Fourier transform infrared conformational investigation of type I collagen aged by in vitro induced dehydration and non-enzymatic glycation treatments

The two main ageing-inducing events in the collagenous tissues are the water loss and the formation of intermolecular crosslinks based on the reaction of collagen with matrix carbohydrates, following a mechanism known as non-enzymatic-glycation. With the aim to mimic the two deleterious processes for the protein structure, rat-tail collagen was submitted to hydration changes and allowed to interact with two sugars characterized by different reducing properties, D-glucose and D-ribose. Fourier transform infrared (FTIR) spectroscopy was employed to investigate the conformational changes induced in the protein by the two treatments by analyzing the subsequent spectra modifications. FTIR spectra monitored: i) the amplitude and position changes of the two characteristic absorption bands OH stretching and Amide I, in dependence on the humidity level: a significant hysteresis effect in the ν(OH) band (ν~3400 cm–1) amplitude of the protein dehydrated and then rehydrated to the initial relative humidity (aw=0.92- 0.06) may be related to the enhancement of the β-sheet fraction in the protein structure as revealed by the parallel modification in the Amide I band (ν~1650 cm–1); ii) the area of the carbohydrate double band peaking at 1080 cm–1 and 1031 cm–1, associated to the accumulation of the glycation products, depending on the sugar concentration and incubation time. The association of both sugars to collagen only minimally affects the protein secondary structure as revealed by Amide I band Gaussian analysis. The whole set of results suggests hints to hypothesize a self-assembly model for collagen molecules induced by ageing

Infrared nanospectroscopy depth-dependent study of modern materials: morpho-chemical analysis of polyurethane/fibroin binary meshes

Infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) and imaging is here exploited together with attenuated total reflection (ATR) IR imaging and scanning electron microscopy (SEM) to depict the chemical composition of fibers in hybrid electrospun meshes. The focus is on a recently developed bio-hybrid material for vascular tissue engineering applications, named Silkothane & REG;, obtained in the form of nanofibrous matrices from the processing of a silk fibroin-polyurethane (SFPU) blend via electrospinning. Morphology and chemistry of single fibers, at both surface and subsurface level, have been successfully characterized with nanoscale resolution, taking advantage of the IR s-SNOM capability to portray the nanoscale depth profile of this modern material working at diverse harmonics of the signal. The applied methodology allowed to describe the superficial characteristics of the mesh up to a depth of about 100 nm, showing that SF and PU do not tend to co-aggregate to form hybrid fibers, at least at the length scale of hundreds of nanometers, and that subdomains other than the fibrillar ones can be present. More generally, in the present contribution, the depth profiling capabilities of IR s-SNOM, so far theoretically predicted and experimentally proven only on model systems, have been corroborated on a real material in its natural conditions with respect to production, opening the room for the exploitation of IR s-SNOM as valuable technique to support the production and the engineering of nanostructured materials by the precise understanding of their chemistry at the interface with the environment

Discovering the coating structure of historic bowed string instruments: an analytical campaign by SR-FTIR microspectroscopy

In many cases, the possibility to analyze a micro sample from an ancient bowed string instrument allows researchers to collect a wealth of information concerning materials used by the great Masters of violin-making and their construction procedures. In fact, the stratigraphic investigation may provide important information about (i) the treatments of the wood, normally involving proteinaceous materials and inorganic fillers such as carbonates, silicates, sulphates, (ii) composition of the different superimposed layers of varnish, and (iii) organic or inorganic pigments dispersed in the binders of the different superimposed layers of varnish [1]. In the present work, a set of four micro-samples - collected from well-preserved violins made by Antonio Stradivari, Francesco Ruggeri and Lorenzo Storioni - have been mounted in cross section and investigated throughout. The analytical approach has been developed on the embedded samples by means of Synchrotron Radiation (SR) FTIR microspectroscopy in reflection mode using a 15X objective, at the Chemical and Life Sciences branch of SISSI beamline (Elettra - Sincrotrone Trieste) [2]. Data have been collected in the MIR range in correspondence of each single layer of the stratigraphy, setting the lateral resolution in order to match the layer thickness (from few microns to tens of microns). A classification model has been constructed and validated to discriminate classes of materials according to the spectral information [3]. Preliminary results obtained from measurements performed by the micro-invasive SR-FTIR approach are elaborated and discussed, together with the analytical procedures, in order to characterize the features of the different samples

The earliest evidence for mechanically delivered projectile weapons in Europe

Microscopic analysis of backed lithic pieces from the Uluzzian technocomplex (45\u201340 thousand yr ago) at Grotta del Cavallo (southern Italy) reveals their use as mechanically delivered projectile weapons, attributed to anatomically modern humans. Use-wear and residue analyses indicate that the lithics were hunting armatures hafted with complex adhesives, while experimental and ethnographic comparisons support their use as projectiles. The use of projectiles conferred a hunting strategy with a higher impact energy and a potential subsistence advantage over other populations and specie

A multidisciplinary study unveils the nature of a Roman ink of the 1st century AD

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Fourier transform infrared conformational investigation of type I collagen aged by in vitro induced dehydration and non-enzymatic glycation treatments

The two main ageing-inducing events in the collagenous tissues are the water loss and the formation of intermolecular crosslinks based on the reaction of collagen with matrix carbohydrates, following a mechanism known as non-enzymatic-glycation. With the aim to mimic the two deleterious processes for the protein structure, rat-tail collagen was submitted to hydration changes and allowed to interact with two sugars characterized by different reducing properties, D-glucose and D-ribose. Fourier transform infrared (FTIR) spectroscopy was employed to investigate the conformational changes induced in the protein by the two treatments by analyzing the subsequent spectra modifications. FTIR spectra monitored: i) the amplitude and position changes of the two characteristic absorption bands OH stretching and Amide I, in dependence on the humidity level: a significant hysteresis effect in the ν(OH) band (ν~3400 cm–1) amplitude of the protein dehydrated and then rehydrated to the initial relative humidity (aw=0.92- 0.06) may be related to the enhancement of the β-sheet fraction in the protein structure as revealed by the parallel modification in the Amide I band (ν~1650 cm–1); ii) the area of the carbohydrate double band peaking at 1080 cm–1 and 1031 cm–1, associated to the accumulation of the glycation products, depending on the sugar concentration and incubation time. The association of both sugars to collagen only minimally affects the protein secondary structure as revealed by Amide I band Gaussian analysis. The whole set of results suggests hints to hypothesize a self-assembly model for collagen molecules induced by ageing

Tissue preservation of 16-18th Century mummies of Roccapelago (Modena, Italy): a SEM and FTIR study

Between 2009 and 2011, during restorative works at the Church of Roccapelago (province of Modena, Italy) a remote mountain village, hundreds of bodies, some of them mummified because of natural processes, were discovered in a forgotten crypt in use from the mid-16th to the 18th centuries. Mummification processes occurred unevenly, with bodies partially skeletonized and bodies only partly articulated [1]. 12 of these mummies, the most complete and representative, were studied with non-invasive methods and replaced in the crypt, set up as a museum (Fig. 1). The objects of this study are fragments of a variety of tissues taken from the mummies of the US 23 of the crypt of Roccapelago: skin pieces taken from different parts of the body, muscle, tendon, lung, bone, hair… The tissues were analyzed by means of FTIR spectroscopy applied in the transmission geometry, on samples obtained by mixing small quantities of the samples with pulverized KBr. In the IR spectrum of the investigated tissues the main absorption bands of the biological components such as proteins, lipids, nucleic acids, and carbohydrates were detected and characterized. The biochemical modifications recorded reveal a partial alteration of the ancient tissues which have been stabilized by the chemical-physical environmental conditions and preserved for hundreds of years. The whole IR spectrum of the tissues reveals traces of these “age-inhibitor” processes: 1) the water OH-stretching band (~3400 cm-1) features are indicative of the twofold role of the dehydration process, as a result of low temperatures and dry air, at the same time responsible for the protein structure modifications and determining the preservation; 2) conformational features of the proteins can be extracted by monitoring Amide I and Amide II bands (1500-1700 cm-1); 3) in the glucid specific spectral range (950-1150 cm-1), an increase was measured in the glucid/protein ratio, a spectroscopic marker for the AGE compounds formation as a consequence of the collagen binding to sugars in tissues [2]; 4) the amount of adipocere formation (2916-2849 cm-1, 1700 cm-1) was correlated with the position of the remains in the pile of the corpses. The bone diagenesis was monitored by means of the mineralization parameters. SEM measurements were carried out with the purpose of a morphological characterization of the tissues, in comparison with the modern ones. SEM images were acquired with Zeiss Supra40-high resolution apparatus using low beams energies. Mummified collagen fibers and collagen network are shown in the skin and compact bones reveal a lot of well preserved ultrastructural features such as Harvesian canals [3]. Septate hyphae and spores, characteristic of a fungal colonization were identified, as well as a lot of burrowing insects, such as Dermestidae, commonly referred to as skin beetles, which feed on dry animal and their predators, staphilinides [4]. [1] G. Gruppioni, D. Labate, L. Mercuri, V. Milani, M. Traversari, B. Vernia, 2010, Gli scavi della Chiesa di San Paolo di Roccapelago nell’Appennino modenese. La cripta con i corpi mummificati naturalmente, Pagani e Cristiani. Forme e attestazioni di religiosità del mondo antico in Emilia, X 2011: 219-245. [2] M. G. Bridelli, A. Dell’Anna, C. Stani, A. Baraldi, R. Boano and S. De Iasio, 2014, FT-IR spectroscopy and microspectroscopy of ancient Egyptian embalmed heads from the Museum of Anthropology and Ethnography of the University of Turin. Yearbook of mummy studies (Verlag Dr. Friedrich Pfeil, Munchen, Germany), Vol. 2, pp. 167-174 – ISBN 978-3-89937-163-5. [3] C. Stani, V. Erokhin, M. Traversari, R. Boano, E. Rabino Massa, M.G. Bridelli. 2014, SEM microscopy of mummified skin. Journal of Biological Research, 87:s1, 14 [4] V. Bugelli, D. Forni, L. Bassi, M. Di Paolo, D. Marra, S. Lenzi, C. Toni, M. Giusiani, R. Domenici, M. Gherardi, and S. Vanin, 2015, Forensic Entomology and the Estimation of the Minimum Time Since Death in Indoor Cases. Journal of Forensic Sciences , 60 (2): 525-531. ISSN 0022-119

Physical and histological investigation of the embalmed skin: application to some Egyptian mummy heads from the Marro collection (Turin)

The work will discuss the materials employed in the mummification of some Egyptian Dynastic embalmed heads (VI – XI Dynasty) and is the summary of a lot of studies performed in this framework in the last two years. The samples are skin fragments removed from mummified heads belonging to the “Giovanni Marro” collection, developed by Professor G. Marro between 1907 and 1913, at the necropolises of Asiut (the old Lycopolis), 380 km south of Cairo, and of Gebelein 28 km south of Luxor (the ancient Thebes), during three of the archaeological campaigns of the Italian Archaeological Mission. The collection is curated at the Museum of Anthropology and Ethnography of the University of Turin. The present work takes part in the number of activities promoted by the Museum for management and conservation of the stored ancient remains [1]. Several physical techniques were employed to detect and analyze the traces of the embalming materials on the mummified remains. FTIR spectroscopy was applied on samples prepared as KBr pellets and allowed to gain information both on the embalming substances spread on the skin and on the preservation state of the skin from the analysis of the characteristic bands of the cutaneous tissue. Micro-FTIR spectroscopy (beam light area about 0.4×0.4 mm2) was employed to identify the chemical nature of the materials on the skin surface and to measure the embalming substance penetration in the skin by focusing the IR beam on tissue layers at different depth. The results were compared with the histological measurements. Skin fragments were Mayer’s hemalum and eosine stained and revealed some brownish-yellow bands inserted among the bluish collagen fibers which may be attributed to the embalming substances soaked through the skin surface. ATR spectroscopy was applied to water and ethanol extracts of one of the embalmed skin samples. Several embalming substances were tested by matching the IR spectra against a library of diagnostic markers and the excellent superposition of the propolis spectrum supports the hypothesis that one of the embalming substances could have been beeswax (Fig. 1) [2], [3]. Finally, SEM images of some skin fragments showing Natron encrustations will be shown, supplied by XRD analyses.[1]R. Boano, D. Meaglia, G. Dutto, E. Costa, and E. Rabino Massa (2011): The biological archive of the Museum of Anthropology and Ethnography of Turin: microscopic analysis to assess the preservation of ancient human hair, in Yearbook of Mummy Study, Vol. 1, eds. H. Gill-Frerking, W. Rosendahl & A. Zink and D. Piombino Mascali, pp. 29-32. [2] S. A Buckley, K. A. Clark, and R. P. Evershed (2004): Complex organic chemical balms of Pharaonic animal mummies. Nature 431: 294-299. [3] C. Stani, A. Baraldi, R. Boano, R. Cinquetti, and M. G. Bridelli (2014) Study of skin degradation in ancient Egyptian mummies: complementarity of Fourier Transform Infrared Spectroscopy and histological analysis. J. Biol. Res. 87: 2133, 26-28