Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war - a 30-year review.

Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.

Histotaphonomic analysis of bone bioerosion reveals a regional framework of diverse deathways in the Neolithic of Southeast Italy.

The wide diversity of Neolithic funerary practices is increasingly recognised. In Southeast Italy, recent studies have drawn attention to the co-existence of multiple ways of treating the dead within single sites and across the region. In this study, we address how such diverse deathways form a regional framework of ritual practice through histotaphonomic analysis of bone bioerosion. Samples were obtained from articulated, semi-articulated and disarticulated remains from four sites in Apulia which each presented different modes of treatment and disposal of the dead. Bone thin sections were analysed by light microscopy to characterise microstructural preservation through features including bacterial bioerosion, staining, inclusions, and Wedl tunnelling. We investigate the early post-mortem histories of individuals whose remains ended up in various states of dis/articulation and diverse depositional contexts. Disarticulated remains frequently displayed arrested or extensive bacterial bioerosion, which was also found in articulated and semi-articulated skeletons. Additionally, remains deposited in similar contexts, as well as articulated and disarticulated remains deposited together in the same context, often showed different histotaphonomic characteristics, suggesting diverse early post-mortem trajectories. As a result, we argue that Neolithic deathways in southeastern Italy incorporated a high level of diversity in the early post-mortem treatment of the body. A framework for funerary practices emerges, whereby disarticulated remains probably originated from bodies which had been buried previously and subjected to varying extents of shelter, exposure to invertebrates, and duration of burial. However, we acknowledge the ongoing research into the origins of bacterial bioerosion and the problem of equifinality, which leaves open the possibility for further scenarios of early post-mortem treatment.

Reassociation of Skeletal Remains Using Laser-Induced Breakdown Spectroscopy.

A major challenge in forensic anthropology and bioarcheology is the development of fast and effective methods for sorting commingled remains. This study assesses how portable laser-induced breakdown spectroscopy (LIBS) can be used to group skeletal remains based on their elemental profiles. LIBS spectra were acquired from the remains of 45 modern skeletons, with a total data set of 8388 profiles from 1284 bones. Spectral feature selection was conducted to reduce the spectral profiles to the peaks exhibiting the highest variation among individuals. Emission lines corresponding to 9 elements (Ca, P, C, K, Mg, Na, Al, Ba, and Sr) were found important for classification. Linear discriminant analysis (LDA) was concurrently used to classify each spectral profile. From the 45 individuals, each LIBS spectrum was successfully sorted to its corresponding skeleton with an average accuracy of 87%. These findings indicate that variation exists among the LIBS profiles of individuals' skeletal remains, highlighting the potential for portable LIBS technology to aid in the sorting of commingled remains.

From Jane Doe to Sofia: DNA Extraction Protocol from Bones and Teeth without Liquid Nitrogen for Identifying Skeletal Remains.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Long-term mental health and resilience of the first responders in Japanese ground self-defense forces engaging body recovery after the great east Japan earthquake.

Handling human remains is extremely difficult and stressful task, and it can contribute to the development of stress-related mental health problems. To prevent disaster from the development of mental disorders in first responders, it will be important to elucidate factors sustaining psychological well-being following the events requiring handling of human remains. Japanese Ground Self-Defense Forces (JGSDF) first responders (n = 146), involved in human remains recovery after the Great East Japan Earthquake (GEJE) participated. We examined the psychological resilience (S-H Resilience Test), depressive symptoms (SDS), and psychological distress (GHQ-28) 6 years after GEJE, in three groups; Group A: no contact no view of human remains, B: view only of human remains, and C: direct handling of human remains. S-H Resilience test evaluated the 3 subclasses of resilience; Social Support, Self Efficacy, Sociality. One-way ANOVA revealed the significant difference in resilience scores between Group B and C without any differences in depressive symptoms or psychological distress among the 3 groups. Multiple regression analyses revealed that depressive symptoms and resilience were associated with psychological distress in all participants. Path analyses showed that whereas one subtype of resilience indirectly reduced psychological distress through lower depressive symptoms in Group A (Social Support) and Group B (Sociality), both subtypes of resilience indirectly reduced psychological distress by lowering depressive symptoms in Group C. These findings suggest that exposure to higher stressful situation may decrease the psychological resilience based on the S-H Resilience test, and two subtypes of resilience may be necessary to sustain the psychological well-being.

Reappraisal of sauropod dinosaur diversity in the Upper Cretaceous Winton Formation of Queensland, Australia, through 3D digitisation and description of new specimens.

Skeletal remains of sauropod dinosaurs have been known from Australia for over 100 years. Unfortunately, the classification of the majority of these specimens to species level has historically been impeded by their incompleteness. This has begun to change in the last 15 years, primarily through the discovery and description of several partial skeletons from the Cenomanian-lower Turonian (lower Upper Cretaceous) Winton Formation in central Queensland, with four species erected to date: Australotitan cooperensis, Diamantinasaurus matildae, Savannasaurus elliottorum, and Wintonotitan wattsi. The first three of these appear to form a clade (Diamantinasauria) of early diverging titanosaurs (or close relatives of titanosaurs), whereas Wintonotitan wattsi is typically recovered as a distantly related non-titanosaurian somphospondylan. Through the use of 3D scanning, we digitised numerous specimens of Winton Formation sauropods, facilitating enhanced comparison between type and referred specimens, and heretofore undescribed specimens. We present new anatomical information on the holotype specimen of Diamantinasaurus matildae, and describe new remains pertaining to twelve sauropod individuals. Firsthand observations and digital analysis enabled previously proposed autapomorphic features of all four named Winton Formation sauropod species to be identified in the newly described specimens, with some specimens exhibiting putative autapomorphies of more than one species, prompting a reassessment of their taxonomic validity. Supported by a specimen-level phylogenetic analysis, we suggest that Australotitan cooperensis is probably a junior synonym of Diamantinasaurus matildae, but conservatively regard it herein as an indeterminate diamantinasaurian, meaning that the Winton Formation sauropod fauna now comprises three (rather than four) valid diamantinasaurian species: Diamantinasaurus matildae, Savannasaurus elliottorum, and Wintonotitan wattsi, with the latter robustly supported as a member of the clade for the first time. We refer some of the newly described specimens to these three species and provide revised diagnoses, with some previously proposed autapomorphies now regarded as diamantinasaurian synapomorphies. Our newly presented anatomical data and critical reappraisal of the Winton Formation sauropods facilitates a more comprehensive understanding of the mid-Cretaceous sauropod palaeobiota of central Queensland.

Phylogenetic analysis of mysterious burials revealed in the former penal labor camp Treblinka I.

The purpose of this paper is to formulate recommendations for the disclosure of biological traces in the laboratory and the handling of forensic evidence submitted for identification tests, recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics. The paper organizes the knowledge of the most relevant stages of preliminary analysis of biological traces based on both literature sources and those resulting from years of research practice. Recommendations formulated in the course of multi-stage expert consultations contained in this study should be used in the development of laboratory procedures applied during the execution. * The research is part of doctoral dissertation of Dagmara Lisman entitled "Genetic analysis of a skeleton site revealed during the works on the premises of the former German Forced Labor Camp Treblinka I."

High-performance liquid chromatography (HPLC) as a means of assessing the presence of uric acid in archeological human remains: Challenges and future directions.

OBJECTIVES: This research aimed to replicate the Swinson, D., Snaith, J., Buckberry, J., & Brickley, M. (2010). High performance liquid chromatography (HPLC) in the investigation of gout in paleopathology. International Journal of Osteoarchaeology, 20, 135-143. https://doi.org/10.1002/oa.1009 method for detecting uric acid in archeological human remains to investigate gout in past populations and to improve the original High Performance Liquid Chromatography-ultraviolet (HPLC-UV) method by using HPLC-mass spectrometry (HPLC-MS), a more sensitive, compound-specific detection method. MATERIALS AND METHODS: We used reference samples of uric acid to create a dilution series to assess the limits of quantification and detection. Samples from individuals with and without gout lesions were taken from foot bones and ribs from the English cemeteries of Tanyard, Hickleton, Gloucester, and Lincoln. RESULTS: We could not replicate the results of Swinson and colleagues using HPLC-UV. Tests using a dilution series of uric acid showed HPLC-MS was approximately 100× more sensitive than HPLC-UV, with the additional benefit of being compound specific. A newly developed hydrophilic interaction chromatography (HILIC) method improved retention characteristics. Fourteen samples from eight individuals, five with skeletal lesions consistent with gout, were analyzed with the final method. None showed evidence of uric acid despite the newly developed method's improved sensitivity and specificity. DISCUSSION: The lack of detectable uric acid extracted from these samples suggests that (1) urate crystals were not present in any of the bone samples, regardless of gout status; (2) urate crystals did not survive these specific archeological conditions; or (3) the concentration of uric acid in our bone extracts was low, and thus larger samples would be required.

Evaluation of the usefulness of insertion-null markers in critical skeletal remains.

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

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.

Smithsonian urged to speed repatriation of human remains.

Task force says museum should return many of its 30,000 remains and seek descendants' consent for research.

Bioanthropological analysis of human remains from the archaic and classic period discovered in Puyil cave, Mexico.

OBJECTIVES: Determine the geographic place of origin and maternal lineage of prehistoric human skeletal remains discovered in Puyil Cave, Tabasco State, Mexico, located in a region currently populated by Olmec, Zoque and Maya populations. MATERIALS AND METHODS: All specimens were radiocarbon (14C) dated (beta analytic), had dental modifications classified, and had an analysis of 13 homologous reference points conducted to evaluate artificial cranial deformation (ACD). Following DNA purification, hypervariable region I (HVR-1) of the mitogenome was amplified and Sanger sequenced. Finally, Next Generation Sequencing (NGS) was performed for total DNA. Mitochondrial DNA (mtDNA) variants and haplogroups were determined using BioEdit 7.2 and IGV software and confirmed with MITOMASTER and WebHome softwares. RESULTS: Radiocarbon dating (14C) demonstrated that the inhabitants of Puyil Cave lived during the Archaic and Classic Periods and displayed tabular oblique and tabular mimetic ACD. These pre-Hispanic remains exhibited five mtDNA lineages: A, A2, C1, C1c and D4. Network analysis revealed a close genetic affinity between pre-Hispanic Puyil Cave inhabitants and contemporary Maya subpopulations from Mexico and Guatemala, as well as individuals from Bolivia, Brazil, the Dominican Republic, and China. CONCLUSIONS: Our results elucidate the dispersal of pre-Hispanic Olmec and Maya ancestors and suggest that ACD practices are closely related to Olmec and Maya practices. Additionally, we conclude that ACD has likely been practiced in the region since the Middle-Archaic Period.

The utility of blow fly (Diptera: Calliphoridae) evidence from burned human remains.

Burning of human remains is a common method to conceal or destroy evidence associated with homicides and illegal activities. However, data regarding blow fly colonization of burned remains are scarce, with all previously published empirical studies focusing only on non-human animals. It is critically important to obtain basic data on blow fly colonization patterns of burned human remains as such evidence may represent the only feasible method for PMI estimation in cases of burning. In this study, we thermally altered six human donors to a Crow-Glassman Scale Level 3 (CGS-3) and placed them at the Anthropology Research Facility at the University of Tennessee in Summer 2021, Spring 2022, and Summer 2022. Six unburned human donors were used as controls. Observations for insect activity began within 24 h of placement and continued twice weekly through decomposition. Age estimations were performed with immature blow flies to estimate the time of colonization (TOC), and accuracy was assessed against the time of placement for each donor. All burned donors examined in this study were colonized by blow flies. No significant difference in species composition was determined between treatments, though TOC estimations from burned donors were slightly (but significantly) less accurate than TOC estimations from unburned donors (80% vs. 83% accuracy; χ2 = 0.041, df = 1, P = 0.840). These results indicate that blow flies can successfully colonize human remains burned to CGS-3 and that accurate TOC estimations can still be generated from larval specimens. Though several limitations to this study exist (e.g., inconsistent donor BMI, lack of donor temperature data), our results underscore the utility of entomological evidence in cases of burned human remains.

Atherosclerosis as the Damocles' sword of human evolution: insights from nonhuman ape-like primates, ancient human remains, and isolated modern human populations.

Atherosclerosis is the leading cause of death worldwide, and the predominant risk factors are advanced age and high-circulating low-density lipoprotein cholesterol (LDL-C). However, the findings of atherosclerosis in relatively young mummified remains and a lack of atherosclerosis in chimpanzees despite high LDL-C call into question the role of traditional cardiovascular risk factors. The inflammatory theory of atherosclerosis may explain the discrepancies between traditional risk factors and observed phenomena in current literature. Following the divergence from chimpanzees several millennia ago, loss of function mutations in immune regulatory genes and changes in gene expression have resulted in an overactive human immune system. The ubiquity of atherosclerosis in the modern era may reflect a selective pressure that enhanced the innate immune response at the cost of atherogenesis and other chronic disease states. Evidence provided from the fields of genetics, evolutionary biology, and paleoanthropology demonstrates a sort of circular dependency between inflammation, immune system functioning, and evolution at both a species and cellular level. More recently, the role of proinflammatory stimuli, somatic mutations, and the gene-environment effect appear to be underappreciated elements in the development and progression of atherosclerosis. Neurobiological stress, metabolic syndrome, and traditional cardiovascular risk factors may instead function as intermediary links between inflammation and atherosclerosis. Therefore, considering evolution as a mechanistic process and atherosclerosis as part of the inertia of evolution, greater insight into future preventative and therapeutic interventions for atherosclerosis can be gained by examining the past.

"Until death do us part". A multidisciplinary study on human- Animal co- burials from the Late Iron Age necropolis of Seminario Vescovile in Verona (Northern Italy, 3rd-1st c. BCE).

Animal remains are a common find in prehistoric and protohistoric funerary contexts. While taphonomic and osteological data provide insights about the proximate (depositional) factors responsible for these findings, the ultimate cultural causes leading to this observed mortuary behavior are obscured by the opacity of the archaeological record and the lack of written sources. Here, we apply an interdisciplinary suite of analytical approaches (zooarchaeological, anthropological, archaeological, paleogenetic, and isotopic) to explore the funerary deposition of animal remains and the nature of joint human-animal burials at Seminario Vescovile (Verona, Northern Italy 3rd-1st c. BCE). This context, culturally attributed to the Cenomane culture, features 161 inhumations, of which only 16 included animal remains in the form of full skeletons, isolated skeletal parts, or food offerings. Of these, four are of particular interest as they contain either horses (Equus caballus) or dogs (Canis lupus familiaris)-animals that did not play a dietary role. Analyses show no demographic, dietary, funerary similarities, or genetic relatedness between individuals buried with animals. Isotopic data from two analyzed dogs suggest differing management strategies for these animals, possibly linked to economic and/or ritual factors. Overall, our results point to the unsuitability of simple, straightforward explanations for the observed funerary variability. At the same time, they connect the evidence from Seminario Vescovile with documented Transalpine cultural traditions possibly influenced by local and Roman customs.

Forensic application of isotope ratio mass spectrometry (IRMS) for human identification.

Application of isotope ratio mass spectrometry (IRMS) to skeletal remains has become an important tool to investigate human behavior and history. Isotopic variations in collagen, enamel, and keratin reflect variations in an individual's diet and drinking water. Since food and water sources typically are geographically linked, isotope testing can assist in forensic identification by classifying remains to a likely geographic or population origin. If remains are commingled, differences in diet or geographic origin also can support their separation. The usefulness of IRMS in forensic science is dependent on the underlying quality and surety of the isotope test results; in other words, we need to understand their reliability in interpretations. To take ownership of isotopic data quality, we recommend asking a series of questions:Here, we use data collected during the buildout and accreditation of an isotope testing program at the Defense POW/MIA Accounting Agency (DPAA) to answer the above questions for the forensic application of IRMS for human identification. While our primary focus is on the preparation and analysis of bone collagen, the questions above should be considered whenever isotope testing is used in forensic casework. Whether the populations of interest are drugs or humans, olives or explosives, users need to evaluate their isotopic data and interpretations to ensure they are scientifically sound and legally defensible.

Forensic anthropologists and estimates of skeletal completeness: The impacts of training and experience.

Forensic anthropologists engage with numerous and diverse stakeholders in their casework. Regarding the recovery of human remains, these stakeholders may be interested in quantifying or qualifying the amount of remains recovered. How forensic anthropologists respond to such questions, whether verbally or in written reporting, has the potential to impact the trajectory of a case. However, communications about skeletal completeness are rarely discussed within the field. Current data-collection procedures recommend the use of inventories. This approach may be less feasible for complicated assemblages involving commingling or high degrees of fragmentation. Numerous methods exist to quantify the amount of skeletal remains present in complex or larger assemblages, but it remains unclear to what extent forensic anthropologists utilize these methods and whether factors like degree of expertise influence analysts' ability to report skeletal completeness consistently and precisely. A study was designed to examine differences between public and professional perceptions of skeletal completeness, presenting images of incomplete bones and skeletal remains. Survey participants were asked to assess the completeness of the remains in each image. Few patterns were observed regarding photographs of skeletal assemblages, but distinct differences were observed among individual bones between respondents with different degrees of expertise. These responses reflect potentially unexamined assumptions underlying assessments of incomplete bones and skeletal assemblages. This highlights the necessity of standardizing how we report estimates of completeness within the forensic anthropology community and how we discuss these results with external stakeholders. Completeness estimates must be either removed from reports and bench notes or annotated and cited clearly, as is standard with other aspects of forensic anthropological analysis. Several methods are summarized, with recommendations for integrating them into casework.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

Nails as optimal source of DNA for molecular identification of 5 decomposed bodies recovered from seawater: from Y-ancestry to personal identification.

Molecular identification of extremely compromised human remains in forensic field is usually performed from DNA typing of bones, which are a difficult sample to work with. Moreover, autosomal STR profiles do not always result in the identification of the donor due to lack of comparisons or non-hit throughout database searching. An attempt to overcome these issues is represented by fingernails as an alternative DNA source and Y-STRs typing to infer both geographical and familial ancestry of the unknown donor. In this study, we analyzed both 24 autosomal and 27 Y-chromosome STRs from unidentified human remains (UHRs) of five males recovered from the water near the southwestern coast of Sardinia by the Italian Harbor Master's Office. Nail clippings provided an optimal source of autologous DNA for molecular identification in a very short time, producing complete autosomal and Y-STR profiles even under conditions of high body degradation. Unfortunately, no match neither compatibility occurred using autosomal STRs (aSTRs), initially. Upon analyzing the Y-haplotypes, we found out they had already been observed in northern Africa, providing us important investigative leads. This prompted the International Criminal Police Organization (INTERPOL) to provide us with references of alleged relatives that were then confirmed to be related. The use of fingernails represents an excellent DNA source especially for genetic identification of decomposed bodies recovered in seawater environment. Notably, DNA extracted from nails gave high-quality Y-STR haplotypes by which predicting paternal ancestry of the unidentified donors may result fundamental in the forensic investigative context.