MALDI-TOF MS as a Novel Tool for the Estimation of Postmortem Interval in Liver Tissue Samples.
ABSTRACT: Estimation of the postmortem interval (PMI) is a complicated task in forensic medicine, especially during homicide and unwitnessed death investigations. Many biological, chemical, and physical indicators can be used to determine the postmortem interval, but most are not accurate. Here, we present a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method that can be used for the estimation of PMI using molecular images and multivariate analyses. In this study, we demonstrate that both rat and human liver tissues of various PMIs (0, 2, 4, and 6days) can be discriminated using MALDI imaging and principal component analysis (PCA). Using genetic algorithm (GA), supervised neural network (SNN), and quick classifier (QC) methods, we built 6 classification models, which showed high recognition capability and good cross-validation. The histological changes in all the samples at different time points were also consistent with the changes seen in MALDI imaging. Our work suggests that MALDI-TOF MS, along with multivariate analysis, can be used to determine intermediate PMIs.
Project description:Postmortem interval (PMI) evaluation remains a challenge in the forensic community due to the lack of efficient methods. Studies have focused on chemical analysis of biofluids for PMI estimation; however, no reports using spectroscopic methods in pericardial fluid (PF) are available. In this study, Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance (ATR) accessory was applied to collect comprehensive biochemical information from rabbit PF at different PMIs. The PMI-dependent spectral signature was determined by two-dimensional (2D) correlation analysis. The partial least square (PLS) and nu-support vector machine (nu-SVM) models were then established based on the acquired spectral dataset. Spectral variables associated with amide I, amide II, COO-, C-H bending, and C-O or C-OH vibrations arising from proteins, polypeptides, amino acids and carbohydrates, respectively, were susceptible to PMI in 2D correlation analysis. Moreover, the nu-SVM model appeared to achieve a more satisfactory prediction than the PLS model in calibration; the reliability of both models was determined in an external validation set. The study shows the possibility of application of ATR-FTIR methods in postmortem interval estimation using PF samples.
Project description:The early postmortem interval (PMI), i.e., the time shortly after death, can aid in the temporal reconstruction of a suspected crime and therefore provides crucial information in forensic investigations. Currently, this information is often derived from an empirical model (Henssge's nomogram) describing posthumous body cooling under standard conditions. However, nonstandard conditions necessitate the use of subjective correction factors or preclude the use of Henssge's nomogram altogether. To address this, we developed a powerful method for early PMI reconstruction using skin thermometry in conjunction with a comprehensive thermodynamic finite-difference model, which we validated using deceased human bodies. PMIs reconstructed using this approach, on average, deviated no more than ±38 minutes from their corresponding true PMIs (which ranged from 5 to 50 hours), significantly improving on the ±3 to ±7 hours uncertainty of the gold standard. Together, these aspects render this approach a widely applicable, i.e., forensically relevant, method for thermometric early PMI reconstruction.
Project description:The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.
Project description:There have been numerous high resolution diffusion tensor imaging studies in fixed animal brains, but relatively few studies in human brains. While animal tissues are generally fixed pre-mortem or directly postmortem, this is not possible for human tissue, therefore there is always some delay between death and tissue fixation. The elapsed time between death and tissue fixation, the postmortem interval (PMI), will most likely adversely affect the tissue's diffusion properties. We studied the effects of PMI on the diffusion properties of rodent brain. Eight mice were euthanized and the brains (kept in the skull) were placed in formalin at PMIs of 0, 1, 4 and 14 days. Post fixation they were placed in a solution of GdDTPA and phosphate buffered saline. Brains were scanned with a 3D EPI DTI sequence at 4.7T. DTI data were processed to generate apparent diffusion coefficient (ADC) and fractional anisotropy (FA) maps. DTI tractography was also performed. The temporal changes in regional ADC and FA values were analyzed statistically using a one-way ANOVA, followed by individual Student's T-tests. Regional FA and ADC of gray and white matter decreased significantly with time (p<0.05). DTI tractography showed a decrease in the number and coherence of reconstructed fiber pathways between PMIs 0 and 14. Elapsed time between death and tissue fixation has a major effect upon the brain's diffusion properties and should be born in mind when interpreting fixed brain DTI.
Project description:Criminal investigations of suspected murder cases require estimating the post-mortem interval (PMI, or time after death) which is challenging for long PMIs. Here we present the case of human remains found in a Swiss forest. We have used a multidisciplinary approach involving the analysis of bones and soil samples collected beneath the remains of the head, upper and lower body and "control" samples taken a few meters away. We analysed soil chemical characteristics, mites and nematodes (by microscopy) and micro-eukaryotes (by Illumina high throughput sequencing). The PMI estimate on hair 14C-data via bomb peak radiocarbon dating gave a time range of 1 to 3 years before the discovery of the remains. Cluster analyses for soil chemical constituents, nematodes, mites and micro-eukaryotes revealed two clusters 1) head and upper body and 2) lower body and controls. From mite evidence, we conclude that the body was probably brought to the site after death. However, chemical analyses, nematode community analyses and the analyses of micro-eukaryotes indicate that decomposition took place at least partly on site. This study illustrates the usefulness of combining several lines of evidence for the study of homicide cases to better calibrate PMI inference tools.
Project description:Estimation of postmortem interval (PMI) is a key issue in the field of forensic pathology. With the availability of quantitative analysis of RNA levels in postmortem tissues, several studies have assessed the postmortem degradation of constitutively expressed RNA species to estimate PMI. However, conventional RNA quantification as well as biochemical and physiological changes employed thus far have limitations related to standardization or normalization. The present study focuses on an interesting feature of the subdomains of certain RNA species, in which they are site-specifically cleaved during apoptotic cell death. We found that the D8 divergent domain of ribosomal RNA (rRNA) bearing cell death-related cleavage sites was rapidly removed during postmortem RNA degradation. In contrast to the fragile domain, the 5' terminal region of 28S rRNA was remarkably stable during the postmortem period. Importantly, the differences in the degradation rates between the two domains in mammalian 28S rRNA were highly proportional to increasing PMI with a significant linear correlation observed in mice as well as human autopsy tissues. In conclusion, we demonstrate that comparison of the degradation rates between domains of a single RNA species provides quantitative information on postmortem degradation states, which can be applied for the estimation of PMI.
Project description:Decomposition of animal bodies in the burial environment plays a key role in the biochemistry of the soil, altering the balance of the local microbial populations present before the introduction of the carcass. Despite the growing number of studies on decomposition and soil bacterial populations, less is known on its effects on fungal communities. Shifts in the fungal populations at different post-mortem intervals (PMIs) could provide insights for PMI estimation and clarify the role that specific fungal taxa have at specific decomposition stages. In this study, we buried pig carcasses over a period of 1- to 6-months, and we sampled the soil in contact with each carcass at different PMIs. We performed metabarcoding analysis of the mycobiome targeting both the internal transcribed spacer (ITS) 1 and 2, to elucidate which one was more suitable for this purpose. Our results showed a decrease in the fungal taxonomic richness associated with increasing PMIs, and the alteration of the soil fungal signature even after 6 months post-burial, showing the inability of soil communities to restore their original composition within this timeframe. The results highlighted taxonomic trends associated with specific PMIs, such as the increase of the Mortierellomycota after 4- and 6-months and of Ascomycota particularly after 2 months, and the decrease of Basidiomycota from the first to the last time point. We have found a limited number of taxa specifically associated with the carrion and not present in the control soil, showing that the major contributors to the recorded changes are originated from the soil and were not introduced by the carrion. As this is the first study conducted on burial graves, it sets the baseline for additional studies to investigate the role of fungal communities on prolonged decomposition periods and to identify fungal biomarkers to improve the accuracy of PMI prediction for forensic applications.
Project description:The necrobiome is the postmortem community that includes bacteria, fungi, arthropods, and other cadaver-associated organisms. It has been suggested as biological evidence for forensic investigation. Fungi form distinctive mildew spots in colonizing decomposing bodies, converting them into moldy cadavers. However, the postmortem fungal community consists of more than these visible species. Characterizing the succession pattern of the fungal community during decomposition is valuable not only for understanding the ecosystem composition of the cadaver decomposition islands but also for contributing to forensic investigations. In the present study, the fungal composition of pig cadavers and succession patterns during decomposition were investigated with high-throughput sequencing. The succession patterns were easier to discern in outdoor cadavers, compared with those that were placed indoors. The metabarcoding approach revealed trends linking particular fungal taxa with specific postmortem intervals (PMIs). Dominant species increased notably in cadavers and soil. Furthermore, the succession of the soil community was driven by the cadaver decomposition. Significant mycoflora differences were observed between environmental and cadaveric soil. The results obtained suggested that postputrefaction mycoflora have considerable potential for PMI estimation, particularly in cases that involve heavily decomposed bodies. In addition, the diversity of fungal communities revealed by the metabarcoding approach allowed us to discriminate the sites of cadaver decomposition, implying that postputrefaction mycoflora may be helpful in identifying the environment in which a cadaver has been placed, or the original location from which a cadaver has been moved. Our results provide an important step towards developing fungal evidence for use in forensic science and add to the growing body of work on postmortem microbial communities.
Project description:The assessment of postmortem degradation of skeletal muscle proteins has emerged as a novel approach to estimate the time since death in the early to mid-postmortem phase (approximately 24 h postmortem (hpm) to 120 hpm). Current protein-based methods are limited to a small number of skeletal muscle proteins, shown to undergo proteolysis after death. In this study, we investigated the usability of a target-based and unbiased system-wide protein analysis to gain further insights into systemic postmortem protein alterations and to identify additional markers for postmortem interval (PMI) delimitation. We performed proteomic profiling to globally analyze postmortem alterations of the rat and mouse skeletal muscle proteome at defined time points (0, 24, 48, 72, and 96 hpm), harnessing a mass spectrometry-based quantitative proteomics approach. Hierarchical clustering analysis for a total of 579 (rat) and 896 (mouse) quantified proteins revealed differentially expressed proteins during the investigated postmortem period. We further focused on two selected proteins (eEF1A2 and GAPDH), which were shown to consistently degrade postmortem in both rat and mouse, suggesting conserved intra- and interspecies degradation behavior, and thus preserved association with the PMI and possible transferability to humans. In turn, we validated the usefulness of these new markers by classical Western blot experiments in a rat model and in human autopsy cases. Our results demonstrate the feasibility of mass spectrometry-based analysis to discover novel protein markers for PMI estimation and show that the proteins eEF1A2 and GAPDH appear to be valuable markers for PMI estimation in humans.
Project description:There are many qualitative indicators for postmortem interval (PMI) of human or animal cadavers. When such indicators are uniformly spaced over PMI, the resultant distribution may be very useful for the estimation of PMI. Existing methods of estimation rely on indicator persistence time that is, however, difficult to estimate because of its dependence on many interacting factors, of which forensic scientists are usually unaware in casework. In this article, an approach is developed for the estimation of PMI from qualitative markers in which indicator persistence time is not used. The method involves the estimation of an interval preceding appearance of a marker on cadaver called the pre-appearance interval (PAI). PMI is delineated by PAI for two consecutive markers: the one being recorded on the cadaver (defining lower PMI) and the other that is next along the PMI timeline but yet absent on the cadaver (defining upper PMI). The approach was calibrated for use with subsequent life stages of carrion insects and tested using results of pig cadaver experiments. Results demonstrate that the presence and absence of the subsequent developmental stages of carrion insects, coupled with the estimation of their PAI, gives a reliable and easily accessible knowledge of PMI in a forensic context.