Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years

Background

Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles.

Methodology/Principal Findings

We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a “clean- laboratory” dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the “unhandled” teeth and there was no indication of contamination, while the latter was the case with half of the “handled” teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians) supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes.

Conclusion

The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied.     

Phosphorus retention as a function of external loading,hydraulic turnover time,area and relative depth in 54 lakes and reservoirs

We analysed phosphorus retention as a function of external loading, hydraulic turnover time, area and relative depth on the basis of published data from 54 lakes and reservoirs in different climate regions around the world. Our analysis demonstrated that reservoirs and lakes that received higher areal loading of phosphorus (TP_in) also retained more P per m² but the proportion of the external P loading retained in the waterbody (retention coefficient, R _P) remained generally independent of TP_in. The waterbodies with longer hydraulic residence times (T _R) retained larger proportions of external P and the correlation between R _P and T _R was much stronger in lakes with areas larger than 25 km² than in the whole data set. TP_in and T _R together determined 78% of the variation in R _P in large lakes. We also partially confirmed our hypothesis that waterbodies with bigger relative depths (Z _R) retain more of the external phosphorus than larger and shallower waterbodies with lower Z _R. The hypothesis was, however, validated only for lakes larger than 25 km² and for those with T _R <0.3 year, where R _P increased significantly with increasing Z _R. In stratified lakes, increasing relative depth correlated with reduced P retention capacity, demonstrating the complex nature of phosphorus biogeochemistry in lake ecosystems.     

An O-acetylated sialyl-Tn is involved in ovarian cancer-associated antigenicity

We reported previously that a monoclonal antibody, 6G9, raised against bovine submaxillary mucin (BSM) reacted with mucinous ovarian cancer and recognized tumor-associated sialylated carbohydrate antigens. To obtain structural information on the carbohydrate antigens recognized by 6G9, the reactivity of several mucins and carbohydrates with the antibody was determined by ELISA. Exoglycosidase digestion of BSM showed that 6G9 recognized Sia as a nonreducing monosaccharide but neither Gal nor GlcNAc. Reactivity of BSM with 6G9 decreased markedly on de-O-acetylation of BSM in mild alkali, and O-acetyl Sia obtained from BSM reacted with the antibody, indicating the presence of O-acetyl groups on Sia in the epitope. A sialyl-Tn structure located in the epitope was also demonstrated by the findings that de-O-acetylated BSM retained weak but significant reactivity with 6G9 and that ovine submaxillary mucin, major sugar chains of which are sialyl-Tn, reacted with 6G9 stronger than de-O-acetylated BSM. Furthermore, weak reactivity of NeuAcalpha2 --> 6GalNAc prepared from BSM demonstrated that 6G9 recognized the sialyl-Tn structure, but the modification of Sia with O-acetyl groups was essential for the recognition. The failure of 9-O-acetyl NeuAc, synthesized chemically, to react with the antibody implied that 6G9 recognized sialyl-Tn with O-acetylation on Sia distinct from C-9 O-acetylation.     

Validation of physiological tests in relation to competitive performances in elite male distance cross-country skiing

The purpose of the present study was to establish which physiological test parameters reflects the distance performances in the Swedish National Championships in cross-country skiing (SNC) and the International Ski Federation's ranking points for distance performances (FISdist). The present study also aimed to create multiple regression models to describe skiing performance for the SNC distance races and International Ski Federation's (FIS) ranking. Twelve male, Swedish, national elite, cross-country skiers (maximal oxygen consumption [·VO?max] = 5.34 ± 0.34 L·min?1) volunteered to participate in the study. Their results in the 2008 SNC (15 km race [SNC15] and 30 km race [SNC30]) and FISdist points were used as performance data. On the week preceding the Championship, subjects completed a test battery consisting of 7 physiological tests: isokinetic knee extension peak torque (PT), vertical jumps (VJ), lactate threshold (LT), ·VO?max, and 3 double poling tests of different durations (DP20, DP60, and DP360). Correlations were established using Pearson's correlation analysis, and models to describe skiing performance were created using standard multiple linear regression analysis. Significant correlations were found between the performance parameters and test parameters derived from LT, ·VO?max, and DP60 tests. No correlations with any performance parameter were found for PT, VJ, DP20, and DP360 tests. For FISdist and SNC15, the models explain 81% and 78% of the variance in performance, respectively. No statistically valid regression model was found for SNC30. The results of this study imply that the physiological demands in male elite distance cross-country skiing performances are different in different events. To adequately evaluate a skier's performance ability in distance cross-country skiing, it is necessary to use test parameters and regression models that reflect the specific performance.     

The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics

A rapid unidirectional method for cloning PCR-amplified cDNA fragments into virtually any fusion protein expression vector is described. The method, termed PRESAT-vector cloning, is based on a T-vector technique that does not require restriction endonuclease digestion of the PCR product. Subsequently, we applied a novel ORF selection method of the ligated plasmid products. This second step involves restriction endonuclease treatment that eliminates the plasmids containing an ORF in the wrong orientation prior to transformation into the bacterial host for further protein expression studies. To achieve this selection, we customized the 5'-sequence of the "rear" PCR primer corresponding to the C terminus of the protein to be expressed. The colonies harbored only the ligated products of the desired orientation at >90% efficiency. This method is applied to a GST fusion expression system, and an HTS system for soluble proteins from an expression library was tested.     

Dependence of phenotypic variance in body size on environmental quality

The recent "overhead threshold" model for optimal age and body size at maturity (Day and Rowe 2002 ) predicts that phenotypic variability in adult body size will be low under inferior environmental quality and will increase with improving conditions. The model is, however, based on a potentially restrictive assumption of a monotone increase of fecundity with increasing body size. On the basis of a numerical model, we show that introducing the concept of maximum adult body size changes the predictions of the model. The dependence of variability in adult body size on environmental quality becomes a concave function with a maximum at intermediate values. Depending on the range of environmental conditions considered, one may therefore expect to observe both increasing and decreasing functions. We test the predictions of our model on a literature-based database of 131 insect species covering all major orders. We demonstrate that, in most species, relative phenotypic variation in body size decreases when environment-specific average of adult body size increases. In the majority of cases at least, such a relationship can be interpreted as a decreased relative variation in better growing conditions. With some potentially meaningful exceptions (e.g., females of capital-breeding insects), the general pattern was largely invariable across different taxa, ecological subdivisions, and sexes.     

The molecular dissection of mtDNA haplogroup H confirms that the Franco-Cantabrian glacial refuge was a major source for the European gene pool

Complete sequencing of 62 mitochondrial DNAs (mtDNAs) belonging (or very closely related) to haplogroup H revealed that this mtDNA haplogroup--by far the most common in Europe--is subdivided into numerous subhaplogroups, with at least 15 of them (H1-H15) identifiable by characteristic mutations. All the haplogroup H mtDNAs found in 5,743 subjects from 43 populations were then screened for diagnostic markers of subhaplogroups H1 and H3. This survey showed that both subhaplogroups display frequency peaks, centered in Iberia and surrounding areas, with distributions declining toward the northeast and southeast--a pattern extremely similar to that previously reported for mtDNA haplogroup V. Furthermore, the coalescence ages of H1 and H3 (~11,000 years) are close to that previously reported for V. These findings have major implications for the origin of Europeans, since they attest that the Franco-Cantabrian refuge area was indeed the source of late-glacial expansions of hunter-gatherers that repopulated much of Central and Northern Europe from ~15,000 years ago. This has also some implications for disease studies. For instance, the high occurrence of H1 and H3 in Iberia led us to re-evaluate the haplogroup distribution in 50 Spanish families affected by nonsyndromic sensorineural deafness due to the A1555G mutation. The survey revealed that the previously reported excess of H among these families is caused entirely by H3 and is due to a major, probably nonrecent, founder event.     

Nutrient runoff dynamics in a rural catchment: Influence of land-use changes, climatic fluctuations and ecotechnological measures

The main trend in land-use changes in the Porijõgi River catchment, south Estonia, is a significant increase in abandoned lands (from 1.7% in 1987 to 10.5% in 1997), and a decrease in arable lands (from 41.8 to 23.9%). Significant climatic fluctuations occurred during the last decades. Milder winters (increase of air temperature in February from −7.9 to −5.5°C during the period 1950–1997) and a change in the precipitation pattern have influenced the mean annual water discharge. This results in more intensive material flow during colder seasons and decreased water runoff in summer. During the period 1987–1997 the runoff of total-N, total-P, SO₄, and organic material (after BOD₅) decreased from 25.9 to 5.1, from 0.32 to 0.13, from 78 to 48, and from 7.4 to 3.5 kg ha⁻¹ year⁻¹, respectively. Most significant was a 4–20-fold decrease in agricultural subcatchments while in the forested upper-course catchment the changes were insignificant. Variations of total-N, and total-P runoff in both the entire catchment and its agricultural subcatchments are well described by the change of land use (including fertilization intensity), soil parameters and water discharge. In small agricultural subcatchments the rate of fertilization was found to be the most important factor affecting nitrogen runoff, while land-use pattern plays the main role in larger mosaic catchments. Ecotechnological measures (e.g. riparian buffer zones and buffer strips, constructed wetlands) to control nutrient flows from agricultural catchments are very important.     

Three in One—Multiple Faunal Elements within an Endangered European Butterfly Species

Ice ages within Europe forced many species to retreat to refugia, of which three major biogeographic basic types can be distinguished: "Mediterranean", "Continental" and "Alpine / Arctic" species. However, this classification often fails to explain the complex phylogeography of European species with a wide range of latitudinal and altitudinal distribution. Hence, we tested for the possibility that all three mentioned faunal elements are represented within one species. Our data was obtained by scoring 1,307 Euphydryas aurinia individuals (46 European locations) for 17 allozyme loci, and sequencing a subset of 492 individuals (21 sites) for a 626 base pairs COI fragment. Genetic diversity indices, F statistics, hierarchical analyses of molecular variance, individual-based clustering, and networks were used to explore the phylogeographic patterns. The COI fragment represented 18 haplotypes showing a strong geographic structure. All but one allozyme loci analysed were polymorphic with a mean F _ST of 0.20, supporting a pronounced among population structure. Interpretation of both genetic marker systems, using several analytical tools, calls for the recognition of twelve genetic groups. These analyses consistently distinguished different groups in Iberia (2), Italy, Provence, Alps (3), Slovenia, Carpathian Basin, the lowlands of West and Central Europe as well as Estonia, often with considerable additional substructures. The genetic data strongly support the hypothesis that E. aurinia survived the last glaciation in Mediterranean, extra-Mediterranean and perialpine refugia. It is thus a rare example of a model organism that combines attributes of faunal elements from all three of these sources. The observed differences between allozymes and mtDNA most likely result from recent introgression of mtDNA into nuclear allozyme groups. Our results indicate discrepancies with the morphologically-based subspecies models, underlining the need to revise the current taxonomy.