Extraction Protocol for Parallel Analysis of Proteins and DNA from Ancient Teeth and Dental Calculus.

Dental calculus is becoming a crucial material in the study of past populations with increasing interest in its proteomic and genomic content. Here, we suggest further development of a protocol for analysis of ancient proteins and a combined approach for subsequent ancient DNA extraction. We tested the protocol on recent teeth, and the optimized protocol was applied to ancient tooth to limit the destruction of calculus as it is a precious and irreplaceable source of dietary, microbiological, and ecological information in the archeological context. Finally, the applicability of the protocol was demonstrated on samples of the ancient calculus.

The Application of Scanning Electron Microscopy with Energy-Dispersive X-Ray Spectroscopy (SEM-EDX) in Ancient Dental Calculus for the Reconstruction of Human Habits.

The great potential of scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) is in detection of unusual chemical elements included in ancient human dental calculus to verify hypotheses about life and burial habits of historic populations and individuals. Elemental spectra were performed from archeological samples of three chosen individuals from different time periods. The unusual presence of magnesium, aluminum, and silicon in the first sample could confirm the hypothesis of high degree of dental abrasion caused by particles from grinding stones in flour. In the second sample, presence of copper could confirm that bronze jewelery could lie near the buried body. The elemental composition of the third sample with the presence of lead and copper confirms the origin of individual to Napoleonic Wars because the damage to his teeth could be explained by the systematic utilization of the teeth for the opening of paper cartridges (a charge with a dose of gunpowder and a bullet), which were used during the 18th and the 19th century AD. All these results contribute to the reconstruction of life (first and third individual) and burial (second individual) habits of historic populations and individuals.

Cost-effective straightforward method for captured whole mitogenome sequencing of ancient DNA.

Working with mitochondrial DNA from highly degraded samples is challenging. We present a whole mitogenome Illumina-based sequencing method suitable for highly degraded samples. The method makes use of double-stranded library preparation with hybridization-based target enrichment. The aim of the study was to implement a new user-friendly method for analysing many ancient DNA samples at low cost. The method combines the Swift 2S™ Turbo library preparation kit and xGen® panel for mitogenome enrichment. Swift allows to use low input of aDNA and own adapters and primers, handles inhibitors well, and has only two purification steps. xGen is straightforward to use and is able to leverage already pooled libraries. Given the ancient DNA is more challenging to work with, the protocol was developed with several improvements, especially multiplying DNA input in case of low concentration DNA extractions followed by AMPure® beads size selection and real-time pre-capture PCR monitoring in order to avoid cycle-optimization step. Nine out of eleven analysed samples successfully retrieved mitogenomes. Hence, our method provides an effective analysis of whole mtDNA, and has proven to be fast, cost-effective, straightforward, with utilisation in population-wide research of burial sites.

Sagittal suture morphological variation in human archaeological populations.

Cranial sutures join the many bones of the skull. They are therefore points of weakness and consequently subjected to the many mechanical stresses affecting the cranium. However, the way in which this impacts their morphological complexity remains unclear. We examine the intrinsic and extrinsic mechanisms of human sagittal sutures by quantifying the morphology from 107 individuals from archaeological populations spanning the Mesolithic to Middle ages, using standardized two-dimensional photographs. Results show that the most important factor determining sutural complexity appears to be the position along the cranial vault from the junction with the coronal suture at its anterior-most point to the junction with the lambdoid suture at its posterior-most point. Conversely, factors such as age and lifeways show few trends in complexity, the most significant of which is a lower complexity in the sutures of Mesolithic individuals who consumed a tougher diet. The simple technique used in this study therefore allowed us to identify that, taken together, structural aspects play a more important role in defining the complexity of the human sagittal suture than extrinsic factors such as the mechanical forces imposed on the cranium by individuals' diet.

Localised enamel hypoplasia of human primary canines (LHPC) in the Necropolis of Great Moravia in Znojmo-Hradiste (the so called Stronghold of Znojmo, 9th - 10th century CE, Czech Republic) and analysis of chemical elements on surface enamel and hypoplastic defect via EDX method.

The present study aimed to evaluate the frequency and time of the development of enamel hypoplasia of the primary canine of the Slavic population from the 9th to the first half of the 10th century CE in the South Moravian burial ground of Znojmo-Hradiste, to establish the association with incidence of dental caries, and to analyse the chemical element content of the enamel. The frequency and time of the development of enamel hypoplasia of the primary canine were established macroscopically. The chemical element content of the enamel was analysed via the EDX method. The incidence of LHPC is very high in this analysed collection in comparison with other Slavic populations. 45.59% individuals (n = 31) displayed at least one hypoplastic defect on their canines. Most individuals were 2-5 years old and more than a quarter of them had multiple hypoplasia. The most frequent shape of enamel defect is irregular. The difference between the defects originating in the perinatal period and those originating in the postnatal or prenatal period is statistically significant. Individuals with LHPC (localised hypoplasia of primary canines) have a higher presence of dental caries in primary teeth than individuals without LHPC, but without statistical significance. Individuals with LHPC have other hypoplasia on the primary teeth more often than individuals without LHPC. The EDX analysis shows the average value of the Ca/P ratio in Spectrum 1 to be highest in teeth with unsolid enamel (LHPC), decreasing to Spectrum 2 and subsequently to Spectrum 3. The concentration of phosphor is increasing from Spectrum 1 to Spectrum 3. Magnesium was most commonly present in Spectrum 1, and less often in Spectrum 2 and Spectrum 3 in mean concentration 0.27 and 0.39 at.%. Values of Mg content rise to on average 0.63 at.% on the base of hypoplastic defects. In agreement with the findings of Robinson et al. (1981), this might mean a lower density of enamel in the place of a hypoplastic defect. However, this cannot be argued clearly, because the difference in concentration could have been caused by diagenetic processes over the time that the samples lay deposited in soil. Sodium is found in our collection of teeth about as often in all three spectra in an almost identical mean concentration (0.51, 0.46 and 0.56 at.%, respectively).

Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.

AIMS: Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA. MATERIALS AND METHODS: Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR® green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test. RESULTS: Methods that measure total DNA present in the sample (NanoDrop™ UV spectrophotometer and Qubit® fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors. CONCLUSIONS: Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.

Detection of metal residues on bone using SEM-EDS. Part I: Blunt force injury.

Previous studies have indicated that metal particles remain on bone after sharp force injury or gunshot and that their detection by scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDS) could greatly help in tool identification. However, the presence of metal particles on bone surfaces in the context of blunt force trauma has never been assessed experimentally. For this reason the present paper represents an experimental study of the behaviour of metal residues on bone following blunt force injury. Ten fresh sub-adult bovine metatarsal bones were manually cleaned of soft tissues. They were then struck by metal bars (copper, iron or aluminium) on the external surface of the mid-diaphysis. All blunt metal instruments used in this study left a sign in the form of single particles, a smear or a powder-like deposit on the bone surface. The residues of all three metal implements were detected on the bone surface, 0.3-10 mm from the fracture border. The presence of metal particles was confirmed in all samples struck with iron and copper and in two of six aluminium samples; no particles were detected on the negative control. Chemical composition of residues highly corresponded with the composition of applied bars.