Bone aerophones from Eynan-Mallaha (Israel) indicate imitation of raptor calls by the last hunter-gatherers in the Levant.

Direct evidence for Palaeolithic sound-making instruments is relatively rare, with only a few examples recorded from Upper Palaeolithic contexts, particularly in European cultures. However, theoretical considerations suggest that such artefacts have existed elsewhere in the world. Nevertheless, evidence for sound production is tenuous in the prehistoric archaeological record of the Levant, the study of music and its evolution being sparsely explored. Here we report new evidence for Palaeolithic sound-making instruments from the Levant with the discovery of seven aerophones made of perforated bird bones in the Final Natufian site of Eynan-Mallaha, Northern Israel. Through technological, use-wear, taphonomic, experimental and acoustical analyses, we demonstrate that these objects were intentionally manufactured more than 12,000 years ago to produce a range of sounds similar to raptor calls and whose purposes could be at the crossroads of communication, attracting hunting prey and music-making. Although similar aerophones are documented in later archaeological cultures, such artificial bird sounds were yet to be reported from Palaeolithic context. Therefore, the discovery from Eynan-Mallaha contributes new evidence for a distinctive sound-making instrument in the Palaeolithic. Through a combined multidisciplinary approach, our study provides important new data regarding the antiquity and development of the variety of sound-making instruments in the Palaeolithic at large and particularly at the dawn of the Neolithic in the Levant.

Mass grave localization prediction with geographical information systems in Guatemala and future impacts.

Conducting physical searches for mass grave locations based on anecdotal evidence is a time-consuming and resource-intensive endeavor in circumstances that often pose a threat to personal safety. The development of tools and procedures to speed such searches can greatly reduce the risk involved, increase the number of individuals whose remains are recovered and identified, and more importantly, reunite these remains with their loved ones to provide them with a proper burial. Geographic information systems (GIS) software, which can analyze and manipulate the spatial characteristics of known mass grave data, represents a powerful tool that can be used to predict new mass grave locations and increase the speed and efficiency with which they are investigated. Using the open source QGIS project, existing mass grave locations in Guatemala were analyzed based on their distance from and change in elevation relative to roads, streets, waterways, points of interest, and possible villages/towns. Statistical and geostatistical analyses performed to detect relationships among the variables resulted in patterns that warrant further study and can be used to further narrow areas of investigation.

Prediction of minimum postmortem submersion interval (PMSImin) based on eukaryotic community succession on skeletal remains recovered from a lentic environment.

Although recent studies explored using microbial succession during decomposition to estimate the postmortem interval (PMI) and postmortem submersion interval (PMSI), there is currently no published research using aquatic eukaryotic community succession to estimate the minimum postmortem submersion interval (PMSImin). The goals of this study were to determine whether eukaryotic community succession occurs on porcine skeletal remains in a lentic environment, and, if so, to develop a statistical model for PMSImin prediction. Fresh porcine bones (rib N = 100, scapula N = 100) were placed in cages (10'' x 10'') attached to floatation devices and submerged in a fresh water lake (Crozet, VA), using waterproof loggers and a YSI Sonde to record temperature and water quality variables, respectively. In addition to baseline samples, one cage, containing five ribs and five scapulae, and water samples (500 mL) were collected approximately every 250 accumulated degree days (ADD). Nineteen sample cohorts were collected over a period of 5200 ADD (579 Days). Variable region nine (V9) of the 18S ribosomal DNA (rDNA) was amplified and sequenced using a dual-index strategy on the MiSeq FGx sequencing platform. Resulting sequences underwent quality control parameters and analysis in mothur v 1.42.3, R v 3.5.3, and R v 3.6.0. Permutational multivariate analysis of variance (PERMANOVA) revealed a significant difference in phylogenetic ß-diversity among ribs, scapulae and water (p = 0.001) and among ADD (p ≤ 0.011), which was supported by distinct clustering of samples associated with each ADD in UniFrac distance based non-metric multidimensional scaling (NMDS) ordinations. Using similarity percentage (SIMPER) analysis of class and family level taxa, differences observed between bone types were attributed to Peronosporomycetes_cl, Eukaryota_unclassified, and Intramacronucleata (e.g., Armophorida), however these differences were not statistically significant. Alpha diversity revealed a non-linear increase in phylogenetic diversity with an increase in ADD. Random forest models for ribs and scapulae predicted PMSImin with an error rate within±104 days (937 ADD) and±63 days (564 ADD), respectively. In conclusion, this study suggests that eukaryotic succession is capable of predicting long term PMSImin in lentic systems.

Postmortem submersion interval (PMSI) estimation from the microbiome of sus scrofa bone in a freshwater lake.

While many studies have developed microbial succession-based models for the prediction of postmortem interval (PMI) in terrestrial systems, similar well-replicated long-term decomposition studies are lacking for aquatic systems. Therefore, this study sought to identify temporal changes in bacterial community structure associated with porcine skeletal remains (n = 198) for an extended period in a fresh water lake. Every ca. 250 ADD, one cage, containing 5 ribs and 5 scapulae, was removed from the lake for a total of nineteen collections. Water was also sampled at each interval. Variable region 4 (V4) of 16S rDNA was amplified and sequenced for all collected samples using Illumina MiSeq FGx Sequencing platform; resulting data were analyzed with the mothur (v1.39.5) and R (v3.6.0). Bacterial communities associated with ribs differed significantly from those associated with scapulae. This difference was mainly attributed to Clostridia, Holophagae, and Spirochaete relative abundances. For each bone type, α-diversity increased with ADD; similarly, ß-diversity bacterial community structure changed significantly with ADD and were explained using environmental parameters and inferred functional pathways. Models developed using 24 rib and 34 scapula family-level taxa allowed the prediction of PMSI with root mean square error of 522.97 ADD (~57 days) and 333.8 ADD (~37 days), respectively.

Development of a quantitative PCR-based method for studying temporal DNA degradation in waterlogged bone.

While several studies have examined temporal DNA degradation in bones collected from terrestrial environments, studies on temporal DNA degradation in bones collected from aquatic environments are limited and mostly based on case studies. The objective of this study was to assess the impact of long-term submersion, aquatic environment, bone type and DNA extraction method on DNA quality and quantity. Bone samples (scapulae and ribs), collected every ~1000 ADD from a freshwater lake and river, underwent DNA extraction via ChargeSwitch® gDNA Plant Kit and organic phenol-chloroform methods, and DNA quantitation using both TaqMan and SYBR Green-based quantitative PCR (qPCR) methods. Results suggest that in both bone types, quality of recovered DNA (i.e., degradation index) declined significantly with increase in submersion time. Among two bone types, quality of recovered DNA from scapulae declined faster than rib samples. There was no significant difference in recovered DNA quantity between bone types, DNA extraction methods, or locations but various interactions between these variables showed significant difference. Overall, it can be concluded that DNA can be extracted from waterlogged bone in sufficient quantity to generate an STR profile up to 4000 ADD.

Postmortem submersion interval (PMSI) estimation from the microbiome of Sus scrofa bone in a freshwater river.

Due to inherent differences between terrestrial and aquatic systems, methods for estimating the postmortem interval (PMI) are not directly applicable to remains recovered from water. Recent studies have explored the use of microbial succession for estimating the postmortem submersion interval (PMSI); however, a non-disturbed, highly replicated and long-term aquatic decomposition study in a freshwater river has not been performed. In this study, porcine skeletal remains (N = 200) were submerged in a freshwater river from November 2017-2018 (6322 accumulated degree days (ADD)/353 days) to identify changes and successional patterns in bacterial communities. One cage (e.g., 5 ribs and 5 scapulae) was collected approximately every 250 ADD for twenty-four collections; baseline samples never exposed to water acted as controls. Variable region 4 (V4) of 16S rDNA, was amplified and sequenced via the Illumina MiSeq FGx sequencing platform. Resulting sequences were analyzed using mothur (v1.39.5) and R (v3.6.0). The abundances of bacterial communities differed significantly between sample types. These differences in relative abundance were attributed to Clostridia, Holophagae and Gammaproteobacteria. Phylogenetic diversity increased with ADD for each bone type; comparably, ß-diversity bacterial community structure ordinated chronologically, which was explained with environmental parameters and inferred functional pathways. Models fit using rib samples provided a tighter prediction interval than scapulae, with a prediction of PMSI with root mean square error of within 472.31 (∼27 days) and 498.47 (∼29 days), respectively.

The Effect of Time and Temperature on the Persistence and Quality of Latent Fingerprints Recovered from 60-Watt Incandescent Light Bulbs.

To understand how time and temperature could affect latent fingerprints, wooden pallets were used to construct five units that each housed ten 60-watt incandescent light bulbs. Fingerprints were deposited on the top, middle, and base of the globes. The bulbs were powered on (except the control bulb in each unit) for 18, 48, 72, 120, 168, 336, 504, and 672 h. Fingerprints recovered from the bulbs by tape lift after black powder processing were given a quality score. A thermal imaging camera determined temperatures on three areas of the bulbs. Fingerprints on the top of the globe (156.3°C) had the lowest quality score, and fingerprints on the middle of the globe (112.6°C) had the highest quality score. The mean quality scores of the three temperature classes were within one standard deviation of one another, meaning there is no way to estimate fingerprint age based on its quality after heat exposure.

A eukaryotic community succession based method for postmortem interval (PMI) estimation of decomposing porcine remains.

Recent, short-term studies on porcine and human models (albeit with few replicates) demonstrated that the succession of the microbial community of remains may be used to estimate time since death. Using a porcine model (N=6) over an extended period of time (1703 ADD, or two months), this study characterized the eukaryote community of decomposing remains. Skin microbial samples were collected from the torso of each set of remains every day during the first week, on alternate days during the second week, and once a week for the remainder of the 60-day period; all collection intervals were recorded in accumulated degree days (ADD). The eukaryote community of each sample was determined using 18S ribosomal DNA (rDNA) MiSeq high throughput sequencing; data were analyzed in the Mothur pipeline (v1.39.5) and in IBM SPSS and R statistical packages. The relative abundance of eukaryote taxa across ADD/Days and an Analysis of Molecular Variance (AMOVA) indicated similarities between sequential ADD/Days, but significant differences in the eukaryote communities as broad stage 'milestones' of decomposition were reached. Fresh remains (0-57 ADD/0-2 Days; exhibiting a total body score (TBS) of 0-10) were characterized by the combined presence of Saccharomycetaceae, Debaryomycetaceae, Trichosporonaceae, Rhabditida, and Trichostomatia. During bloat and active decay (87-209 ADD/3-7 Days; exhibiting TBS of 11-20), Diptera was the most abundant eukaryotic taxa. During advanced decay stage (267-448 ADD/9-15 Days; exhibiting TBS of 21-25), Rhabditida was the most dominant eukaryote. Dry/skeletal remains (734-1703 ADD/26-61 Days; TBS≥26) were dominated by fungal families Dipodascaceae, Debaryomycetaceae, Trichosporonaceae, and Sporidiobolaceae. Using the family-level eukaryote taxonomic data for the entire study, random forest modelling explained 89.58% of the variation in ADD/Days, with a root mean square error (RMSE) of 177.55 ADD (≈6 days). Overall, these results highlight the importance of the microbial eukaryote community during the process of decomposition and in estimation of PMI.

Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study.

When deaths occur in water, soft tissue decomposes after a temperature-dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol-chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.

The effect of range and ammunition type on fracture patterns in porcine postcranial flat bones.

Pig half-carcasses were shot in scapulae, ribs and mandibles with either 0.243 hunting rifle using high velocity expanding ammunition (N = 30) or AK47 using full metal jacketed (FMJ) ammunition (N = 12) from a range of either 5 or 20 m. Fracture patterns related to distance of fire and ammunition type were compared on de-fleshed, macerated, and reconstructed bones. For expanding ammunition, location of fracture on ribs affected the resulting pattern. Scapulae shot from 5 m presented a comminuted pattern different from those shot from 20 m. Mandibles shot from 20 m showed a characteristic radiating pattern at entrance with the opposite ramus un-fractured; those shot from 5 m exhibited fractures to both rami. Using decision tree analysis provided accuracies of 93.8% for scapulae and 87.5% for mandibles. For FMJ, no distance dependent fracture differences were apparent in any bone. Decision tree analysis facilitated the interpretation of fracture patterns caused by projectile trauma.

An Improved Equation for TBS and ADD: Establishing a Reliable Postmortem Interval Framework for Casework and Experimental Studies.

Megyesi et al.'s (J Forensic Sci, 2005, 50, 618) paper was important to forensic anthropology as it introduced a quantitative framework for estimating time since death in human cadavers, based upon physical appearance by way of scoring on a novel scale. However, errors concerning rounding, temperature scale, and incorrect use of a statistical regression model render their predictive formula unusable. Based upon only their more reliable data, a more appropriate regression model to predict accumulated degree days (ADD) from total body score (TBS) is presented. The new model is also a superior fit (r(2)  = 0.91) and produces markedly narrower confidence intervals than the original, which also allowed impossible, negative ADD values. Explanations of the shortcomings in the original analysis and calculations are presented, which it is hoped will help forensic scientists avoid making similar mistakes.

Insights into the Social Structure of the PPNB Site of Kfar HaHoresh, Israel, Based on Dental Remains.

One of the central questions of the transition from mobile hunter-gatherers to sedentary farming communities concerns the establishment of new social structures and group identities. Along with other important factors, such as territory, ideology or economy, biological relationships might have played a decisive role in defining social groups. We therefore systematically analyzed teeth and jaw remains from nine sites in the Near East dating from the Natufian to the Late PPNB as primary proxy data for the reconstruction of familial relationships. This paper presents the results of morphological analyses on the teeth of the individuals from Kfar HaHoresh, one of the investigated Pre-Pottery Neolithic B sites. Kfar HaHoresh is located in the Nazareth hills of Galilee (32°42'20'' N 35°16'28'' E), Israel. Different statistical methods were applied to our data of epigenetic traits with the aim of determining biological relationships within the community, whereby the data of the eight other sites were used as cross-references. Our comparison of the traits of all individuals from Kfar HaHoresh indicates a rather heterogeneous community, but clearly shows one cluster belonging to a quite homogenous group, suggesting close biological relations between females and sub-adults. Interestingly, none of the male individuals belongs to this cluster, although their number outweighs that of the female individuals. This might suggest matrilocal residence patterns. However, due to the incomplete preservation of the teeth along with several other uncertainties, our conclusion must be seen as preliminary. A cross-examination of the results on skeletons excavated after our investigation should also be taken into consideration.

Interpreting Injury Mechanisms of Blunt Force Trauma from Butterfly Fracture Formation.

According to biomechanics of fracture production during blunt impact, tubular bones are subject to compressive (impact site) and tensile (opposite impact site) forces; this causes bones to break in tension before compression, producing Y-shaped fracture patterns with breakaway (butterfly) fragments. In current forensic models, the side of the bone exhibiting the breakaway fragment is designated the impact side, with initial breakage occurring opposite. Fracture production and patterning of blunt impacts to 255 sheep femora were analyzed. Contra the existing model, only 60% of complete butterfly fractures exhibited impact side breakaway fragments. Although fractures initiated on the tension, nonimpact side, butterfly fragments formed on either compression or tension sides. Using newly defined breakaway fragment shape criteria, impact side was estimated with 98% accuracy for both complete and partial butterfly fractures. Furthermore, the results suggest that the impact site is the located on one of the Y-fracture's arms, not the butterfly fragment's center, as previously modeled.

Decomposition Rate and Pattern in Hanging Pigs.

Accurate prediction of the postmortem interval requires an understanding of the decomposition process and the factors acting upon it. A controlled experiment, over 60 days at an outdoor site in the northwest of England, used 20 freshly killed pigs (Sus scrofa) as human analogues to study decomposition rate and pattern. Ten pigs were hung off the ground and ten placed on the surface. Observed differences in the decomposition pattern required a new decomposition scoring scale to be produced for the hanging pigs to enable comparisons with the surface pigs. The difference in the rate of decomposition between hanging and surface pigs was statistically significant (p=0.001). Hanging pigs reached advanced decomposition stages sooner, but lagged behind during the early stages. This delay is believed to result from lower variety and quantity of insects, due to restricted beetle access to the aerial carcass, and/or writhing maggots falling from the carcass.

The Effect of Clothing on the Rate of Decomposition and Diptera Colonization on Sus scrofa Carcasses.

Twenty Sus scrofa carcasses were used to study the effect the presence of clothing had on decomposition rate and colonization locations of Diptera species; 10 unclothed control carcasses were compared to 10 clothed experimental carcasses over 58 days. Data collection occurred at regular accumulated degree day intervals; the level of decomposition as Total Body Score (TBSsurf ), pattern of decomposition, and Diptera present was documented. Results indicated a statistically significant difference in the rate of decomposition, (t427  = 2.59, p = 0.010), with unclothed carcasses decomposing faster than clothed carcasses. However, the overall decomposition rates from each carcass group are too similar to separate when applying a 95% CI, which means that, although statistically significant, from a practical forensic point of view they are not sufficiently dissimilar as to warrant the application of different formulae to estimate the postmortem interval. Further results demonstrated clothing provided blow flies with additional colonization locations.

Oral inflammation, tooth loss, risk factors, and association with progression of Alzheimer's disease.

Periodontitis is a polymicrobial chronic inflammatory disease of tooth-supporting tissues with bacterial etiology affecting all age groups, becoming chronic in a subgroup of older individuals. Periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola are implicated in the development of a number of inflammatory pathologies at remote organ sites, including Alzheimer's disease (AD). The initial inflammatory hypothesis proposed that AD hallmark proteins were the main contributors of central nervous system (CNS) inflammation. This hypothesis is expanding to include the role of infections, lifestyle, and genetic and environmental factors in the pathogenesis of AD. Periodontal disease (PD) typifies a condition that encompasses all of the above factors including pathogenic bacteria. These bacteria not only are the source of low-grade, chronic infection and inflammation that follow daily episodes of bacteremia arising from everyday tasks such as brushing, flossing teeth, chewing food, and during dental procedures, but they also disseminate into the brain from closely related anatomical pathways. The long-term effect of inflammatory mediators, pathogens, and/or their virulence factors, reaching the brain systemically or otherwise would, over time, prime the brain's own microglia in individuals who have inherent susceptibility traits. Such susceptibilities contribute to inadequate neutralization of invading agents, upon reaching the brain. This has the capacity to create a vicious cycle of sustained local inflammatory milieu resulting in the loss of cytoarchitectural integrity and vital neurons with subsequent loss of function (deterioration in memory). The possible pathways between PD and AD development are considered here, as well as environmental factors that may modulate/exacerbate AD symptoms.

Quantifying the temperature of maggot masses and its relationship to decomposition.

Numerous Calliphoridae species have been observed to form larval aggregations during the feeding stage of development, resulting in localized increases in temperature. This study investigates the relationship between maggot numbers in a mass and heat generation. Single-species aggregations (Lucilia sericata) of various sizes (50-2500 individuals) were reared in the laboratory at a constant ambient temperature of 22°C. Internal mass temperatures were recorded every 5 min throughout the feeding stage of development. Results showed that mass temperatures increased with mass numbers (p-value < 0.001), ranging from 2.5 to 14°C above ambient. A minimum mass size of 1200 produced overall temperatures that were significantly warmer than ambient, diverging away from 22°C after c. 26 h. These results indicate that the microclimate of a mass has the potential to differ significantly from ambient, which may be influencing larval development rates and should therefore be factored into mPMI estimates to increase accuracy.

A preliminary examination of differential decomposition patterns in mass graves.

Five pairs of mass graves, each containing carcasses of 21 rabbits, were used to examine differential decomposition at four locations within the burial: surface, deep, mid-outer, and core. Every 100 accumulated degree days (ADD), a pair of graves was exhumed, and total body score (TBS) and internal carcass temperature of each rabbit were recorded. Decomposition did not differ for core- and deep-positioned carcasses (p = 0.13); differences were significant (p < 0.001) for all other location comparisons. Decomposition occurred fastest in shallow carcasses, followed by mid-outer carcasses; deep and core carcasses decomposed slowest and at rates not significantly different from one another. Adipocere formation was minimal and confined to deep carcasses. Carcass location within the mass grave significantly influenced internal carcass temperature (p < 0.001); a mean internal temperature difference of ca. 1°C existed between deep and shallow carcasses (30 cm apart). Effects of mass compactness and oxygenation require further investigation.

The formation of early stage adipocere in submerged remains: a preliminary experimental study.

In some circumstances, the presence of adipocere may retard decomposition and complicate postmortem interval estimation. This article explores the correlation between Accumulated Degree Days (ADD) and early stage formation of adipocere. Sixty wild rabbit (Oryctolagus cuniculus) carcasses were used in this experiment; a control group (N = 30) deposited directly on the ground surface and an experimental group (N = 30) completely submersed in water in individual buckets. Data (water and inner body temperature, pH, and total body score) were collected every 100 ADD. Results indicated that early stage adipocere is correlated to ADD and that its formation on submersed remains is more likely to occur after 630 ADD. Skin sloughing promoted the formation of adipocere. No adipocere was formed on any of the control group rabbits. This study also highlights the fact that multiple factors influence adipocere formation and it is suggested that further research needs to be conducted into this area.