The genomic origins of the Bronze Age Tarim Basin mummies.

The identity of the earliest inhabitants of Xinjiang, in the heart of Inner Asia, and the languages that they spoke have long been debated and remain contentious1. Here we present genomic data from 5 individuals dating to around 3000-2800 BC from the Dzungarian Basin and 13 individuals dating to around 2100-1700 BC from the Tarim Basin, representing the earliest yet discovered human remains from North and South Xinjiang, respectively. We find that the Early Bronze Age Dzungarian individuals exhibit a predominantly Afanasievo ancestry with an additional local contribution, and the Early-Middle Bronze Age Tarim individuals contain only a local ancestry. The Tarim individuals from the site of Xiaohe further exhibit strong evidence of milk proteins in their dental calculus, indicating a reliance on dairy pastoralism at the site since its founding. Our results do not support previous hypotheses for the origin of the Tarim mummies, who were argued to be Proto-Tocharian-speaking pastoralists descended from the Afanasievo1,2 or to have originated among the Bactria-Margiana Archaeological Complex3 or Inner Asian Mountain Corridor cultures4. Instead, although Tocharian may have been plausibly introduced to the Dzungarian Basin by Afanasievo migrants during the Early Bronze Age, we find that the earliest Tarim Basin cultures appear to have arisen from a genetically isolated local population that adopted neighbouring pastoralist and agriculturalist practices, which allowed them to settle and thrive along the shifting riverine oases of the Taklamakan Desert.

Tracking the transition to agriculture in Southern Europe through ancient DNA analysis of dental calculus.

Archaeological dental calculus, or mineralized plaque, is a key tool to track the evolution of oral microbiota across time in response to processes that impacted our culture and biology, such as the rise of farming during the Neolithic. However, the extent to which the human oral flora changed from prehistory until present has remained elusive due to the scarcity of data on the microbiomes of prehistoric humans. Here, we present our reconstruction of oral microbiomes via shotgun metagenomics of dental calculus in 44 ancient foragers and farmers from two regions playing a pivotal role in the spread of farming across Europe-the Balkans and the Italian Peninsula. We show that the introduction of farming in Southern Europe did not alter significantly the oral microbiomes of local forager groups, and it was in particular associated with a higher abundance of the species Olsenella sp. oral taxon 807. The human oral environment in prehistory was dominated by a microbial species, Anaerolineaceae bacterium oral taxon 439, that diversified geographically. A Near Eastern lineage of this bacterial commensal dispersed with Neolithic farmers and replaced the variant present in the local foragers. Our findings also illustrate that major taxonomic shifts in human oral microbiome composition occurred after the Neolithic and that the functional profile of modern humans evolved in recent times to develop peculiar mechanisms of antibiotic resistance that were previously absent.

Environmental implications and evidence of natural products from dental calculi of a Neolithic-Chalcolithic community (central Italy).

In this contribution, we investigated the role of plants in the prehistoric community of Casale del Dolce (Anagni, FR, central Italy), through microparticles recovered from dental calculus. The finding of a great amount of pollen types, even in form of compact lumps, could indicate use of natural substances, such as honeybee products and/or conifer resins. This plant-microremain record also suggested environmental implications relative to the Neolithic and Chalcolithic period. Additionally, the stability of the tartar microenvironment had preserved starches and other microparticles, such as one epidermal trichome, a sporangium, and fragments of plant tissue, rarely detected in ancient dental calculus. The detection of secondary metabolites in the ancient matrix confirmed the familiarity of this community with plant resources. All these data supply various interesting food for thought and expand the knowledge about the potential of dental calculus in archaeological and archaeobotanical fields with a special focus on palaeoecology.

Exploring late Paleolithic and Mesolithic diet in the Eastern Alpine region of Italy through multiple proxies.

OBJECTIVES: The analysis of prehistoric human dietary habits is key for understanding the effects of paleoenvironmental changes on the evolution of cultural and social human behaviors. In this study, we compare results from zooarchaeological, stable isotope and dental calculus analyses as well as lower second molar macrowear patterns to gain a broader understanding of the diet of three individuals who lived between the end of the Late Pleistocene and the Early Holocene (ca., 17-8 ky cal BP) in the Eastern Alpine region of Italy. MATERIALS AND METHODS: We analyze individuals buried at the sites of Riparo Tagliente (Verona), Riparo Villabruna, and Mondeval de Sora (Belluno). The three burials provide a unique dataset for diachronically exploring the influence of climatic changes on human subsistence strategies. RESULTS: Isotopic results indicate that all individuals likely relied on both terrestrial and freshwater animal proteins. Even though dental calculus analysis was, in part, hindered by the amount of mineral deposit available on the teeth, tooth macrowear study suggests that the dietary habits of the individuals included plant foods. Moreover, differences in macrowear patterns of lower second molars have been documented between Neanderthals and modern humans in the present sample, due to a prevalence of Buccal wear among the former as opposed to higher values of Lingual wear in modern human teeth. DISCUSSION: Isotopic analyses have emphasized the contribution of animal proteins in the diet of the three foragers from the Eastern Alpine region. The possible intake of carbohydrate-rich plant foods, suggested by the retrieval of plant remains in dental calculus, is supported by the signal of macrowear analysis. Moreover, the latter method indicates that the distribution of macrowear in lower second molars (M2 s) allows us to discriminate between Neanderthals and modern humans within the present reference sample. Overall, our results show these three prehistoric hunter-gatherers were well adapted to the environment in which they lived exploiting many natural resources.

Exploitation and utilization of tropical rainforests indicated in dental calculus of ancient Oceanic Lapita culture colonists.

Remote Oceania, which largely consists of islands covered in tropical forests, was the last region on earth to be successfully colonized by humans, beginning 3,000 years ago. We examined human dental calculus from burials in an ancient Lapita culture cemetery to gain insight into the early settlement of this previously untouched tropical environment, specifically on the island of Efate in Vanuatu. Dental calculus is an ideal material to analyse questions of human and plant interactions due to the ingestion of plant-derived microparticles that become incorporated into the calculus as it forms throughout a person's life. Most of the microparticles identified here are from tree and shrub resources, including a ~2,900 calibrated (cal) BP example of banana in Remote Oceania, providing direct evidence for the importance of forests and arboriculture during the settlement of Remote Oceania.

Lifestyle of a Roman Imperial community: ethnobotanical evidence from dental calculus of the Ager Curensis inhabitants.

BACKGROUND: The analysis of ancient calcified dental plaque is a powerful archaeobotanical method to elucidate the key role of the plants in human history. METHODS: In this research, by applying both optic microscopy and gas chromatography mass spectrometry on this matrix, a detailed qualitative investigation for reconstructing the lifestyle of a Roman imperial community of the Ager Curensis (Sabina Tiberina, Central Italy) was performed. RESULTS: The detection of animal micro-remains and molecules (e.g., hairs, feather barbules, markers of dairy products), starch granules of several cereals and legumes, pollen (e.g., Juglans regia L., Hedera sp. L.) and other plant micro-debris (e.g., trichome of Olea sp., hemp fibers), and phytochemicals (e.g., Brassicaceae, Lamiaceae herbs, Ferula sp., Trigonella foenum-graecum L., wine, and Humulus lupulus L.) in the dental calculus sample demonstrated that plant-derived foods were regularly consumed together with animal resources. CONCLUSIONS: This nutritional plan, consistent with the information reported in ancient written texts, suggested that the studied population based its own subsistence on both agriculture and husbandry, probably also including beekeeping and hunting activities. All together, these results represent proofs for the comprehension of food habits, phytotherapeutic practices, and cultural traditions of one of the first Roman settlements in the Sabina Tiberina area.

Proteomic evidence of dietary sources in ancient dental calculus.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein ß-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Dental calculus reveals Mesolithic foragers in the Balkans consumed domesticated plant foods.

Researchers agree that domesticated plants were introduced into southeast Europe from southwest Asia as a part of a Neolithic "package," which included domesticated animals and artifacts typical of farming communities. It is commonly believed that this package reached inland areas of the Balkans by ∼6200 calibrated (cal.) BC or later. Our analysis of the starch record entrapped in dental calculus of Mesolithic human teeth at the site of Vlasac in the Danube Gorges of the central Balkans provides direct evidence that already by ∼6600 cal. BC, if not earlier, Late Mesolithic foragers of this region consumed domestic cereals, such as Triticum monococcum, Triticum dicoccum, and Hordeum distichon, which were also the main crops found among Early Neolithic communities of southeast Europe. We infer that "exotic" Neolithic domesticated plants were introduced to southern Europe independently almost half a millennium earlier than previously thought, through networks that enabled exchanges between inland Mesolithic foragers and early farming groups found along the Aegean coast of Turkey.

Mineral density volume gradients in normal and diseased human tissues.

Clinical computed tomography provides a single mineral density (MD) value for heterogeneous calcified tissues containing early and late stage pathologic formations. The novel aspect of this study is that, it extends current quantitative methods of mapping mineral density gradients to three dimensions, discretizes early and late mineralized stages, identifies elemental distribution in discretized volumes, and correlates measured MD with respective calcium (Ca) to phosphorus (P) and Ca to zinc (Zn) elemental ratios. To accomplish this, MD variations identified using polychromatic radiation from a high resolution micro-computed tomography (micro-CT) benchtop unit were correlated with elemental mapping obtained from a microprobe X-ray fluorescence (XRF) using synchrotron monochromatic radiation. Digital segmentation of tomograms from normal and diseased tissues (N=5 per group; 40-60 year old males) contained significant mineral density variations (enamel: 2820-3095 mg/cc, bone: 570-1415 mg/cc, cementum: 1240-1340 mg/cc, dentin: 1480-1590 mg/cc, cementum affected by periodontitis: 1100-1220 mg/cc, hypomineralized carious dentin: 345-1450 mg/cc, hypermineralized carious dentin: 1815-2740 mg/cc, and dental calculus: 1290-1770 mg/cc). A plausible linear correlation between segmented MD volumes and elemental ratios within these volumes was established, and Ca/P ratios for dentin (1.49), hypomineralized dentin (0.32-0.46), cementum (1.51), and bone (1.68) were observed. Furthermore, varying Ca/Zn ratios were distinguished in adapted compared to normal tissues, such as in bone (855-2765) and in cementum (595-990), highlighting Zn as an influential element in prompting observed adaptive properties. Hence, results provide insights on mineral density gradients with elemental concentrations and elemental footprints that in turn could aid in elucidating mechanistic processes for pathologic formations.

Neanderthal medics? Evidence for food, cooking, and medicinal plants entrapped in dental calculus.

Neanderthals disappeared sometime between 30,000 and 24,000 years ago. Until recently, Neanderthals were understood to have been predominantly meat-eaters; however, a growing body of evidence suggests their diet also included plants. We present the results of a study, in which sequential thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) were combined with morphological analysis of plant microfossils, to identify material entrapped in dental calculus from five Neanderthal individuals from the north Spanish site of El Sidrón. Our results provide the first molecular evidence for inhalation of wood-fire smoke and bitumen or oil shale and ingestion of a range of cooked plant foods. We also offer the first evidence for the use of medicinal plants by a Neanderthal individual. The varied use of plants that we have identified suggests that the Neanderthal occupants of El Sidrón had a sophisticated knowledge of their natural surroundings which included the ability to select and use certain plants.

Calcium, vitamin D supplements with or without alendronate and supragingival calculus formation in osteoporotic women: a preliminary study.

OBJECTIVES: Long-term calcium intake is related to the formation of urinary stones. Structure and composition of kidney and gallstones are similar to dental calculus. Saliva is the source of calcium for supragingival dental calculus formation. The aim of this preliminary study was to evaluate the possible effects of long-term calcium and vitamin D supplementation with or without alendronate administration on salivary electrolyte concentrations and supragingival calculus formation in osteoporotic women. METHODS: Thirty-one female patients with osteoporosis for at least 3 years participated in this study. Eighteen women were taking calcium plus vitamin D plus alendronate, while 13 women were taking only calcium plus vitamin D supplements. Eleven systemically healthy women volunteered for the control group. Whole saliva samples were collected from all women before initiation of any periodontal intervention. Plaque index, probing depth, clinical attachment level, bleeding on probing, and calculus index were recorded at six sites/tooth. Salivary concentrations of ionic calcium, potassium, magnesium and sodium were determined by atomic absorption spectrophotometer. Results were evaluated statistically by non-parametric tests. RESULTS: No significant differences were found in clinical parameters or results of saliva analysis between the study groups (p > 0.05). CONCLUSION: Within the limits of this preliminary study, it is suggested that long-term calcium and vitamin D supplementation with or without alendronate does not appear to have a significant effect on supragingival calculus formation or saliva total calcium, potassium, magnesium and sodium concentrations. Larger-scale studies investigating the possible effects of various treatment modalities of osteoporosis on supragingival calculus formation are required to better clarify this issue.

[Fluoride content in supragingival dental calculus measured with X-ray microanalysis]. / Badania zawartosci fluorków w próbkach naddziaslowego kamienia nazebnego metoda mikroanalizy rentgenowskiej.

PURPOSE: Sparse reports on the composition of mineralized deposits forming in the child's oral cavity generally reveal their lower degree of mineralization. As reports on the formation of dental calculus in children are incomplete, we decided to study some conditions in which this process takes place. The aim of the study was to determine whether fluorine is present in supragingival dental calculus. We enrolled 12-year-old children (17 girls, 11 boys) living in a large city. RESULTS: X-ray microanalysis of dental calculus revealed the presence of carbon, oxygen, calcium, phosphorus, sodium, magnesium, potassium, sulphur, chlorine, silicon, and fluorine. Chemical analysis of 10 sites on each specimen showed large variations as to the number of elements found and their content. Fluorine was present in one specimen only (3.6%) at a very high concentration of 10.27%+/-1.27 (w/w). Chemical composition of mineralized dental deposits shows marked individual variability and the presence of fluorine is not a constant finding.

Dental calculus composition following use of essential-oil/ZnCl2 mouthrinse.

PURPOSE: To test the hypothesis that anticalculus agents cannot completely inhibit calculus formation but can influence the types of calcium phosphate which form, i.e., they can influence the composition of the inorganic component of human dental calculus (HDC). MATERIALS AND METHODS: The composition of HDC specimens obtained from a 16-week multi-center clinical study using three regimens were analyzed, investigators blinded. The treatment regimens were: (a) standard dentifrice (SD), (b) pyrophosphate antitartar dentifrice, and (c) SD with Tartar Control Listerine Antiseptic mouthrinse (containing essential oils and 0.09% zinc chloride). 25 individual samples and eight pooled samples from each group were analyzed using X-ray diffraction, infrared spectroscopy, and scanning electron microscopy. RESULTS: (1) relative frequency of occurrence for: (a) bacteria: Group A = 100%, Group B = 60%, and Group C = 25%; (b) Carbonate hydroxyapatite (CHA): Groups A, B, and C = 100%; (c) dicalcium phosphate dihydrate (DCPD): Group A = 55%; Group B = 45%; Group C = 80%; (2) The relative amount of DCPD is inversely proportional to that of CHA in HDC: the higher the amount of DCPD, the lower the amount of CHA. Group C regimen with essential oil/ZnCl2 mouthrinse and standard dentifrice showed a significant anti-microbial effect and favored the formation of DCPD, the most soluble Ca-P.

Spectrochemical analysis of dental calculus by synchrotron radiation X-ray fluorescence.

This work presents elemental composition studies of human dental calculus by X-ray fluorescence analysis using synchrotron radiation. The intrinsic characteristics of synchrotron light allow for a quantitative analysis of major, minor, and trace elements of very small samples in a single measurement. At present, several theories of calculus formation exist, but none of them can describe completely all the complicated mechanisms involved in the mineralization processes. For this reason, and taking into account that experimental data are long overdue, several calculi with certain degrees of formation were collected from adult patients for analysis. The ratio of calcium/phosphorus was used as an indicator of the major crystalline structure and the state of formation of the calculus. The results demonstrate a clear correlation between the concentrations of certain elements and the type of calculus (supra- or sub-gingival). In addition, the possible correlation between the elements was analyzed.

Fluoride profiles in dental calculus from Japanese, Chinese and British residents.

Whether the fluoride concentrations and profiles differ in human dental calculus obtained from different countries was investigated. A total of 203 dental calculus deposits on 203 permanent teeth from residents (mean age, 52.1 years) of Nagoya (Japan), Shanghai (China), Leeds (Great Britain) and the Wuhan mountainous area (China, fluoridated area) were analysed. An abrasive microsampling procedure was used to examine fluoride distribution. There were five types of fluoride profiles in dental calculus in each area/country (designated types L, J, U, T, W). In supragingival calculus, type L (highest in the outermost layers) and type J (highest in the innermost layers) both had significantly higher values than type U (high in the surface and innermost layers) but were relatively identical. In subgingival calculus, type W (high in the outermost, mid and innermost layers) was characteristics. Calculus from the Wuhan mountainous area (fluoridated) had the highest fluoride concentration, followed by Leeds (non-fluoridated), and Nagoya and Shanghai (non-fluoridated) calculus had the lowest. Fluoride concentrations in supragingival calculus were higher in teeth extracted because of periodontal diseases than dental caries. It is concluded that fluoride concentrations and distribution in dental calculus differ from country to country, probably due to different fluoride environments.

Fluoride distribution in human dental calculus obtained from different sites on the tooth surface.

We examined the site specificity of fluoride (F) distribution in human dental calculus. Teeth with supra- and subgingival calculus were obtained from patients who resided in non-fluoridated areas in Japan and China. Sequential layers of the dental calculus (30 microns thick) were abraded by an abrasive micro-sampling technique and fluoride and phosphorus in the powdered samples were analyzed. Fluoride concentrations were highest in the outer, lowest in the middle and intermediate in the inner layers of dental calculus in general. In the outermost layers fluoride concentrations were highest in calculus found near the tooth cervix both in supra- and subgingival calculus. Fluoride concentrations decreased markedly toward the apical region in subgingival calculus, while it did not change toward the incisal or occlusal region in supragingival calculus. In the inner layers, fluoride concentrations in both supra- and subgingival calculus were not affected by position on the teeth. Fluoride concentrations in subgingival calculus near the apex were lower than in supragingival calculus near the incisal or occlusal region. It was concluded that the fluoride concentrations differ in different regions of dental calculus, probably due to their different mechanisms of formation.

Localized silicon distribution in supragingival calculus from Japanese monkeys (Macaca fuscata).

Using an electron-probe microanalyser, the distribution of silicon and other elements in supragingival dental calculus in domestic Japanese monkeys (Macaca fuscata) was studied. In two out of four monkeys kept in animals centres, a localized silicon distribution was found in both fracture and oral surfaces of the calculi. The silicon-rich area consisting of silicon alone resembled opal, but the areas containing silicon and other metal ions such as magnesium, aluminium, potassium and iron resembled clay minerals. In eight domestic monkeys, including the four animals described above, abundant calculus deposits were found. However, in four captured wild monkeys and in one which had been kept for less than a year at an animal centre, no dental calculus was found. There was almost no dental plaque accumulation in captured wild monkeys. It is suggested that calculus formation in Japanese monkeys is dependent on length of exposure to a commercial diet.

Possible regulatory roles of silicic acid, silica and clay minerals in the formation of calcium phosphate precipitates.

The effects of silicic acid, silica and clay minerals on the conversion of amorphous calcium phosphate to hydroxyapatite (HAP) were studied in vitro by a pH drop method. At a concentration range of 0.01-0.1 mM. silicic acid stimulated the rate of HAP transformation by about 30-40%. Silica stimulated the rate of HAP transformation by 33-43% at a concentration range of 0.05-1.5 mg/ml. The clay minerals, i.e. kaolin and talc, also stimulated the rate of HAP transformation by 40-90% at a concentration range of 0.4-10 mg/ml, but mica inhibited the reaction markedly at 10 mg/ml. The distribution of silicon in human supragingival dental calculus was studied by an electron-probe microanalyser. We found localized silicon distribution on the oral surface of the calculus. Such silicon-rich areas always contained silicon either alone or together with magnesium, aluminium, potassium, calcium and iron. This implies that the silicon-rich area may be opal and mica. Because silicic acid, silica, kaolin and talc stimulated and mica inhibited the in vitro calcium phosphate precipitation, it is possible that these silicon-rich areas may regulate the formation of the dental calculus.

Scanning electron microscopy and energy-dispersive X-ray microanalysis studies of early dental calculus on resin plates exposed to human oral cavities.

Dental calculus formed after 10 days on resin plates, applied to the lingual sides of the mandibular gingival regions in eight human subjects, was investigated by means of scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX). The mineral deposits were mainly divided into three types: A, B, and C. The type A deposits showing an average Ca/P molar ratio of 1.42 were densely packed with fine needle-shaped crystals formed by the intra- and extra-cellular calcification. The type A deposits, probably composed of Ca-deficient apatites and the transitional forms between apatite and octacalcium phosphate (OCP), were observed in all subjects. The type B deposits showing an average Ca/P molar ratio of 0.96 were aggregated with polygonal column, triangular plate-shaped, and rhombohedral crystals. These crystals identified as brushite (CaHPO4-2H2O:dicalcium phosphate dihydrate: DCPD) were found in four subjects. Platelet-shaped crystals of the type C deposits were observed in three subjects. Their Ca/P molar ratio of 1.26 and the crystal shape were similar to those of OCP. Whitlockite crystals were not found although Mg-containing hexagonal disk-like crystals were observed in two subjects.