Structure and stability of biological materials – characterisation at the nanoscale

Mummies are witnesses of the past harbouring information about the lives and fates of our ancestors. By examining them, the conditions of living, dietary, lifestyle and cultural habits as well as maladies in ancient times can be revealed. Knowledge of these maladies can be used to ascertain the evolution of diseases and may be helpful in characterising and treating them today. Uncovering information from mummies, however, depends on the preservation of the mummy tissue. Once degradation sets in, the molecular structure of the tissue is changed, and much information is lost. Favourable environmental conditions can slow down the process of decay and, hence, preserve organic material for long periods of time. As discussed in this work, biological tissue, which has substructural arrangements that are advantageous for withstanding mechanical load, might also be particularly favourable for preservation after the organism’s death. To address the question concerning the degree of preservation and to retrieve additional information from ancient tissue, two quasi-non-invasive analysis techniques, atomic force microscopy and Raman spectroscopy, were used. With these methods, the submicron structure, chemical composition, and nanomechanical properties of small mummified tissue samples were determined. In preliminary tests on recent collagen, the main connective tissue protein of vertebrates, results showed that in addition to imaging by atomic force microscopy, Raman spectroscopy is able to verify the alignment of this protein. Based on this knowledge, the arrangement and degree of collagen preservation in mummified human skin was investigated. Samples extracted from a 5300-year-old glacier mummy, the Iceman, were analysed. Extremely well-preserved collagen fibrils, in which the micro, ultra, and molecular structure were largely unaltered, were found. These results were in contrast, to the collagen fibrils found in the dermis of the Zweeloo mummy, a bog body of a female dating to the Roman period (78–233 AD). The Zweeloo mummy collagen fibrils showed moderate decomposition likely due to the acidic environment in the bog. Therefore, mummification due to freeze-drying, as in the Iceman, seems to be particularly beneficial for tissue preservation. The Iceman collagen, moreover, was found to be slightly stiffer than recent collagen, indicating that dehydration due to freeze-drying changed the mechanical properties of the tissue. This change likely improves the resilience of the freeze-dried collagen, stiffens the skin, and in turn maintains the skin’s protective function that prevents the underlying tissue from decomposing. Finally, also the preservation of red blood cells in wound tissue samples from the Iceman was observed. Single and clustered red blood cells were found whose morphological and molecular characteristics were similar to those of recent red blood cells. The ancient corpuscles moreover featured the typical red blood cell structure that indicates the preservation of healthy cells in Iceman tissue. Because fibrin, a protein formed during blood coagulation, was also detected, it appears that the clustered cells resembled remnants of a blood clot. The structure of the blood clot, stabilised by fibrin, may have been a protective envelope, which prevented the red blood cells from decomposing. Nonetheless, Raman spectra of the cells provided first indications of slight red blood cell degradation. These investigations emphasise the fundamental importance of the substructure and molecular arrangement of tissues, indicating that a tissue’s overall function and stability correlate with its molecular properties, in particular, the degree of cross-linking and the arrangement of the tissue molecular constituents. Last but not least the results show that ancient tissue can be preserved and its molecular properties probed and addressed even after millennia

Structure and stability of biological materials – characterisation at the nanoscale

Mummies are witnesses of the past harbouring information about the lives and fates of our ancestors. By examining them, the conditions of living, dietary, lifestyle and cultural habits as well as maladies in ancient times can be revealed. Knowledge of these maladies can be used to ascertain the evolution of diseases and may be helpful in characterising and treating them today. Uncovering information from mummies, however, depends on the preservation of the mummy tissue. Once degradation sets in, the molecular structure of the tissue is changed, and much information is lost. Favourable environmental conditions can slow down the process of decay and, hence, preserve organic material for long periods of time. As discussed in this work, biological tissue, which has substructural arrangements that are advantageous for withstanding mechanical load, might also be particularly favourable for preservation after the organism’s death. To address the question concerning the degree of preservation and to retrieve additional information from ancient tissue, two quasi-non-invasive analysis techniques, atomic force microscopy and Raman spectroscopy, were used. With these methods, the submicron structure, chemical composition, and nanomechanical properties of small mummified tissue samples were determined. In preliminary tests on recent collagen, the main connective tissue protein of vertebrates, results showed that in addition to imaging by atomic force microscopy, Raman spectroscopy is able to verify the alignment of this protein. Based on this knowledge, the arrangement and degree of collagen preservation in mummified human skin was investigated. Samples extracted from a 5300-year-old glacier mummy, the Iceman, were analysed. Extremely well-preserved collagen fibrils, in which the micro, ultra, and molecular structure were largely unaltered, were found. These results were in contrast, to the collagen fibrils found in the dermis of the Zweeloo mummy, a bog body of a female dating to the Roman period (78–233 AD). The Zweeloo mummy collagen fibrils showed moderate decomposition likely due to the acidic environment in the bog. Therefore, mummification due to freeze-drying, as in the Iceman, seems to be particularly beneficial for tissue preservation. The Iceman collagen, moreover, was found to be slightly stiffer than recent collagen, indicating that dehydration due to freeze-drying changed the mechanical properties of the tissue. This change likely improves the resilience of the freeze-dried collagen, stiffens the skin, and in turn maintains the skin’s protective function that prevents the underlying tissue from decomposing. Finally, also the preservation of red blood cells in wound tissue samples from the Iceman was observed. Single and clustered red blood cells were found whose morphological and molecular characteristics were similar to those of recent red blood cells. The ancient corpuscles moreover featured the typical red blood cell structure that indicates the preservation of healthy cells in Iceman tissue. Because fibrin, a protein formed during blood coagulation, was also detected, it appears that the clustered cells resembled remnants of a blood clot. The structure of the blood clot, stabilised by fibrin, may have been a protective envelope, which prevented the red blood cells from decomposing. Nonetheless, Raman spectra of the cells provided first indications of slight red blood cell degradation. These investigations emphasise the fundamental importance of the substructure and molecular arrangement of tissues, indicating that a tissue’s overall function and stability correlate with its molecular properties, in particular, the degree of cross-linking and the arrangement of the tissue molecular constituents. Last but not least the results show that ancient tissue can be preserved and its molecular properties probed and addressed even after millennia

Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman

Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as ‘the Iceman’. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 ± 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young's modulus of single mummified fibrils was 4.1 ± 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 ± 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years

The Iceman's Last Meal Consisted of Fat, Wild Meat, and Cereals

The history of humankind is marked by the constant adoption of new dietary habits affecting human physiology, metabolism, and even the development of nutrition-related disorders. Despite clear archaeological evidence for the shift from hunter-gatherer lifestyle to agriculture in Neolithic Europe [1], very little information exists on the daily dietary habits of our ancestors. By undertaking a complementary -omics approach combined with microscopy, we analyzed the stomach content of the Iceman, a 5,300-yearold European glacier mummy [2, 3]. He seems to have had a remarkably high proportion of fat in his diet, supplemented with fresh or dried wild meat, cereals, and traces of toxic bracken. Our multipronged approach provides unprecedented analytical depth, deciphering the nutritional habit, meal composition, and food-processing methods of this Copper Age individual

Many Labs 2: Investigating Variation in Replicability Across Samples and Settings

We conducted preregistered replications of 28 classic and contemporary published findings, with protocols that were peer reviewed in advance, to examine variation in effect magnitudes across samples and settings. Each protocol was administered to approximately half of 125 samples that comprised 15,305 participants from 36 countries and territories. Using the conventional criterion of statistical significance (p < .05), we found that 15 (54%) of the replications provided evidence of a statistically significant effect in the same direction as the original finding. With a strict significance criterion (p < .0001), 14 (50%) of the replications still provided such evidence, a reflection of the extremely highpowered design. Seven (25%) of the replications yielded effect sizes larger than the original ones, and 21 (75%) yielded effect sizes smaller than the original ones. The median comparable Cohen’s ds were 0.60 for the original findings and 0.15 for the replications. The effect sizes were small (< 0.20) in 16 of the replications (57%), and 9 effects (32%) were in the direction opposite the direction of the original effect. Across settings, the Q statistic indicated significant heterogeneity in 11 (39%) of the replication effects, and most of those were among the findings with the largest overall effect sizes; only 1 effect that was near zero in the aggregate showed significant heterogeneity according to this measure. Only 1 effect had a tau value greater than .20, an indication of moderate heterogeneity. Eight others had tau values near or slightly above .10, an indication of slight heterogeneity. Moderation tests indicated that very little heterogeneity was attributable to the order in which the tasks were performed or whether the tasks were administered in lab versus online. Exploratory comparisons revealed little heterogeneity between Western, educated, industrialized, rich, and democratic (WEIRD) cultures and less WEIRD cultures (i.e., cultures with relatively high and low WEIRDness scores, respectively). Cumulatively, variability in the observed effect sizes was attributable more to the effect being studied than to the sample or setting in which it was studied.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Sociales::Instituto de Investigaciones Psicológicas (IIP

Preservation of 5300 year old red blood cells in the Iceman

Changes in elasticity and structures of red blood cells (RBCs) are important indicators of disease, and this makes them interesting for medical studies. In forensics, blood analyses represent a crucial part of crime scene investigations. For these reasons, the recovery and analysis of blood cells from ancient tissues is of major interest. In this study, we show that RBCs were preserved in Iceman tissue samples for more than 5000 years. The morphological and molecular composition of the blood corpuscle is verified by atomic force microscope and Raman spectroscopy measurements. The cell size and shape approximated those of healthy, dried, recent RBCs. Raman spectra of the ancient corpuscle revealed bands that are characteristic of haemoglobin. Additional vibrational modes typical for other proteinaceous fragments, possibly fibrin, suggested the formation of a blood clot. The band intensities, however, were approximately an order of magnitude weaker than those of recent RBCs. This fact points to a decrease in the RBC-specific metalloprotein haemoglobin and, thus, to a degradation of the cells. Together, the results show the preservation of RBCs in the 5000 year old mummy tissue and give the first insights into their degradatio

Blood platelet adhesion to printed von Willebrand factor

Von Willebrand factor (vWF), a glycoprotein in blood, mediates the adhesion of blood platelets and thus plays a crucial role in hemostasis and thrombosis. Functional coating of surfaces with vWF allows the investigation of in vitro adhesion of blood platelet. We used soft lithography to create a functional patterned substrate. vWF was printed on plasma-treated glass and mica surfaces, producing elongated network-like fibril structures. A minimum layer thickness of 3 nm was observed, corresponding to the height of a monolayer of vWF. The stability of the patterns was verified in a laminar fluid flow, and the bioactivity of the structures was tested with platelet adhesion experiments. Platelets adhered to and spread on printed vWF. These results indicate that printed vWF substrates are stable and functional in typical perfusion experiments, and thus provide a useful tool for studying thrombus formation in vitro