Victoria E. Gibbon

Novel methods of molecular sex identification from skeletal tissue using the amelogenin gene

Sex identification from skeletal material is of vital importance in order to reconstruct the demographic variables of an individual in forensic genetics and ancient DNA (aDNA) analysis. When the use of conventional methods of sex identification are impossible, molecular analysis of the X and Y chromosomes provides an expedient solution. Two novel systems of molecular sex identification suitable for skeletal material using the amelogenin gene are described, beginning in intron 2-3, spanning exon 3 and ending in intron 3-4. This area was optimal for sexing, as it includes 14 sex-specific polymorphic regions in addition to an indel (insertion or deletion of nucleotides). Once optimised and working with 100% efficiency on the controls, these procedures were applied to a collection of miscellaneous archaeological skeletons (ex situ) sourced from the Raymond Dart Collection of Human Skeletons (Dart Collection). This collection was used to optimise these techniques for skeletal remains derived from an archaeological context. These methods produced 46.66% sex results for the ex situ sample, which is within the normal range for aDNA studies. These new techniques are optimal for sex identification, with both the inherent control of isolating many sex-specific features and combined with the use of sensitive micro-fluidic electrophoresis.

Mseleni joint disease: A potential model of epigenetic chondrodysplasia

OBJECTIVE In this paper past research on the natural history of Mseleni joint disease, a crippling endemic osteoarthritis, its socio-economic impacts, the demographics, diet, geology and the genetic background of affected people are reviewed. In addition, some new research ideas are suggested to continue the search for etiological avenues for this disease such as stable isotope analysis and epigenetic mechanisms. RESULTS Mseleni joint disease is a chondrodysplasia first described in 1970. It is geographically confined to a remote area in the Maputaland region in northern Kwazulu Natal, South Africa. This disease affects most joints but primarily those of the hip; it is a progressive condition beginning with pain and stiffness until the patients ability to walk becomes compromised. Mseleni joint disease is characterized by two distinct abnormalities, protrusio acetabuli that mainly affects females and increases in frequency with age, and hip dysplasia that is more frequent with age. Much research has been conducted on the people with the disease and their surrounding environment. CONCLUSION Despite intensive investigations into the etiology of Mseleni joint disease, it remains unknown. As a result the examination of epigenetic mechanisms and stable isotope analysis of teeth are suggested as a means of providing information on the etiology of the disease. These methods can also be applied to other chondroplasias of unknown etiology.

CD133/EpCAM Cancer Stem Cell Markers of Tumour Stage in Colorectal Cancer Cells

In solid tumours, a discreet population of tumour associated cancer stem cells (CSCs) are proposed to drive and sustain tumour development and be responsible for tumour relapse. Colorectal cancer stem cells express cellspecific surface markers, including amongst others, CD133, EpCAM, CD44, CD166, and CD94f. In the present study, we aimed to characterisecellpopulations in the human colon adenocarcinoma cell lines, SW1116, HT29 and DLD1, expressing both CSC markers CD133 and EpCAM. These cell lines represent early, mid and late stages of colorectal tumours, respectively. Up to 107 SW1116, HT29 and DLD1 cells, co-stained with anti-CD133 and anti-EpCAM, were evaluated using flow cytometry. We report here progressive increasing proportions of cells coexpressing the CD133/EpCAM epitopes in the respective cell lines. In the SW1116 cell line, 2.42 ± 0.20 percent of cells were CD133+EpCAM+, in the HT29 cell line, 5.13 ± 0.17 percent of cells were CD133+EpCAM+, and in the DLD1 cell line, 10.30 ± 0.2 percent of cells were CD133+EpCAM+. These data suggest the frequency of CD133/ EpCAM marker expression may be associated with tumour stage and aggression.

Brief communication: Minimally invasive bone sampling method for DNA analysis.

Obtaining a bone sample for DNA analysis has traditionally been a destructive practice, which has resulted in reluctance on behalf of curators for skeletal collections to allow invasive testing. A novel minimally invasive bone sampling method for DNA analysis is presented here. This method uses a conventional hand drill wherein the bone sample is extracted from the intercondylar fossa of the femur; it does not interfere with any known anthropometric landmarks and only leaves a small hole on the surface of the bone. The temperature of the drill is documented and it was established due to the minor increase in temperature, that this should not affect the molecular integrity of the sample. This method is easily replicated and is suitable for both human and other animal skeletal material and can be applied to rare specimens with little risk.

A diachronic examination of biomechanical changes in skeletal remains from Tombos in ancient Nubia

Using morphometric assessment, we diachronically analyse mechanical stress and limb function at the Tombos (modern Sudan) archaeological site through time and changing socioeconomic circumstances. Based on previous research, we expect that during the Third Intermediate/Napatan (c. 1070-656 BCE) people were larger and more physically active than in the New Kingdom (∼1400-1070 BCE). On the appendicular skeleton of adults 57 measurements were obtained on individuals from 67 discrete burials and 370 commingled skeletal elements. These raw measurements were analysed statistically. Individuals from the discrete burials were used to calculate body mass and estimate mechanical behaviour (torsional and bending rigidity of long bones) modeled using beam theory across several bones of the upper and lower limbs. Body mass estimates for both sexes show people during the Third Intermediate/Napatan period were statistically significantly larger. On the upper limb for both sexes, variation reflects joint stabilisation and actions of flexion and extension at the elbow and/or supination/pronation of the forearm. On the lower limb, females show variation related to weight bearing activities and foot flexion; male variation is related to weight bearing activities, joint movement and stabilisation. These data point to altered habitual behaviour and physical activity for both sexes at Tombos through time. Suggested causes for both sexes include increased agricultural activities, and for males increased granite quarrying and equestrian activities. Using analyses of multiple bones of the upper and lower limbs in conjunction with biomechanical analyses, this study demonstrates the importance of the examination of physical activities in past populations, highlighting changes that can occur with sociopolitical transitions.

Forensic taphonomy: Vertebrate scavenging in the temperate southwestern Cape, South Africa

Vertebrate scavenging can significantly accelerate the rate of decomposition, which can hinder estimating the post-mortem interval (PMI). Patterns of decomposition and scavenging are highly specific to different environments in a forensic context, with no known data for South Africa. A better understanding of local decomposition patterns, taking scavenging into account, could increase the accuracy of PMI estimation and improve identification of human remains. Using a porcine model in the forensically significant thicketed Cape Flats Dune Strandveld habitat, the effect of vertebrate scavenging on the decomposition process was examined. This part of Cape Town suffers from poor socioeconomic conditions and a high murder rate, which is due in part to the dense population. Human decomposition was simulated using three small (∼20kg) domestic pig (Sus scrofa domesticus) carcasses, with ethical clearance. One pig served as a control and was caged to prevent vertebrate but not invertebrate access; the other two served as experimental treatments. They were deployed in Delft, Cape Town, and observed by motion-activated, time-lapse remote photography to record scavenger species and activity. A rapid increase in the decomposition rate was observed due to Cape grey mongoose (Galerella pulverulenta) scavenging, with early skeletonisation reached by both experimental pigs by day 14, compared to the control remaining in advanced decomposition after 93 days. Mongoose is the primary scavenger in this habitat, and showed notable patterns of feeding behaviour, exclusively within daylight hours. Scavenging activity was only influenced by rainfall later in the cycle. This research provides knowledge on locally relevant decomposition patterns and highlights the necessity for PMI estimation methods to consider vertebrate scavengers. This may improve human skeletal identification in forensic cases. There is scope for expansion of this study, with an investigation of seasonal effects, the interaction between invertebrate and vertebrate activity, as well as, the effect of clothing on scavenger access.

Forensic taphonomy: Scavenger-induced scattering patterns in the temperate southwestern Cape, South Africa — A first look

The effect of vertebrate scavenging can drastically alter the rate of decomposition and cause skeletal scatter, which hinders human forensic recovery and identification. Patterns of scavenging, disarticulation and scatter in a forensic context are specific to different environments with no known data for South Africa. A better understanding of these patterns can increase the chances of full body recovery and improve identification of human remains. In this preliminary study, the effect of wild vertebrate scavenging on skeletal scatter was examined using a porcine model in the forensically significant thicketed Cape Flats Dune Strandveld habitat. This area is a densely populated part of Cape Town, which suffers from poor socioeconomic conditions and a high murder rate. Ethics was granted for the use of three small (∼20kg) domestic pig (Sus scrofa domesticus) carcasses as proxies for human decomposition. They were deployed in Delft, Cape Town, and observed by motion-activated cameras to record wild scavenger activity. One pig served as a control and was caged to prevent vertebrate but not invertebrate access; the other two served as experimental treatments. Scatter was recorded every second day by marking the location of skeletal elements and measuring the distance and angle from the centre of each deposition site. No scattering was observed in the control, but notable scattering patterns were observed in the experimental pigs due to Cape grey mongoose (Galerella pulverulenta) scavenging, the only vertebrate scavenger species observed. No clear pattern of scatter distance over time was followed. Scatter began in the skeletonisation phase (day 25-30), resulting in a maximum distance of 12.67m and scatter/search area of 504.32m2. Mongoose-induced skeletal scatter followed a distinct pattern of movement into dense undergrowth, a previously unobserved behaviour and a key finding of this study. These results provide baseline data for sub-adult human scatter, or scatter of smaller components of an adult human skeleton, as demonstrated in the forensic case example provided. Knowledge is provided on locally relevant decomposition patterns and informs search methods for improved human skeletal recovery in forensic cases. There is scope for expansion of this study, with an investigation of seasonal effects, the interaction between invertebrate and vertebrate activity, as well as, the effect of clothing on scavenger access.

Use of high resolution computed tomography to diagnose ante-mortem dental root fractures in archaeological samples

Dental root fractures are rarely documented in past human populations, but when they are observed, diagnosing ante-mortem events as causal factors can be difficult due to postmortem alteration. Can high resolution X-ray computed tomography (CT) improve our ability to diagnose if a dental fracture was caused ante- or post-mortem? To test this, a case study of two individuals with previously diagnosed dental root fractures were re-examined with high resolution CT to confirm or refute the original diagnoses. For individual 4170, use of high resolution CT supported the original diagnosis of an ante-mortem root fracture on the right mandibular central incisor that was made based on planar X-ray. With the new analyses, the root fracture was found to be associated with evidence of calcification, and with radicular and alveolar bone resorption, which are positive correlates of an ante-mortem origin. Resolving this level of detail of bone modification was not possible without using high resolution CT. For individual 4172, the use of high resolution CT enabled visualisation of fracturing and cracking throughout several molars and crowns of other teeth that were not evident in planar X-ray, suggesting that these fractures were likely produced by post-mortem taphonomic processes rather than ante-mortem events as originally diagnosed. In this case study, high resolution CT offered critical advantages compared to a planar X-ray approach, such as using 3D multiple slice views without superimposing alveolar bone and teeth as in planar X-ray imaging. This study demonstrates the potential of high resolution CT in confirming and refuting ante-mortem processes, and that visualisation of 3D structures is crucial for arriving at definitive diagnoses.

Predictive factors for alveolar fenestration and dehiscence

The purpose of this study was to evaluate the distribution and incidence of two forms of alveolar bone resorption known as fenestration and dehiscence across time and space. To accomplish this a Medieval French population was studied and the results were compared with other studies to examine incidence and distribution of alveolar bone resorption. Thus, 1175 teeth were analysed for 81 individuals, from an agropastoral Medieval (12th-14th century) archaeological site of Vilarnau located in the South of France. Tooth presence and absence as well as dental alveolar resorption were recorded. A new standardised methodological approach to record alveolar resorption is presented and can be used for any skeletonised series. Measurements of dehiscence were made in the midline on each root in relation to the cemento-enamel junction and fenestration was considered as resorption restricted to alveolar bone. Through analyses of the distribution and incidence of alveolar bone resorption over-time in a Medieval French population, along with nine other studies, we present a list of predictive factors for alveolar bone resorption. Among these factors tooth position and function were the most important; anterior teeth were more commonly affected, bone resorption was more common on the labial/buccal versus palatal/lingual surfaces, fenestration was also more common on the maxilla and dehiscence on the mandible (p≤0.001). These patterns do not vary through time or space, and therefore, provide predictive factors for health practitioners in oral therapy to improve patient recovery and post oral treatment success.

Dental wear quantity and direction in Chalcolithic and Medieval populations from southwest France

The aim of this study was to verify if dental wear changed due to the dietary shift between the Chalcolithic and Middle Ages from relatively hard and fibrous foods to soft cooked cereals. This was accomplished by comparing dental wear quantity and direction between people from two archaeological sites, Les Treilles during the Chalcolithic (mixed subsistence farmers) and Marsan from the Middle Ages (agriculturalists) in southwest France. The materials studied include 65 mandibles, 32 from Les Treilles and 33 from Marsan; 549 teeth were studied. The results show statistically significant difference in wear quantity and direction, the Chalcolithic population (Les Treilles) had the greatest levels of wear in a mainly oblique direction, with the anterior teeth heavily affected by wear. Comparatively, the Medieval sample (Marsan) had lesser levels of wear in a mainly horizontal direction, and the most heavily worn teeth were the molars and incisors. The quantity of wear seems to correlate well with changes in diet, the high level of wear on the anterior teeth in the Chalcolithic sample corresponds with the consumption of a mixed diet of fibrous and tough foods. At Marsan, the lower wear quantity was likely due to a diet of soft boiled cereals, requiring less mastication. However, wear direction appears dependent on several factors and may correlate with more mixed subsistence practices. This study demonstrates the need for additional research into the complex actions of mastication and its effect on dental wear, as well as standardised methodology for the examination of dental wear in archaeological samples.