Successive Protein Extraction Using Hydroxylamine to Increase the Depth of Proteome Coverage in Fresh, Forensic, and Archaeological Bones.

Proteomics is continually being applied to a wider range of applications, now including the analysis of archaeological samples and anatomical specimens, particularly collagen-containing tissues such as bones and teeth. Here, we present the application of a chemical digestion-based proteomics sample preparation protocol to the analysis of fresh, anatomical, and archaeological samples. We describe and discuss two protocols: one that uses hydroxylamine as an additional step of the proteomic workflow, applied to the insoluble fraction, and another that applies hydroxylamine directly on demineralized bones and teeth. We demonstrate the additional information that can be extracted using both protocols, including an increase in the sequence coverage and number of peptides detected in modern and archaeological samples and an increase in the number of proteins identified in archaeological samples. By targeting research related to collagens or extracellular matrix proteins, the use of this protocol will open new insights, considering both fresh and ancient mineralized samples.

A sudden death, an aortic rupture, and an unexpected cause: a report about suspected child abuse.

All unexpected deaths of children require an autopsy to determine the cause of death. In cases of aortic rupture, the immediate cause of death is easily identified at autopsy. Although the majority of aortic ruptures are caused by high-energy trauma, other causes should not be missed.We present and discuss the case of a 29-month-old child who died suddenly at home. Her recent medical history and the ecchymotic lesions observed on external examination of the body appeared potentially suspicious of physical abuse. The autopsy concluded that death was due to complete rupture of the abdominal aorta with associated vertebral disjunction. At first glance, the overall forensic picture could suggest a traumatic death. However, careful inspection of the retroperitoneum revealed a discrete atypical mass of infiltrative tissue within the hematoma. Histopathological examinations confirmed tumor proliferation of the soft tissues, triggering vascular and spinal injuries. Other paraneoplastic elements or metastases were ultimately revealed (orbital and subcutaneous). Overall, this was a rare and fatal case of abdominal aortic rupture induced by tumors. Due to the mechanisms and the forces needed to cause vertebral dislocations and aortic rupture, the combination of the two is highly suggestive of child abuse when an accidental traumatic history is absent or inconsistent with the injuries. Nevertheless, this case illustrates the importance of a systematic and rigorous forensic examination, rather than ignoring other possible diagnoses.

Effect of density and species preferences on collective choices: an experimental study on maggot aggregation behaviours.

Collective decisions have been extensively studied in arthropods, but they remain poorly understood in heterospecific groups. This study was designed to (1) assess the collective behaviours of blow fly larvae (Diptera: Calliphoridae) in groups varying in density and species composition, and (2) relate them to the costs and benefits of aggregating on fresh or decomposed food. First, experiments testing conspecific groups of Lucilia sericata and Calliphora vicina larvae, two species feeding at the same time on fresh carcasses, demonstrated decreases in growth and survival on rotten beef liver compared with fresh liver. However, mixing species together reduced this adverse impact of decomposition by increasing the mass of emerged adults. Second, larval groups were observed in binary choice tests between fresh and rotten liver (i.e. optimal and sub-optimal food sources). The results showed that larvae interacted with each other and that these interactions influenced their food preferences. We observed that (1) larvae were able to collectively choose the optimal food, (2) their choice accuracy increased with larval density and (3) the presence of another species induced a reversal in larval preference towards rotten food. These results highlight the ubiquity of collective decision properties in gregarious insects. They also reveal an unexpected effect of interspecific association, suggesting the colonization of new resources through a developmental niche construction.

Traumatic rectovaginal fistula after sexual intercourse following a non-consensual anal penetration: a case report and a review of the literature.

Rectovaginal fistulas are rare genital lesions, often due to obstetric causes. More rarely they are a consequence of sexual intercourse. We present the case of a 36-year-old woman who presented to a gynecological emergency department with pelvic pain and feces coming from the vagina. This symptomatology appeared after non-consensual anal intercourse following consensual penile vaginal penetration. She was treated for a perforation of the rectovaginal septum. On the basis of our results, we reviewed the cases published in the literature, with a summary of the elements that may favor the appearance of this lesion, such as "virginity", obstetrics and gynecological history, or coitus position, its classification by size and localization, and the therapeutic indications (suture repair of the wall or a colostomy). Better knowledge of this type of lesion would improve the practice of forensic pathologists for screening and management.

Temperature: the weak point of forensic entomology.

Measuring temperature is a key factor in forensic entomology. While noting factors to consider for a posteriori temperature estimation, many studies lack detailed methods or general rules allowing their integration into insect development-time calculations. This article proposes tools for determining the adequacy of weather station temperature datasets versus the local temperature experienced by carrion breeders. The idea is to start from a local scale (i.e., the cadaver) and gradually move to larger scales: at each step, the temperature can be increased, decreased or smoothed by environmental or biological factors. While a one-size-fits-all solution is not feasible for a complex and sensitive issue such as forensic meteorology, this checklist increases the reliability of minimum post-mortem interval (PMImin) estimation and the traceability of the proposed assumption.

Histological dating of subdural hematoma in infants.

BACKGROUND: After infant deaths due to non-accidental head injury (NAHI) with subdural hematoma (SDH), the magistrates ask experts to date the traumatic event. To do so, the expert only has tools based on adult series of NAHI. We aimed to develop an SDH dating system applicable to infants aged under 3 years. METHODS AND RESULTS: We studied a retrospective multicenter collection of 235 infants who died between the ages of 0 and 36 months, diagnosed with SDH by forensic pathological examination and with known posttraumatic interval (PTI). Two pathologists assessed blindly and independently 12 histomorphological criteria relating to the clot and 14 relating to the dura mater in 73 victims (31 girls, 42 boys) whose median age was 3.8 months. Histopathological changes were significantly correlated with PTI for the appearance of red blood cells (RBCs) and the presence or absence of siderophages, and regarding the dura mater, the quantity of lymphocytes, macrophages, and siderophages; presence or absence of hematoidin deposits; collagen and fibroblast formation; neomembrane thickness; and presence or absence of neovascularization. Dating systems for SDH in adults are not applicable to infants. Notably, neomembrane of organized connective tissue is formed earlier in infants than in adults. CONCLUSION: Our dating system improves the precision and reliability of forensic pathological expert examination of NAHI, particularly for age estimation of SDH in infants. However, the expert can only define a time interval. Histopathology is indispensable to detect repetitive trauma.

Age at death estimation by cementochronology: Too precise to be true or too precise to be accurate?

OBJECTIVES: Cementochronology based on annual deposition of acellular cementum is acknowledged for its superior performance for estimating age-at-death but is also disregarded because of its suspicious effectiveness. This article aims to provide a standardized framework for the validation of the technique and to define and test cementochronology's performance and limitations. MATERIALS AND METHODS: To determine the boundaries of the cementum aging technique, we applied a certified protocol on a sample of 200 healthy canines from individuals of known age, sex, postmortem, and inhumation intervals from anthropological and anatomical collections. We scored readability and preservation of cementum and measured the agreement between estimates, i.e., the precision, and assessed the quality of the accordance between estimates and chronological age, i.e., the accuracy. To investigate the applicability on ancient material, 200 additional canines extracted from archeological assemblages were included. Accuracy and precision were analyzed for each age group in considering postmortem intervals and taphonomical conditions. RESULTS: A strong correlation was found between chronological age and estimates (r = .927; p = .000) but results reveal an association between readability of incremental lines and chronological age (p < .05) and a notable difference in both precision and accuracy between individuals under and over 50 years. Results also demonstrate that taphonomy can be a serious obstacle increasing imprecision by a factor of three. DISCUSSION: Improperly adopted, cementochronology can lead to precise but inaccurate estimations. If methodological, physiological, and taphonomical factors are taken into account, then, and only then, cementochronology will serve as a versatile and powerful tool for age-at-death estimation.

Medico-legal considerations for Morel-Lavallée lesions.

The Morel-Lavallée lesion is an infrequent traumatic lesion that the forensic physician may need to evaluate during examination of a victim. Using a review of the literature and an illustrative case, the objective of this paper is to discuss the medico-legal issues (accountability, functional recovery and healing times, and aftermath) associated with Morel-Lavallée lesions. We describe the case of a 24-year-old motorcycle driver who was hit by a car. Clinically, the right lumbar region had a large 16 × 15 cm ecchymosis with subcutaneous fluid swelling found by palpation. A body scan revealed a Morel-Lavallée lesion among other lesions. On day 7, its thickness was decreased by half. Morel-Lavallée lesions are specifically induced by shearing force with moderate-to-high kinetics in an anatomical area with an underlying fascia. The associated functional disability is globally moderate, but a large lesion or recurrences can extend the time needed to recover. Apart from some exceptions, care without any delay in diagnosis and treatment allows evolution of the lesion without functional consequences, but these lesions are frequently unnoticed in the initial phase.

Is bone analysis with µCT useful for short postmortem interval estimation?

The evaluation of the postmortem interval (PMI) is an important consideration in forensic medicine. The time between death and the discovery thereof is extremely important, not only in terms of the legal process, but also for the course of the investigation. At the time of the discovery of human bones, the evaluation of the PMI is extremely difficult. To date, there is no really effective tool for this evaluation. The objective of our study is to characterize the architectural modifications of human bones using micro-tomography (µCT) in the postmortem period. Ten bone samples were extracted from a single fresh human cranial vault (no freezing, no embalming) from a body donation. The samples were conserved in a controlled environment (a laboratory hood at 20 °C with 60% humidity) for 10 weeks. Every 2 weeks after the death and the sample extraction from the vault, the samples were analyzed with µCT (resolution 10 µm, Bruker HR1172 ®). The 3D analysis focused on general 3D data related to the trabeculae: the total volume (TV), the bone volume (BV), and the bone surface (BS), as well as specific trabeculae data; namely: the number of trabeculae (TbN), trabecular thickness (TbTh), the average distance between the trabeculae (TbSp), and the trabecular pattern factor (TbPf). Each sample was observed separately and compared to itself over time; no comparisons among samples were made. We used a linear mixed model for repeated measures (an unstructured covariance pattern model) and post hoc comparisons between each consecutive time using linear contrast with an SAS software package, release 9.3 (SAS Institute, Cary, NC). We showed a statistical difference for BS/BV (p = 0.046) over time, with a significant decrease during the first 2 weeks (p = 0.003; mean decrease, - 0.99, 95% CI [- 1.61; - 0.36]), while TbSp (p < 0.0001) showed a significant increase during the first 2 weeks (p < 0.0001; mean increase, 0.022, 95% CI, [- 0.016; - 0.028]).Our study highlighted architectural trabecular modifications on postmortem bones using the µCT. These modifications occurred at an early stage, during the first 2 weeks of conservation. Complementary work will need to be performed to allow for better understanding of the biological phenomena observed and to create databases of osseous taphonomy, starting with various individuals, mediums of conservation, types of bone, and with varying techniques for analysis.

Characterization of bone diagenesis by histology in forensic contexts: a human taphonomic study.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

Technical note: early post-mortem changes of human bone in taphonomy with µCT.

Post-mortem interval (PMI) estimation is an important issue in forensic medicine, particularly for criminal purposes and legal limitation periods. The goal of the present study is to examine the evolution of the trabecular cranial vault bone after 4 weeks of conservation in a controlled environment with micro-tomography (µCT) analyses.Four bone samples were extracted from a fresh human cranial vault (a donation to science according to the French law) and conserved in an air-controlled environment. The samples were weighed and µCT scanned at a 10-µm resolution every week after death for a month. The µCT features were identical for every sample. Each set of data from the µCTs was reconstructed, registered, and analyzed in terms of the total volume, bone volume, bone surface, number of trabeculae, trabeculae thickness, and mean distance of the trabeculae. The samples were conserved in a glass box in 20 °C air with 60% humidity in a laboratory hood between each µCT acquisition. Descriptive statistics were determined. Each sample was observed and compared to itself over time.After 1 month of conservation, the mean bone volume (-1.9%), bone surface (-5.1%), and trabecular number (-12.35%) decreased, whereas the mean trabecular separation (+5.55%) and trabecular thickness (+12.7%) increased. Many variations (i.e., increases and decreases) were observed between the extraction of the sample and the end of the 4 weeks of conservation. The present observations may be explained by bone diagenesis. Previous observations have indicated that protein and lipid losses occur with bone weight and volume losses. These diagenesis effects may explain the trabecular modifications observed in the present work. We observed many bone variations with the µCT scans between the beginning and the end of the conservation that had no explanations. Additional studies, particularly studies involving statistics, need to be performed to confirm our observations and explain these results more clearly.

Interspecific shared collective decision-making in two forensically important species.

To date, the study of collective behaviour has mainly focused on intraspecific situations: the collective decision-making of mixed-species groups involving interspecific aggregation-segregation has received little attention. Here, we show that, in both conspecific and heterospecific groups, the larvae of two species (Lucilia sericata and Calliphora vomitoria, calliphorid carrion-feeding flies) were able to make a collective choice. In all groups, the choice was made within a few minutes and persisted throughout the period of the experiment. The monitoring of a focal individual within a group showed that these aggregations were governed by attractive and retentive effects of the group. Furthermore, the similarity observed between the conspecific and heterospecific groups suggested the existence of shared aggregation signals. The group size was found to have a stronger influence than the species of necrophagous larvae. These results should be viewed in relation to the well-known correlation between group size and heat generation. This study provides the first experimental examination of the dynamics of collective decision-making in mixed-species groups of invertebrates, contributing to our understanding of the cooperation-competition phenomenon in animal social groups.

The biology and ecology of Necrodes littoralis, a species of forensic interest in Europe.

Necrodes littoralis (Linnaeus, 1758) (Coleoptera: Silphidae), also known as the "shore sexton beetle," is a common silphid beetle that visits and breeds on large vertebrate cadavers. This study describes, for the first time, the involvement of N. littoralis on human corpses based on a large dataset of 154 French forensic cases. Various parameters regarding corpse location, decomposition stages, and entomofauna were extracted from each file. Compared to all of the forensic entomology cases analyzed between 1990 and 2013 (1028), N. littoralis was observed, on average, in one case out of eight; most of these cases occurred during spring and summer (73.5%). More than 90% of the cases were located outdoors, especially in woodlands, bushes, and fields. The decomposition stage of the corpse varied among cases, with more than 50% in the advanced decomposition stage, 36% in the early decomposition stage, and less than 10% in the fresh, mummified, or skeletonized stages. Regarding other necrophagous species sampled with N. littoralis, Calliphorid flies were found in 94% of the cases and Fanniidae/Muscidae in 65% of the cases. Chrysomya albiceps, a heliophilic species mostly located in the Mediterranean area, was present in 34% of the cases (only 20% in the whole dataset). The most common coleopteran species were Necrobia spp. (Coleoptera: Cleridae) and Creophilus maxillosus (Coleoptera: Staphylinidae); these beetles were observed in 27% of the cases. The over-representation of these species is likely due to similar requirements regarding the climate and decomposition stage. As N. littoralis is frequently observed and tends to become more common, we conclude that the developmental data for this species would be a precious tool for forensic entomologists in Europe.

Cyanide Suicide After Deep Web Shopping: A Case Report.

Cyanide is a product that is known for its use in industrial or laboratory processes, as well as for intentional intoxication. The toxicity of cyanide is well described in humans with rapid inhibition of cellular aerobic metabolism after ingestion or inhalation, leading to severe clinical effects that are frequently lethal. We report the case of a young white man found dead in a hotel room after self-poisoning with cyanide ordered in the deep Web. This case shows a probable complex suicide kit use including cyanide, as a lethal tool, and dextromethorphan, as a sedative and anxiolytic substance. This case is an original example of the emerging deep Web shopping in illegal drug procurement.

Organ Procurement in Forensic Deaths: Specific Features of Nonheart-Beating Donors.

The need for organs has increased in the recent years, and this shortage will only worsen. In addition to the organs procured from brain-dead donors, a procedure for non-heart-beating donors was therefore developed. When deaths involve legal proceedings, the medical examiner makes the decision of whether or not to remove organs. To assist medical examiner in their decision-making, a number of forensic scientific societies aimed to develop recommendations, and legal adjustments were adopted. Nevertheless, these do present certain limits in special cases of procurement: Maastricht class I and II non-heart-beating donors. The peculiarity of this procedure stems from the urgency of the process. Compliance with temporal criteria would require that the deceased subject be removed from the crime scene even before the arrival of the authorities. This is extremely problematic legally and technically because forensic teams must collect evidence at crime scenes. Developments will have to be made to further minimize the restrictions on donations in forensic deaths.

Homemade Firearm Suicide With Dumbbell Pipe Triggering by an Air-Compressed Gun: Case Report and Review of Literature.

Firearm suicides are frequent and well described in the forensic literature, particularly in Europe and the United States. However, the use of homemade and improvised firearms is less well described. The present case reports a suicide with an original improvised gun created using an air-compressed pellet gun and a dumbbell pipe. The aims of this study were to describe the scene, the external examination of the corpse, the body scan, and the autopsy; to understand the mechanism of death; and to compare the results with a review of the forensic literature to highlight the epidemiology of homemade firearm use, the tools used for homemade and improvised firearms in suicides versus homicides, and the manners in which homemade firearms are used (homicide or suicide, particularly in complex suicide cases).

Involvement of larder beetles (Coleoptera: Dermestidae) on human cadavers: a review of 81 forensic cases.

From 1994 to 2013, French forensic entomology laboratories investigated 1,093 cases. Larder beetles (Coleoptera: Dermestidae) were observed in 81 (7.5%) of these cases. To describe and analyze these 81 cases, eight parameters were used: city, location (indoor or outdoor), decay stage (fresh, decay, or dry), dermestid species and instar (adults and/or larvae), presence of living calliphorid larvae, presence of calliphorid pupae or adults, and presence of other necrophagous species. Eight Dermestidae species were observed: Dermestes frischii (42% of cases), Dermestes undulatus (35.8%), Dermestes peruvianus (12.3%), Dermestes lardarius (9.9%), Dermestes haemorrhoidalis (8.6%), Dermestes maculatus (7.4%), Dermestes bicolor (3.7%), and Dermestes ater (1.2%). Larder beetles primarily developed on human cadavers in outdoor locations in areas with a dry climate and were never reported in oceanic areas (which are characterized by frequent rainfall and high ambient humidity). The number of dermestid species on a single corpse never exceeded three. Typically, one species was found per corpse. Species differed between indoor and outdoor cases, with D. frischii and D. undulatus dominant in outdoor cases, while D. peruvianus dominant in indoor cases. Calliphoridae was found in 88% of the cases, while Hydrotaea and Piophilidae were observed 40% of the time. Regarding Coleoptera, Necrobia spp. (Coleoptera: Cleridae) was observed in 46% of the cases. Lastly, we observed a typical decomposition pattern, with preferential feeding areas on the face, hands, and feet (i.e., the extremities). Pupation chambers on or inside the bones were not observed.

[Performing autopsies of all sudden deaths: a public health prevention strategy]. / Pratiquer l'autopsie de toutes les morts subites : un moyen de prévention en santé publique.

INTRODUCTION: In France, the management of sudden death (including nion-forensic autopsies) among adults could be improved. Thee number of sudden deaths may require a change in practice to promote screening for hereditary diseases. The purpose of this study was to identify areas for improvement and to assess the prevalence of non-forensic autopsies. METHODS: A retrospective study (2005-2010) conducted at Lille University Hospital resulted in the selection of 174 non-forensic autopsies of adults who died suddenly. The criteria used were: the geographical origin of the bodies, indication, age, gender and the diagnosis ofdeath. RESULTS: The study found that the annual number of non-forensic autopsies was very low, with proportions ranging from 1.06 to 1.73 per 1,000 deaths in the case of hospital deaths and from 0 to 0.43 per 1,000 deaths in the case of non-hospital deaths. Only 11 non-forensic autopsies of people who died at home were recorded over the study period, including 4 heart disease-related deaths. Overall, the results indicate that sudden death autopsies are poorly managed. DISCUSSION: Non-forensic autopsies are in decline, particularly in the case of deaths outside hospital, despite the fact that they are a crucial consideration for screening among relatives of a person who died sud!denly. The findings suggest that there needs to be an improvement in practice, a fact made possible by recent legislative developlments in France. A number of initiatives have been successful arId give hope that, like the reference centers specializing in unexpected infant deaths, reference centers specializing in sudden de'aths, which allow for more systematic non-forensic autopsies ofadults who die suddenly, might be created.

Study of Root Transparency in Different Postmortem Intervals Using Scanning Electron Microscopy.

In the fields of forensics, the identification of human remains is a recurrent problem. The estimated age at death is one of the copious criteria to be evaluated. In adult teeth, the height of the root dentin transparency is used to estimate age. However, in archaeological material, this phenomenon appears inconstant. The aim of this work was to observe the structural modifications of the sclerotic dentin in the teeth for different postmortem intervals. The study included two parts (retrospective and prospective study) with 21 human monoradicular teeth, from bodies donated to medical science with postmortem intervals (PMIs) of 0, 1, 2 and 5 years and archeological excavation. After inclusion based on resin, section and polishing, the samples were analyzed with a scanning electron microscope (SEM) JSM-7800F®, and the procedure was completed via a semiquantitative analysis of calcium and phosphorus using EDX microanalysis. The analysis showed the existence of tubular and chemical modifications of sclerotic dentin at different PMIs. Our SEM study allowed us to observe a difference in tubule aspects linked to an increased PMI: the loss of peritubular collar and the lumen obstruction of tubules with a hyperdense material. Microanalysis highlighted variations in phosphocalcic ratios among the different groups, especially in the pulp area and the canine. Our hypotheses that explain these differences are based on the postmortem modifications of the crystals of the mineral phase of sclerotic dentin under the influence of chemical and/or bacterial action.

Interpretation of hair and nails findings in an infant death case related to maternal addiction to tramadol.

An 11-month-old boy was found dead. Autopsy findings (cyanosis and polyvisceral congestion) and blood tramadol (TR) concentration of 6240 µg/L were consistent with an acute TR intoxication. In this poisoning situation, owing to the mother's statements (TR addiction leading to daily TR-orange juice mixture preparation accidentally used for the baby bottle preparation by the mother's partner), and the question of possible previous TR administrations to the infant, hair and/or nails (infant, mother, partner, 6-year-old sister) analysis was performed. Hair (2-cm-long hair segments from proximal [S1] to distal [S3]) and nails concentrations (pg/mg; nd: not detected) were as follows: Infant (hair: TR 1420 [S1], 1622 [S2], 2736 [S3]; O-DMT 16-38; N-DMT 34-100 [TR in significant quantities in the hair decontamination bath]-toenails: TR 584; O-DMT 8; N-DMT 15), mother (hair: TR 2340 [S1], 2150 [S2], 2500 [S3]; O-DMT 704-1170; N-DMT 827-1360), mother's partner (fingernails: TR 72; O-DMT nd; N-DMT nd) and sister (hair: TR 261 [S1], 524 [S2]; O-DMT 15 [S1], 16 [S2]; N-DMT 20 [S1], 38 [S2]). Metabolite ratio (infant and sister hair) was comparable to those observed in hair of pharmaceutical industry employees manufacturing tramadol. TR in washing baths, low observed nail concentrations (infant and partner) confirm (i) TR-related mother's addiction and (ii) external contamination issues (TR in sweat of the child at the time of death and in living environment) to explain the infant's keratinized samples results. This case report illustrates the interest of analyzing keratinized matrices of the whole family in such a situation.