From flesh to bones: Multi-omics approaches in forensic science.

Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high-throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state-of-the-art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi-omics techniques for forensic investigations.

Parasitic and predatory behavior of Alysia manducator (Hymenoptera: Braconidae) on blow fly larvae feeding on an adult pig carcass in the Western Cape Province of South Africa: preliminary observations and forensic implications.

Wasps are part of the entomofauna associated with vertebrate carrion. They are known to parasitize and prey on specific life stages of insect hosts such as eggs, larvae, pupae, and/or adults associated with vertebrate carrion. However, reports of parasitic behavior of wasps on carrion-associated insect life stages and their possible forensic implications are non-existent in the Western Cape Province of South Africa. This study is part of ongoing research investigating the entomofauna and their pattern of succession on an adult pig carcass in Cape Town, South Africa. During this study, the parasitic wasp Alysia manducator was noted parasitizing and preying on blow fly larvae associated with the decomposing carcass. The arrival of A. manducator coincides with the occurrence of blow fly eggs and/or larvae on the carcass. These wasps were seen in close association with the eggs and larvae of blow flies on various parts of the carcass and some wasps were seen dragging fly larvae attached to their ovipositors away from one part of the carcass to another. Some A. manducator were also observed walking over several larvae on the carcass while exhibiting a stabbing behavior presumably in search of a host for oviposition. We suggest that the observations recorded in this study are of considerable forensic importance as the dragging effect and predatory and stabbing behavior exhibited by A. manducator could potentially disrupt the feeding and development of the fly larvae on the carcass. This could subsequently alter the process of carcass decomposition and/or affect minimum post-mortem interval estimations.

The influence of cooling on biomechanical time since death estimations using ovine brain tissue.

The significance of biomechanical analyses for forensic time since death estimations has recently been demonstrated. Previous biomechanical analyses successfully discriminated post-mortem brain tissue from tissue with a post-mortem interval of at least one day when held at 20 °C. However, the practical utility of such analyses beyond day one at 20 °C was limited. This study investigates the storage, loss, and complex shear modulus of various brain regions in sheep stored at 4 °C in 24-hour intervals over four days post-mortem using rheometry tests. The aim is to identify the critical biomechanical tissue property values to predict post-mortem time and assess the temperature sensitivity of the rheometry method by comparing results to recent findings at 20 °C. Thirty sheep brains were examined, including the frontal lobe, parietal lobe, anterior and posterior deep brain, superior colliculi, pons, medulla, and cerebellum. Rheometry tests were conducted, and receiver operator characteristic analyses were employed to establish cut-off values. At 4 °C storage, all investigated biomechanical properties of the examined brain regions remained stable for at least one day post-mortem. Using cerebellar samples stored at 4 °C, a post-mortem interval of at least two days could be determined with excellent diagnostic ability. Complex shear modulus values below 1435 Pa or storage modulus values below 1313 Pa allowed prediction of two or more days post-mortem. Comparisons between 4 °C and 20 °C revealed brain region-specific results. For instance, the complex shear moduli of the anterior deep brain at 4 °C were significantly higher on all individual testing days when compared to 20 °C. In contrast, the combined medulla and pons samples were similar on each day. Rheometry testing of brain tissue consistently stored at 4 °C since death proved valuable for forensic time since death estimations starting from two days after death.

Catch me if you can-emission patterns of human bodies in relation to postmortem changes.

The present study examines for the first time the emission patterns and olfactory signatures of 9 complete human corpses of different stages of decomposition. Air sampling was performed inside the body bags with solid sorbents and analysed by coupled gas chromatography-mass spectrometry after thermal desorption (TD-GC-MS). Furthermore, odour-related substances were detected by gas chromatography-olfactometry (GC-O). Sulfurous compounds (mainly dimethyl di- and trisulfide) were identified as most important to the odour perception. Around 350 individual organic substances were detected by TD-GC-MS, notably sulfurous and nitrogenous substances as well as branched alkanes, aldehydes, ketones, alcohols, carboxylic acids, carboxylic acid esters and ethers. A range of terpenes was detected for the first time in a characteristic emission pattern over all decomposition stages. Concentrations of the substances varied greatly, and no correlation between the emission patterns, the stage of decomposition and the cause of death could be found. While previous studies often analysed pig cadavers or only parts of human tissue, the present study shows the importance of analysing complete human corpses over a range of decomposition stages. Moreover, it is shown that using body bags as a kind of "emission test chamber" is a very promising approach, also because it is a realistic application considering the usual transport and store of a body before autopsy.

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Forensic taphonomic experimental design matters: a study assessing clothing and carrion biomass load on scavenging in Cape Town, South Africa.

The identification of unknown human remains is a significant and ongoing challenge in South Africa, worsened by the country's high murder rate. The rate of decomposition in South Africa is significantly influenced by vertebrate scavenging, which, if not considered, can impede the accurate estimation of the post-mortem interval. Scavenging patterns vary greatly depending on the environment and ecological region, and there is limited data for the Western Cape province. To address this gap, two clothed and uncaged pig carcasses weighing 60 kg each were placed in the field in July 2021 and January 2022, respectively. Motion-activated infrared-capable trail cameras were used to observe decomposition, scavenger species, and their activities. Additionally, a comparative sample of 16 unclothed carcasses deployed between 2014 and 2016 in the same habitat were analyzed to assess the impact of clothing and biomass load. The study found three main results: (1) Regardless of habitat or biomass load, it took significantly less time to reach decomposition milestones (25%, 50%, and 75%) during the summer season; (2) the presence of mongoose scavengers had a greater impact on the time required to reach milestones during winter compared to summer; and (3) single carcass deployments reached the milestones faster than multi-carcass deployments in both seasons. This research highlights the potential inaccuracy of current methods for estimating the post-mortem interval when scavenging activity is not considered or documented in the underlying experimental data, particularly for environments or ecological biomes where scavengers actively impact decomposition rates.

Detrimental effects of scene manipulations on temperature-based time since death estimation.

In forensic casework, time since death (TSD) estimations may play a crucial role to establish chains of events as well as for alibi assessment in homicide cases. Classical TSD estimation relies on reasonably stable ambient temperatures and a correct documentation of ambient and rectal temperatures. This constancy is in some cases disturbed by post-discovery alterations of the crime scene, e.g. opening a window. In order to develop a better understanding of this alteration-based detrimental impact on TSD estimation as well as to identify feasible recommendations for casework, the present pilot study examined ambient temperature effects of different window opening scenarios regarding various time intervals (5 to 360 min) in a furnished 10 m2 apartment during winter. In this context, in addition to the ambient temperature and thus the cooling rate of the room, re-approximation to initial room temperature, potential influences on a nomogram-based time since death estimation using a fictitious case, and the impact of the measurement height above the ground were investigated. Our data indicate a significant reduction of the mean temperature decrease rate after 15 min regardless of the remaining opening time and a correlation with the size of the respective opening surfaces. Re-approximation to initial room temperatures was observed with up to three times longer than the initial opening time. There was no evidence of a substantial advantage of temperature measurements above the level of the corpse (> 0.1 m). The limitations of the study and its applicability for forensic casework are critically reviewed.

Carrion insects living within the bones of large mammals: insect conservation and forensic entomology implications.

Succession patterns of carrion insects on large mammal's carrion has been widely studied, notably to estimate the post-mortem interval in forensic investigations as accurately as possible. However, little attention has been paid to the carrion insects living inside these bones once a carcass is skeletonized. One very recent study documented flies emerging from pig carcasses, and only scarce authors reported the presence of other carrion insects taking advantage of the bone marrow. We, thus, aimed to (1) estimate the frequency of inner-bone space colonization by carrion insects, with particular attention to bone-skipper flies; (2) identify the insects living inside the carrion bones; and (3) determine whether or not carrion insects found within the bones can successfully exit the bones and complete their development. We extensively sampled 185 large mammals' bones collected from twelve vulture feeding stations and four isolated carcasses in southwest France and northern Spain. Sampled bones were opened, and the insects found inside were identified. For two bones, foramen, i.e., the holes providing a natural entrance and exit to the bone's inner cavity, was monitored with a camera to assess the insect's putative exit. We describe the entomofauna, i.e., the set of insect species, living within the bones, and illustrate insects' ability to exit the bones for their subsequent development and maturity. These results are discussed in the framework of carrion insect conservation and forensic entomology perspectives.

Counting the days: Exploring the post-mortem interval factors in sexual homicides.

This study explores the offender, victim, and environmental characteristics that significantly influence the number of days a sexual homicide victim remains undiscovered. Utilizing a sample of 269 cases from the Homicide Investigation Tracking System database an in-depth analysis was conducted to unveil the factors contributing to the delay in the discovery of victims' bodies. The methodological approach involves applying a negative binomial regression analysis, which allows for the examination of count data, specifically addressing the over-dispersion and excess zeros in the dependent variable - the number of days until the victim is found. The findings reveal that certain offender characteristics, victim traits, and spatio-temporal factors play a pivotal role in the time lag experienced in locating the bodies of homicide victims. These findings have crucial implications for investigative efforts in homicide cases, offering valuable insights that can inform and enhance the efficacy and efficiency of future investigative procedures and strategies.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Protophormia terraenovae (Diptera: Calliphoridae).

Protophormia terraenovae is a colonizer of decomposing bodies and is known to cause pre-mortem myiasis as the female flies lay eggs in uncleaned wounds. In this study the effects of different concentrations of antibiotics levofloxacin and ceftriaxone on maggot development, weight, length, and mortality were examined. The maggot length and weight were significantly increased by therapeutical doses of levofloxacin and ceftriaxone. The maggot development time was significantly decreased in every levofloxacin treatment compared to the control. The time to start pupation was significantly increased in the control compared to the antibiotic treatments. Levofloxacin significantly increased the survivability of the maggots. Every levofloxacin treatment significantly improved the rearing conditions for the maggots. Reaching the third instar was delayed by 24 h in the control compared to the Levo 3.57 treatment. The Pupation in the control was delayed by an average of 48 h compared to the Levo 3.57 treatment. The significantly reduced development time of the maggots in the antibiotic treatments might lead to an overestimation of the post-mortem interval and therefore an incorrect time of death determination. The improved rearing conditions may be an indication of the potential of a combined application of antibiotics and maggot therapy.

Immunohistochemical expression of HMGB1 and related proteins in the skin as a possible tool for determining post-mortem interval: a preclinical study.

Determining the post-mortem interval (PMI) is one of forensic pathology's primary objectives and one of its most challenging tasks. Numerous studies have demonstrated the accuracy of histomorphology and immunohistochemical investigations in determining the time of death. Nevertheless, the skin, a robust and easy-to-remove tissue, has only been partially analyzed so far. By studying 20 adult male mice, we tried to determine whether post-mortem immunohistochemical detection in the skin of HMGB1 proteins and associated components (Beclin1 and RAGE) could be used for this purpose. We discovered that nuclear HMGB1 overexpression indicates that death occurred within the previous 12 h, nuclear HMGB1 negativization with high cytoplasmic HMGB1 intensity indicates that death occurred between 12 and 36 h earlier and cytoplasmic HMGB1 negativization indicates that more than 48 h have passed since death. RAGE and Beclin1 levels in the cytoplasm also decreased with time. The latter proteins' negativization might indicate that more than 24 and 36 h, respectively, have passed from the time of death. These indicators might potentially be helpful in forensic practice for determining the PMI using immunohistochemistry.

Effect of diazinon (organophosphate) on the composition and succession of Calliphoridae assemblages in rabbit carcasses in the Eastern Amazon.

Fatal poisonings with diazinon may occur both accidentally or intentionally in suicide cases. Forensic entomotoxicology can aid in understanding these deaths by detecting and analyzing the interference of toxic substances in the biology of necrophagous insects. Hence, this study sought to evaluate diazinon's effect on the composition and succession of calliphorid species in the tropical savannas of the Amazon. Nine rabbit carcasses were divided into three groups: one control and two diazinon treatments (100 mg/kg and 300 mg/kg); each group had three replicates. Three fragments of the Amazon tropical savanna were selected for the experiments. Daily collections of adult and immature calliphorids were performed. Five decomposition stages were observed: fresh, bloated, active decay co-occurring, advanced decay, and dry. Eight species of Calliphoridae were identified among the collected adults: Chloroprocta idioidea (0.1%), Chrysomya albiceps (58.3%), Chrysomya megacephala (14.2%), Chrysomya putoria (2.6%), Cochliomyia hominivorax (1.3%), Cochliomyia macellaria (0.5%), Lucilia eximia (19.8%), and Paralucilia paraensis (3.3%). The adult specimens in the control group with the highest abundance were observed only from the advanced decay stage onward. In the dry stage, abundance was higher in control than in treated carcasses. From the sampled 941 Calliphorid immatures, three species were identified: C. albiceps (76,3%), C. putoria (1%), and L. eximia (22,7). The number of immatures was higher in control than in treated carcasses. Therefore, diazinon interferes with the putrefaction timeframe in carcasses, slowing the decomposition stages and affecting their colonization by immature forms of Calliphoridae.

Initial laboratory validation of temperature development models for Necrodes littoralis L. (Staphylinidae: Silphinae).

Development models of necrophagous insects are applied in forensic entomology for post-mortem interval estimation. Such estimates may be used as scientific evidence in legal investigations. For this reason, it is important that the models are valid and that the expert witness is aware of their limitations. Necrodes littoralis L. (Staphylinidae: Silphinae) is a necrophagous beetle species that frequently colonizes human cadavers. Temperature models of development for the Central European population of these beetles were recently published. In this article, we present results of the laboratory validation study for these models. Errors of beetle age estimation differed significantly between the models. Thermal summation models yielded the most accurate estimates, and the isomegalen diagram least accurate estimates. Errors of the beetle age estimation varied across beetle developmental stages and rearing temperatures. In general, most development models of N. littoralis were satisfactorily accurate in estimating beetle age under laboratory conditions; therefore, the study provides initial evidence to support their validity in forensic cases.

Mummification in a forensic context: an observational study of taphonomic changes and the post-mortem interval in an indoor setting.

The objective of this study was to evaluate the presence of mummification in an indoor setting, with an emphasis on the forensic perspective. A dataset of 102 forensic autopsy cases was assessed for distribution of desiccation of skin and soft tissue (i.e., subcutaneous fat and musculature) and for moist decompositional (i.e., putrefactive) changes. Further, possible correlation with the post-mortem interval (PMI) was evaluated, as well as the effects of clothing coverage of the body. The results indicated that yellow to orange parchment-like desiccated skin was found at significantly shorter PMIs than reddish brown to black leathery desiccated skin, even when soft tissue desiccation was included in the comparative analysis. Clothing appeared to have a significant decelerating effect on the extent of desiccation on the legs, but findings in regard to whole body or torso/arms were inconclusive. A large variation in PMIs was evident as regards fully desiccated skin (PMI 18-217 days), indicating difficulties in PMI estimation due to a variable repressive effect on the decompositional process per se in an indoor setting. For the specific case in forensic practice, no definite conclusion can be drawn from the observed desiccation changes to the PMI. One way forward might be creating a systematic and standardized method for describing different desiccation types, as well as other cooccurring decompositional changes and how they relate to the PMI, as a foundation for a future quantification model.

The use of brain tissue mechanics for time since death estimations.

Time since death estimation is a vital part of forensic pathology. Despite the known tissue degradation after death, the efficacy of using biomechanical tissue properties to estimate time since death remains unexplored. Here, eight brain tissue localizations were sampled from the frontal lobe, parietal lobe, anterior and posterior deep brain, superior colliculi, pons, medulla, and cerebellum of 30 sheep; were then stored at 20 °C; and subsequently subjected to rheometry tests on days zero to four after death. Overall, the measured tissue storage modulus, loss modulus, and complex shear modulus decreased after death for all of the tested regions in a site-specific manner. Day zero to day one changes were the only 24-h interval, for which statistically significant differences in tissue mechanical moduli were observed for some of the tested brain regions. Based on receiver operator characteristic analyses between day zero and the pooled data of days one to four, a post mortem interval of at least 1 day can be determined with a sensitivity of 90%, a specificity of 92%, and a positive likelihood ratio of 10.8 using a complex shear modulus cut-off value of 1461 Pa for cerebellar samples. In summary, biomechanical properties of brain tissue can discriminate between fresh and at least 1-day-old samples stored at 20 °C with high diagnostic accuracy. This supports the possible value of biomechanical analyses for forensic time since death estimations. A striking advantage over established methods to estimate the time since death is its usability in cases of disintegrated bodies, e.g. when just the head is found.

Effects of antibiotics ceftriaxone and levofloxacin on the growth of Lucilia sericata (Diptera: Calliphoridae).

Lucilia sericata is one of the most studied species in forensic entomology due to its widespread distribution, forensic importance as well as medical use. The growth and development stage of maggots is often used to determine the post-mortem interval in forensic cases. L. sericata can cause myiasis in humans who are not able to maintain personal hygiene due to age or medical condition and can therefore be used to determine the time period of neglect. The influence of the temperature on the maggot development has been examined in various studies. Different examinations on the effects of toxic substances on the maggot development and survival have been conducted in order to test the influence and resulting deviations. In this study, the effects of different therapeutical doses of the antibiotics ceftriaxone and levofloxacin were examined on L. sericata in order to determine and compare deviations in maggot development and survival. The used antibiotics did not significantly influence the maggot weight and length. A significant delay in the time of pupation has been determined in the treatments with high concentrations of levofloxacin. The mortality was significantly increased in all treatments with antibiotics compared to the control, whereby the survivability of all treatments remained over 80%. Because of the high doses needed to produce an effect, we concluded that an overestimation of the post-mortem interval is unlikely but higher antibiotic concentrations or possible interactions with other medications could increase the maggot development time.

Calibrating insect age at eclosion by size in a gregarious carrion beetle Thanatophilus sinuatus (Staphylinidae: Silphinae).

Recent discoveries have shown that the physiological age at eclosion of forensically useful beetles differs between males and females and between beetles of various sizes. Accordingly, it was postulated that the size and sex of the beetles at eclosion may be used to calibrate their age, which may improve the accuracy of age (and post-mortem interval) estimates in forensic entomology. In this study, we derived thermal summation models for the eclosion for the Central European population of carrion beetles Thanatophilus sinuatus (Fabricius, 1775), (Staphylinidae: Silphinae), and tested the usefulness of sex and size for the calibration of beetle age at eclosion. Although in previous developmental studies, the beetles were reared individually, we reared them in larval aggregations, since in natural conditions T. sinuatus beetles are gregarious. Weak (r2 between 5% and 13%) negative correlations were observed between the size and age of T. sinuatus males or females at eclosion, demonstrating that calibration of age by beetle size and sex may bring only minimal benefits regarding the accuracy of age estimation in this species. However, it may still be worthwhile in the case of extremely large or small beetles. Moreover, the total development times recorded in this study were much shorter than in the previous T. sinuatus study, at 14°C by about 15 days and at 26°C by about 2 days. These differences emphasise the importance of gregariousness for the development of carrion beetles, and at the same time highlight the need for the ecologically-relevant protocols of development studies in forensic entomology.

Thanatomicrobiome in forensic medicine.

The circumstances of death and the estimation of the post-mortem interval (PMI) are often a great challenge for scientific and judicial investigators, especially when some time has elapsed since death. Several techniques are used; nevertheless, each presents its own limitations. In the quest for new techniques that are more reliable or at least complementary to those existing and sometimes less expensive, researchers have in recent years turned toward exploring the dynamics of the different microbial communities of a corpse according to their different stages of decomposition. This article summarizes the various works done in the field and shows the different sources of microorganisms in the different parts of the human corpse and their potential interest in the field of forensic medicine.

Forensic Proteomics for the Discovery of New post mortem Interval Biomarkers: A Preliminary Study.

Estimating the time since death (post mortem interval, PMI) represents one of the most important tasks in daily forensic casework. For decades, forensic scientists have investigated changes in post mortem body composition, focusing on different physical, chemical, or biological aspects, to discover a reliable method for estimating PMI; nevertheless, all of these attempts remain unsuccessful considering the currently available methodical spectrum characterized by great inaccuracies and limitations. However, recent promising approaches focus on the post mortem decomposition of biomolecules. In particular, significant advances have been made in research on the post mortem degradation of proteins. In the present study, we investigated early post mortem changes (during the first 24 h) in the proteome profile of the pig skeletal muscle looking for new PMI specific biomarkers. By mass spectrometry (MS)-based proteomics, we were able to identify a total of nine potential PMI biomarkers, whose quantity changed constantly and progressively over time, directly or inversely proportional to the advancement of post mortem hours. Our preliminary study underlines the importance of the proteomic approach in the search for a reliable method for PMI determination and highlights the need to characterize a large number of reliable marker proteins useful in forensic practice for PMI estimation.

The biochemistry of the vitreous humour in estimating the post-mortem interval-a review of the literature, and use in forensic practice in Galicia (northwestern Spain).

The K+ and hypoxanthine (Hx) concentrations of the vitreous humour (VH) rise gradually after death, providing a means of estimating the post-mortem interval (PMI). The correlation between these analytes and the PMI is good since the vitreous chamber is partially isolated from autolytic events occurring elsewhere; the [K +] and [Hx] recorded is thus the result of changes within the eye. The present work provides a systematic review, following PRISMA recommendations, of 36 articles (3 reviews and 33 retrospective cohort studies) discussing the many procedures and regression models that have been developed for improving PMI estimates involving VH analytes. The results of a descriptive study are also provided, highlighting the causes and distribution of mortality as registered in medico-legal autopsies performed in 2019 in Galicia (northwestern Spain), and revealing the use of these PMI estimation methods in real forensic practice. Great heterogeneity was detected in the collection of VH samples, the treatments to which they were subjected before examination, and in their conservation and analysis. A lack of reproducibility in the analytical methods employed to estimate [K +] and [Hx] was noted, as well as an absence of external validation for most of the regression formulae used to determine the PMI from analyte values. The use of methods based on high-performance liquid chromatography, focal electrophoresis, or thermogravimetric/chemometric procedures might solve the problems encountered with traditional analytical techniques, offering reliable results more quickly and effectively (even when samples are contaminated). This study recommends using flexible multiple regression models that combine physical and chemical variables, and that population databases be constructed so that models can be properly validated.