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The growth of proteomics-based methods in archaeology prompted an investigation of the survival of non-collagenous proteins, specifically immunoglobulin G (IgG), in archaeological human bone and dentine. Over a decade ago reports were published on extracted, immunoreactive archaeological IgG, and the variable yields of IgG molecules detected by Western blots of 1D and 2D SDS-PAGE gels. If IgG can indeed be recovered from archaeological skeletal material, it offers remarkable opportunities for exploring the history of disease - for example in applying functional anti-malarial IgGs to study past patterns of malaria. More recently, the field has seen a move away from immunological approaches and towards the use of shotgun proteomics via mass spectrometry. Using previously published techniques, this study attempted to extract and characterize archaeological IgG proteins. In only one extraction method were immunoglobulin derived peptides identified, and these displayed extensive evidence of degradation. The failure to extract immunoglobulins by all but one method, along with observed patterns of protein degradation, suggests that IgG may be an unsuitable target for detecting disease-associated antigens. This research highlights the importance of revisiting previously ‘successful’ biomolecular methodologies using emerging technologies.

Shamanic belief systems represent the first form of religious practice visible within the global archaeological record. Here we report on the earliest known evidence of shamanic costume: modified red deer crania headdresses from the Early Holocene site of Star Carr (c. 11 kya). More than 90% of the examples from prehistoric Europe come from this one site, establishing it as a place of outstanding shamanistic/cosmological significance. Our work, involving a programme of experimental replication, analysis of macroscopic traces, organic residue analysis and 3D image acquisition, metrology and visualisation, represents the first attempt to understand the manufacturing processes used to create these artefacts. The results produced were unexpected-rather than being carefully crafted objects, elements of their production can only be described as expedient.

We experimentally demonstrate topologically protected optical waveguiding in silicon at the interface between two topologically distinct dimer chains. Further, we propose and demonstrate beating between topological and trivial defect modes.

East African elephants have been hunted for their ivory for millennia but the nineteenth century witnessed strongly escalating demand from Europe and North America. It has been suggested that one consequence was that by the 1880s elephant herds along the coast had become scarce, and to meet demand, trade caravans trekked farther into interior regions of East Africa, extending the extraction frontier. The steady decimation of elephant populations coupled with the extension of trade networks have also been claimed to have triggered significant ecological and socio-economic changes that left lasting legacies across the region. To explore the feasibility of using an isotopic approach to uncover a ‘moving frontier’ of elephant extraction, we constructed a baseline isotope data set (δ13C, δ15N, δ18O and 87Sr/86Sr) for historic East African elephants known to have come from three distinct regions (coastal, Rift Valley, and inland Lakes). Using the isotope results with other climate data and geographical mapping tools, it was possible to characterise elephants from different habitats across the region. This baseline data set was then used to provenance elephant ivory of unknown geographical provenance that was exported from East Africa during the late nineteenth and early twentieth centuries to determine its likely origin. This produced a better understanding of historic elephant geography in the region, and the data have the potential to be used to provenance older archaeological ivories, and to inform contemporary elephant conservation strategies.

International audience; One contribution of 16 to a theme issue 'From DNA barcodes to biomes'. Over the last few centuries, many cetacean species have witnessed dramatic global declines due to industrial overharvesting and other anthropogenic influences, and thus are key targets for conservation. Whale bones recovered from archaeological and palaeontological contexts can provide essential baseline information on the past geographical distribution and abundance of species required for developing informed conservation policies. Here we review the challenges with identifying whale bones through traditional anatomical methods, as well as the opportunities provided by new molecular analyses. Through a case study focused on the North Sea, we demonstrate how the utility of this (pre)historic data is currently limited by a lack of accurate taxonomic information for the majority of ancient cetacean remains. We then discuss current opportunities presented by molecular identification methods such as DNA barcoding and collagen peptide mass fingerprinting (zooarchaeology by mass spectrometry), and highlight the importance of molecular identifications in assessing ancient species' distributions through a case study focused on the Mediterranean. We conclude by considering high-throughput molecular approaches such as hybridization capture followed by next-generation sequencing as cost-effective approaches for enhancing the ecological informativeness of these ancient sample sets. This article is part of the themed issue 'From DNA barcodes to biomes'.

We experimentally demonstrate topological waveguiding of correlated photon pairs generated by four-wave mixing in a bipartite one-dimensional lattice of silicon waveguides.

We employ planar silicon photonic crystal “photonic boron nitride” to observe Dirac physics in guided optical modes, by breaking the inversion symmetry of a honeycomb lattice of air holes to open an observable optical bandgap.

Ratione: Non-enzymatic deamidation accumulates in aging tissues in vivo and has been proposed to be potentially useful as a molecular clock. The process continues post mortem, and here we explore the increase in levels of deamidation in archaeological collagen, as measured during Zooarchaeology by Mass Spectrometry (ZooMS) analysis. Methods: With the high sensitivity of current generation mass spectrometers, ZooMS provides a non-destructive and highly cost-effective method to characterise collagen peptides. Deamidation can be detected by mass spectrometry as a +0.984 Da mass shift; therefore, aside from its original purpose, peptide mass-fingerprinting for bone identification, ZooMS concurrently yields a 'thermal indicator' of the samples. RESULTS: By analysis of conventional ZooMS spectra, we determined the deamidation rate for glutamine residues in 911 bone collagen samples from 50 sites, with ages varying from medieval to Palaeolithic. The degree of deamidation was compared to diagenetic parameters and nearby sequence properties. CONCLUSIONS: The extent of deamidation was found to be influenced more by burial conditions and thermal age than, for example, chronological age, the extent of bioerosion or crystallinity. The method lends itself mostly to screening heterogenic deposits of bone to identify outliers. Copyright © 2012 John Wiley & Sons, Ltd.

The last few years have seen an enormous proliferation of ancient biomolecules research, especially in the field of ancient DNA. Ancient DNA studies have been transformed by the advent of next generation sequencing, with the first Pleistocene sample being analysed in 2005, and several complete and draft genomes that have been compiled from ancient DNA to date. At the same time, although less conspicuous, research on ancient proteins has also seen advances, with the time limit for research on ancient biomolecules now extending to over 1 million years. Here we review which effects these developments have on research in Quaternary science. We identify several lines of research that have the potential to profit substantially from these recent developments in ancient biomolecules research. First, the identification of taxa can be made using ancient biomolecules, and in the case of ancient DNA, specimens can even be assigned to specific populations within a species. Second, increasingly large DNA data sets from Pleistocene animals allow the elucidation of ever more precise pictures of the population dynamic processes whereby organisms respond to climate and environmental change. With the accompanying better understanding of process in the Quaternary, past ecologies can also more realistically be interpreted from proxy data sets. The dominant message from this research so far is that the Quaternary saw a great deal more dynamism in populations than had been forecast by conventional palaeoecology. This suggests that reconstructions of past environmental conditions need to be done with caution. Third, ancient DNA can also now be obtained directly from sediments to elucidate the presence of both plant and animal species in an area even in the absence of identifiable fossils, be it macro- or micro-fossils. Finally, the analysis of proteins enables the identification of bone remains to genus and sometimes species level far beyond the survival time of DNA, at least in temperate regions, illustrating that precise data is now forthcoming from seemingly unlikely sources. Together, these approaches allow the study of environmental dynamics throughout a substantial part, and perhaps even the entire Quaternary (the last 2.6 million years).

In this work, two sets of experiments were carried out to assess the potential of soil proteomics for archaeological site interpretation. First, we examined the effects of various protein isolation reagents and soil constituents on peptide mass fingerprinting (PMF) of soil-like materials spiked with bovine serum albumin (BSA). In a subsequent case study, we assessed the relative age of soils from an ancient clay floor of a Roman farmhouse using amino acid racemization and then applied MALDI-TOF-MS-MS to detect and identify biomarkers for human occupation. The results from the first experiments indicate that BSA isolation efficiencies are hampered by the presence of clays and reactive organic matter (glucose), whereas these effects are, to some extent, reversed by the presence of humic acids. We also show that specific regions of the BSA molecule (the out most α-helical regions of domain 2 and 3) are more susceptible to isolation than other regions and this suggest that soil proteins can be only partly isolated. Soil-protein interactions were also found to inhibit tryptic cleavage of BSA, resulting in an enhanced specificity of BSA peptides. Our results further stress the importance of multiple or sequential protein isolation protocols with a focus on citrate and hydrofluoric acid. Results from the case study indicate that the ancient floor samples contain degraded and most likely aged proteinaceous matter, probably of keratinaceous origin. © 2012 Elsevier Ltd.

The authors acknowledge Science Foundation Ireland European Research Council (ERC) Support Award 12/ERC/B2227, Valeria Mattiangeli, and the Trinity Genome Sequencing Laboratory (TrinSeq) for MiSeq support. This work was supported by the Marie Curie International Fellowship PALIMPSEST FP7-PEOPLE-2011-IEF 299101, a University of Manchester Research Institute seedcorn grant, British Academy Postdoctoral Fellowship funding, and ERC Investigator Grant 295729-CodeX. Tissue-thin parchment made it possible to produce the first pocket Bibles: Thousands were made in the 13th century. The source of this parchment, often called “uterine vellum,” has been a long-standing controversy in codicology. Use of the Latin term abortivum in many sources has led some scholars to suggest that the skin of fetal calves or sheep was used. Others have argued that it would not be possible to sustain herds if so many pocket Bibles were produced from fetal skins, arguing instead for unexpected alternatives, such as rabbit. Here, we report a simple and objective technique using standard conservation treatments to identify the animal origin of parchment. The noninvasive method is a variant on zooarchaeology by mass spectrometry (ZooMS) peptide mass fingerprinting but extracts protein from the parchment surface by using an electrostatic charge generated by gentle rubbing of a PVC eraser on the membrane surface. Using this method, we analyzed 72 pocket Bibles originating in France, England, and Italy and 293 additional parchment samples that bracket this period. We found no evidence for the use of unexpected animals; however, we did identify the use of more than one mammal species in a single manuscript, consistent with the local availability of hides. These results suggest that ultrafine vellum does not necessarily derive from the use of abortive or newborn animals with ultrathin hides, but could equally well reflect a production process that allowed the skins of maturing animals of several species to be rendered into vellum of equal quality and fineness. Postprint

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.

Identifying animals to species from relict proteins is a powerful new archaeological tool. Here the authors apply the method to answer questions relating to the Salish of west coast North America. Did they weave their blankets out of dog hair? The proteomic analysis shows that they did, interweaving it with goat, and that the woolly dog was increasingly superseded by sheep in the later nineteenth century.

It is generally a challenge to interpret incomplete and degraded skeletal assemblages found during archaeological excavations. Several events after the death of animals and humans, before and during burial, will have influenced the situation as seen upon excavation. This postmortem sequence of events can be called the taphonomic history of bones. Taphonomic signatures as detected using histology can provide additional data on deposition/burial and the evolution of the burial environment. This article reports on the results of a histological characterisation of diagenetic alterations in a bone assemblage from the Roman period burial and settlement site of Castricum, located in the province of Noord-Holland in the Netherlands. The aim is to assess the relationship between bone histology and known taphonomic events. Both transmitted light microscopy and scanning electron microscopy were used to investigate histological changes. In 1995, the excavation at Castricum uncovered the skeletal material of humans and a variety of animal species that had received different burial treatments. The humans, as well as some dogs and cattle, were buried as complete inhumations. The skeletal remains of horses provided evidence of surface exposure before deposition. In addition, both environmental changes (e.g. variations in groundwater levels) and disturbances by later human activity are known to have occurred since the internment of the bones. Together with the animal refuse bones from the site, this assemblage provides an excellent opportunity to investigate the relationship between early taphonomy, burial conditions and histological appearance. The complex taphonomic history of the Castricum skeletal material was found to be reflected in several characteristic alterations of the bone microstructure such as the extent of bioerosion, cracking, staining and inclusions of framboidal pyrite crystals. This allowed for the reconstruction of a postmortem sequence of events. Copyright © 2011 John Wiley & Sons, Ltd.

Fish are a large, highly diverse, and anthropologically important group of vertebrates. However, fish bones are underrepresented in the archaeological literature because they are less stable than those of other taxa and identification of bone to species is often difficult or impossible. We explore a new identification system, ZooMS (Zooarchaeology by Mass Spectrometry), which is based upon protein barcoding. As proteins can be cleaved enzymatically and analyzed by mass spectrometry in a repeatable way, protein barcoding is used widely in microbiological contexts for quick and inexpensive protein identification; mass spectra reflect the differences in protein sequence and can therefore be reproducibly linked to a particular protein or protein fragment. ZooMS uses peptide fingerprinting of bone collagen as a method for rapid identification of archaeological bone. This has involved the identification of masses related to peptides of known sequence. For mammals, sufficient sequence information is available for this approach but for groups, such the teleost fish, species are highly diverse and there are few available collagen sequences. Here we report a preliminary investigation into the identification of fish species by peptide mass fingerprinting that does not require sequence information. Collagen mass spectra are used to identify eight species of bony fishes (Osteichthyes) from four orders: Clupeiformes, Salmoniformes, Gadiformes, and Perciformes. The method is applied to both modern and archaeological fish remains and offers the capacity to identify traditionally unidentifiable fish fragments, thus increasing the Number of Identified Specimens (NISP) and providing invaluable information in specialized contexts.

Bone collagen is found throughout most of the archaeological record. Under experimental conditions, collagen is apparently preserved as an intact molecule, with amino acid compositions and isotopic profiles only changing when almost all of the protein is lost. The ubiquity of collagen in archaeological bone has lead to the development of the use of collagen peptide mass fingerprints for the identification of bone fragments—Zooarchaeology by Mass Spectrometry (ZooMS). We report a novel, but a simple method for the partial extraction of collagen for ZooMS that uses ammonium bicarbonate buffer but avoids demineralisation. We compared conventional acid demineralisation with ammonium bicarbonate buffer extraction to test ZooMS in a range of modern and archaeological bone samples. The sensitivity of the current generation of mass spectrometers is high enough for the non-destructive buffer method to extract sufficient collagen for ZooMS. We envisage that a particular advantage of this method is that it leaves worked bone artefacts effectively undamaged post-treatment, suitable for subsequent analysis or museum storage or display. Furthermore, it may have potential as a screening tool to aid curators in the selection of material for more advanced molecular analysis—such as DNA sequencing.

Near-complete collagen (I) sequences are proposed for elephantid and mammutid taxa, based upon available African elephant genomic data and supported with LC–MALDI-MS/MS and LC–ESI-MS/MS analyses of collagen digests from proboscidean bone. Collagen sequence coverage was investigated from several specimens of two extinct mammoths (Mammuthus trogontherii and Mammuthus primigenius), the extinct American mastodon (Mammut americanum), the extinct straight-tusked elephant (Elephas (Palaeoloxodon) antiquus) and extant Asian (Elephas maximus) and African (Loxodonta africana) elephants and compared between the two ionization techniques used. Two suspected mammoth fossils from the British Middle Pleistocene (Cromerian) deposits of the West Runton Forest Bed were analysed to investigate the potential use of peptide mass spectrometry for fossil identification. Despite the age of the fossils, sufficient peptides were obtained to identify these as elephantid, and sufficient sequence variation to discriminate elephantid and mammutid collagen (I). In-depth LC–MS analyses further failed to identify a peptide that could be used to reliably distinguish between the three genera of elephantids (Elephas, Loxodonta and Mammuthus), an observation consistent with predicted amino acid substitution rates between these species.

As part of an ongoing project at the World Heritage Site of Bukhara, we investigated the glue used in the construction of the Khoja Zaynuddin mosque. Analysis by a range of techniques confirmed that it consisted of a collagen-based glue. However, the glue contained many non-protein constituents. The presence of lipid material suggests that the glue was produced by a relatively unsophisticated process. More surprisingly, various marker compounds of urine were found. Study of the viscosity of a mixture of a modern collagen-based glue and urea showed that the presence of urea at a concentration typical of human urine reduced the viscosity of the glue, thus improving its spreading characteristics and enabling its use at a lower temperature. Whilst the advantages of adding urea to glue were known to craftsmen in Europe and America in the 19th century, it would appear that the builders of Bukhara were aware of the technique, using less pure components, at a significantly earlier date.

Archaeological work in advance of construction at a site on the edge of York, UK, yielded human remains of prehistoric to Romano-British date. Amongst these was a mandible and cranium, the intra-cranial space of which contained shrunken but macroscopically recognizable remains of a brain. Although the distinctive surface morphology of the organ is preserved, little recognizable brain histology survives. Though rare, the survival of brain tissue in otherwise skeletalised human remains from wet burial environments is not unique. A survey of the literature shows that similar brain masses have been previously reported in diverse circumstances. We argue for a greater awareness of these brain masses and for more attention to be paid to their detection and identification in order to improve the reporting rate and to allow a more comprehensive study of this rare archaeological survival.

Over the past 30 years, ancient DNA studies have grown from a scientific curiosity into a powerful research tool with applications in molecular evolution, archaeology, paleontology, conservation genetics, and forensics. The analysis of ancient proteins has lagged behind that of ancient DNA, but recent developments in high-throughput, high-resolution mass spectrometry (MS) have the potential to provide the accuracy and robustness required for confident and reliable sequencing of ancient proteins. This methodology may allow biomolecular recovery to be extended further back in time, because DNA chains fall apart 10 times faster ( 1 ) than proteins ( 2 ). Furthermore, mass spectrometry enables investigation of protein expression specific for different tissues, developmental phases, or biological processes, including disease states.

During the late Pleistocene, a large number of species colonized North America from Eurasia through the Bering Land Bridge. At least four different genera within the tribe Ovibovini are known from this time, the holarctically distributed Ovibo s moschatus (the extant musk ox) and Praeovibos , and the nearctically distributed Bootherium and Euceratherium . Questions about the taxonomic relationships of the four North American musk oxen arose soon after their description and continue to be unresolved at the present. In this study we present the first DNA and protein analyses, of the four mentioned genera. Phylogenetic analysis at the mitochondrial level indicates that Praeovibos falls within the diversity of modern Ovibos , which might indicate that Praeovibos is simply an earlier morphotype of the extant species O. moschatus . In contrast Euceratherium and Bootherium are clearly distinct genera, in agreement with the findings of previous morphological studies.

Image analysis techniques have been used to investigate the likelihood of being able to classify and assign a probability regarding the plant origin of individual starch granules in a collection of granules. Quantifiable variables were used to characterize the granules, and the assignments and probabilities were calculated objectively. We consider the classification of images containing granules of a single species and of mixed species and the possibility of assigning a class to granules of unknown species in an image of a slide obtained from the dental calculus of chimpanzees.

Aminostratigraphic studies of continental deposits in the UK have hitherto relied almost exclusively on data from the aragonitic shells of non-marine molluscs for dating Pleistocene sequences. This is usually based on the d/l value of a single amino acid, d-alloisoleucine/l-isoleucine (A/I), in the total shell proteins. Two genera of freshwater gastropods (Valvata and Bithynia) are used to explore the value of using multiple amino acids from the intra-crystalline fraction, which should be more protected from the effects of diagenesis than the inter-crystalline component. Results are compared from both the aragonitic shells and opercula composed of calcite, a more stable form of calcium carbonate. In order to put the amino acid data from the West Runton Freshwater Bed into perspective, statistical analyses are used to compare them with results from the Hoxnian (MIS 11) site at Clacton-on-Sea, Essex. Twelve protein decomposition indicators revealed that the results from the shells were not as clear-cut as those from the opercula. Five indicators from the Valvata shell suggest that West Runton is older than Clacton (at a 95% significance level), but two actually suggested a younger age. Seven indicators show that the Bithynia shells from West Runton are older than congeneric shells from Clacton. In marked contrast, all 12 indicators isolated from the opercula demonstrate that West Runton is significantly older than Clacton. The data are also compared with results from Waverley Wood, an important archaeological site in the English Midlands falling within the ‘Cromerian Complex’. Contrary to earlier interpretations, the new amino acid data from Bithynia opercula indicate that West Runton is older than Waverley Wood, a relationship now consistent with the available biostratigraphy.

Is it possible to identify cooked, rather than burnt, bone? Mild heating (≤100 °C,1 h) – typical of cooking – does not lead to detectable changes in any biochemical parameter of bone yet measured. If it is only possible to detect charred bone, how is it possible to detect cooking in the archaeological record? In a previous paper (Koon et al., 2003, J. Arch. Sci.), we used a Transmission Electron Microscopy (TEM) based approach to investigate changes in the organization of the bone protein, collagen, as it is heated, using bone from heating experiments and short term burials. The work revealed that mineralized collagen, despite requiring aggressive treatment to gelatinise the protein (e.g. 90 °C, 240+ h), readily accumulates minor damage. We believe that the presence of mineral matrix stabilises the collagen enabling the damage to accumulate, but preventing it from causing immediate gelatinisation. Once the mineral is removed, the damage can be observed using appropriate visualization methods. In this paper the visualization technique was tested in a blind study of bovine bone from the Anglo-Scandinavian site of Coppergate, York. The purpose of the study was to determine if the method could discriminate between bones thought likely, on the basis of zoo-archaeological and spatial evidence, to have been cooked (high meat yield bones from a domestic context) and those which were butchered but unlikely to have been cooked (low yield bones from a butchery site). The results of the TEM analysis identified two clear groups of bones, one set more damaged than the other. This finding was consistent with archaeozoological interpretation, with the exception of one bone from the domestic context, which was not identified as having been cooked.

We report here the first integrated investigation of both ancient DNA and proteins in archaeobotanical samples: medieval grape (Vitis vinifera L.) seeds, preserved by anoxic waterlogging, from an early medieval (seventh–eighth century A.D.) Byzantine rural settlement in the Salento area (Lecce, Italy) and a late (fourteenth–fifteenth century A.D.) medieval site in York (England). Pyrolysis gas chromatography mass spectrometry documented good carbohydrate preservation, whilst amino acid analysis revealed approximately 90% loss of the original protein content. In the York sample, mass spectrometry-based sequencing identified several degraded ancient peptides. Nuclear microsatellite locus (VVS2, VVMD5, VVMD7, ZAG62 and ZAG79) analysis permitted a tentative comparison of the genetic profiles of both the ancient samples with the modern varieties. The ability to recover microsatellite DNA has potential to improve biomolecular analysis on ancient grape seeds from archaeological contexts. Although the investigation of five microsatellite loci cannot assign the ancient samples to any geographic region or modern cultivar, the results allow speculation that the material from York was not grown locally, whilst the remains from Supersano could represent a trace of contacts with the eastern Mediterranean. Electronic supplementary material The online version of this article (doi:10.1007/s00114-009-0629-3) contains supplementary material, which is available to authorized users.

We describe a method of isolating and analyzing a single collagen peptide able to distinguish between sheep and goat bone collagen. The 33 amino acid peptide from both sheep and goat collagen was sequenced and shown to differ between the two species at two positions. Analysis of a range of caprines indicated that the sequence changes occurred between the divergence of the Himalayan tahr (Hemitragus jemlahicus) and the ibex (Capra ibex) and that the proposed goat marker is diagnostic of all Capra species and breeds. The survival of these markers in archaeological bones was tested using a set of 26 ovicaprid specimens from Domuztepe, a Neolithic site in south central Turkey. These markers were used to test the osteological determination of 24 of the Domuztepe bones, and determine the species for two immature specimens. The collagen-peptide method has advantages over other non-morphological methods of sheep/goat distinction because of the long-term survival of collagen over other biomolecules such as ancient DNA. The results also highlighted the problems in relying upon one morphological criterion, in this case on the distal radius, to distinguish between sheep and goat bones.

Species identification of fragmentary bone, such as in rendered meat and bone meal or from archaeological sites, is often difficult in the absence of clear morphological markers. Here we present a robust method of analysing genus-specific collagen peptides by mass spectrometry simply by using solid-phase extraction (a C18 ZipTip) for peptide purification, rather than liquid chromatography/mass spectrometry (LC/MS). Analysis of the collagen from 32 different mammal species identified a total of 92 peptide markers that could be used for species identification, for example, in processed food and animal feed. A set of ancient (>100 ka@10 degrees C) bone samples was also analysed to show that the proposed method has applications to archaeological bone identification.

The detection and identification of protein residues from archaeological artefacts has been the subject of considerable controversy over the past two decades, this controversy spurring the application of a range of different analytical methods. Part of the debate surrounds the ability of the methods to uniquely identify original proteins. Another is the apparent contradiction between the ease of extraction in the laboratory of residues that have managed to survive in sediment over millennia; some studies use simple solvents including water, whilst others claim that efficient extraction requires the destruction of the artefact. Desorption electrospray ionisation mass spectrometry (DESI-MS) offers the potential to directly analyse proteins bound to the surface of artefacts. The method permits desorption of analytes under ambient conditions directly from a wide range of surfaces with little or no sample preparation. This has already led to its application in a range of areas, including forensic analysis of trace levels of narcotic drugs. It does not require destruction of the artefact (e.g. by drilling or dissolution) and can work in concert with other methods (for instance it could be used as a screening tool prior to lipid extraction). Here we report the construction of a DESI-MS interface and consider the potential of the method to contribute to the debate surrounding the preservation of proteins on artefacts. The DESI-MS interface was fitted to a conventional ion trap mass spectrometer and tested using peptides and proteins artificially applied to the surface of flint flakes and potsherd samples. A range of intact proteins was analysed; the DESI interface identified test proteins up to a maximum molecular weight of approximately 20 kDa. Peptides generated from these and larger proteins could be detected using DESI-MS following tryptic digestion in situ. The collected data allowed identification of the originating proteins. The potential applications to archaeological science of this new analytical tool are discussed.

DNA appears to decay by random chain scission resulting in a predictable range of fragment lengths. Collagen decay has also been modelled in this same way, although it has become increasingly evident that collagen decay does not follow this same pattern. Radiocarbon and stable isotope analysis now use ultra-filtration to isolate large fragments (>30% of original polymer length) even in Pleistocene bone. How then does collagen decay? This study contrasts experimentally degraded samples with collagen extracted from forensic, archaeological and fossil bone. In experimentally degraded bone, values for amino acid and elemental (C:N) composition, bulk δ 13C, δ 15N, and aspartic acid racemisation (AAR) changed very little until 99% of the collagen was lost, suggesting that the collagen triple helix and polypeptide chains remained remarkably intact. This suggestion was demonstrated directly by examining the integrity of individual polypeptide chains using cyanogen bromide (CNBr) cleavage followed by SDS-PAGE electrophoresis. In ancient samples, AAR values remain remarkably stable and the pattern of CNBr-cleavage was only replaced with a smear of smaller polypeptides in the oldest (Pleistocene) bones investigated. Smearing may reflect both modification of the methionine resides (the sites of CNBr-cleavage) and/or partial hydrolysis of the collagen molecule. The findings reveal why it is not usually necessary to worry about collagen diagenesis; it is mostly intact. However, evidence of partial deterioration of the oldest bone samples suggests that alternative purification strategies may increase yields in some samples.

Recent work in various parts of the world has suggested the possibility of ancient starch granules surviving and adhering to archaeological artefacts. Often this information is used to infer aspects of diet. One additional source for recovery of archaeological starch granules is dental calculus. The presence of plant food debris in dental calculus is well known but has not been not widely investigated using archaeological material. The extraction of starch granules from dental calculus represents a direct link to the consumption of starchy food by humans or animals. Using dental calculus also sidesteps many other questions still inherent in using starch granules to reconstruct aspects of ancient diet, such as the effects of diagenesis on their morphology; as the starches are trapped inside a concreted matrix they are less likely to alter over time. We used amylase digestion by a starch-specific enzyme to confirm the material as starch.

We present a novel method for the isolation and analysis of the bone collagen (I) α2 chain carboxytelopeptide as a species biomarker. Conventional methods for the analysis and sequencing of mixtures of proteins and peptides commonly involve using the protease trypsin to cleave proteins present in the sample. However, in the study of collagen, these methods result in very complex mixtures of peptides that are difficult to analyze and the acquired results are not reproducible. Here we use bacterial collagenase (from Clostridium histolyticum) for its ability to cleave the highly unusual Gly-Xaa-Yaa repeating sequence of collagen, where Xaa usually is Pro and Yaa often is Hyp. Followed by a simple isolation step using a reverse phase solid phase extraction cartridge, the α2 (I) chain carboxytelopeptide can be readily analyzed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and the results can be used to distinguish between different species of origin. © 2007 Elsevier Inc. All rights reserved.

Although the recent history of human colonisation and impact on Mauritius is well documented, virtually no records of the pre-human native ecosystem exist, making it difficult to assess the magnitude of the changes brought about by human settlement. Here, we describe a 4000-year-old fossil bed at Mare aux Songes (MAS) in south-eastern Mauritius that contains both macrofossils (vertebrate fauna, gastropods, insects and flora) and microfossils (diatoms, pollen, spores and phytoliths). With >250 bone fragments/m(2) and comprising 50% of all known extinct and extant vertebrate species (n(s)=44) of Mauritius, MAS may constitute the first Holocene vertebrate bone Concentration-Lagerstatte identified on an oceanic volcanic island. Fossil remains are dominated by extinct giant tortoises Cylindraspis spp. (63%), passerines (similar to 10%), small bats (7.8%) and dodo Raphus cucullatus (7.1%). Twelve radiocarbon ages [four of them duplicates] from bones and other material suggest that accumulation of fossils took place within several centuries. An exceptional combination of abiotic conditions led to preservation of bones, bone collagen, plant tissue and microfossils. Although bone collagen is well preserved, DNA from dodo and other Mauritian vertebrates has proved difficult. Our analysis suggests that from ca 4000 years ago (4 ka), rising sea levels created a freshwater lake at MAS, generating an oasis in an otherwise dry environment which attracted a diverse vertebrate fauna. Subsequent aridification in the south-west Indian Ocean region may have increased carcass accumulation during droughts, contributing to the exceptionally high fossil concentration. The abundance of floral and faunal remains in this Lagerstatte offers a unique opportunity to reconstruct a pre-human ecosystem on an oceanic island, providing a key foundation for assessing the vulnerability of island ecosystems to human impact. (C) 2008 Elsevier Ltd. All rights reserved.

Evolutionary biologists are increasingly relying on ancient DNA from archaeological animal bones to study processes such as domestication and population dispersals. As many animal bones found on archaeological sites are likely to have been cooked, the potential for DNA preservation must be carefully considered to maximise the chance of amplification success. Here, we assess the preservation of mitochondrial DNA in a medieval cattle bone assemblage from Coppergate, York, UK. These bones have variable degrees of thermal alterations to bone collagen fibrils, indicative of cooking. Our results show that DNA preservation is not reliant on the presence of intact collagen fibrils. In fact, a greater number of template molecules could be extracted from bones with damaged collagen. We conclude that moderate heating of bone may enhance the retention of DNA fragments. Our results also indicate that ancient DNA preservation is highly variable, even within a relatively recent assemblage from contexts conducive to organic preservation, and that diagenetic parameters based on protein diagenesis are not always useful for predicting ancient DNA survival.

Aspartic acid racemization has been found to be an accurate measure of age at death for recent forensic material. This paper examines the practicality of using acid etching of the tooth surface to extract amino acids from the enamel for racemization analysis. By serial etching of the tooth and contamination of the teeth with bovine serum albumin prior to etching, the ability of etching to remove contamination was assessed. The destructiveness of the method was visualized and quantified using micro-computed tomography (micro-CT). By bleaching the teeth and by deeper etching it was possible to obtain more consistent values. While etching had little effect on the enamel at the macroscale, it did have an impact at the microscale. The quantities of enamel removed varied depending upon the tooth morphology, but were not large. Acid etching of enamel thus appears to be a promising new method for extracting proteins for amino acid racemization age estimation noninvasively. © 2008 American Academy of Forensic Sciences.

Although the application of sequencing-by-synthesis techniques to DNA extracted from bones has revolutionized the study of ancient DNA, it has been plagued by large fractions of contaminating environmental DNA. The genetic analyses of hair shafts could be a solution: We present 10 previously unexamined Siberian mammoth ( Mammuthus primigenius ) mitochondrial genomes, sequenced with up to 48-fold coverage. The observed levels of damage-derived sequencing errors were lower than those observed in previously published frozen bone samples, even though one of the specimens was >50,000 14 C years old and another had been stored for 200 years at room temperature. The method therefore sets the stage for molecular-genetic analysis of museum collections.

The integration of microfluidics and microphotonics brings the ability to tune and reconfigure ultra-compact optical devices. This flexibility is essentially provided by three characteristics of fluids that are scalable at the micron-scale: fluid mobility, large ranges of index modulation, and abrupt interfaces that can be easily reshaped. Several examples of optofluidic devices are presented here to illustrate the achievement of flexible devices on (semi) planar and compact platforms. First, we report an integrated geometry for a compact and tunable interferometer that exploits a sharp and mobile air/water interface. We then describe a class of optically controlled devices that rely on the actuation of optically trapped micron-sized objects within a fluid environment. The last architecture results from the infiltration of photonic crystal devices with fluids. This produces tunable and reconfigurable photonic devices, like optical switches. Higher degrees of functionality could be achieved with sophisticated optofluidic platforms that associate complex microfluidic delivery and mixing schemes with microphotonic devices. Moreover, optofluidics offers new opportunities for realizing highly responsive and compact sensors.

The thermal behaviour of the animal by-product meat and bone meal (MBM) has been investigated in order to assess how it is affected structurally and chemically by incineration. Initially composed of intergrown collagen and hydroxyapatite (HAP), combustion of the organic component is complete by 650 °C, with most mass loss (50–55%) occurring by 500 °C. No original proteins were detected in samples heated at 400 °C or above. Combustion of collagen is accompanied by an increase in HAP mean crystallite size at temperatures greater than 400 °C, from 10 nm to a constant value of 120 nm at 800 °C or more. Newly formed crystalline phases appear beyond 400 °C, and include β-tricalcium phosphate, NaCaPO4, halite (NaCl) and sylvite (KCl). Crystallite thickness as judged by small angle X-ray scattering (SAXS) increases from 2 nm (25–400 °C) to 8–9 nm very rapidly at 550 °C, and then gradually increases to approximately 10 nm. The original texture of HAP within a collagen matrix is progressively lost, producing a porous HAP dominated solid at 700 °C, and a very low porosity sintered HAP product at 900 °C.

We have applied cluster analysis to mercury intrusion porosimetry data from 219 archaeological bones (121 human and 98 animal) and soil chemistry data from 219 accompanying soil samples (1 per bone sample), to investigate the influence of soil chemistry on bone preservation. The samples chosen for the study were obtained from sites ranging in time from the pre-modern to the Mesolithic and were representative of burial environments across Europe (from the Baltic to the Mediterranean). These results represent the single largest database for archaeological bone preservation in the European Holocene to date and demonstrate the potential for large-scale diagenetic studies to help develop long term preservation strategies for our European heritage. Despite the variety of sites and environments, bones could be categorised into only four main diagenetic types. Furthermore, soil chemistry appears to significantly affect only one type of preservation, the pathway characterised by loss of mineral. In neutral to basic soils, taphonomy and in particular the differences between the treatment of human and animal remains, becomes the dominating factor in determining preservation. Using these results, strategies for heritage management of archaeological sites can be suggested; grouping sites into those requiring immediate excavation and those where in situ preservation is viable. © 2006 Elsevier Ltd. All rights reserved.

We investigate the generation of random numbers via the quantum process of spontaneous Raman scattering. Spontaneous Raman photons are produced by illuminating a highly nonlinear chalcogenide glass ( As2S3) fiber with a CW laser at a power well below the stimulated Raman threshold. Single Raman photons are collected and separated into two discrete wavelength detuning bins of equal scattering probability. The sequence of photon detection clicks is converted into a random bit stream. Postprocessing is applied to remove detector bias, resulting in a final bit rate of ∼650 kb/s. The collected random bit-sequences pass the NIST statistical test suite for one hundred 1 Mb samples, with the significance level set to α=0.01. The fiber is stable, robust and the high nonlinearity (compared to silica) allows for a short fiber length and low pump power favourable for real world application.

The growing requirement for photon pairs with specific spectral correlations in quantum optics experiments has created a demand for fast, high resolution and accurate source characterization. A promising tool for such characterization uses the classical stimulated process, in which an additional seed laser stimulates photon generation yielding much higher count rates, as recently demonstrated for a $\chi^{(2)}$ integrated source in A.~Eckstein \emph{et al.}, Laser Photon. Rev. \textbf{8}, L76 (2014). In this work we extend these results to $\chi^{(3)}$ sources, demonstrating spectral correlation measurements via stimulated four-wave mixing for the first time in a integrated optical waveguide, namely a silicon nanowire. We directly confirm the speed-up due to higher count rates and demonstrate that additional resolution can be gained when compared to traditional coincidence measurements. As pump pulse duration can influence the degree of spectral entanglement, all of our measurements are taken for two different pump pulse widths. This allows us to confirm that the classical stimulated process correctly captures the degree of spectral entanglement regardless of pump pulse duration, and cements its place as an essential characterization method for the development of future quantum integrated devices., Comment: 10 pages, 4 figures

Xiang et al. (1) assert that chickens were domesticated on the North China plain 10,000 y ago. Although a great deal remains unknown about the temporal and geographic origins of poultry husbandry, this claim is extraordinary. We welcome the increasing application of modern bioarcheological techniques to questions pertaining to animal domestication in China, but we are skeptical about these conclusions for several reasons.

Parchment represents an invaluable cultural reservoir. Retrieving an additional layer of information from these abundant, dated livestock-skins via the use of ancient DNA (aDNA) sequencing has been mooted by a number of researchers. However, prior PCR-based work has indicated that this may be challenged by cross-individual and cross-species contamination, perhaps from the bulk parchment preparation process. Here we apply next generation sequencing to two parchments of seventeenth and eighteenth century northern English provenance. Following alignment to the published sheep, goat, cow and human genomes, it is clear that the only genome displaying substantial unique homology is sheep and this species identification is confirmed by collagen peptide mass spectrometry. Only 4% of sequence reads align preferentially to a different species indicating low contamination across species. Moreover, mitochondrial DNA sequences suggest an upper bound of contamination at 5%. Over 45% of reads aligned to the sheep genome, and even this limited sequencing exercise yield 9 and 7% of each sampled sheep genome post filtering, allowing the mapping of genetic affinity to modern British sheep breeds. We conclude that parchment represents an excellent substrate for genomic analyses of historical livestock.

Very recently, we discovered a vast new microbial self: the human microbiome. Our native microbiota interface with our biology and culture to influence our health, behavior, and quality of life, and yet we know very little about their origin, evolution, or ecology. With the advent of industrialization, globalization, and modern sanitation, it is intuitive that we have changed our relationship with microbes, but we have little information about the ancestral state of our microbiome, and therefore, we lack a foundation for characterizing this change. High-throughput sequencing has opened up new opportunities in the field of paleomicrobiology, allowing us to investigate the evolution of the complex microbial ecologies that inhabit our bodies. By focusing on recent coprolite and dental calculus research, we explore how emerging research on ancient human microbiomes is changing the way we think about ancient disease and how archaeological studies can contribute to a medical understanding of health and nutrition today.

We use classical stimulated four-wave mixing to directly characterize quantum spectral correlations generated in a silicon nanowire for two different pump durations. Signal to noise is increased and acquisition time reduced compared to coincidence detection.

By harnessing quantum mechanical effects we can create technology with greatly improved functionality including enhanced sensing, exponentially faster computing, the simulation of previously inaccessible quantum systems, and the secure transfer of information. In our ever more connected world, it is important that we can communicate securely, and as technology develops into the quantum regime it will become important for distant quantum processors to exchange information over a quantum network. Here we discuss how quantum information can be encoded and transferred around a such a network. We concentrate on the use of nonlinear optics to generate and manipulate photons, and present schemes for fully secure quantum communication.

Background: As high utilizers of health care resources, frequent attenders to medical practices pose a significant issue for primary health care. Studies documenting content of visits and characteristics of frequent attenders have paid limited attention to community health center populations. This study profiles these high utilizers comparing them to non-frequent attenders. Methods: Through medical record abstraction, retrospective and longitudinal patient data were obtained for a 30-month time period for 382 established patients. Results: Older patients 45 to 64 years (OR = 1.85; 95% CI: 0.92, 3.70) and Medicaid recipients (OR = 3.22; 95% CI: 1.54, 6.72) were more likely to be frequent attenders. Patients residing furthest away from the CHC were also the most frequent of attenders (OR = 3.68; 95% CI: 1.22, 11.13), as were those with a diagnosis of diabetes (OR = 5.03; 95% CI: 2.17, 11.70) or depression (OR = 1.85; 95% CI: 0.96, 3.56). Patients who used the Emergency Department more (OR = 1.54; 95% CI: 1.23,1.91) had the highest number of visits, although those patients who missed more appointments as a proportion of the number of scheduled appointments were less likely to be frequent attenders (OR = 0.49; 95% CI: 0.36, 0.66). Conclusions: Through developing interventions such as customized social report cards and applying elements of the Chronic Care Model, these results can help administrators and policy makers ensure that frequent attenders are cared for adequately and that safety net providers’ resources are appropriate to the tasks demanded of them.

In order to assess the distribution of African Palaeolithic sites and hominin remains, we need an understanding of the factors that may affect the preservation of archaeological material in the fossil record. One of the main factors that is thought to affect seriously the global distribution of fossil remains is taphonomy, but it would seem that taphonomic processes may themselves be overprinted by collagen degradation. The rate of collagen loss is highly temperature dependent, and it has been shown that bone is more likely to be preserved in areas of lower temperature and so lower collagen loss. Hence, it would be expected that fossil fauna remains would be distributed in areas of low collagen loss, i.e. low temperature. Conversely, lithics will not be subject to a temperature bias in their preservation. We have tested this hypothesis through the use of archaeological material. Our results show that the distributions of both lithic and faunal assemblages are non-random across the African continent, with all archaeological sites being found in areas of relatively low collagen degradation. This implies that bone collagen degradation is not the only factor affecting the distribution of African Palaeolithic sites and hominin remains. We suggest that the site distribution is not a reflection of bias in excavation history, but is a real phenomenon reflecting hominin habitat choice.

The field of palaeomicrobiology is dramatically expanding thanks to recent advances in high-throughput biomolecular sequencing, which allows unprecedented access to the evolutionary history and ecology of human-associated and environmental microbes. Recently, human dental calculus has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, preserving not only microbial and host biomolecules but also dietary and environmental debris. Modern investigations of native human microbiota have demonstrated that the human microbiome plays a central role in health and chronic disease, raising questions about changes in microbial ecology, diversity and function through time. This paper explores the current state of ancient oral microbiome research and discusses successful applications, methodological challenges and future possibilities in elucidating the intimate evolutionary relationship between humans and their microbes.