Bone Density Assessment Through Sodium Poly-Tungstate Gradient Centrifugation: A Preliminary Study on Decades-Old Human Samples

Soluções de iodeto de sódio (NaI) e de politungstato de sódio (PTS) têm sido utilizadas, de forma indistinta, no fracionamento densimétrico da matéria orgânica (MO) do solo. O objetivo deste estudo foi avaliar o efeito desses dois produtos em soluções de mesma densidade (1,80 kg L-1), bem como do aumento da densidade da solução de PTS (2,0 e 2,2 kg L-1), na obtenção de C na fração leve (FL) da MO do solo. As características estruturais da FL obtida com as diferentes soluções também foram avaliadas por ressonância magnética nuclear do 13C (RMN-13C) neste estudo, o qual foi realizado com amostras da camada de 0-5 cm de dois solos brasileiros (PVd-Argissolo Vermelho e LVd-Latossolo Vermelho). A dispersão do solo foi realizada com ultra-som (250 J mL-1 no PVd e 450 J mL-1 no LVd); a suspensão de 20 g de solo e 80 mL-1 de solução foi centrifugada (2.000 g, 90 min), e a FL, obtida pela filtragem do sobrenadante em filtro de fibra de vidro, sendo o C desta fração analisado por combustão seca (Shimadzu TOC-V CSH). O uso da solução de PTS aumentou a recuperação de C da FL em 152 % no PVd e em 166 % no LVd, em comparação à solução de NaI de mesma densidade (1,8 kg L-1). O incremento da densidade da solução de PTS também aumentou a obtenção de C na FL, o qual foi mais expressivo na densidade de 1,8 para 2,0 kg L-1 (57 % em ambos os solos) do que de 2,0 para 2,2 kg L-1 (21 % no PVd e 5 % no LVd). Com base nos teores de Fe e C da fração argila, estimou-se que a contaminação da FL com C da fração argila, quando do uso das soluções de PTS, variou de 1,9 a 3,5 % no LVd e de 6,8 a 10,4 % no PVd, os quais foram considerados baixos perante o alto percentual de incremento na recuperação de C na FL. O incremento da razão C Alquil/C-O-alquil e do C-carbonila na análise de RMN-13C sugere incremento na recuperação de FL em estádios mais avançados de decomposição com o uso de PTS e incremento da densidade da solução, tendo sido esses resultados mais expressivos no LVd. Com base no incremento da obtenção de C e na baixa contaminação da FL com C da fração argila, recomenda-se o uso da solução de PTS 2,0 kg L-1 em estudos de fracionamento densimétrico da MO do solo.

Recycling of sodium polytungstate used in soil organic matter studies

Evaluation of potentially labile soil organic carbon and nitrogen fractionation procedures

The mysteries of DNA preservation in bone: A comparative study of petrous bones and metacarpal epiphyses using ATR-FTIR spectroscopy

Different skeletal elements as a source of DNA for genetic identification of Second World War victims

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

This review paper presents a comprehensive overview of DNA preservation in hard tissues (bones and teeth) for applications in forensic and archaeogenetic analyses. It presents bone structure, DNA location in bones and teeth, and extensive information about postmortem DNA location and preservation. Aged bones are a challenging biological material for DNA isolation due to their low DNA content, degraded DNA, and the potential presence of PCR inhibitors. In addition, the binding of DNA to the mineral matrix necessitates the inclusion of a demineralization process in extraction, and its contribution to the resulting increase in both DNA quality and quantity is explained. Guidelines and recommendations on bone sample selection to obtain higher DNA yields are discussed in terms of past, recent, and possible future recommendations. Interskeletal and intraskeletal differences in DNA yield are also explained. Recent studies have shown that current recommendations for the genetic identification of skeletal remains, including femurs, tibias, and teeth, may not be the most effective sampling approach. Moreover, when mass disasters and mass graves with commingled skeletal remains are considered, there is a greater possibility that the recommended set of skeletal elements will not be available for sampling and subsequent genetic testing. This review highlights interskeletal and intraskeletal variability in DNA yield, with a focus on studies conducted on poorly preserved skeletal remains, including both postwar (1945) victims from Slovenia and ancient human skeletons. Special emphasis is placed on anatomical differences and potential mechanisms influencing DNA preservation, as demonstrated in research on both modern and historical skeletons. Finally, the petrous part of the temporal bone and tooth cementum were reviewed in greater detail because they have been recognized as an optimal sampling type in both ancient DNA studies and routine forensic case analyses. Our experiences with the Second World War and archaeological petrous bones are discussed and compared to those of other bone types.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.

Background: Identifying the optimal bone regions for DNA analysis is critical, as DNA preservation and quality vary significantly across bone types and structures and is defined as intra-bone variability. This study aimed to evaluate the correlation between computed tomography (CT)-measured bone density and DNA preservation in small skeletal elements to identify optimal regions for DNA analysis. Methods: 137 bones from six skeletal elements excavated from a single burial site were analysed using Dual-Source CT (DSCT) to map compact and cancellous regions. DNA was extracted using a demineralisation method and quantified via real-time PCR to assess DNA quantity and degradation. Results: Among 461 bone regions analysed (137 bones; patellae, calcaneus, talus, the navicular bones, the cuboid bone, and the medial cuneiform bone), a significant difference in DNA quantity was observed only in the calcaneus, where the sulcus contained more DNA than the body. No significant differences in the degradation index were detected among bone segments or skeletal elements. Correlations between CT-measured bone density and DNA quantity or degradation index were region-specific. Higher bone density correlated positively with DNA quantity in compact regions of the calcaneus and talus. Regarding degradation, a positive correlation (higher bone density → higher degradation) was observed in the patella's anterior surface, while a negative correlation (higher bone density → lower degradation) was found in the talus's sulcus, the opposite side of the talar sulcus, the posterior calcaneal articular facet, and the cuboid's tuberosity. No significant correlations were found in other bone segments. Conclusions: Our study identified small skeletal elements, particularly the patella and the navicular bone, as promising sources for DNA analysis. While bone density correlated with DNA preservation in some cases, the relationship was inconsistent. Our findings support the use of small bones in forensic and archaeological research and warrant further investigation.

Skeletonized human remains from Second World War mass graves in Slovenia are a major challenge in genetic identification, and bones with a high DNA yield must be selected for successful identification. The goal of this study was to construct skeletal sampling strategy recommendations through comparison of the most appropriate groups of skeletal elements. Altogether, 566 bones and teeth from the same mass grave were compared, half analyzed in this study and half in previous studies performed by our group. After anthropological examination, mechanical and chemical cleaning was performed, followed by bone and tooth powdering. Total demineralization of 0.5 g of bone and tooth was followed by extraction and purification of DNA with a Biorobot EZ1 device (Qiagen). The qPCR PowerQuant kit (Promega) was used to measure the amount of DNA, and statistical analysis was performed. Skeletal elements were selected according to known better preservation of DNA in the human body, and they were arranged in seven groups: petrous bone, long bones (femur and tibia), torso bones (first rib and 12th vertebra), metacarpals, metatarsals, short and sesamoid bones (talus, navicular, medial cuneiform, cuboid, calcaneus, and patella), and teeth. Sampling strategy recommendations were constructed based on DNA quantity and quality results. The petrous bone group, metacarpal group, torso bone group, and short and sesamoid bone group produced the highest DNA yields. Accordingly, in addition to standard sampling of long bones (femurs and tibias) and teeth, those additional bone types should be collected for Slovenian Second World War victim identification.

Gravitational separation of 137Cs contaminated soil in Fukushima environment: Density dependence of 137Cs activity and application to volume reduction

Well-crystallized groundmass fractions are commonly used to obtain reliable K-Ar ages for volcanic rocks, as phenocrysts potentially contain excess 40Ar. In general, phenocrysts are removed by magnetic separator, heavy liquid separation, and hand picking. Heavy liquid separation utilizes sodium polytungstate (SPT) solution, which replaced toxic heavy liquids such as bromoform in the 1980s. Recently, a vacuum pump has been used to reduce the filtration time for heavy liquid separation using SPT solution, which has a high viscosity. This note describes the separation procedure for gentle suction filtration using a hand vacuum pump, which can be safely completed in 50-60 minutes per sample. The procedure was tested on andesite and basalt samples from Hakusan, Miyakejima, and Etna Volcanos. Separation of the groundmass fraction from the phenocryst fraction was successful for all andesite samples, whereas separation was incomplete in some of the basalt samples, probably because the gravity of groundmass grains was heavier than the maximum gravity of the SPT solution (3.11 g/cm3).

The characterization of ancient DNA in fossil bones is providing invaluable information on the genetics of past human and other animal populations. These studies have been aided enormously by the discovery that ancient DNA is relatively well preserved in the petrous bone compared to most other bones. The reasons for this better preservation are however not well understood. Here we examine the hypothesis that one reason for better DNA preservation in the petrous bone is that fresh petrous bone contains more DNA than other bones. We therefore determined the concentrations of osteocyte cells occluded inside lacunae within the petrous bone and compared these concentrations to other bones from the domestic pig using high resolution microCT. We show that the concentrations of osteocyte lacunae in the inner layer of the pig petrous bone adjacent to the otic chamber are about three times higher (around 95,000 lacunae per mm3) than in the mastoid of the temporal bone (around 28,000 lacunae per mm3), as well as the cortical bone of the femur (around 27,000 lacunae per mm3). The sizes and shapes of the lacuna in the inner layer of the petrous bone are similar to those in the femur. We also show that the pig petrous bone lacunae do contain osteocytes using a histological stain for DNA. We therefore confirm and significantly expand upon previous observations of osteocytic lacuna concentrations in the petrous bone, supporting the notion that one possible reason for better preservation of ancient DNA in the petrous bone is that this bone initially contains at least three times more DNA than other bones. Thus during diagenesis more DNA is likely to be preserved in the petrous bone compared to other bones.

A solution of sodium polytungstate (SPT) is used as a heavy liquid in global soil studies. This reagent has found wide use because it is nontoxic, incombustible, well soluble, environmentally safe, and of low viscosity. There is a relatively simple and accessible method for its regeneration. The reagent contains no carbon compounds, which is important for studying organic matter with the use of carbon isotopes. However, some chemical properties of SPT should be taken into consideration to ensure the obtainment of incoherent and sorbed solid-phase forms of organic matter. A modification of granulodensimetric fractionation is described that allows separating the solid-phase fractions of soils without changes in the content and isotope composition of the organic carbon and organic nitrogen in the separated material.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains