Automatic removal of soft tissue from 3D dental photo scans; an important step in automating future forensic odontology identification.

The potential of intraoral 3D photo scans in forensic odontology identification remains largely unexplored, even though the high degree of detail could allow automated comparison of ante mortem and post mortem dentitions. Differences in soft tissue conditions between ante- and post mortem intraoral 3D photo scans may cause ambiguous variation, burdening the potential automation of the matching process and underlining the need for limiting inclusion of soft tissue in dental comparison. The soft tissue removal must be able to handle dental arches with missing teeth, and intraoral 3D photo scans not originating from plaster models. To address these challenges, we have developed the grid-cutting method. The method is customisable, allowing fine-grained analysis using a small grid size and adaptation of how much of the soft tissues are excluded from the cropped dental scan. When tested on 66 dental scans, the grid-cutting method was able to limit the amount of soft tissue without removing any teeth in 63/66 dental scans. The remaining 3 dental scans had partly erupted third molars (wisdom teeth) which were removed by the grid-cutting method. Overall, the grid-cutting method represents an important step towards automating the matching process in forensic odontology identification using intraoral 3D photo scans.

Statistical Modelling Investigation of MALDI-MSI-Based Approaches for Document Examination.

Questioned document examination aims to assess if a document of interest has been forged. Spectroscopy-based methods are the gold standard for this type of evaluation. In the past 15 years, Matrix-Assisted Laser Desorption Ionisation-Mass Spectrometry Imaging (MALDI-MSI) has emerged as a powerful analytical tool for the examination of finger marks, blood, and hair. Therefore, this study intended to explore the possibility of expanding the forensic versatility of this technique through its application to questioned documents. Specifically, a combination of MALDI-MSI and chemometric approaches was investigated for the differentiation of seven gel pens, through their ink composition, over 44 days to assess: (i) the ability of MALDI MSI to detect and image ink chemical composition and (ii) the robustness of the combined approach for the classification of different pens over time. The training data were modelled using elastic net logistic regression to obtain probabilities for each pen class and assess the time effect on the ink. This strategy led the classification model to yield predictions matching the ground truth. This model was validated using signatures generated by different pens (blind to the analyst), yielding a 100% accuracy in machine learning cross-validation. These data indicate that the coupling of MALDI-MSI with machine learning was robust for ink discrimination within the dataset and conditions investigated, which justifies further studies, including that of confounders such as paper brands and environmental factors.

Intimate partner homicides in Denmark 1992-2016.

Intimate partner homicide is one of the most common types of homicide and a significant contributor to domestic homicides worldwide, especially affecting females. We focus on the intimate partner homicides in Denmark during 1992-2016. Though gender identity data was unavailable, sex data from official documents enabled critical analysis. Of the 1417 homicides in the period, 26.5% were intimate partner homicides, i.e., 55.6% of female and 8.9% of male victims. The annual intimate partner homicide rate was 0.28 per 100,000 (0.44 for female victims and 0.12 for male victims), declining at a lower rate than other types of homicide. Most victims of intimate partner homicides were females (79.3%). The demographics of the victims and the characteristics of the homicides were markedly different depending on victim sex. Female victims were killed by more varied methods, with more severe injuries and followed by suicide in 26.5% and with multiple homicide victims in 8.1%.

Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.

Untargeted metabolomics is the study of all detectable small molecules, and in geroscience, metabolomics has shown great potential to describe the biological age-a complex trait impacted by many factors. Unfortunately, the sample sizes are often insufficient to achieve sufficient power and minimize potential biases caused by, for example, demographic factors. In this study, we present the analysis of biological age in ~10,000 toxicologic routine blood measurements. The untargeted screening samples obtained from ultra-high pressure liquid chromatography-quadruple time of flight mass spectrometry (UHPLC- QTOF) cover + 300 batches and + 30 months, lack pooled quality controls, lack controlled sample collection, and has previously only been used in small-scale studies. To overcome experimental effects, we developed and tested a custom neural network model and compared it with existing prediction methods. Overall, the neural network was able to predict the chronological age with an rmse of 5.88 years (r2  = 0.63) improving upon the 6.15 years achieved by existing normalization methods. We used the feature importance algorithm, Shapley Additive exPlanations (SHAP), to identify compounds related to the biological age. Most importantly, the model returned known aging markers such as kynurenine, indole-3-aldehyde, and acylcarnitines along with a potential novel aging marker, cyclo (leu-pro). Our results validate the association of tryptophan and acylcarnitine metabolism to aging in a highly uncontrolled large-s cale sample. Also, we have shown that by using robust computational methods it is possible to deploy large LC-MS datasets for metabolomics studies to reduce the risk of bias and empower aging studies.

Blunt force homicides in Denmark 1992-2016.

Blunt force trauma is a common homicide method, inflicted in three different ways: bodily force, assault with blunt objects of various types and falls from height. The objective of this study is to provide thorough information on blunt force homicides with data on the victims, the offenders, the surrounding circumstances, the injury methods, the extent of injuries, and survival time, which will help inform the inexperienced as well as the seasoned forensic pathologist in their daily work with death investigation and as expert witnesses in court. We have analyzed autopsy reports and available case files of 311 blunt force homicides, making up 21.9% of all homicides in Denmark during 1992-2016. Most victims and offenders were male. Altercation in the setting of nightlife and intoxication was common in male victims, while most female victims were killed in a domestic setting. Bodily force was the most common primary homicide method, followed by assault with a blunt object and fall from height. The head was the region that most often had external injuries, with no noteworthy difference between cases with bodily force and blunt objects. Two out of three victims had one or more lacerations, most often located on the head and more often on the front. Brain injury was the primary cause of death in at least 72.0% victims. Compared to bodily force victims of blunt object assault were especially prone to skull and brain injuries, had a higher trauma score, and more died at the crime scene and had a shorter survival time.

From genotype to phenotype: Genetic redundancy and the maintenance of an adaptive polymorphism in the context of high gene flow.

A central question in evolution is how several adaptive phenotypes are maintained within a species. Theory predicts that the genetic determination of a trait, and in particular the amounts of redundancy in the mapping of genotypes to phenotypes, mediates evolutionary outcomes of phenotypic selection. In Mediterranean wild thyme, numerous discrete chemical phenotypes (chemotypes) occur in close geographic proximity. Chemotypes are defined by the predominant monoterpene produced by individual plants in their essential oil. In this study, we analyze the ecological genetics of six chemotypes nested within two well-established chemical families (hereafter ecotypes). Ecotypes, and chemotypes within ecotypes, are spatially segregated, and their distributions track local differences in the abiotic environment. By combining population genomic, phenotypic, and environmental data from 700 individuals, we show how the genetics of ecotype determination mediates this evolutionary response. Variation in three terpene-synthase loci explains variation in ecotype identity, with one single locus accounting for as much as 78% of this variation. Phenotypic selection combined with low segregating genotypic redundancy of ecotypes leaves a clear footprint at the genomic level: alleles associated with ecotype identity track environmental variation despite extensive gene flow. Different chemotypes within each ecotype differentially track environmental variation. Their identity is determined by multiple loci and displays a wider range of genotypic redundancy that dilutes phenotypic selection on their characteristic alleles. Our study thus provides a novel illustration of how genetic redundancy of a phenotype modulates the ability of selection to maintain adaptive differentiation. Identifying the precise genetics of the chemical polymorphism in thyme is the next crucial step for our understanding of the origin and maintenance of a polymorphism that is present in many aromatic plants.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Asphyxia homicides in Denmark 1992-2016.

In this retrospective study , we present the findings in 250 homicides by asphyxia in Denmark in a 25-year period, with a particular focus on the autopsy findings in strangulation. Our intention is for the results to be used in future death investigations, where difficulties in interpretation of findings in potential asphyxial deaths arise. Asphyxia homicides showed a strong bias with respect to sex, age, and homicide type. The frequent female victim was typically an adult, whereas the rarer male victim was most often a child. Female offenders most often killed their children, and male offenders most often killed their female partner. Generally, most asphyxia homicides took place in a domestic setting. Manual strangulation and ligature strangulation were the most common mechanisms of asphyxia homicides (81.6%). A lack of petechial hemorrhages, especially in the conjunctiva, was rare in homicidal strangulation, but there were exceptions, especially when there was postmortem decomposition, making it impossible to verify them. Most victims of strangulation had skin lesions in the face (including the jawline) or on the neck, with accompanying hemorrhages in muscle and connective tissue, but the findings could be subtle or compounded by decomposition. Fractures of the laryngo-hyoid complex were common in strangulation, particularly in manual strangulation (chi-sq = 4.0993, df = 1, P < 0.05) and were clearly related to the age of the victim (chi-sq = 82.193, df = 4, P < 0.001). In children and young adults dying from homicidal strangulation, a lack of fractures is to be expected, while a lack of fractures is unusual, but not entirely unexpected, for adults and aged people.

Assessment of XCMS Optimization Methods with Machine-Learning Performance.

The metabolomics field is under rapid development. In particular, biomarker identification and pathway analysis are growing, as untargeted metabolomics is usable for discovery research. Frequently, new processing and statistical strategies are proposed to accommodate the increasing demand for robust and standardized data. One such algorithm is XCMS, which processes raw data into integrated peaks. Multiple studies have tried to assess the effect of optimizing XCMS parameters, but it is challenging to quantify the quality of the XCMS output. In this study, we investigate the effect of two automated optimization tools (Autotuner and isotopologue parameter optimization (IPO)) using the prediction power of machine learning as a proxy for the quality of the data set. We show that optimized parameters outperform default XCMS settings and that manually chosen parameters by liquid chromatography-mass spectrometry (LC-MS) experts remain the best. Finally, the machine-learning approach of quality assessment is proposed for future evaluations of newly developed optimization methods because its performance directly measures the retained signal upon preprocessing.

Gunshot homicides in Denmark 1992-2016.

The majority of homicides in the world are caused by firearms and a good understanding of gunshot wounds is important for the forensic pathologist. We have analyzed all 315 homicides by gunshots in Denmark during 1992-2016 with focus on information relevant to forensic pathologists. Of the 1417 homicides in Denmark in 1992-2016, 315 (22.2%) had gunshot trauma as the primary homicide method. During 1992-2016, there has been a significant decrease in the number of gunshot homicides overall, of homicides committed with hunting weapons, and of domestic gunshot homicides. Most victims (70.5%) and offenders (93.7% of homicides with known offenders) were males. Male victims were significantly younger than female victims. Most victims were killed with handguns (43.8%) or shotguns (41.0%). Compared to all other weapons, the victims killed with shotguns had fewer entry wounds, a higher New Injury Severity Score (NISS), and a higher proportion of contact/close-range entry wounds. The head (58.1%) and thorax (46.7%) were often injured, with injuries to the head being more common in contact/close-range entry wounds. The results show that gunshot injuries in homicides are different from suicides and accidents and clearly are affected by the homicide type, the sex of the victim, and the weapon used and that these entities are intertwined. Our findings provide an evidence-based foundation for use in death investigations and for policy development in the area of interpersonal violence.

Improved medical treatment could explain a decrease in homicides with a single stab wound.

Since the 1990s, there has been a reduction in the homicide rate in Denmark and other Western countries. Our hypothesis is that part of the decrease in the sharp force homicide rate can be explained by better and faster medical treatment over time, and we explore this via stab wound homicides, the largest group of homicides in Denmark. To investigate our hypothesis we undertook an epidemiological study of 428 stab wound homicides in Denmark 1992-2016 based on autopsy reports with registration of stab wounds, quantification of injury severity, treatment intensity and survival time. During 1992-2016, there was a significant reduction in the annual number of victims with a single stab wound, but no reduction in victims with multiple stab wounds. Victims with single stab wounds reached the hospital more often, survived longer and had less severe injuries (New Injury Severity Score (NISS)) than victims with multiple stab wounds. Higher NISS correlated with shorter survival time for all the stab wound victims and for the subgroup that underwent medical treatment. During the 25-year study period, the proportion of victims who underwent surgery before dying increased threefold. The victims in the first half of the study period had shorter survival times than the victims in the last half. We concluded that better and faster medical treatment could partly be responsible for the observed decrease in the number of single stab wound homicides and thereby possibly also in the total number of stab wound homicides.

Sharp Force Homicide in Denmark 1992-2016.

Sharp force trauma is a common homicide method. The weapon is typically a knife, which is easily accessible and does not require special skills. We have analyzed all 471 sharp force homicides in Denmark during 1992-2016 with special focus on aspects that are relevant to forensic pathologists, including the distribution of wounds and organ injuries. Most homicides were committed inside with a kitchen knife. The front left thorax was the most common area to be affected by sharp force trauma. In 18.9% of the victims, there was only one sharp injury, the majority on the thorax. The most common trajectory for stab wounds was directly posterior with no deviation to the sides or up/down followed by directly anterior. The heart (including pericardium) and lungs (including hemo- and pneumothorax) had injuries in more than 75% of the victims. 67% of victims were males. Female victims had more sharp force injuries and defense wounds than male victims. Most females were killed in domestic homicides (73.7%), most commonly in partner killings (56.4%). In contrast, many male victims were killed in a setting of nightlife/intoxication (34.0%) most by a friend/acquaintance delivering a few stab wounds. The results clearly show strong sex differences in both victims and offenders. This could be useful for shaping policies and public opinion, and as a route for understanding the developments in interpersonal violence. In the narrow setting of death investigation, our results will provide an evidence-based approach to understanding the injury patterns in sharp force homicide.

Homicide in Denmark 1992-2016.

We present the findings for homicides in Denmark for 1992-2016. There were 1417 homicide victims (62.2% males, 37.8% females) that were killed in 1321 homicide events. The most common methods were sharp force trauma (33.2%), gunshot (22.2%), blunt force trauma (21.9%) and asphyxia (17.6%), and all methods exhibited a reduction during the study period. The homicide rate was 1.05 per 100,000, 1.32 per 100,000 for males, and 0.78 per 100,000 for females. Domestic homicides were the largest main group of homicides (76.5% of all female victims vs. 23.6% of male victims). Of the non-domestic homicides, 84.2% of victims were male, the largest group being in the setting of nightlife and/or intoxication. Most female victims (76.9%) were killed by someone in their family, while the largest share of male victims (34.5%) were killed by a friend or acquaintance. The offenders were males in 87.9% of all homicides.

Transcriptome Analysis of Long Non-coding RNAs and Genes Encoding Paraspeckle Proteins During Human Ovarian Follicle Development.

Emerging evidence indicated that many long non-coding (lnc)RNAs function in multiple biological processes and dysregulation of their expression can cause diseases. Most regulatory lncRNAs interact with biological macromolecules such as DNA, RNA, and protein. LncRNAs regulate gene expression through epigenetic modification, transcription, and posttranscription, through DNA methylation, histone modification, and chromatin remodeling. Interestingly, differential lncRNA expression profiles in human oocytes and cumulus cells was recently assessed, however, lncRNAs in human follicle development has not previously been described. In this study, transcriptome dynamics in human primordial, primary and small antral follicles were interrogated and revealed information of lncRNA genes. It is known that some lncRNAs form a complex with paraspeckle proteins and therefore, we extended our transcriptional analysis to include genes encoding paraspeckle proteins. Primordial, primary follicles and small antral follicles was isolated using laser capture micro-dissection from ovarian tissue donated by three women having ovarian tissue cryopreserved before chemotherapy. After RN sequencing, a bioinformatic class comparison was performed and primordial, primary and small antral follicles were found to express several lncRNA and genes encoding paraspeckle proteins. Of particular interest, we detected the lncRNAs XIST, NEAT1, NEAT2 (MALAT1), and GAS5. Moreover, we noted a high expression of FUS, TAF15, and EWS components of the paraspeckles, proteins that belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity, and mRNA/microRNA processing. We also interrogated the intra-ovarian localization of the FUS, TAF15, and EWS proteins using immunofluorescence. The presence and the dynamics of genes that encode lncRNA and paraspeckle proteins may suggest that these may mediate functions in the cyclic recruitment and differentiation of human follicles and could participate in biological processes known to be associated with lncRNAs and paraspeckle proteins, such as gene expression control, scaffold formation and epigenetic control through human follicle development. This comprehensive transcriptome analysis of lncRNAs and genes encoding paraspeckle proteins expressed in human follicles could potentially provide biomarkers of oocyte quality for the development of non-invasive tests to identify embryos with high developmental potential.

Stability of amphetamine impurity profiles during 12 months of storage.

Impurity profiling is a well described forensic tool that may be applied to gain information about the illegal drug market. However, it requires experience to assess the correlation between chemical profiles thereby separating linked from unlinked samples. One of the challenges in this context is that the chemical profiles may change over time, thus complicating an assessment if samples are stored under different conditions. In this study, the impact of different storage conditions on the stability of amphetamine impurity profiles was investigated. We examined the influence of storage time, temperature, sample purity, sample quantity and the presence of methanol on the amphetamine profile stability when stored for up to 12 months. We find that the target compounds in amphetamine impurity profiles are susceptible to all the examined storage conditions. Consequently, this unstable nature of amphetamine profiles may complicate the assessment when comparing amphetamine seizures that has been separated for longer time periods or stored under different conditions prior to seizure. Knowledge about the seizure history is rarely available to the forensic analyst. Therefore, sample stability issues should be taken into account when comparisons of chemical profiles are made.

Whole-Genome Sequencing of Aggregatibacter Species Isolated from Human Clinical Specimens and Description of Aggregatibacter kilianii sp. nov.

Aggregatibacter species are commensal bacteria of human mucosal surfaces that are sometimes involved in serious invasive infections. During the investigation of strains cultured from various clinical specimens, we encountered a coherent group of 10 isolates that could not be allocated to any validly named species by phenotype, mass spectrometry, or partial 16S rRNA gene sequencing. Whole-genome sequencing revealed a phylogenetic cluster related to but separate from Aggregatibacter aphrophilus The mean in silico DNA hybridization value for strains of the new cluster versus A. aphrophilus was 56% (range, 53.7 to 58.0%), whereas the average nucleotide identity was 94.4% (range, 93.9 to 94.8%). The new cluster exhibited aggregative properties typical of the genus Aggregatibacter Key phenotypic tests for discrimination of the new cluster from validly named Aggregatibacter species are alanine-phenylalanine-proline arylamidase, N-acetylglucosamine, and ß-galactosidase. The name Aggregatibacter kilianii is proposed, with PN_528 (CCUG 70536T or DSM 105094T) as the type strain.

Variation in chemical profiles within large seizures of cocaine bricks.

Cocaine is usually trafficked from South America throughout the world in packages of approximately one kilogram shaped as bricks and imprinted with a logo. Seizures consisting of multiple cocaine bricks gives the opportunity to examine the variation in the chemical profile within cocaine bricks assumed to originate from the same manufacturer and maybe even the same production batch. This knowledge may be important to the forensic investigator when chemical profiles from cocaine samples of unknown origin are compared. In the present study, the alkaloid and residual solvent profiles from three large cocaine seizures each containing identical cocaine bricks was examined. The three cases consisted of 36, 84 and 100 cocaine bricks, respectively. Each cocaine brick was profiled according to its cocaine alkaloid and residual solvent content using gas chromatography-mass spectrometry (GC-MS) and headspace GC-MS. The study showed that each of the three identical looking seizures consisted of up to four groups of cocaine bricks displaying the same cocaine alkaloid and residual solvent profile. The size of the groups varied from 2.4 to 63.3kg cocaine. The study also showed that the residual solvent profile within each of the three large seizures exhibited very little variation whereas the alkaloid profile varied considerably more. This finding suggest that the same organic solvent is used for the production of several batches of cocaine HCl. Therefore, the residual solvent profile may be a tool to link different production batches from the same manufacturer even though the alkaloid profile are different.

Profiling of cocaine using ratios of GC-MS peaks.

Illicit cocaine seizures are often compared to each other by using gas chromatography-mass spectrometry (GC-MS) data from cocaine alkaloid compounds to determine whether two specimens originate from the same production batch or not. This can provide intelligence or investigative information at the early stages of an investigation or evidence in court. Traditional classification methods assume high stability of all alkaloids, use all of them to calculate the correlation between two profiles and use a threshold to classify samples. Unstable alkaloids will have a strong influence on the performance. We show that comparing each alkaloid target compound individually improves the classification. Unfortunately, it requires normalization and is also sensitive to the stability. Instead we suggest to use ratios of all possible pairwise combinations of the GC-MS peaks. These ratios are scale free and directly comparable between samples. The peaks can be given different weights in the comparison of profiles using appropriate classification methods and we show that randomForest classification using these ratios have a high and reproducible performance in comparison with other methods. The performance of this method is not affected by noise, transformation or normalization and should be considered for future comparison of chromatographic profiles in general.

Assembly and analysis of 100 full MHC haplotypes from the Danish population.

Genes in the major histocompatibility complex (MHC, also known as HLA) play a critical role in the immune response and variation within the extended 4-Mb region shows association with major risks of many diseases. Yet, deciphering the underlying causes of these associations is difficult because the MHC is the most polymorphic region of the genome with a complex linkage disequilibrium structure. Here, we reconstruct full MHC haplotypes from de novo assembled trios without relying on a reference genome and perform evolutionary analyses. We report 100 full MHC haplotypes and call a large set of structural variants in the regions for future use in imputation with GWAS data. We also present the first complete analysis of the recombination landscape in the entire region and show how balancing selection at classical genes have linked effects on the frequency of variants throughout the region.

Sequencing and de novo assembly of 150 genomes from Denmark as a population reference.

Hundreds of thousands of human genomes are now being sequenced to characterize genetic variation and use this information to augment association mapping studies of complex disorders and other phenotypic traits. Genetic variation is identified mainly by mapping short reads to the reference genome or by performing local assembly. However, these approaches are biased against discovery of structural variants and variation in the more complex parts of the genome. Hence, large-scale de novo assembly is needed. Here we show that it is possible to construct excellent de novo assemblies from high-coverage sequencing with mate-pair libraries extending up to 20 kilobases. We report de novo assemblies of 150 individuals (50 trios) from the GenomeDenmark project. The quality of these assemblies is similar to those obtained using the more expensive long-read technology. We use the assemblies to identify a rich set of structural variants including many novel insertions and demonstrate how this variant catalogue enables further deciphering of known association mapping signals. We leverage the assemblies to provide 100 completely resolved major histocompatibility complex haplotypes and to resolve major parts of the Y chromosome. Our study provides a regional reference genome that we expect will improve the power of future association mapping studies and hence pave the way for precision medicine initiatives, which now are being launched in many countries including Denmark.