Innovative nanomaterials for bone remains consolidation: performance evaluation and impact on 14C dating and on palaeogenetic analysis

An innovative protocol for the consolidation of ancient bone remains based on the use of nanometric hydroxyapatite (HAP) was set up and tested through a multidisciplinary approach. A new protocol for the synthesis of HAP nanoparticles was developed, and the composition of the obtained nanomaterial were investigated through Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD); sizes, shape and morphology of the synthesized particles were studied by Scanning Electron Microscopy (SEM). The consolidation performance was evaluated by testing the new nanomaterial on degraded ancient bone findings. An increase of the mineral density and of the micro-hardness of the bone were observed. The new consolidation method was also tested to assess possible effects on the palaeogenetic analysis and radiocarbon dating on the treated bones. The consolidation treatment has no substantial impact on the genetic characterisation of the skeletal remains and does not introduce any contaminations that could affect radiocarbon dating. This consolidation procedure represents a more compatible conservation tool with respect to traditional procedures: it has been shown that the treatment is effective, easily-applicable and not detrimental for 14C dating and palaeogenetic analysis.

The effect of eraser sampling for proteomic analysis on Palaeolithic bone surface microtopography

Bone surface modifications are crucial for understanding human subsistence and dietary behaviour, and can inform about the techniques employed in the production and use of bone tools. Permission to destructively sample such unique artefacts is not always granted. The recent development of non-destructive proteomic extraction techniques has provided some alternatives for the analysis of rare and culturally significant artefacts, including bone tools and personal ornaments. The Eraser Extraction Method (EEM), first developed for ZooMS analysis of parchment, has recently been applied to bone and ivory specimens. To test the potential impact of the EEM on ancient bone surfaces, we analyse six anthropogenically modified Palaeolithic bone specimens from Bacho Kiro Cave (Bulgaria) through a controlled sampling experiment using qualitative and 3D quantitative microscopy. Although the overall bone topography is generally preserved, our findings demonstrate a slight flattening of the microtopography alongside the formation of micro-striations associated with the use of the eraser for all bone specimens. Such modifications are similar to ancient use-wear traces. We therefore consider the EEM a destructive sampling approach for Palaeolithic bone surfaces. Together with low ZooMS success rates in some of the reported studies, the EEM might not be a suitable approach to taxonomically identify Pleistocene bone specimens.

The impact of moderate heating on human bones: an infrared and neutron spectroscopy study

This study aims to analyse human bones exposed to low/medium temperatures (200–650°C) under experimentally controlled conditions, both oxidizing and reducing, using complementary optical and neutron vibrational spectroscopy techniques. Clear differences were observed between the aerobically and anaerobically heated bones. The organic constituents disappeared at lower temperatures for the former ( ca 300°C), while they lingered for higher temperatures in anaerobic environments ( ca 450–550°C). Unsaturated non-graphitizing carbon species (chars) were detected mainly for anaerobically heated samples, and cyanamide formation occurred only at 650°C in reducing settings. Overall, the main changes were observed from 300 to 400°C in anaerobic conditions and from 450 to 500°C in aerobic environments. The present results enabled the identification of specific spectroscopic biomarkers of the effect of moderate temperatures (less than or equal to 650°C) on human bone, thus contributing to a better characterization of forensic and archaeological skeletal remains subject to heating under distinct environmental settings. In particular, these data may provide information regarding cannibalism or ancient bone boiling and defleshing rituals.

Author Correction: Artificial intelligence provides greater accuracy in the classification of modern and ancient bone surface modifications

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Artificial intelligence provides greater accuracy in the classification of modern and ancient bone surface modifications

Bone surface modifications are foundational to the correct identification of hominin butchery traces in the archaeological record. Until present, no analytical technique existed that could provide objectivity, high accuracy, and an estimate of probability in the identification of multiple structurally-similar and dissimilar marks. Here, we present a major methodological breakthrough that incorporates these three elements using Artificial Intelligence (AI) through computer vision techniques, based on convolutional neural networks. This method, when applied to controlled experimental marks on bones, yielded the highest rate documented to date of accurate classification (92%) of cut, tooth and trampling marks. After testing this method experimentally, it was applied to published images of some important traces purportedly indicating a very ancient hominin presence in Africa, America and Europe. The preliminary results are supportive of interpretations of ancient butchery in some places, but not in others, and suggest that new analyses of these controversial marks should be done following the protocol described here to confirm or disprove these archaeological interpretations.

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

A method for the taphonomic assessment of bone tools using 3D surface texture analysis of bone microtopography

Increasingly researchers have employed confocal microscopy and 3D surface texture analysis to assess bone surface modifications in an effort to understand ancient behavior. However, quantitative comparisons between the surfaces of purported archaeological bone tools and experimentally manufactured and used bones are complicated by taphonomic processes affecting ancient bone. Nonetheless, it may be reasonable to assume that bones within the same deposits are altered similarly and thus these alterations are quantifiable. Here we show how unworked bones can be used to quantify the taphonomic effect on bone surfaces and how this effect can then be controlled for and incorporated into an analysis for evaluating the modified surfaces of purported bone tools. To assess the baseline taphonomy of Middle Paleolithic archaeological deposits associated with typologically identified bone artifacts, specifically lissoirs, we directly compare the surface textures of ancient and modern unworked ribs. We then compare the ancient unworked ribs and lissoirs to assess their differences and predict the ancient artifacts’ original surface state using a multilevel multivariate Bayesian model. Our findings demonstrate that three of five tested surface texture parameters (Sa, Spc, and IsT) are useful for distinguishing surface type. Our model predictions show that lissoirs tend to be less rough, have more rounded surface peaks, and exhibit more directionally oriented surfaces. These characteristics are likely due to anthropogenic modifications and would have been more pronounced at deposition. Quantifying taphonomic alterations moves us one step closer to accurately assessing how bone artifacts were made and used in the ancient past.