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.

Comparison of Morphological Sex Assessment and Genetic Sex Determination on Adult and Sub-Adult 17th-19th Century Skeletal Remains.

The first step in the analysis of human skeletal remains is the establishment of the biological profile of an individual. This includes sex assessment, which depends highly on the age of the individual and on the completeness and preservation state of the remains. Macroscopic methods only provide the assessment of sex, while for sex determination, molecular methods need to be included. However, poor preservation of the remains can make molecular methods impossible and only assessment can be performed. Presented research compares DNA-determined and morphologically assessed sex of adult and non-adult individuals buried in a modern-age cemetery (17th to late 19th century) in Ljubljana, Slovenia. The aim of the study was to assess the accuracy of commonly used macroscopic methods for sex assessment on a Slovenian post-medieval population. Results demonstrate that for adults, macroscopic methods employed are highly reliable and pelvic morphology, even the sciatic notch alone, is more reliable than skull. In non-adults, macroscopic methods are not as reliable as in adults, which agrees with previous research. This study shows how morphological and molecular methods can go hand in hand when building a biological profile of an individual. On their own, each methodology presented some individuals with undetermined sex, while together, sex of all the individuals was provided. Results confirm suitability of sex assessment based on skull and especially pelvic morphology in Slovenian post-medieval adults, while in the non-adult population molecular methods are advised.

Sex assessment using the radius bone in a French sample when applying various statistical models.

Sex estimation of skeletal remains is one of the most important tasks in forensic anthropology. The radius bone is useful to develop standard guidelines for sex estimation across various populations and is an alternative when coxal or femoral bones are not available.The aim of the present study was to assess the sexual dimorphism from radius measurements in a French sample and compare the predictive accuracy of several modelling techniques, using both classical statistical methods and machine learning algorithms.A total of 78 left radii (36 males and 42 females) were used in this study. Sixteen measurements were made. The modelling techniques included a linear discriminant analysis (LDA), flexible discriminant analysis (FDA), regularised discriminant analysis (RDA), penalised logistic regression (PLR), random forests (RF) and support vector machines (SVM).The different statistical models showed an accuracy of classification that is greater than 94%. After selection of variables, the accuracies increased to 97%. The measurements made at the proximal part of the radius (sagittal and transversal diameters of the head, and sagittal diameter of the neck), at distal part (maximum width of the distal epiphysis) and of the entire bone (maximum length) stand out among the various models.The present study suggests that the radius bone constitutes a valid alternative for sex estimation of skeletal remains with comparable classification accuracies to the pelvis or femur and that the non-classical statistical models may provide a novel approach to sex estimation from the radius bone. However, the extrapolation of the current results cannot be made without caution because our sample was composed of very aged individuals.

Evaluation of the obturator foramen as a sex assessment trait.

Correctly assessing sex from skeletal remains is one of the main elements of creating a biological profile. Many traits allow for this, the obturator foramen being one. However, research on its accuracy has provided mixed results. This study examines the obturator foramen using a 5-point grading scale to assess the degree of sexual dimorphism in four known age and sex skeletal collections from the UK and South Africa. Overall, sexual dimorphism was found in the obturator foramen when using the new scoring system; however, accuracies for correct sex classification ranged from ~ 46 to ~ 75%. Considering its wide range in accuracy rates across the four samples and difficulty in identifying the subtle changes in morphology, the obturator foramen should only be used as part of a multifactorial assessment of sex.

The Oshu Fujiwara-An interdisciplinary study on the history, culture and medical assessment of the oldest known mummified human remains in Japan (late Heian, 12th century AD).

This study documents a rare case of mummified human remains from Japan, dating to the late Heian period, 12th Century AD. The remains have only been scientifically investigated once in 1950 so far. The results of this investigation were translated, analyzed, and interpreted using methods of the 21st century. The remains have been traditionally identified as the four ruling generations of the Oshu Fujiwara clan, who built a cultural and economic center in Hiraizumi. Accordingly, this paper will first examine the historical and cultural significance of Hiraizumi and its ruling class before re-evaluating the findings of the 1950 investigation. This study is the first in the Western scientific literature to provide a comprehensive historical, cultural, and medical evaluation of these mummies.

The biological index of frailty: A new index for the assessment of frailty in human skeletal remains.

OBJECTIVE: Frailty is the physiological stress that individuals suffer during their life. In past populations, frailty is conventionally assessed through the occurrence of different biomarkers of biological stress. Some efforts have been made to propose indexes that combine all biomarkers. However, these indices have some critical limitations: they cannot be used on incomplete skeletons, do not consider the severity and/or healing of lesions, and assign equal importance to different biomarkers. To address these limitations, we propose a new index to assess frailty in skeletal individuals. MATERIAL AND METHODS: By statistically analyzing a large amount of osteological data available from the Museum of London, and using a Logit model, we were able to define a different weight for each reported biomarker of frailty, based on their importance in increasing the risk of premature death for the individuals. RESULTS: The biological index of frailty (BIF) is the weighted mean of all biomarkers scored on the individuals, according to a different degree of importance assigned to each one. It also considers the severity and healing of the biomarkers when this is relevant to diagnose frailty. We applied BIF on a sample of Monastics and Non-Monastics from medieval England and compared it with the skeletal index of frailty (SFI). DISCUSSION: BIF is the first frailty index that gives a different weight to each skeletal biomarker of stress, considers both severity and healing of the lesions, and can be applied on partial skeletal remains. The comparison with SFI showed that BIF is applicable to a larger number of skeletal individuals, revealing new differences between the Monastic and the Non-Monastic groups.

The recovery and repatriation of the remains of Japanese war dead and the roles of physical anthropologists.

During and after World War II, around 2.4 million Japanese died overseas. The bodies of nearly half of them are still missing as they remain in the field where they fell and have never been repatriated. The tasks of recovering and repatriating the remains of Japanese war dead started in 1953 by the former Ministry of Health and Welfare, and are now carried out by the Social Welfare and War Victims' Relief Bureau of the Ministry of Health, Labour and Welfare (MHLW). In 2016, the "Act on Promoting the Recovery of the Remains of Japanese War Dead (Act No. 12 of 2016)" was enacted. The Act designates Fiscal Year (FY) 2016 (from April 2016 to March 2017) to FY 2024 as the period of intensive implementation and stipulates that the state shall establish a process to promote the identification of the war dead. In line with this Act, physical anthropologists were employed as full-time experts by the MHLW to conduct scientific analysis on the remains in the field, and since then, they have accompanied all overseas delegations for repatriation. The authors of this paper have been sent to the sites in the partner countries overseas such as the former Soviet Union, the Philippines, Papua New Guinea, and the Solomon Islands, and nationally to Ioto (Iwo Jima) to analyze the minimum number of individuals (MNI), ancestry, age at death, and sex of the remains. Along with the morphological investigations, DNA analyses of mitochondrial polymorphism and Y-chromosomal/autosomal short tandem repeat (STR) have been applied for estimation of the ancestry and identification of the individual. By narrowing down the possible candidates based on the historical records such as name list of the missing, if individual identification of the remains is achieved, the remains are returned to the bereaved families, and if not, they are placed in the Chidorigafuchi National Cemetery in Tokyo to rest in peace. Also, the implementation of single nucleotide polymorphism (SNP) analyses with next generation sequencing (NGS) for ancestry is under discussion. This paper provides an overview of the process of recovery and identification of the missing bodies from World War II in Japan.

Bile acids and oxo-metabolites as markers of human faecal input in the ancient Pompeii ruins.

Small organic molecules, lipids, proteins, and DNA fragments can remain stable over centuries. Powerful and sensitive chemical analysis can therefore be used to characterize ancient remains for classical archaeological studies. This bio-ecological dimension of archaeology can contribute knowledge about several aspects of ancient life, including social organization, daily habits, nutrition, and food storage. Faecal remains (i.e. coprolites) are particularly interesting in this regard, with scientists seeking to identify new faecal markers. Here, we report the analysis of faecal samples from modern-day humans and faecal samples from a discharge pit on the site of the ruins of ancient Pompeii. We propose that bile acids and their gut microbiota oxo-metabolites are the most specific steroid markers for detecting faecal inputs. This is due to their extreme chemical stability and their exclusive occurrence in vertebrate faeces, compared to other ubiquitous sterols and steroids.

Mitochondrial DNA analysis of the putative skeletal remains of Sieur de Marle: Genetic support for anthropological assessment of biogeographic ancestry.

In 1932, seven burials were discovered on a Texas plantation that was originally the site of a 17th-century Caddo Indian village. Of the seven excavated graves, one set of remains (an adult male) was notably buried in a manner inconsistent with traditional Caddoan burial practices and has long been purported to be the remains of Sieur de Marle (a member of the French explorer La Salle's last expedition). Diary accounts of La Salle's expedition scribe report that Sieur de Marle died along a river near an Indian village during a trek to Canada to find help for colonists left behind at the ill-fated Fort St. Louis. Additionally, two lead projectiles recovered from the grave were ballistically analyzed and determined to be consistent with ammunition used in 17th-century weaponry. In the 1980s, anthropologists requested access to the remains for study, but the skull was missing. Cranial measurements recorded in 1940 and 1962 (by two independent anthropologists) were used to investigate the ancestry of this individual; and the Giles-Elliot (G-E) discriminant function was calculated to be 18.1, within the Anglo-European range. Dietary isotope testing on non-cranial skeletal elements determined that this unknown male's diet was rich in animal/marine protein sources, which differs appreciably from Caddo Indian populations of that time period. In order to genetically assess this individual's biogeographic ancestry and to provide further support that this individual is of European descent, mitochondrial DNA (mtDNA) sequencing was performed using the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel. mtDNA sequencing of multiple sections from two different long bones yielded compiled results consistent with either Haplogroup H or R, both predominantly European mtDNA haplogroups. Further anthropological calculations were conducted using cranial measurements, FORDISC™ software, and discriminant function analysis. Two-way, four-way, and multigroup discriminant function analyses further classify this set of unidentified remains as being White (European) in origin, with posterior probabilities of 0.999, 0.881 and 0.986, respectively. Combined with historical records of Sieur de Marle's death, as well as overlays of historical and contemporary maps which demonstrate that the plantation site aligns with Joutel's diary accounts of de Marle's burial, these collective results support that these remains are of a European male and may possibly belong to this prominent member of La Salle's expedition team.

Sex assessment with the radius in Portuguese skeletal populations (late 19th - early to mid 20th centuries).

The assessment of sex is of immense relevance in the analysis of human skeletal remains, as other parameters of the biological profile are usually sex-specific (e.g., age at death or stature). The radius can be used to estimate sex when more dimorphic bones are not available or in the case of incomplete and fragmentary remains. Ten radius measurements collected in a sample of 364 individuals (166 females and 198 males) from the Coimbra Identified Skeletal Collection (late 19th - early 20th centuries) were employed to generate univariable and multivariable models for sex estimation. All models were evaluated with a 10-fold cross-validation method and an independent holdout sample from the Luís Lopes Collection (late 19th - mid 20th centuries) encompassing 50 individuals (25 females and 25 males). Univariable models show an accuracy ranging from 77.7% to 89.8% (cross-validation), and from 70% to 86% (test sample), while accuracy in the multivariable models varies from 88.7% to 93.4% (cross-validation), and 84.0% to 90.0% (test sample). Results suggest that measurements of the radius are useful to develop standard guidelines for sex estimation of anonymous skeletal remains.

The prehistoric roots of Chinese cuisines: Mapping staple food systems of China, 6000 BC-220 AD.

We conducted a meta-analysis of published carbon and nitrogen isotope data from archaeological human skeletal remains (n = 2448) from 128 sites cross China in order to investigate broad spatial and temporal patterns in the formation of staple cuisines. Between 6000-5000 cal BC we found evidence for an already distinct north versus south divide in the use of main crop staples (namely millet vs. a broad spectrum of C3 plant based diet including rice) that became more pronounced between 5000-2000 cal BC. We infer that this pattern can be understood as a difference in the spectrum of subsistence activities employed in the Loess Plateau and the Yangtze-Huai regions, which can be partly explained by differences in environmental conditions. We argue that regional differentiation in dietary tradition are not driven by differences in the conventional "stages" of shifting modes of subsistence (hunting-foraging-pastoralism-farming), but rather by myriad subsistence choices that combined and discarded modes in a number of innovative ways over thousands of years. The introduction of wheat and barley from southwestern Asia after 2000 cal BC resulted in the development of an additional east to west gradient in the degree of incorporation of the different staple products into human diets. Wheat and barley were rapidly adopted as staple foods in the Continental Interior contra the very gradual pace of adoption of these western crops in the Loess Plateau. While environmental and social factors likely contributed to their slow adoption, we explored local cooking practice as a third explanation; wheat and barley may have been more readily folded into grinding-and-baking cooking traditions than into steaming-and-boiling traditions. Changes in these culinary practices may have begun in the female sector of society.

The dietary impact of the Norman Conquest: A multiproxy archaeological investigation of Oxford, UK.

Archaeology has yet to capitalise on the opportunities offered by bioarchaeological approaches to examine the impact of the 11th-century AD Norman Conquest of England. This study utilises an integrated multiproxy analytical approach to identify and explain changes and continuities in diet and foodways between the 10th and 13th centuries in the city of Oxford, UK. The integration of organic residue analysis of ceramics, carbon (δ13C) and nitrogen (δ15N) isotope analysis of human and animal bones, incremental analysis of δ13C and δ15N from human tooth dentine and palaeopathological analysis of human skeletal remains has revealed a broad pattern of increasing intensification and marketisation across various areas of economic practice, with a much lesser and more short-term impact of the Conquest on everyday lifestyles than is suggested by documentary sources. Nonetheless, isotope data indicate short-term periods of instability, particularly food insecurity, did impact individuals. Evidence of preferences for certain foodstuffs and cooking techniques documented among the elite classes were also observed among lower-status townspeople, suggesting that Anglo-Norman fashions could be adopted across the social spectrum. This study demonstrates the potential for future archaeological research to generate more nuanced understanding of the cultural impact of the Norman Conquest of England, while showcasing a method which can be used to elucidate the undocumented, everyday implications of other large-scale political events on non-elites.

Chagas disease: Historic perspective.

This review is a perspective on the history of Chagas disease, and it adopts a novel approach from literary studies, historical documents and the science and epidemiology of the nature of the disease. From this analysis, comes the review's working definition of the Contact Zone (CZ): "the space in which geographically and historically separated people come into contact with each other and establish long-lasting relationships, which usually involve coercive conditions, radical inequality and intolerable conflict." In the Patient-Physician CZ, we verified the triple transition phenomena: the American trypanosomiasis shifted from a rural, acute, and vectorial transmitted disease to an urban, chronic and non-vectorial disease. In the Academic CZ, we describe the original disagreements which denied the existence of the disease and the current controversies about pathogenic mechanisms and etiological treatment. From the News from Latin America, and in the Original CZ, we will review the evolution of different forms of transmission. As in any good story, research across broad disciplines is necessary to reveal historical perspectives, scientific approaches, and the epidemiology of the disease, which has a prequel of 9000 years and an open ending: thus, we explore across the Global CZ, with its multiple and unexpected actors.

Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success.

Skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.

An economical and efficient method for postmortem DNA sampling in mass fatalities.

In mass fatality events, the need to identify large numbers of deceased persons using DNA can be a significant drain on already overburdened forensic practitioners, both in the field setting and the laboratory. The laboratory may be required to extract DNA from a variety of postmortem sample types, family or direct reference samples related to the missing, and perform matching of these results in a short period of time. While most forensic institutions are well equipped to handle both family and direct reference samples, postmortem samples such as bone or heterogeneous tissue samples can be difficult for labs to analyze. We have devised an easily deployable, efficient, and inexpensive method for collecting postmortem DNA samples on commercially available DNA preservation cards ("FTA®" cards). FTA® cards are already widely used in forensic labs and are convenient for shipping due to their small volume and stability at room temperature. We evaluated the suitability of a protocol involving swabbing of incisions made on cadavers and sample deposition onto FTA® cards over various postmortem intervals and under different environmental conditions. Each trial took place during a different point in the calendar year to evaluate the effects of seasonal weather patterns and temperature on decomposition, DNA yield, and rates of DNA degradation. To further account for the effects of seasonality (temperature and humidity), the progression of body decomposition was recorded following the Total Body Score (TBS) method [1]. DNA degradation was assessed either through STR amplification of 1.2 mm FTA punches or DNA extraction from 3.0 mm punches followed by real-time PCR quantification and STR amplification and genotyping. No consistent relationship was observed between postmortem interval and DNA degradation. Instead, the TBS score, which captures the stage of body decomposition, was shown to correlate well with DNA quantity. A TBS of 15 and below consistently yielded strong partial or full profiles (20 STR loci and Amelogenin using the PowerPlex 21 System) from all individuals from either 1.2 mm or 3.0 mm punches. Transfer of sample swabs to FTA cards is shown to be a simple and effective method for both field and laboratory operations over a range of conditions that can be evaluated by field forensic practitioners based on a body decomposition score. The approach could be beneficially integrated into mass fatality response plans.

Radiographic assessment of the skeletons of Dolly and other clones finds no abnormal osteoarthritis.

Our recent report detailing the health status of cloned sheep concluded that the animals had aged normally. This is in stark contrast to reports on Dolly (first animal cloned from adult cells) whose diagnoses of osteoarthritis (OA) at 5½ years of age led to considerable scientific concern and media debate over the possibility of early-onset age-related diseases in cloned animals. Our study included four 8-year old ewes derived from the cell line that gave rise to Dolly, yet none of our aged sheep showed clinical signs of OA, and they had radiographic evidence of only mild or, in one case, moderate OA. Given that the only formal record of OA in Dolly is a brief mention of a single joint in a conference abstract, this led us to question whether the original concerns about Dolly's OA were justified. As none of the original clinical or radiographic records were preserved, we undertook radiographic examination of the skeletons of Dolly and her contemporary clones. We report a prevalence and distribution of radiographic-OA similar to that observed in naturally conceived sheep, and our healthy aged cloned sheep. We conclude that the original concerns that cloning had caused early-onset OA in Dolly were unfounded.

Radiological assessment of the skeletal remains from Hiraizumi, Japan: review of results from the 1950 investigation.

Scientific investigations on the skeletal remains of four generations of the Fujiwara clan enshrined at the Chusonji World Heritage Site were carried out in March 1950. Although an official report on this investigation was published at the time, it did not merit much public attention. Thus, the purpose of this review is to describe the radiological aspects of the investigation, which may be of interest in the context of eleventh and twelfth century Japanese history.

Large-scale forensic investigations into the missing: Challenges and considerations.

Large-scale forensic investigations may follow episodes of mass violence and disasters where hundreds or thousands of people have died or are missing. A number of unique challenges for forensic science, different from domestic investigations, arise in these contexts. The setting and situation of these investigations regularly force forensic scientists into practices not regularly encountered while working in a standard criminal justice system. These practices can entail activities not specific to a practitioner's particular field or necessarily be scientific in nature, but are still needed in order for the investigation to move forward. These activities can include (1) establishing the number of and who exactly is missing after mass violence and disaster, (2) the creation of working protocols to deal with the scale of the loss of life that often overwhelm domestic practices and institutions, (3) negotiating the form that the investigation will take with various stakeholders, (4) addressing cultural beliefs of the affected society regarding the dead and missing, and (5) working within prescribed economic, political, and time constraints, among others. Forensic scientific responses to these challenges have proven to be flexible, innovative, and continually evolving.

Desempenho, características de carcaça e viabilidade econômica de coelhos sexados abatidos em diferentes idades / Performance, carcass characteristics and economic viability of sexed rabbits slaughtered at different ages

Avaliaram-se o desempenho, as características de carcaça e a viabilidade econômica de coelhos sexados abatidos em diferentes idades. Foram usados 100 coelhos (50 machos e 50 fêmeas), em delineamento de blocos inteiramente ao acaso e arranjo fatorial 2×2 (dois sexos e duas idades ao abate), com quatro tratamentos e cinco repetições. Os coelhos foram desmamados aos 35 e abatidos aos 75 ou 90 dias de idade. Não houve diferença entre sexo (P>0,05) nos parâmetros avaliados. Os animais abatidos aos 90 dias (P<0,001) eram mais pesados e consumiram (diário) mais ração. Os abatidos aos 75 dias (P<0,001) ganharam mais peso (diário) e apresentaram melhor conversão alimentar. Coelhos abatidos aos 90 dias de idade tiveram maiores pesos relativos de carcaça (P<0,04), de coxa (P<0,001), de gordura interna (P<0,001) e de fígado (P<0,01) e, ainda, apresentaram maior peso final e melhores características de carcaça, cuja venda foi mais lucrativa quando realizada aos 75 dias ou quando foram abatidos aos 90 dias de idade.

Performance, carcass characteristics and economic viability of sexed rabbits slaughtered at different ages were evaluated. One-hundred rabbits (50 males and 50 females) were assigned to a completely randomized block design in a factorial arrangement of 2×2 (two sexes and two slaughter ages), with four treatments and five replicates. The rabbits weaned at 35 were slaughtered at 75 and 90 days of age. No effect of sex (P>0.05) on the evaluated parameters was observed. Animals slaughtered at 90 days were heavier(P<0.001), had a higher daily feed intake and a higher carcass weight than animals slaughtered at 75 days which showed higher daily weight gain and better feed: gain ratio. Rabbits slaughtered at 90 days of age had higher carcass weight (P<0.04), thigh (P<0.005), internal fat (P<0.001) and liver (P<0.01). They also showed better carcass characteristics. High rentability was obtained alternatively for animals sold alive at 25 days of age or slaughtered at 90 days of age.