Neuroanatomy and pneumaticity of the extinct Malagasy "horned" crocodile Voay robustus and its implications for crocodylid phylogeny and palaeoecology.

Voay robustus, the extinct Malagasy "horned" crocodile, was originally considered to be the only crocodylian representative in Madagascar during most part of the Holocene. However, Malagasy crocodylian remains have had confused taxonomic attributions and recent studies have underlined that Crocodylus and Voay populations coexisted on the island for at least 7500 years. Here, we describe the inner braincase anatomy of Voay robustus using x-ray computed tomography on four specimens, to provide new anatomical information that distinguishes Voay from Crocodylus, especially features of the brain endocast and the paratympanic sinuses. Geometric morphometric analyses are performed on 3D models of the internal organs to compare statistically Voay with a subset of extant Crocodylidae. Following these comparisons, we build an endocranial morphological matrix to discuss the proposed phylogenetic affinities of Voay with Osteolaeminae from an endocranial point of view. Additionally, we discuss the use of internal characters in systematic studies and find that they can have a major impact on morphological analyses. Finally, new radiocarbon data on Voay and subfossil Crocodylus specimens are recovered between 2010 and 2750 cal BP, which confirm the cohabitation of the two species in the same area for a long period of time. We thus assess several extinction scenarios, and propose a slightly different ecology of Voay compared to Crocodylus, which could have allowed habitat partitioning on the island. Our approach complements information obtained from previous molecular and morphological phylogenies, as well as previous radiocarbon dating, together revealing past diversity and faunal turnovers in Madagascar.

Scurvy complicated with Capnocytophaga sputigena sepsis as a possible cause of death of king Saint-Louis of France (1270 AD).

The cause of death of Saint-Louis is not known, but recent findings indicated that he presented scurvy and inflammatory jaw disease, which has been associated with infection by oral commensals. Here, we have the exceptional opportunity to analyze the relics of the viscera of King Saint-Louis. A 4.3 g sample from the viscera relics of King Saint-Louis conserved in Versailles' cathedral was subjected to radiocarbon dating, electronic and optic microscopy, and elementary, palynological, molecular, proteomics and microbiological analyses including specific PCR and v3v4 16 S rRNA gene amplification prior to large-scale sequencing using an Illumina MiSeq instrument. The measured radiocarbon age was Cal 1290 CE-1400, which was compatible with that of the viscera of St Louis viscera, considering the addition of lime, incense and vegetables within the human organs. Elemental and palynological analyses confirmed a medieval embalming process. Proteomics analysis identified mainly human muscle and blood proteins. Specific PCR for plague, amoebiasis, shigellosis and typhoid fever was negative. C. sputigena was identified as the main pathogenic species representing 10.8 % of all microbial sequences. In contrast, C. sputigena was found in only 0.001 % of samples sequenced in our center, and the 23 positive human samples showed a dramatically lower abundance (0.02-2.6 %). In the literature, human infections with C. sputigena included odontitis, dental abscess, sinusitis, thoracic infections and bacteremia, particularly in immunocompromised patients with oral and dental diseases consistent with recent analysis of King Saint-Louis' jaw. C. sputigena, a commensal of the mouth that is potentially pathogenic and responsible for fatal bacteremia, may have been the cause of the king's death.

Isotopic systematics point to wild origin of mummified birds in Ancient Egypt.

Millions of mummified birds serving for religious purpose have been discovered from archeological sites along the Nile Valley of Egypt, in majority ibises. Whether these birds were industrially raised or massively hunted is a matter of heavy debate as it would have a significant impact on the economy related to their supply and cult, and if hunted it would have represented an ecological burden on the birds populations. Here we have measured and analysed the stable oxygen, carbon and radiogenic strontium isotope compositions as well as calcium and barium content of bones along with the stable carbon, nitrogen and sulfur isotope composition of feathers from 20 mummified ibises and birds of prey recovered from various archeological sites of Ancient Egypt. If these migratory birds were locally bred, their stable oxygen, radiogenic strontium and stable sulfur isotopic compositions would be similar to that of coexisting Egyptians, and their stable carbon, nitrogen and oxygen isotope variance would be close, or lower than that of Egyptians. On one hand, isotopic values show that ibises ingested food from the Nile valley but with a higher isotopic scattering than observed for the diet of ancient Egyptians. On the other hand, birds of prey have exotic isotopic values compatible with their migratory behaviour. We therefore propose that most mummified ibises and all the birds of prey analysed here were wild animals hunted for religious practice.

Omics for Precious Rare Biosamples: Characterization of Ancient Human Hair by a Proteomic Approach.

Omics technologies have far-reaching applications beyond clinical medicine. A case in point is the analysis of ancient hair samples. Indeed, hair is an important biological indicator that has become a material of choice in archeometry to study the ancient civilizations and their environment. Current characterization of ancient hair is based on elemental and structural analyses, but only few studies have focused on the molecular aspects of ancient hair proteins-keratins-and their conservation state. In such cases, applied extraction protocols require large amounts of raw hair, from 30 to 100 mg. In the present study, we report an optimized new proteomic approach to accurately identify archeological hair proteins, and assess their preservation state, while using a minimum of raw material. Testing and adaptation of three protocols and of nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) parameters were performed on modern hair. On the basis of mass spectrometry data quality, and of the required initial sample amount, the most promising workflow was selected and applied to an ancient archeological sample, dated to about 3880 years before present. Finally, and importantly, we were able to identify 11 ancient hair proteins and to visualize the preservation state of mummy's hair from only 500 µg of raw material. The results presented here pave the way for new insights into the understanding of hair protein alteration processes such as those due to aging and ecological exposures. This work could enable omics scientists to apply a proteomic approach to precious and rare samples, not only in the context of archeometrical studies but also for future applications that would require the use of very small amounts of sample.

Cluster TOF-SIMS imaging of human skin remains: analysis of a South-Andean mummy sample.

A skin sample from a South-Andean mummy dating back from the XI(th) century was analyzed using time-of-flight secondary ion mass spectrometry imaging using cluster primary ion beams (cluster-TOF-SIMS). For the first time on a mummy, skin dermis and epidermis could be chemically differentiated using mass spectrometry imaging. Differences in amino-acid composition between keratin and collagen, the two major proteins of skin tissue, could indeed be exploited. A surprising lipid composition of hypodermis was also revealed and seems to result from fatty acids damage by bacteria. Using cluster-TOF-SIMS imaging skills, traces of bio-mineralization could be identified at the micrometer scale, especially formation of calcium phosphate at the skin surface. Mineral deposits at the surface were characterized using both scanning electron microscopy (SEM) in combination with energy-dispersive X-ray spectroscopy and mass spectrometry imaging. The stratigraphy of such a sample was revealed for the first time using this technique. More precise molecular maps were also recorded at higher spatial resolution, below 1 µm. This was achieved using a non-bunched mode of the primary ion source, while keeping intact the mass resolution thanks to a delayed extraction of the secondary ions. Details from biological structure as can be seen on SEM images are observable on chemical maps at this sub-micrometer scale. Thus, this work illustrates the interesting possibilities of chemical imaging by cluster-TOF-SIMS concerning ancient biological tissues.

Identification of different copper green pigments in Renaissance paintings by cluster-TOF-SIMS imaging analysis.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging using cluster primary ion beams is used for the identification of a green painting layer on the scene The Angels Concert from the Issenheim Altarpiece (painted in 1516) from a German Renaissance painter, Matthias Grünewald. Copper carboxylate clusters inside a basic copper chloride (atacamite) layer have been identified and located in the copper green layer. The mechanisms of transformation of atacamite into copper carboxylates may be initiated by an aging of the paint layers. The combination of the high mass resolution of the technique together with a micrometer spatial resolution and the possibility to simultaneously identify both minerals and organics, has proven to be the method of choice for the study of the stratigraphy of a paint cross-section.

Unexpected materials in a Rembrandt painting characterized by high spatial resolution cluster-TOF-SIMS imaging.

The painting materials of the Portrait of Nicolaes van Bambeeck (Royal Museums of Fine Arts of Belgium, Brussels, inv. 155) painted by Rembrandt van Rijn in 1641 has been studied using high resolution cluster-TOF-SIMS imaging. In the first step, a moderate spatial resolution (2 µm) was used to characterize the layer structure and the chemical composition of each layer on account of a high mass resolution. Then, in the second step, and despite a low mass resolution, the cluster primary ion beam was focused well below 1 µm in order to reveal smaller structures in the painting sample. The study confirmed the presence of starch in the second ground layer, which is quite surprising and, at least for Rembrandt paintings, has never been reported before. TOF-SIMS also indicated the presence of proteins, which, added to the size and shape of lake particles, suggests that it was manufactured from shearings (waste of textile manufacturing) of dyed wool, used as the source of the dyestuff. The analyses have also shown various lead carboxylates, being the products of the interaction between lead white and the oil of the binding medium. These findings considerably contribute to the understanding of Rembrandt's studio practice and thus demonstrate the importance and potential of cluster-TOF-SIMS imaging in the characterization on a submicrometer scale of artist painting materials.

Animal urine as painting materials in African rock art revealed by cluster ToF-SIMS mass spectrometry imaging.

The rock art site at the village of Songo in Mali is a very important Dogon ritual place where, since the end of the nineteenth century until today, takes place the ceremony of circumcision. During these ceremonies, paintings are performed on the walls of the shelter with mainly three colors: red, black and white. Ethnological literature mentions the use of animal urine of different species such as birds, lizards or snakes as a white pigment. Urine of these animals is mainly composed of uric acid or urate salts. In this article, time-of-flight secondary ion mass spectrometry (ToF-SIMS) is used to compare uric acid, snake urine and a sample of a white pigment of a Dogon painting coming from the rock art site of Songo. ToF-SIMS measurements in both positive and negative ion modes on reference compounds and snake urine proved useful for the study of uric acid and urate salts. This method enables to identify unambiguously these compounds owing to the detection in negative ion mode of the ion corresponding to the deprotonated molecule ([M-H](-) at m/z 167.01) and its fragment ions. Moreover, the mass spectra obtained in positive ion mode permit to differentiate uric acid and urate salts on the basis of specific ions. Applying this method to the Dogon white pigments sample, we show that the sample is entirely composed of uric acid. This proves for the first time, that animal urine was used as a pigment by the Dogon. The presence of uric acid instead of urate salts as normally expected in animal urine could be explained by the preparation of the pigment for its application on the stone.

Analysis of ancient Greco-Roman cosmetic materials using laser desorption ionization and electrospray ionization mass spectrometry.

Microsamples of pink cosmetic powders from the Greco-Roman period were analyzed using two complementary analytical approaches for identification of the colouring agents (lake pigments originally manufactured from madder plants with an inert binder, usually a metallic salt) present in the samples. The first technique was a methanolic acidic extraction of the archaeological samples with an additional ethyl acetate extraction of the anthraquinone-type colouring agents which were identified using high performance liquid chromatography coupled to electrospray ionization with high resolution mass spectrometry (LC-ESI-HRMS), and the second was direct analysis of a microsample by laser desorption ionization-mass spectrometry (LDI-MS). The latter technique is well suited when the quantity of samples is very low. This soft ionization technique enables the detection of very small quantities of compounds using the combination of positive and negative-ion modes. It was also successfully applied for the direct analysis of some laboratory-made reference compounds. However, the presence of lead in one of these ancient samples induced a spectral suppression phenomenon. In this case and conditional on a sufficient quantity of available sample, the former method is better adapted for the characterization of these anthraquinone-type molecules. This study also confirmed that purpurin, munjistin, and pseudopurpurin are the principal colouring agents present in these ancient cosmetic powders constituted from madder plants.

Identification of ritual blood in African artifacts using TOF-SIMS and synchrotron radiation microspectroscopies.

A new protocol is implemented to demonstrate the presence of blood in the patina of African art objects from Mali. Divided into three steps, the protocol first consists in demonstrating the presence of proteins and localizing them in the sample's cross sections using time-of-flight secondary ion mass spectrometry (TOF-SIMS) and synchrotron-based infrared microspectrometry (microFT-IR). In a second time, TOF-SIMS is used to investigate heme, which is a blood marker. If heme is missing, which could mean that it is too degraded to be detected, X-ray microfluorescence (microXRF) and X-ray absorption near-edge microspectroscopy (microXANES) are used to prove the presence of iron in the protein area and to get a fingerprint of its chemical environment. This permits us thus to demonstrate that iron is indeed linked with proteins and not with mineral phases of the sample. Coupled with the ritual context of the objects, this constitutes a proof of the use of blood. Thanks to this protocol, which has the major advantage of avoiding false positive results, the presence of blood has been demonstrated in seven out of the eight studied samples.