope ratios are difficult to recover in hairs derived from decomposing whole bodies.

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Multi-isotope analysis (e.g., Sr-Pb-O-H-C-N) of human scalp hair is routinely used in forensic investigations of human remains to constrain the geographic origin of unidentified bodies, and to investigate antemortem mobility patterns. However, while it is known that postmortem processes can affect the preservation of, or even overprint, the biogenic isotopic signatures in hair, the speed and nature of these processes have rarely been studied. This study investigates the effects of decomposition and environment on the H-Pb-Sr isotope compositions of human hair as well as the relationship between structural hair shaft degradation and isotopic signature change over time. Human scalp hair samples from four body donations were collected at different stages throughout gross body decomposition. The willed-donated bodies were placed to decompose outdoors at the Forensic Anthropology Research Facility (FARF) at Texas State University. Hair fibers from two of the donations were examined using scanning electron microscopy (SEM) and high-resolution light microscopy (HRLM). Chemical and microbiological degradation of hair fibers occurred rapidly after placement of the body outdoors. Measurements of scalp hair isotopic composition demonstrated that H-Pb-Sr isotope ratios were altered within days after environmental exposure, presumably by deposition, leaching and/or exchange with the local bioavailable soil, and vapor. The degree of physical hair degradation and changes in H-Pb-Sr isotope composition were not correlated. We conclude that antemortem isotopic H-Pb-Sr isotope ratios are difficult to recover in hairs derived from decomposing whole bodies.

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