PALEORADIOLOGICAL STUDY ON TWO INFANTS DATED TO THE 17th AND 18th CENTURIES

During an excavation campaign in the Church of the Conversion of Saint Paul in Roccapelago (North Italy), a hidden crypt was discovered, which yielded the remains of more than 400 individuals. The crypt was used as a cemetery by the inhabitants of the village of Roccapelago between the 16th and 18th centuries. Along the north side of the crypt, an area apparently separated from the rest of the burials was found, bordered by stones, where several burials of newborns and infants were concentrated. From here, five fabric rolls containing bones were recovered, and it was decided not to carry out destructive analyses, allocating the two best examples to a thorough radiological investigation to try to define the type of burial and the complete biological profile of the infant. The two rolls, subjects of this study, can be dated archaeologically between the 17th and 18th centuries. CT analysis shows a varied group of bones with a fairly good state of conservation. The paleoradiological study carried out had the primary objective of avoiding the destruction of the two rolls, ensuring their conservation; but at the same time, providing essential data to understand their nature, defining the biological profile and the type of deposition.

Ancient DNA re-opens the question of the phylogenetic position of the Sardinian pika Prolagus sardus (Wagner, 1829), an extinct lagomorph.

Palaeogenomics is contributing to refine our understanding of many major evolutionary events at an unprecedented resolution, with relevant impacts in several fields, including phylogenetics of extinct species. Few extant and extinct animal species from Mediterranean regions have been characterised at the DNA level thus far. The Sardinian pika, Prolagus sardus (Wagner, 1829), was an iconic lagomorph species that populated Sardinia and Corsica and became extinct during the Holocene. There is a certain scientific debate on the phylogenetic assignment of the extinct genus Prolagus to the family Ochotonidae (one of the only two extant families of the order Lagomorpha) or to a separated family Prolagidae, or to the subfamily Prolaginae within the family Ochotonidae. In this study, we successfully reconstructed a portion of the mitogenome of a Sardinian pika dated to the Neolithic period and recovered from the Cabaddaris cave, an archaeological site in Sardinia. Our calibrated phylogeny may support the hypothesis that the genus Prolagus is an independent sister group to the family Ochotonidae that diverged from the Ochotona genus lineage about 30 million years ago. These results may contribute to refine the phylogenetic interpretation of the morphological peculiarities of the Prolagus genus already described by palaeontological studies.

Pope Leo XII's death: the twist to a longstanding dispute by novel historical documents and paleopathographic analysis.

Although the practice of autopsy on the Pope's corpse was performed from the 16th century, autopsy reports are only rarely analysed, and never with the aim of investigating the real causes of the death from a concomitant medical and historical point of view. Here, for the first time, we report on the discovery of new unpublished documents from the Vatican Secret Archives and their investigation by a scientific and inter-disciplinary approach. This analysis allows us to draw new conclusions on the true cause of Leo XII's mysterious death. His sudden death, that occurred on February 10th, 1829 after a short illness, particularly struck the public. Suspicions of poisoning or surgeon's guilt or inexperience and even the shadow of a venereal disease, contributed to create a "black legend" on his pontificate and death. On the contrary, the present paleopathographic analysis points toward a new conclusion. The regular use of catheterization with a silver syringe provided an easy access for bacterial superinfection, confirmed by the observed early emphysematous stage of the corpse. So, the most substantiated hypothesis concerning the cause of Leo XII's death indicates a severe form of sepsis, exacerbated by a weakened state due to chronic hemorrhoids.

Multi-analytic study of a probable case of fibrous dysplasia (FD) from certosa monumental cemetery (Bologna, Italy).

OBJECTIVE: To evaluate, via a multidisciplinary approach, a distinctive paleopathological condition believed to be fibrous dysplasia, found on a 19th/20th century skeleton from Certosa Monumental Cemetery, Bologna, Italy. MATERIALS: A skeletonized cranium and mandible recovered from an ossuary in 2014. METHODS: Pathological alterations were analysed by radiological examination, dental macrowear, histopathological and genetic analyses. RESULT: The skeleton is believed to be an adult male. Differential diagnoses include Paget's disease, McCune-Albright syndrome, osteochondroma and osteosarcoma. The radiographic findings, along with the solitary nature of the lesions, are strong evidence for the diagnosis of fibrous dysplasia (FD). Genetic analysis further revealed a frequency of ˜1% of mutant alleles with the R201C substitution, one of the post-zygotic activating mutation frequently associated with FD. CONCLUSIONS: The multi-analytical method employed suggests a diagnosis of monostotic form of FD. The diagnostic design incorporates multiple lines of evidence, including macroscopic, histopathological, and genetic analyses. SIGNIFICANCE: Through the use of a multi-analytic approach, robust diagnoses can be offered. This case serves as one of the oldest examples of FD from an historical context. The genetic mutation detected, associated with FD, has not been previously reported in historical/ancient samples.

The genetic legacy of the Yaghnobis: A witness of an ancient Eurasian ancestry in the historically reshuffled central Asian gene pool.

OBJECTIVES: The Yaghnobis are an ethno-linguistic minority historically settled along the Yaghnob River in the Upper-Zarafshan Valley in Tajikistan. They speak a language of Old Sogdian origin, which is the only present-day witness of the Lingua Franca used along the Silk Road in Late Antiquity. The aim of this study was to reconstruct the genetic history of this community in order to shed light on its isolation and genetic ancestry within the Euro-Asiatic context. MATERIALS AND METHODS: A total of 100 DNA samples were collected in the Yaghnob and Matcha Valleys during several expeditions and their mitochondrial, Y-chromosome and autosomal genome-wide variation were compared with that from a large set of modern and ancient Euro-Asiatic samples. RESULTS: Findings from uniparental markers highlighted the long-term isolation of the Yaghnobis. Mitochondrial DNA ancestry traced an ancient link with Middle Eastern populations, whereas Y-chromosome legacy showed more tight relationships with Central Asians. Admixture, outgroup-f3, and D-statistics computed on autosomal variation corroborated Y-chromosome evidence, pointing respectively to low Anatolian Neolithic and high Steppe ancestry proportions in Yaghnobis, and to their closer affinity with Tajiks than to Iranians. DISCUSSION: Although the Yaghnobis do not show evident signs of recent admixture, they could be considered a modern proxy for the source of gene flow for many Central Asian and Middle Eastern groups. Accordingly, they seem to retain a peculiar genomic ancestry probably ascribable to an ancient gene pool originally wide spread across a vast area and subsequently reshuffled by distinct demographic events occurred in Middle East and Central Asia.

Poisoning histories in the Italian renaissance: The case of Pico Della Mirandola and Angelo Poliziano.

Giovanni Pico della Mirandola and Angelo Poliziano were two of the most important humanists of the Italian Renaissance. They died suddenly in 1494 and their deaths have been for centuries a subject of debate. The exhumation of their remains offered the opportunity to study the cause of their death through a multidisciplinary research project. Anthropological analyses, together with documentary evidences, radiocarbon dating and ancient DNA analysis supported the identification of the remains attributed to Pico. Macroscopic examination did not reveal paleopathological lesions or signs related to syphilis. Heavy metals analysis, carried out on bones and mummified tissues, showed that in Pico's remains there were potentially lethal levels of arsenic, supporting the philosopher's poisoning theory reported by documentary sources. The arsenic concentrations obtained from analysis of Poliziano's remains, are probably more related to an As chronic exposure or diagenetic processes rather than poisoning.

Unravelling biocultural population structure in 4th/3rd century BC Monterenzio Vecchio (Bologna, Italy) through a comparative analysis of strontium isotopes, non-metric dental evidence, and funerary practices.

The 4th century BC marks the main entrance of Celtic populations in northern Italy. Their arrival has been suggested based on the presence of Celtic customs in Etruscan mortuary contexts, yet up to now few bioarchaeological data have been examined to support or reject the arrival of these newcomers. Here we use strontium isotopes, non-metric dental traits and funerary patterns to unravel the biocultural structure of the necropolis of Monterenzio Vecchio (Bologna, Italy). Subsamples of our total sample of 38 individuals were analyzed based on different criteria characterizing the following analyses: 1) strontium isotope analysis to investigate migratory patterns and provenance; 2) non-metric dental traits to establish biological relationships between Monterenzio Vecchio, 13 Italian Iron age necropolises and three continental and non-continental Celtic necropolises; 3) grave goods which were statistically explored to detect possible patterns of cultural variability. The strontium isotopes results indicate the presence of local and non-local individuals, with some revealing patterns of mobility. The dental morphology reveals an affinity between Monterenzio Vecchio and Iron Age Italian samples. However, when the Monterenzio Vecchio sample is separated by isotopic results into locals and non-locals, the latter share affinity with the sample of non-continental Celts from Yorkshire (UK). Moreover, systematic analyses demonstrate that ethnic background does not retain measurable impact on the distribution of funerary elements. Our results confirm the migration of Celtic populations in Monterenzio as archaeologically hypothesized on the basis of the grave goods, followed by a high degree of cultural admixture between exogenous and endogenous traits. This contribution shows that combining different methods offers a more comprehensive perspective for the exploration of biocultural processes in past and present populations.

Iron Age Italic population genetics: the Piceni from Novilara (8th-7th century BC).

BACKGROUND: Archaeological data provide evidence that Italy, during the Iron Age, witnessed the appearance of the first communities with well defined cultural identities. To date, only a few studies report genetic data about these populations and, in particular, the Piceni have never been analysed. AIMS: To provide new data about mitochondrial DNA (mtDNA) variability of an Iron Age Italic population, to understand the contribution of the Piceni in shaping the modern Italian gene pool and to ascertain the kinship between some individuals buried in the same grave within the Novilara necropolis. SUBJECTS AND METHODS: In a first set of 10 individuals from Novilara, we performed deep sequencing of the HVS-I region of the mtDNA, combined with the genotyping of 22 SNPs in the coding region and the analysis of several autosomal markers. RESULTS: The results show a low nucleotide diversity for the inhabitants of Novilara and highlight a genetic affinity of this ancient population with the current inhabitants of central Italy. No family relationship was observed between the individuals analysed here. CONCLUSIONS: This study provides a preliminary characterisation of the mtDNA variability of the Piceni of Novilara, as well as a kinship assessment of two peculiar burials.

CT Scan of Thirteen Natural Mummies Dating Back to the XVI-XVIII Centuries: An Emerging Tool to Investigate Living Conditions and Diseases in History.

OBJECTIVES: To correlate the radiologic findings detected with computed tomography scan with anthropological data in 13 naturally mummified bodies discovered during works of recovery of an ancient church in a crypt in Roccapelago, in the Italian Apennines. METHODS: From a group of about sixty not-intentionally mummified bodies, thirteen were selected to be investigated with volumetric computed tomography (CT). Once CT scan was performed, axial images were processed to gather MPR and Volume Rendering reconstructions. Elaborations of these images provided anthropometric measurements and a non-invasive analysis of the residual anatomical structures. For each body the grade of preservation and the eventual pathological changes were recorded. Furthermore, in order to identify nutritional and occupational markers, radiologic signs of bone tropism and degenerative changes were analysed and graded. RESULTS: Mummies included seven females and six males, with an estimated age ranging from 20 to 60 years. The first relevant finding identified was a general low grade of preservation, due to the lack of anatomic tissues different from bones, tendons and dehydrated skin. The low grade of preservation was related to the natural process of mummification. Analysing bone degenerative changes on CT scan, the majority of the bodies had significant occupational markers consisting of arthritis in the spine, lower limbs and shoulders even in young age. Few were the pathological findings identified. Among these, the most relevant included a severe bilateral congenital hip dysplasia and a wide osteolytic lesion involving left orbit and petrous bone that was likely the cause of death. CONCLUSIONS: Although the low grade of preservation of these mummies, the multidisciplinary approach of anthropologists and radiologists allowed several important advances in knowledge for the epidemiology of Roccapelago. First of all, a profile of living conditions was delineated. It included occupational and nutritional conditions. Moreover, identification of some causes of death and, most importantly the definition of general living conditions.

Inferring chronological age from DNA methylation patterns of human teeth.

OBJECTIVE: Current methods to determine chronological age from modern and ancient remains rely on both morphological and molecular approaches. However, low accuracy and the lack of standardized protocols make the development of alternative methods for the estimation of individual's age even more urgent for several research fields, such as biological anthropology, biodemography, forensics, evolutionary genetics, and ancient DNA studies. Therefore, the aim of this study is to identify genomic regions whose DNA methylation level correlates with age in modern teeth. METHODS: We used MALDI-TOF mass spectrometry to analyze DNA methylation levels of specific CpGs located in the ELOVL2, FHL2, and PENK genes. We considered methylation data from cementum, dentin and pulp of 21 modern teeth (from 17 to 77 years old) to construct a mathematical model able to exploit DNA methylation values to predict age of the individuals. RESULTS: The median difference between the real age and that estimated using DNA methylation values is 1.20 years (SD = 1.9) if DNA is recovered from both cementum and pulp of the same modern teeth, 2.25 years (SD = 2.5) if DNA is recovered from dental pulp, 2.45 years (SD = 3.3) if DNA is extracted from cementum and 7.07 years (SD = 7.0) when DNA is recovered from dentin only. DISCUSSION: We propose for the first time the evaluation of DNA methylation at ELOVL2, FHL2, and PENK genes as a powerful tool to predict age in modern teeth for anthropological applications. Future studies are needed to apply this method also to historical and relatively ancient human teeth.

The pla gene, encoding plasminogen activator, is not specific to Yersinia pestis.

Here we present evidence to show that the pla gene, previously thought to be specific to Yersinia pestis, occurs in some strains of Citrobacter koseri and Escherichia coli. This means that detection of this gene on its own can no longer be taken as evidence of detection of Y. pestis.

A hyper-realistic method for facial approximation: the case of the Italian humanist Angelo Poliziano.

Frequently, identification of individuals is problematical due to the level of associated decomposition and even more when the skeletal remains are incomplete or fragmented. The identikit, which includes a sketch or a facial reconstruction, could assist investigators with determining the identity of the decedent. Similarly, in archeology and physical anthropology it gives a realistic appearance to a historical character known only through iconography. We examined the skull of Angelo Poliziano, an Italian humanist of the 15(th) century. Previously, his facial approximation was completed in clay according to the Manchester protocol and then a duplication was prepared in ultra-realistic materials. This technique returns a long lasting 3D model of the individual and provides the perception to be in front of a real person and, although expensive, applied in forensic context could it improve the recognition of the individual.

Technical Note: Guidelines for the digital computation of 2D and 3D enamel thickness in hominoid teeth.

The study of enamel thickness has received considerable attention in regard to the taxonomic, phylogenetic and dietary assessment of human and non-human primates. Recent developments based on two-dimensional (2D) and three-dimensional (3D) digital techniques have facilitated accurate analyses, preserving the original object from invasive procedures. Various digital protocols have been proposed. These include several procedures based on manual handling of the virtual models and technical shortcomings, which prevent other scholars from confidently reproducing the entire digital protocol. There is a compelling need for standard, reproducible, and well-tailored protocols for the digital analysis of 2D and 3D dental enamel thickness. In this contribution we provide essential guidelines for the digital computation of 2D and 3D enamel thickness in hominoid molars, premolars, canines and incisors. We modify previous techniques suggested for 2D analysis and we develop a new approach for 3D analysis that can also be applied to premolars and anterior teeth. For each tooth class, the cervical line should be considered as the fundamental morphological feature both to isolate the crown from the root (for 3D analysis) and to define the direction of the cross-sections (for 2D analysis).

Technical note: virtual reconstruction of KNM-ER 1813 Homo habilis cranium.

A very limiting factor for paleoanthropological studies is the poor state of preservation of the human fossil record, where fragmentation and deformation are considered normal. Although anatomical information can still be gathered from a distorted fossil, such specimens must typically be excluded from advanced morphological and morphometric analyses, thus reducing the fossil sample size and, ultimately, our knowledge of human evolution. In this contribution we provide the first digital reconstruction of the KNM-ER 1813 Homo habilis cranium. Based on state of-the-art three-dimensional digital modeling and geometric morphometric (GM) methods, the facial portion was aligned to the neurocranium, the overall distortion was removed, and the missing regions were restored. The reconstructed KNM-ER 1813 allows for an adjustment of the anthropometric measurements gathered on the original fossil. It is suitable for further quantitative studies, such as GM analyses focused on skull morphology or for finite element analysis to explore the mechanics of early Homo feeding behavior and diet.

Comparison of occlusal loading conditions in a lower second premolar using three-dimensional finite element analysis.

OBJECTIVES: This study aimed to compare the patterns of stress distribution in a lower second premolar using three conventional occlusal loadings and two more realistic loading scenarios based on occlusal contact areas. MATERIALS AND METHODS: The teeth of a dried modern human skull were micro-CT scanned in maximum intercuspation contact with a Viscom X8060 NDT X-ray system. A kinematic analysis of the surface contacts between antagonistic right upper and lower teeth during the power stroke was carried out in the Occlusal Fingerprint Analyser (OFA) software. Stress distribution in the lower right second premolar was analysed using three-dimensional finite element (FE) methods, considering occlusal information taken from OFA results (cases 4-5). The output was compared to that obtained by loading the tooth with a single point force (cases 1-3). RESULTS: Results for cases 1-3 differ considerable from those of cases 4-5. The latter show that tensile stresses might be concentrated in grooves and fissures of the occlusal surface, in the marginal ridges, in the disto-lingual and in the distal side of the root. Moreover, the premolar experiences high tensile stresses in the buccal aspect of the crown, supporting the idea that abfraction might be a dominant factor in the aetiology of non-carious cervical lesions. CONCLUSIONS: The application of FE methods in dental biomechanics can be advanced considering individual wear patterns. CLINICAL RELEVANCE: More realistic occlusal loadings are of importance for both new developments in prosthetic dentistry and improvements of materials for tooth restoration, as well to address open questions about the worldwide spread problem of dental failure.

The evolutionary paradox of tooth wear: simply destruction or inevitable adaptation?

Over the last century, humans from industrialized societies have witnessed a radical increase in some dental diseases. A severe problem concerns the loss of dental materials (enamel and dentine) at the buccal cervical region of the tooth. This "modern-day" pathology, called non-carious cervical lesions (NCCLs), is ubiquitous and worldwide spread, but is very sporadic in modern humans from pre-industrialized societies. Scholars believe that several factors are involved, but the real dynamics behind this pathology are far from being understood. Here we use an engineering approach, finite element analysis (FEA), to suggest that the lack of dental wear, characteristic of industrialized societies, might be a major factor leading to NCCLs. Occlusal loads were applied to high resolution finite element models of lower second premolars (P2) to demonstrate that slightly worn P2s envisage high tensile stresses in the buccal cervical region, but when worn down artificially in the laboratory the pattern of stress distribution changes and the tensile stresses decrease, matching the results obtained in naturally worn P2s. In the modern industrialized world, individuals at advanced ages show very moderate dental wear when compared to past societies, and teeth are exposed to high tensile stresses at the buccal cervical region for decades longer. This is the most likely mechanism explaining enamel loss in the cervical region, and may favor the activity of other disruptive processes such as biocorrosion. Because of the lack of dental abrasion, our masticatory apparatus faces new challenges that can only be understood in an evolutionary perspective.

Individual tooth macrowear pattern guides the reconstruction of Sts 52 (Australopithecus africanus) dental arches.

The functional restoration of the occlusal relationship between maxillary and mandibular tooth rows is a major challenge in modern dentistry and maxillofacial surgery. Similar technical challenges are present in paleoanthropology when considering fragmented and deformed mandibular and maxillary fossils. Sts 52, an Australopithecus africanus specimen from Sterkfontein Member 4, represents a typical case where the original shape of the dental arches is no longer preserved. It includes a partial lower face (Sts 52a) and a fragmented mandible (Sts 52b), both incomplete and damaged to such an extent to thwart attempts at matching upper and lower dentitions. We show how the preserved macro wear pattern of the tooth crowns can be used to functionally reconstruct Sts 52's dental arches. High-resolution dental stone casts of Sts 52 maxillary and mandibular dentition were mounted and repositioned in a dental articulator. The occlusal relationship between antagonists was restored based on the analysis of the occlusal wear pattern of each preserved tooth, considering all dental contact movements represented in the occlusal compass. The reconstructed dental arches were three-dimensional surface scanned and their occlusal kinematics tested in a simulation. The outcome of this contribution is the first functional restoration of A. africanus dental arches providing new morphometric data for specimen Sts 52. It is noteworthy that the method described in this case study might be applied to several other fossil specimens.