Association between ancient bone preservation and dna yield: A multidisciplinary approach

Ancient molecular typing depends on DNA survival in archaeological bones. Finding valuable tools to predict DNA presence in ancient samples, which can be measured prior to undertaking a genetic study, has become an important issue as a consequence of the peculiarities of archaeological samples. Since the survival of DNA is explained by complex interrelations of multiple variables, the aim of the present study was to analyze morphological, structural, chemical, and biological aspects of a set of medieval human bones, to provide an accurate reflection of the state of preservation of the bony components and to relate it with DNA presence. Archaeological bones that yielded amplifiable DNA presented high collagen content (generally more than 12%), low racemization values of aspartic acid (lesser than 0.08), leucine and glutamic acid, low infrared splitting factor, small size of crystallite, and more compact appearance of bone in the scanning electron micrographs. Whether these patterns are characteristic of ancient bones or specific of each burial site or specimen requires further investigation. Am J Phys Anthropol 151:102–109, 2013. © 2013 Wiley Periodicals, Inc.     

Is amino acid racemization a useful tool for screening for ancient DNA in bone?

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone.     

Cremation practices and the survival of ancient DNA: burnt bone analyses via RAPD-mediated PCR

Numerous burial rites have been developed in different time periods of human cultural evolution. One of the most interesting burial practices was the ritual cremation of human bodies. Due to the respective cultural and religious background, brand graves are known where human remains had been buried together with burnt bones of animal origin. To date, burnt bone samples have been refractory to PCR-mediated amplification. D/L values of aspartic acid far greater than 80 x 10(-3) were measured in the samples thus indicating the presence of severely nicked and fragmented nucleic acids. In order to differentiate between burial gift of animal origin and burnt human specimens we established a highly sensitive protocol that addresses all the shortcomings connected to degraded ancient DNA. With the novel procedure it was possible to classify 4 specimens ranging from 2,000-5,000 BP on the basis of mitochondrial DNA sequences.     

Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy

Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization.     

The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10^–6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R² = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.     

Comparaison de l'état de conservation des phases minérales et organiques d'os fossiles. Implications pour les reconstitutions paléoenvironnementales et phylétiques

Bones have been collected from different geological sites, the sediment and the age of which being different. Comparizon between the mineral and organic phases shows the very fast alteration of the organic matrix in recent bones. Fossil bones show a loss in the organic matrices. Moreover, insoluble and soluble organic matrix alterations differ in a single bone. Implications of the diagenesis in palaeoenvironmental reconstruction and phylogeny are evoked.     

An elemental analysis of archaeological bone from Sicily as a test of predictability of diagenetic change

Cortical human bone samples from three tightly dated components of a single Sicilian site were chemically analyzed employing the highly sensitive technique of inductively coupled plasma emission spectroscopy. Although the skeletons appeared to be excellently preserved, significant diagenesis was detected. Moreover, a majority of the elements tested showed no constant or linear variation over time, implying that diagenetic change tends not to be a predictable function of duration of interment. Variation among major long bones of a single skeleton was quite high, as was variation across the cortex. The latter may reflect chemical inhomogeneity in bone tissue or may be an artifact of postmortem change. The results demonstrate the hazards of unsuspected and unpredictable diagenesis, which must be controlled before reliable dietary inferences can be drawn.     

Reliable differential diagnosis between osteosarcoma and regenerative bone cells in rats through simultaneous analysis of nuclear DNA content and size

OBJECTIVE: To evaluate the difference between benign proliferative lesions of regenerative bone calluses and malignant tumors in rats by simultaneous analysis of nuclear DNA content and size. STUDY DESIGN: Nuclei were stained with DAPI and measured by cytofluorometry. Five regenerative bones and five osteosarcoma samples were used. Two-dimensional dispersion diagrams between relative nuclear DNA content and nuclear size were drawn up and their regression lines were compared. RESULTS: It was possible to distinguish between osteosarcomas and regenerative bones by simultaneous analysis of nuclear size and DNA content during the cell cycle. The results yielded coefficients of correlation and regression. All five regression lines of osteosarcomas were situated dramatically higher on dispersiongrams than those of regenerative bones. CONCLUSION: Differential diagnosis between regenerative lesions and malignant tumors may now be possible using simultaneous measurement of nuclear DNA content and size.     

New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains

Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples.     

Problems in determination of skeletal lead burden in archaeological samples: an example from the First African Baptist Church population

Human bone lead content has been demonstrated to be related to socioeconomic status, occupation and other social and environmental correlates. Skeletal tissue samples from 135 individuals from an early nineteenth century Philadelphia cemetery (First African Baptist Church) were studied by electrothermal atomic absorption spectrometry and X-ray fluorescence for lead content. High bone lead levels led to investigation of possible diagenetic effects. These were investigated by several different approaches including distribution of lead within bone by X-ray fluorescence, histological preservation, soil lead concentration and acidity as well as location and depth of burial. Bone lead levels were very high in children, exceeding those of the adult population that were buried in the cemetery, and also those of present day adults. The antemortem age-related increase in bone lead, reported in other studies, was not evidenced in this population. Lead was evenly deposited in areas of taphonomic bone destruction. Synchrotron X-ray fluorescence studies revealed no consistent pattern of lead microdistribution within the bone. Our conclusions are that postmortem diagenesis of lead ion has penetrated these archaeological bones to a degree that makes their original bone lead content irretrievable by any known method. Increased bone porosity is most likely responsible for the very high levels of lead found in bones of newborns and children.     

Historical and non‐invasive samples: a study case of genotyping errors in newly isolated microsatellites for the lesser anteater (Tamandua tetradactyla L., Pilosa)

Tamandua tetradactyla (Pilosa), the lesser anteater, is a medium‐size mammal from South America. Its wide distribution through different landscapes, solitary and nocturnal habits, and the difficulty to capture and contain specimens limit the amount of individuals and populations sampled during fieldworks. These features along with the lack of specific molecular markers for the lesser anteater might be the causes for paucity in population genetic studies for the species. Historical samples from museum specimens, such as skins, and non‐invasive samples, such as plucked hair, can be supplementary sources of DNA samples. However, the DNA quantity and quality of these samples may be limiting factors in molecular studies. In this study, we describe nine microsatellite loci for T. tetradactyla and test the amplification success, data reliability and estimate errors on both historical and non‐invasive sample sets. We tested nine polymorphic microsatellites and applied the quality index approach to evaluate the relative performance in genotype analysis of 138 historical samples (study skin) and 19 non‐invasive samples (plucked hair). The observed results show a much superior DNA quality of non‐invasive over historical samples and support the quality index analysis as a practical tool to exclude samples with doubtful performance in genetic studies. We also found a relationship between the age of non‐invasive samples and DNA quality, but lack of evidence of this pattern for historical samples.     

Fluctuating asymmetry and morphometric variation of hand bones

The major aim of this study was to test three hypotheses: 1) more complex traits of the hand are less prone to developmental insults and therefore show lower fluctuating asymmetry (FA) as compared with simple traits; 2) the manifestation of FA correlates with the variability of the trait (i.e., CV); and 3) FA is an organ-wide property, and therefore a concordance exists between the FA measures of different traits in hand bones. Seventy-two bilateral measurements of hand bones, were made from plain-film radiographs of 365 cadavers. A complex trait was considered as the total length of the three phalanges of a finger and their contiguous metacarpals. Simple traits were considered to be the lengths of individual bone that made up the complex trait. The following results were obtained: 1) on the average simple traits, composing the complex trait, show much higher FA than the corresponding complex trait, but this result is expected if there is no correlation (or low correlation) between FA of simple traits within the complex trait, due to random direction of right-left differences; 2) strong and highly significant correlation was observed between FA and CV of studied traits, regardless of sex and age of individuals; and 3) the majority of FA measurements of hand bones showed no correlation. However, correlations between some sets of FA traits were highly significant. They were interpreted, although not specifically tested, as the result of a tight relationship between traits related not only developmentally but also by active performance of the same function. Am J Phys Anthropol 107:125–136, 1998. © 1998 Wiley-Liss, Inc.     

A developmental constraint on the fledging time of birds

We examined the hypothesis that the rate of bone growth limits the minimum fledging time of birds. Previous observations in California gulls indicate that linear growth of wing bones may be the rate limiting factor in wing development. If bone growth is rate limiting, then birds with relatively long bones for their size could be expected to have longer fledging periods than birds with relatively short bones. We tested this by comparing the length of wing bones, relative to body mass, to the relative length of fledging periods among 25 families. The results support the hypothesis. A strong correlation exists between relative fledging period and relative bone length. Species which have relatively long bones for their body size tend to take longer to fly. In contrast, parameters that influence flight style and performance, such as size of the pectoralis muscle and wing loading, show little or no correlation with fledging time. The analysis also indicates that, when altricial and precocial species are considered together, bone length is more highly correlated with fledging time than is body mass or rate of increase in body mass during growth. These observations suggest that linear growth of bones does limit the growth of avian wings and that it is one of the factors that influences the fledging time of birds.     

Ancient pathogen DNA in human teeth and petrous bones

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petrous bone which has become the most desired skeletal element in ancient DNA research due to its high endogenous DNA content. To compare the potential for pathogenic aDNA retrieval from teeth and petrous bones, we sampled these elements from five ancient skeletons, previously shown to be carrying Yersinia pestis. Based on shotgun sequencing data, four of these five plague victims showed clearly detectable levels of Y. pestis DNA in the teeth, whereas all the petrous bones failed to produce Y. pestis DNA above baseline levels. A broader comparative metagenomic analysis of teeth and petrous bones from 10 historical skeletons corroborated these results, showing a much higher microbial diversity in teeth than petrous bones, including pathogenic and oral microbial taxa. Our results imply that although petrous bones are highly valuable for ancient genomic analyses as an excellent source of endogenous DNA, the metagenomic potential of these dense skeletal elements is highly limited. This trade‐off must be considered when designing the sampling strategy for an aDNA project.     

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics

BACKGROUND: Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. METHODOLOGY/PRINCIPALS FINDINGS: To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. CONCLUSIONS: We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5' and 3' ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.     

The influence of the storage temperature and cryopreservation conditions on the extent of human sperm DNA fragmentation

We explored the influence of different storage temperature conditions, including different methods of cryopreservation, on the structure of DNA organization in human sperm using a direct labeling procedure for detecting DNA fragmentation. Nineteen sperm samples that were obtained from healthy men with normozoospermia (according to the criteria of the World Health Organization) were used for the investigation. A significant increase in human sperm DNA fragmentation was observed 8 h after the incubation at +39°C (by 76.7%) and at +37°C (by 68.9%). It was found that cooling the sperm with a cryoprotectant immediately after thawing did not produce a significant difference in the extent of DNA fragmentation; however, the samples that contained cryoprotectants showed a sharp increase in the DNA fragmentation 24 h after the incubation, which could suggest cryoprotectant cytotoxicity.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Some studies on the composition of bovine cortical-bone sialoprotein

1. An analysis of bovine bone sialoprotein, a homogeneous glycoprotein isolated from cortical bone, is presented. 2. Analytical results agree with earlier physical measurements indicating a molecular weight of about 23000. 3. Mild acid hydrolysis and treatment with neuraminidase showed that fucose and sialic acid occupy terminal positions on oligosaccharide chains. 4. Treatment of the sialic acid-free glycoprotein with beta-galactosidase showed that much of the galactose occupies a sub-terminal location in the intact glycoprotein. 5. The polypeptide chain is rich in aspartic acid, glutamic acid, serine, threonine and glycine, and has no detectable free terminal amino group. 6. Glycopeptides were studied after proteolytic digestion. 7. It is considered that the carbohydrate moiety is highly branched and is probably linked by an acid- and alkali-stable glycosylamine bond involving aspartic acid.     

The limitation of DEXA analysis for bone mass determination in mice

An increase in femoral and tibio/fibular bone mass following periosteal membrane stimulation by Moloney sarcoma virus inoculation into thigh muscles of mice was measured in situ on formalin fixed excised hind limbs using a Hologic 4500A Fan Beam X-ray bone densitometer adapted for small bone samples. These results were verified by measurements of constant dry bone mass of the same bones liberated from soft limb tissues by NaOH hydrolysis. There was no consistent data correlation found between the DEXA scan and dry bone mass evaluations. It is concluded that the sensitivity of the DEXA measurement is unsuitable when assessing very small bone samples, weighing merely 20-30 mg.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.