Stable Isotope and Radiocarbon Analysis for Diet, Climate and Mobility Reconstruction in Agras (Early Iron Age) and Edessa (Roman Age), Northern Greece

In this article we present an isotopic analysis of human bone collagen (δ13Ccol, and δ15Ncol) and bone apatite (δ13C) for diet reconstruction, as well as δ18Oap of human bone apatite for climate reconstruction, using samples from Northern Greece. Radiocarbon dating analysis was conducted on three of the Agras samples and the results (from 1000 to 800 BC) correspond to the Early Iron Age. Isotopic values for δ13Ccol range from −20.5‰ to −16‰ and for δ15Ncol from 6‰ to 11.1‰—a strong indication of a C3-based diet, with contributions by C4 and freshwater fish elements. The results were compared to the ones from Roman Edessa, and Alexandreia (a contemporary city near Edessa), as well as to other Early Iron sites in Greece and wider Europe. In general, the results from Agras are in good agreement with the results from Northern Greece, with the exception of the Makriyalos site, and are quite close to those of Croatia’s and Hungary’s sites. Additionally, from the δ18Oap results we calculated the oxygen isotopic composition of consumed water for Agras (from −9.6‰ to −10.9‰) and for Roman Edessa (from −9.6‰ to −11.2‰) for the palaeoclimate and palaeomobility reconstruction.

Reconstructed Paleodiets and Subsistence Strategies of the Central Ciscaucasian Population (1000 BC to 1000 AD), Based on Collagen Isotope Analysis of Bone Samples from the Kichmalka II Burial Ground

Settlement and economy patterns of the Iron Age and early medieval population of the Central North Caucasus evidence complex cultural processes in the region. The ecological approach including the evaluation of carbon and nitrogen isotopes in the local biota opens up new prospects in the study of environments, climate, anthropogenic effect, land use, and nutrition. We analyze the isotopic composition of collagen in 19 human and 11 animal bone samples from Kichmalka II—a cemetery successively used by the Koban people, those of the Sarmatian stage, and Alans. The isotopic composition of the Alanian sample indicates a heavy predominance of plants with the C3-type photosynthesis in the diet of humans and animals. People who lived during the Koban and Sarmatian stages consumed also C4-plants, such as common millet (Panicum miliaceum), suggesting the rise of the trophic step for carbon (Δδ13Chuman-animal). Statistically signifi cant differences in the isotopic composition of carbon were found within the Koban population, apparently evidencing two dietary models. The Δδ15Nhuman-animal values fall within the trophic step, mirroring a focus on meat and dairy products in the diet of all groups. Comparison with respective data on the Klin-Yar III cemetery revealed differences in isotopic signatures in the diet of both humans and domestic animals during the Koban period. The possible reason is climatic change in the Iron Age and the variable share of millet in the diet of the Koban people. The low proportion of δ15N (below 4 ‰) in the bone collagen of goat, sheep, and horse of the Alanian period may attest to vertical transhumance.

A Novel Gelatinase from Marine Flocculibacter collagenilyticus SM1988: Characterization and Potential Application in Collagen Oligopeptide-Rich Hydrolysate Preparation

Although the S8 family in the MEROPS database contains many peptidases, only a few S8 peptidases have been applied in the preparation of bioactive oligopeptides. Bovine bone collagen is a good source for preparing collagen oligopeptides, but has been so far rarely applied in collagen peptide preparation. Here, we characterized a novel S8 gelatinase, Aa2_1884, from marine bacterium Flocculibacter collagenilyticus SM1988T, and evaluated its potential application in the preparation of collagen oligopeptides from bovine bone collagen. Aa2_1884 is a multimodular S8 peptidase with a distinct domain architecture from other reported peptidases. The recombinant Aa2_1884 over-expressed in Escherichia coli showed high activity toward gelatin and denatured collagens, but no activity toward natural collagens, indicating that Aa2_1884 is a gelatinase. To evaluate the potential of Aa2_1884 in the preparation of collagen oligopeptides from bovine bone collagen, three enzymatic hydrolysis parameters, hydrolysis temperature, hydrolysis time and enzyme-substrate ratio (E/S), were optimized by single factor experiments, and the optimal hydrolysis conditions were determined to be reaction at 60 ℃ for 3 h with an E/S of 400 U/g. Under these conditions, the hydrolysis efficiency of bovine bone collagen by Aa2_1884 reached 95.3%. The resultant hydrolysate contained 97.8% peptides, in which peptides with a molecular weight lower than 1000 Da and 500 Da accounted for 55.1% and 39.5%, respectively, indicating that the hydrolysate was rich in oligopeptides. These results indicate that Aa2_1884 likely has a promising potential application in the preparation of collagen oligopeptide-rich hydrolysate from bovine bone collagen, which may provide a feasible way for the high-value utilization of bovine bone collagen.

Therapeutic Mechanism of Chinese Medicine on the Healing of Early and Middle Fractures in Rabbits Under the Expression Level of Bone Morphogenetic Protein-2 (BMP-2) in Bone Tissue

In order to explore the therapeutic mechanism of Chinese medicine on the healing of rabbits early and middle fractures, a rabbit fracture model was established in this study. The study was divided into several groups, i.e., treatment group (TG) (fed with Chinese medicine Capsule) and control group (CG) (fed with normal saline (NS)). The materials were collected at 1, 3, and 5 weeks after the start of the experiment for analysis. The experiment content included: callus Hematoxylin-Eosin staining (HE staining); Bone Morphogenetic protein-2 (BMP-2) protein level detection; Type I and type II bone collagen (BC) detection; and serum biochemical factors detection. The experimental results showed that the formation of callus in the TG was better than in the CG; the BMP-2 protein expression level in the TG was higher than in the CG, and there were statistically significant differences (SSDs); the type I and type II BC levels in the TG were higher than the CG, there were SSDs; the levels of serum calcium (SC), phosphorus ion (PI), and alkaline phosphatase (ALP) in the TG were also higher than in the CG, and there were SSDs.

A modern baseline for the paired isotopic analysis of skin and bone in terrestrial mammals

We present the isotopic discrimination between paired skin and bone collagen from animals of known life history, providing a modern baseline for the interpretation of archaeological isotopic data. At present, the interpretation of inter-tissue variation (Δ (skin–bone) ) in mummified remains is based on comparisons with other archaeological material, which have attributed divergence to their contrasting turnover rates, with rapidly remodelling skin collagen incorporating alterations in environmental, cultural and physiological conditions in the months prior to death. While plausible, the lack of baseline data from individuals with known life histories has hindered evaluation of the explanations presented. Our analysis of a range of animals raised under a variety of management practices showed a population-wide trend for skin collagen to be depleted in 13 C by –0.7‰ and enriched in 15 N by +1.0‰ relative to bone collagen, even in stillborn animals. These results are intriguing and difficult to explain using current knowledge; however, on the basis of the findings reported here, we caution any results which interpret simply on differing turnover rates. We hypothesize that there may be a consistent difference in the routing of dietary protein and lipids between skin and bone, with potentially on-site synthesis of non-essential amino acids using carbon and nitrogen that have been sourced via different biochemical pathways.

Combining δ13C and δ15N from bone and dentine in marine mammal palaeoecological research: insights from toothed whales

Rationale Stable carbon (δ13C) and nitrogen (δ15N) isotope compositions of bone and dentine collagen extracted from subfossil specimens of extinct and extant mammalian species have been widely used to study the paleoecology of past populations. Due to possible systematic differences in stable isotope values between bone and dentine, dentine values can be transformed into bone-collagen equivalent using a correction factor. This approach has been applied to terrestrial species, but correction factors specifically for marine mammals are lacking. Here, we provide correction factors to transform dentine δ13C and δ15N values into bone-collagen equivalent for two toothed whale sister species: narwhal and beluga. Methods We sampled bone and tooth dentine from the skulls of 11 narwhals and 26 belugas. In narwhals, dentine was sampled from tusk and embedded tooth; in beluga, dentine was sampled from tooth. δ13C and δ15N were measured using an elemental analyzer coupled to a continuous flow isotope ratio mass spectrometer. Intraindividual bone and dentine isotopic compositions were used to calculate correction factors for each species, and to translate dentine isotopic values into bone-collagen equivalent. Results Our analysis revealed differences in δ13C and δ15N between bone and dentine. In narwhals, we found (i) lower average δ13C in bone compared with dentine from tusk and embedded tooth; (ii) no difference in dentine δ13C between tusk and embedded tooth; (iii) lower average δ15N in bone compared with dentine, with the highest values found in embedded tooth. For belugas, we also detected lower δ13C and δ15N in bone compared with tooth dentine. Conclusions Based on our analysis, we provide bone/dentine correction factors for narwhals (both at species and population level), and for belugas. The correction factors, when applied to dentine δ13C and δ15N values, enable the combined analysis of stable isotope data from bone and dentine.

Diagnosis of Bone Mineral Density Based on Backscattering Resonance Phenomenon Using Coregistered Functional Laser Photoacoustic and Ultrasonic Probes

Dual-energy X-ray absorptiometry (DXA) machines based on bone mineral density (BMD) represent the gold standard for osteoporosis diagnosis and assessment of fracture risk, but bone strength and toughness are strongly correlated with bone collagen content (CC). Early detection of osteoporosis combined with BMD and CC will provide improved predictability for avoiding fracture risk. The backscattering resonance (BR) phenomenon is present in both ultrasound (US) and photoacoustic (PA) signal transmissions through bone, and the peak frequencies of BR can be changed with BM and CC. This phenomenon can be explained by the formation of standing waves within the pores. Simulations were then conducted for the same bone µCT images and the resulting resonance frequencies were found to match those predicted using the standing wave hypothesis. Experiments were performed on the same bone sample using an 808 nm wavelength laser as the PA source and 3.5 MHz ultrasonic transducer as the US source. The backscattering resonance effect was observed in the transmitted waves. These results verify our hypothesis that the backscattering resonance phenomenon is present in both US and PA signal transmissions and can be explained using the standing waves model, which will provide a suitable method for the early detection of osteoporosis.

Asynchronous ecological upheavals on the Western Mediterranean islands: New insights on the extinction of their autochthonous small mammals

Comparative studies on extinction scenarios are an invaluable contribution to enhance our understanding of their patterns and mechanisms underpinning them. This paper presents new radiocarbon dates based on specimens of five extinct mammal species from Mallorca and Sardinia. The new evidence permits to reanalyse the extinction dynamics on both islands. Radiocarbon ages directly obtained on bone collagen from these species show evidence of different extinction patterns on Mallorca and Sardinia. For Mallorca the most reliable scenario is a mass extinction of all non-volant mammal species as an immediate consequence of the first human irruption on the island. However, for Sardinia, the extinction of autochthonous mammals lasted over several millennia. The new radiocarbon dates of the last occurrence of endemic mammals suggest a sequence of punctuated extinction events throughout the late Sardinian Holocene. These events are here tentatively related to successive human colonisation waves. The current lack of chronological dates for some Sardinian fossil mammals impedes to outline a more accurate pattern of extinction events. The present paper points that Mallorca have been more vulnerable than Sardinia to the external disturbances introduced by humans. We suggest that the capacity of each island to absorb external perturbations could be related to the island area, the duration of the isolated evolution and the degree of faunal complexity.

Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme–substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.