Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

The efficacy of whole human genome capture on ancient dental calculus and dentin.

OBJECTIVES: Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. MATERIALS AND METHODS: Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in-solution hybridization capture and high-throughput sequencing. RESULTS: Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four-fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on-target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum-level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off-target sequences in human genome-enriched calculus libraries may still be useful for oral microbiome reconstruction. DISCUSSION: While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.

Bronze Age population dynamics and the rise of dairy pastoralism on the eastern Eurasian steppe.

Recent paleogenomic studies have shown that migrations of Western steppe herders (WSH) beginning in the Eneolithic (ca. 3300-2700 BCE) profoundly transformed the genes and cultures of Europe and central Asia. Compared with Europe, however, the eastern extent of this WSH expansion is not well defined. Here we present genomic and proteomic data from 22 directly dated Late Bronze Age burials putatively associated with early pastoralism in northern Mongolia (ca. 1380-975 BCE). Genome-wide analysis reveals that they are largely descended from a population represented by Early Bronze Age hunter-gatherers in the Baikal region, with only a limited contribution (∼7%) of WSH ancestry. At the same time, however, mass spectrometry analysis of dental calculus provides direct protein evidence of bovine, sheep, and goat milk consumption in seven of nine individuals. No individuals showed molecular evidence of lactase persistence, and only one individual exhibited evidence of >10% WSH ancestry, despite the presence of WSH populations in the nearby Altai-Sayan region for more than a millennium. Unlike the spread of Neolithic farming in Europe and the expansion of Bronze Age pastoralism on the Western steppe, our results indicate that ruminant dairy pastoralism was adopted on the Eastern steppe by local hunter-gatherers through a process of cultural transmission and minimal genetic exchange with outside groups.

Differential preservation of endogenous human and microbial DNA in dental calculus and dentin.

Dental calculus (calcified dental plaque) is prevalent in archaeological skeletal collections and is a rich source of oral microbiome and host-derived ancient biomolecules. Recently, it has been proposed that dental calculus may provide a more robust environment for DNA preservation than other skeletal remains, but this has not been systematically tested. In this study, shotgun-sequenced data from paired dental calculus and dentin samples from 48 globally distributed individuals are compared using a metagenomic approach. Overall, we find DNA from dental calculus is consistently more abundant and less contaminated than DNA from dentin. The majority of DNA in dental calculus is microbial and originates from the oral microbiome; however, a small but consistent proportion of DNA (mean 0.08 ± 0.08%, range 0.007-0.47%) derives from the host genome. Host DNA content within dentin is variable (mean 13.70 ± 18.62%, range 0.003-70.14%), and for a subset of dentin samples (15.21%), oral bacteria contribute > 20% of total DNA. Human DNA in dental calculus is highly fragmented, and is consistently shorter than both microbial DNA in dental calculus and human DNA in paired dentin samples. Finally, we find that microbial DNA fragmentation patterns are associated with guanine-cytosine (GC) content, but not aspects of cellular structure.

Is coronary calcium scoring too late? Total body arterial calcium burden in patients without known CAD and normal MPI.

BACKGROUND: Patients with normal myocardial perfusion imaging (MPI) have a good prognosis. However, pre-clinical coronary and extracoronary atherosclerosis may exist in the absence of myocardial ischemia. METHODS: 154 Egyptian patients (mean age 53 years) underwent whole-body non-contrast CT following normal MPI. RESULTS: Atherosclerosis in the form of calcification was observed in ≥1 vascular bed in 115 of 154 (75%) patients. This included the iliofemoral (62%), abdominal aorta (53%), thoracic aorta (47%), coronary (47%), and carotid (25%) vascular beds. Mean total body calcium score was 3172 ± 530 AU. Extracoronary atherosclerosis in patients with a zero coronary artery calcium (CAC) score was common, occurring in the above-listed beds 42%, 36%, 29%, and 7% of the time, respectively. CAC was rarely present without iliofemoral or abdominal aortic calcification. CONCLUSION: Quantitative assessment of calcification in different vascular beds demonstrates that extracoronary atherosclerosis is common in patients who have normal MPI. Atherosclerotic calcifications are most common in the iliofemoral arteries and abdominal aorta, which typically predate coronary calcifications. An imaging strategy to detect extracoronary atherosclerosis could lead to greater understanding of the natural history of atherosclerosis in its long pre-clinical phase and possibly to earlier preventive strategies.

Coronary atherosclerosis in indigenous South American Tsimane: a cross-sectional cohort study.

BACKGROUND: Conventional coronary artery disease risk factors might potentially explain at least 90% of the attributable risk of coronary artery disease. To better understand the association between the pre-industrial lifestyle and low prevalence of coronary artery disease risk factors, we examined the Tsimane, a Bolivian population living a subsistence lifestyle of hunting, gathering, fishing, and farming with few cardiovascular risk factors, but high infectious inflammatory burden. METHODS: We did a cross-sectional cohort study including all individuals who self-identified as Tsimane and who were aged 40 years or older. Coronary atherosclerosis was assessed by coronary artery calcium (CAC) scoring done with non-contrast CT in Tsimane adults. We assessed the difference between the Tsimane and 6814 participants from the Multi-Ethnic Study of Atherosclerosis (MESA). CAC scores higher than 100 were considered representative of significant atherosclerotic disease. Tsimane blood lipid and inflammatory biomarkers were obtained at the time of scanning, and in some patients, longitudinally. FINDINGS: Between July 2, 2014, and Sept 10, 2015, 705 individuals, who had data available for analysis, were included in this study. 596 (85%) of 705 Tsimane had no CAC, 89 (13%) had CAC scores of 1-100, and 20 (3%) had CAC scores higher than 100. For individuals older than age 75 years, 31 (65%) Tsimane presented with a CAC score of 0, and only four (8%) had CAC scores of 100 or more, a five-fold lower prevalence than industrialised populations (p≤0·0001 for all age categories of MESA). Mean LDL and HDL cholesterol concentrations were 2·35 mmol/L (91 mg/dL) and 1·0 mmol/L (39·5 mg/dL), respectively; obesity, hypertension, high blood sugar, and regular cigarette smoking were rare. High-sensitivity C-reactive protein was elevated beyond the clinical cutoff of 3·0 mg/dL in 360 (51%) Tsimane participants. INTERPRETATION: Despite a high infectious inflammatory burden, the Tsimane, a forager-horticulturalist population of the Bolivian Amazon with few coronary artery disease risk factors, have the lowest reported levels of coronary artery disease of any population recorded to date. These findings suggest that coronary atherosclerosis can be avoided in most people by achieving a lifetime with very low LDL, low blood pressure, low glucose, normal body-mass index, no smoking, and plenty of physical activity. The relative contributions of each are still to be determined. FUNDING: National Institute on Aging, National Institutes of Health; St Luke's Hospital of Kansas City; and Paleocardiology Foundation.

Quantitative analysis of the morphological changes of the pubic symphyseal face and the auricular surface and implications for age at death estimation.

Age estimation methods are often based on the age-related morphological changes of the auricular surface and the pubic bone. In this study, a mathematical approach to quantify these changes has been tested analyzing the curvature variation on 3D models from CT and laser scans. The sample consisted of the 24 Suchey-Brooks (SB) pubic bone casts, 19 auricular surfaces from the Buckberry and Chamberlain (BC) "recording kit" and 98 pelvic bones from the Terry Collection (Smithsonian Institution). Strong and moderate correlations between phases and curvature were found in SB casts (ρ 0.60-0.93) and BC "recording kit" (ρ 0.47-0.75), moderate and weak correlations in the Terry Collection bones (pubic bones: ρ 0.29-0.51, auricular surfaces: ρ 0.33-0.50) but associated with large individual variability and overlap of curvature values between adjacent decades. The new procedure, requiring no expert judgment from the operator, achieved similar correlations that can be found in the classic methods.

Is atherosclerosis fundamental to human aging? Lessons from ancient mummies.

Case reports from Johan Czermak, Marc Ruffer, and others a century or more ago demonstrated ancient Egyptians had atherosclerosis three millennia ago. The Horus study team extended their findings, demonstrating that atherosclerosis was prevalent among 76 ancient Egyptian mummies and among 61 mummies from each of the ancient cultures of Peru, the American Southwest, and the Aleutian Islands. These findings challenge the assumption that atherosclerosis is a modern disease caused by present day risk factors. An extensive autopsy of an ancient Egyptian teenage male weaver named Nakht found that he was infected with four parasites: Schistosoma haematobium, Taenia species, Trichinella spiralis, and Plasmodium falciparum. Modern day patients with chronic inflammatory disease such as rheumatoid arthritis, systemic lupus erythematosus, and human immunodeficiency virus experience premature atherosclerosis. Could the burden of chronic inflammatory disease have been a risk factor for atherosclerosis in these ancient cultures? The prevalence of atherosclerosis in four diverse ancient cultures is consistent with atherosclerosis being fundamental to aging. The impact of risk factors in modern times, and potentially in ancient times, suggests a strong gene-environmental interplay: human genes provide a vulnerability to atherosclerosis, the environment determines when and if atherosclerosis becomes manifest clinically.

Computed tomographic evidence of atherosclerosis in the mummified remains of humans from around the world.

Although atherosclerosis is widely thought to be a disease of modernity, computed tomographic evidence of atherosclerosis has been found in the bodies of a large number of mummies. This article reviews the findings of atherosclerotic calcifications in the remains of ancient people-humans who lived across a very wide span of human history and over most of the inhabited globe. These people had a wide range of diets and lifestyles and traditional modern risk factors do not thoroughly explain the presence and easy detectability of this disease. Nontraditional risk factors such as the inhalation of cooking fire smoke and chronic infection or inflammation might have been important atherogenic factors in ancient times. Study of the genetic and environmental risk factors for atherosclerosis in ancient people may offer insights into this common modern disease.

Atherosclerosis in ancient and modern Egyptians: the Horus study.

BACKGROUND: Although atherosclerosis is usually thought of as a disease of modernity, the Horus Team has previously reported atherosclerotic vascular calcifications on computed tomographic (CT) scans in ancient Egyptians. OBJECTIVES: The purpose of this study was to compare patterns and demographic characteristics of this disease among Egyptians from ancient and modern eras. METHODS: We compared the presence and extent of vascular calcifications from whole-body CT scans performed on 178 modern Egyptians from Cairo undergoing positron emission tomography (PET)/CT for cancer staging to CT scans of 76 Egyptian mummies (3100 bce to 364 ce). RESULTS: The mean age of the modern Egyptian group was 52.3 ± 15 years (range 14 to 84) versus estimated age at death of ancient Egyptian mummies 36.5 ± 13 years (range 4 to 60); p < 0.0001. Vascular calcification was detected in 108 of 178 (60.7%) of modern patients versus 26 of 76 (38.2%) of mummies, p < 0.001. Vascular calcifications on CT strongly correlated to age in both groups. In addition, the severity of disease by number of involved arterial beds also correlated to age, and there was a very similar pattern between the 2 groups. Calcifications in both modern and ancient Egyptians were seen peripherally in aortoiliac beds almost a decade earlier than in event-related beds (coronary and carotid). CONCLUSIONS: The presence and severity of atherosclerotic vascular disease correlates strongly to age in both ancient and modern Egyptians. There is a striking correlation in the distribution of the number of vascular beds involved. Atherosclerotic calcifications are seen in the aortoiliac beds almost a decade earlier than in the coronary and carotid beds.

Genomic correlates of atherosclerosis in ancient humans.

Paleogenetics offers a unique opportunity to study human evolution, population dynamics, and disease evolution in situ. Although histologic and computed x-ray tomographic investigations of ancient mummies have clearly shown that atherosclerosis has been present in humans for more than 5,000 years, limited data are available on the presence of genetic predisposition for cardiovascular disease in ancient human populations. In a previous whole-genome study of the Tyrolean Iceman, a 5,300-year-old glacier mummy from the Alps, an increased risk for coronary heart disease was detected. The Iceman's genome revealed several single nucleotide polymorphisms that are linked with cardiovascular disease in genome-wide association studies. Future genetic studies of ancient humans from various geographic origins and time periods have the potential to provide more insights into the presence and possible changes of genetic risk factors in our ancestors. The study of ancient humans and a better understanding of the interaction between environmental and genetic influences on the development of heart diseases may lead to a more effective prevention and treatment of the most common cause of death in the modern world.

Funerary artifacts, social status, and atherosclerosis in ancient peruvian mummy bundles.

BACKGROUND: Evidence of atherosclerotic plaques in ancient populations has led to the reconsideration of risk factors for heart disease and of the common belief that it is a disease of modern times. METHODS: Fifty-one wrapped mummy bundles excavated from the sites of Huallamarca, Pedreros, and Rinconada La Molina from the Puruchuco Museum collection in Lima, Peru, were scanned using computed tomography to investigate the presence of atherosclerosis. Funerary artifacts contained within the undisturbed mummy bundles were analyzed as an attempt to infer the social status of the individuals to correlate social status with evidence of heart disease in this ancient Peruvian group. This work also provides an inventory of the museum mummy collection to guide and facilitate future research. RESULTS: Statistical analysis concluded that there is little association between the types of grave goods contained within the bundles when the groups are pooled together. However, some patterns of artifact type, material, atherosclerosis, and sex emerge when the 3 excavation sites are analyzed separately. CONCLUSIONS: From the current sample, it would seem that social class is difficult to discern, but those from Huallamarca have the most markers of elite status. We had hypothesized that higher-status individuals may have had lifestyles that would place them at a higher risk for atherogenesis. There seems to be some indication of this within the site of Huallamarca, but it is inconclusive in the other 2 archeological sites. It is possible that a larger sample size in the future could reveal more statistically significant results.

Why did ancient people have atherosclerosis?: from autopsies to computed tomography to potential causes.

Computed tomographic findings of atherosclerosis in the ancient cultures of Egypt, Peru, the American Southwest and the Aleutian Islands challenge our understanding of the fundamental causes of atherosclerosis. Could these findings be true? Is so, what traditional risk factors might be present in these cultures that could explain this apparent paradox? The recent computed tomographic findings are consistent with multiple autopsy studies dating as far back as 1852 that demonstrate calcific atherosclerosis in ancient Egyptians and Peruvians. A nontraditional cause of atherosclerosis that could explain this burden of atherosclerosis is the microbial and parasitic inflammatory burden likely to be present in ancient cultures inherently lacking modern hygiene and antimicrobials. Patients with chronic systemic inflammatory diseases of today, including systemic lupus erythematosus, rheumatoid arthritis, and human immunodeficiency virus infection, experience premature atherosclerosis and coronary events. Might the chronic inflammatory load of ancient times secondary to infection have resulted in atherosclerosis? Smoke inhalation from the use of open fires for daily cooking and illumination represents another potential cause. Undiscovered risk factors could also have been present, potential causes that technologically cannot currently be measured in our serum or other tissue. A synthesis of these findings suggests that a gene-environmental interplay is causal for atherosclerosis. That is, humans have an inherent genetic susceptibility to atherosclerosis, whereas the speed and severity of its development are secondary to known and potentially unknown environmental factors.

Anemia or scurvy: A pilot study on differential diagnosis of porous and hyperostotic lesions using differential cranial vault thickness in subadult humans.

Metabolic disorders, such as scurvy, manifested in human skeletal remains provide insight into health, nutrition, and environmental quality in past populations. Porous cranial vault lesions are often used to diagnose metabolic conditions in subadult remains, but overlapping gross lesion expressions have led to over-diagnosis of anemia and under-diagnosis of scurvy. Studies by Ortner and colleagues have suggested that specific porous cranial lesions are pathognomonic of scurvy, but additional diagnostic tools are necessary. In this technical report, we offer a preliminary assessment of cranial vault thickness (CVT) at the site of porous lesions (sensu lato porotic hyperostosis, cribra orbitalia) as a method for distinguishing between scurvy and anemia in subadult crania. Computed Tomography (CT) was used to measure CVT at various landmarks associated with porotic hyperostosis and cribra orbitalia, complemented by lesion scores, from scorbutic (N=11), anemic (N=3), and non-pathological (N=28) subadult crania used as a control group. Results indicate that CVT consistently distinguishes scorbutic from non-pathological individuals, while anemic individuals overlap with both - likely a function of small sample size in this study. Despite current limitations, CVT has the potential to be an objective diagnostic tool for distinguishing scurvy and expanding reconstructions of nutritional adequacy over the life course in past populations.

Atherosclerosis across 4000 years of human history: the Horus study of four ancient populations.

BACKGROUND: Atherosclerosis is thought to be a disease of modern human beings and related to contemporary lifestyles. However, its prevalence before the modern era is unknown. We aimed to evaluate preindustrial populations for atherosclerosis. METHODS: We obtained whole body CT scans of 137 mummies from four different geographical regions or populations spanning more than 4000 years. Individuals from ancient Egypt, ancient Peru, the Ancestral Puebloans of southwest America, and the Unangan of the Aleutian Islands were imaged. Atherosclerosis was regarded as definite if a calcified plaque was seen in the wall of an artery and probable if calcifications were seen along the expected course of an artery. FINDINGS: Probable or definite atherosclerosis was noted in 47 (34%) of 137 mummies and in all four geographical populations: 29 (38%) of 76 ancient Egyptians, 13 (25%) of 51 ancient Peruvians, two (40%) of five Ancestral Puebloans, and three (60%) of five Unangan hunter gatherers (p=NS). Atherosclerosis was present in the aorta in 28 (20%) mummies, iliac or femoral arteries in 25 (18%), popliteal or tibial arteries in 25 (18%), carotid arteries in 17 (12%), and coronary arteries in six (4%). Of the five vascular beds examined, atherosclerosis was present in one to two beds in 34 (25%) mummies, in three to four beds in 11 (8%), and in all five vascular beds in two (1%). Age at time of death was positively correlated with atherosclerosis (mean age at death was 43 [SD 10] years for mummies with atherosclerosis vs 32 [15] years for those without; p<0·0001) and with the number of arterial beds involved (mean age was 32 [SD 15] years for mummies with no atherosclerosis, 42 [10] years for those with atherosclerosis in one or two beds, and 44 [8] years for those with atherosclerosis in three to five beds; p<0·0001). INTERPRETATION: Atherosclerosis was common in four preindustrial populations including preagricultural hunter-gatherers. Although commonly assumed to be a modern disease, the presence of atherosclerosis in premodern human beings raises the possibility of a more basic predisposition to the disease. FUNDING: National Endowment for the Humanities, Paleocardiology Foundation, The National Bank of Egypt, Siemens, and St Luke's Hospital Foundation of Kansas City.

Ecological divergence and medial cuneiform morphology in gorillas.

Gorillas are more closely related to each other than to any other extant primate and are all terrestrial knuckle-walkers, but taxa differ along a gradient of dietary strategies and the frequency of arboreality in their behavioral repertoire. In this study, we test the hypothesis that medial cuneiform morphology falls on a morphocline in gorillas that tracks function related to hallucial abduction ability and relative frequency of arboreality. This morphocline predicts that western gorillas, being the most arboreal, should display a medial cuneiform anatomy that reflects the greatest hallucial abduction ability, followed by grauer gorillas, and then by mountain gorillas. Using a three-dimensional methodology to measure angles between articular surfaces, relative articular and nonarticular areas, and the curvatures of the hallucial articular surface, the functional predictions are partially confirmed in separating western gorillas from both eastern gorillas. Western gorillas are characterized by a more medially oriented, proportionately larger, and more mediolaterally curved hallucial facet than are eastern gorillas. These characteristics follow the predictions for a more prehensile hallux in western gorillas relative to a more stable, plantigrade hallux in eastern gorillas. The characteristics that distinguish eastern gorilla taxa from one another appear unrelated to hallucial abduction ability or frequency of arboreality. In total, this reexamination of medial cuneiform morphology suggests differentiation between eastern and western gorillas due to a longstanding ecological divergence and more recent and possibly non-adaptive differences between eastern taxa.

Assessment of age at death by microscopy: unbiased quantification of secondary osteons in femoral cross sections.

The microscopic method of age at death determination was introduced by Kerley in 1965 [E.R. Kerley, The microscopic determination of age in human bone, Am. J. Phys. Anthropol, 23 (1965) 149-163.]. However, even though the method has been revised several times, there remain some fundamental issues concerning the reliability of the methods. This is because several basic histological features seen in a cross section of a bone have to be quantified according to the method, but the definition, and hence quantification, of these features leaves room for subjectivity. In a previous study we found that some of these features (osteon fragments and Haversian canals) could not be identified reliably. Only secondary osteons could be identified with a low inter and intra observer error. Furthermore, since the histological features are quantified in only parts of an entire bone cross section, the selection of these parts or areas is a potential source of bias. Finally, unless an unbiased method is used for addressing features on the borders of the selected areas, this will also introduce error. These issues have not been addressed specifically in previous studies. In this study, we used the methods of stereology to choose the regions of interest, as well as for dealing with border phenomena, and we only counted secondary osteons. Our results show a statistically significant increase in the median number of osteons per area unit with increasing age at death. However, this was after exclusion of one outlier. This result is probably due to the limited sample size (N = 24). As such, this study is preliminary, but does warrant applying the described techniques to a larger sample.