IntroductionHuman donations are often used in forensic research as they can provide unique insigh... more IntroductionHuman donations are often used in forensic research as they can provide unique insights into post-mortem research that cannot be obtained with animal proxies. This is especially true for forensic microbiome research, as human circumstances such as drug-use or health conditions may influence the post-mortem microbiome. However, it is not always feasible to conduct such research immediately after death. Donors are often stored frozen in human taphonomy facilities, pending the start of any experimental study, yet little is known about how freezing may affect their microbiome.MethodsWe assessed the effects of freezing on the post-mortem human microbiome by analysing the microbial diversity and abundance of seven human donors at the Forensic Anthropology Center at Texas State (FACTS) before and after freezing. Swab samples were taken from five locations on each corpse upon arrival to FACTS and again after they had been frozen in storage for a period ranging between 11 and 40 ...
Tooth Cementum Annulation (or TCA) is a technique that relies on the analysis of the incremental ... more Tooth Cementum Annulation (or TCA) is a technique that relies on the analysis of the incremental growth of dental cementum for age and season at death estimation. It has been extensively adopted as a “black box technique”, despite numerous controversies. Its potential in forensics called for this review, which aims to provide the reader with an overarching critical synthesis of what has been done and what is known about the TCA from different perspectives. Results have been divided according to three different themes: human, animal and evolutionary studies, as well as biological studies. The summary and comparison of these show the complexity of this topic, its limits, and how the scientific community can collectively collaborate for improvements. Overall, it is clear that the potential of the TCA is significant in terms of age and season at death estimation and that (as long as its biology is still addressed and researched for) there should not be any reason to not consider this te...
Bone is a hard biological tissue and a precious reservoir of information in forensic investigatio... more Bone is a hard biological tissue and a precious reservoir of information in forensic investigations as it retains key biomolecules commonly used for identification purposes. Bone proteins have recently attracted significant interest for their potential in estimating postmortem interval (PMI) and age at death (AAD). However, the preservation of such proteins is highly dependent on intrinsic and extrinsic factors that can hinder the potential application of molecular techniques to forensic sciences. The present study aims at investigating the effects that two commonly used types of burial practices (entombment and inhumation) have on bone protein survival. The sample consists of 14 exhumed individuals from cemeteries in Southern Italy with different AADs (29-85 years) and PMIs (1-37 years). LC-MS/MS analyses show that 16 proteins are better preserved under the entombed conditions and 4 proteins are better preserved under the inhumed conditions, whereas no clear differences are detected for post-translational protein modifications. Furthermore, several potential "stable" protein markers (i.e., proteins not affected by the burial environment) are identified for PMI and AAD estimation. Overall, these results show that the two burial environments play a role in the differential preservation of noncollagenous proteins, confirming the potential of LC-MS/MS-based proteomics in forensic sciences.
Human DNA samples can remain unaltered for years and preserve important genetic information for f... more 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
The combined use of multiple omics allows to study complex interrelated biological processes in t... more 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 t...
The combined use of multiple omics methods to answer complex system biology questions is growing ... more The combined use of multiple omics methods to answer complex system biology questions is growing in biological and medical sciences, as the importance of studying interrelated biological processes in their entirety is increasingly recognized. We applied a combination of metabolomics, lipidomics and proteomics to human bone to investigate the potential of this multi-omics approach to estimate the time elapsed since death (i.e., the post-mortem 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 in a fresh stage of decomposition before placement of the bodies to decompose outdoors at the human taphonomy facility managed by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PM...
The study of post-mortem changes is a crucial component of forensic investigation. Human forensic... more The study of post-mortem changes is a crucial component of forensic investigation. Human forensic taphonomic facilities (HFTFs) are the only institutions allowing the design and execution of controlled human decomposition experiments. When bodies are skeletonized, bones are normally stored in skeletal collections and used for anthropological studies. However, HFTFs apply chemical and/or thermal treatments to the remains prior bone long-term storage. These treatments are believed to alter heavily the original biochemical and molecular signature of bone material. The present study aims to evaluate the effect of these procedures on the bone metabolome and lipidome by using an animal bone model. Three intact bovine tibiae were processed using three protocols routinely applied at HFTFs, and their three counterparts were used as non-treated controls. Bone powder samples were subjected to biphasic extraction and both metabolites and lipids were analysed via liquid chromatography tandem mas...
Bacteria are considered one of the major driving forces of the mammalian decomposition process an... more Bacteria are considered one of the major driving forces of the mammalian decomposition process and have only recently been recognised as forensic tools. At this point, little is known about their potential use as 'post-mortem clocks'. This study aimed to establish the proof of concept for using bacterial identification as post-mortem interval (PMI) indicators, using a multi-omics approach. Pieces of pork were placed in the University's outdoor facility and surface swabs were taken at regular intervals up to 60 days. Terminal restriction fragment length polymorphism (T-RFLP) of the 16S rDNA was used to identify bacterial taxa. It succeeded in detecting two out of three key contributors involved in decomposition and represents the first study to reveal Vibrionaceae as abundant on decomposing pork. However, a high fraction of present bacterial taxa could not be identified by T-RFLP. Proteomic analyses were also performed at selected time points, and they partially succeeded...
Proceedings of the National Academy of Sciences, 2017
Significance Radiocarbon dating of Neanderthal remains recovered from Vindija Cave (Croatia) init... more Significance Radiocarbon dating of Neanderthal remains recovered from Vindija Cave (Croatia) initially revealed surprisingly recent results: 28,000–29,000 B.P. This implied the remains could represent a late-surviving, refugial Neanderthal population and suggested they could have been responsible for producing some of the early Upper Paleolithic artefacts more usually produced by anatomically modern humans. This article presents revised radiocarbon dates of the human bones from this site obtained using a more robust purification method targeting the amino acid hydroxyproline. The data show that all the Neanderthal remains are from a much earlier period (>40,000 cal B.P.). These revised dates change our interpretation of this important site and demonstrate that the Vindija Neanderthals probably did not overlap temporally with early modern humans.
Proteomics methods are being increasingly used to study archaeological and palaeontological bone,... more Proteomics methods are being increasingly used to study archaeological and palaeontological bone, assisting in species identification and phylogenetic studies as well as improving our understanding of bone diagenesis. More recently, there are developing interests in the study of post-translational modifications, some of which are potentially diagnostic of decay, but none of the previous extraction methods have been developed in light of this. To be able to record close to natural deamidation levels of samples, an extraction procedure should minimize laboratory-induced decay, such as asparagine and glutamine deamidations, which are considered most strongly related with decay and known to occur frequently with standard laboratory procedures. Here we tested numerous methods to identify an optimal approach of extracting proteins from bone while minimizing artificial decay. Using a weak acid to partially demineralize the bone sample, then subsequent incubation of the acid insoluble fraction with guanidine hydrochloride and enzymatic digestion in ammonium acetate, we observed an ∼50% reduction in deamidation while also substantially decreasing the protocol length. We propose this optimized method as appropriate for studies of archaeological, palaeontological, as well as potentially forensic investigations using proteomics where decay measurements could act as "molecular timers".
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