Collagen Samples Research Articles

Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation.

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.

Heterogeneity in proline hydroxylation of fibrillar collagens observed by mass spectrometry.

Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.

The effects of washing a collagen sample prior to TEM examination.

Transmission electron microscopy (TEM) is an important analysis technique to visualize (bio)macromolecules and their assemblies, including collagen fibers. Many protocols for TEM sample preparation of collagen involve one or more washing steps to remove excess salts from the dispersion that could hamper analysis when dried on a TEM grid. Such protocols are not standardized and washing times as well as washing solvents vary from procedure to procedure, with each research group typically having their own protocol. Here, we investigate the influence of washing with water, ethanol, but also methanol and 2‐propanol, for both mineralized and unmineralized collagen samples via a protocol based on centrifugation. Washing with water maintains the hydrated collagen structure and the characteristic banding pattern can be clearly observed. Conversely, washing with ethanol results in dehydration of the fibrils, often leading to aggregation of the fibers and a less obvious banding pattern, already within 1 min of ethanol exposure. As we show, this process is fully reversible. Similar observations were made for methanol and propanol. Based on these results, a standardized washing protocol for collagenous samples is proposed.

Subsistence shift and socio-economic response to cultural and climate changes among north-central Iberian megalithic groups

The insufficient attention traditionally paid to the complex mortuary biographies of megalithic graves has long obscured a significant amount of synchronic and diachronic information. The Rioja Alavesa region of north-central Iberia holds a number of megalithic graves with large skeletal assemblages that can generally be ordered by internal stratigraphy and/or relatively fine-grained radiocarbon chronologies, providing a rare opportunity to assess the potential of such information. Here, we undertake stable carbon and nitrogen isotope analyses of 125 human and seven animal bone collagen samples from six late prehistoric megalithic graves (La Cascaja, El Sotillo, San Martín, Alto de la Huesera, Chabola de la Hechicera and Longar) which, together with 107 previously published Late Neolithic/Early Chalcolithic (ca. 3600-2900 BC) bone collagen samples (100 humans and six animals) from the same tombs, are used to explore subsistence trends over time. This approach provides a means to address the socio-economic response of northern Iberian megalithic communities to the cultural and climatic changes occurring in late prehistory. Isotopic data are contextualized using palaeoenvironmental and osteoarchaeological information. The results show a significant shift in diet between the late Middle Neolithic and the Late Neolithic/Early Chalcolithic that coincides with marked changes in burial practices that are likely related to the transition from mainly pastoral to mixed farming economies. There may also be a change in subsistence between the Late Neolithic/Early Chalcolithic and the following periods, suggested by differences in δ15N values between the layers of the Alto de la Huesera megalithic grave, as well as in population and funerary dynamics. By contrast, an apparent continuity in subsistence is seen, at least isotopically, from the Middle Chalcolithic to the Bronze Age, despite clear changes in beliefs and socio-economic practices. These findings support the existence of substantial asynchronous changes in lifeways and ideology among Iberian megalithic groups and challenge the traditional idea of a long and uniform stability in late prehistoric northern Iberia.

Development of the technology of juice fruit and vegetable products enriched with collagen

The beauty and aging of the human body is associated with such a protein substance as collagen. Our body’s ability to produce this biologically active component naturally and in the right amount becomes limited as we age. Collagen slows down the aging process of the skin, prevents injury, promotes wound healing, improves the absorption of amino acids, can help maintain or improve the biophysical properties of the skin (elasticity, moisture), reduce transepidermal water loss and solve the problem of skin roughness. The purpose of the research work is the development of the technology, recipes for food products — fruit and vegetable juices, drinks, enriched with animal or plant collagen. As a result, the assortment and quality of different types of collagen (tomato, beef, pork and fish) were studied. The organoleptic characteristics of this food additive were studied and the most preferred collagen sample for drinks was selected. The required amount of this substance has been determined, while ensuring the required quality of the drinks. The preservation of the active properties of various types of collagen in a drinking product was investigated. It was proved that beef collagen in an amount of 5% to the mass of the drink retains its active properties as much as possible, in terms of fractional composition — salt, alcohol, alkaline protein fractions. It has been shown that the amino acids methionine, tryptophan and hydroxyproline, which confirm the presence of collagen in the product, are contained in ready-made juice drinks enriched with collagen of beef origin. This type of collagen makes it possible to obtain drinks of “potable” quality, which is confirmed by organoleptic and physicochemical indicators. In general, the studies have shown that a new assortment of juice-containing fruit and vegetable products supplies the important biologically active component (collagen) to the human body.

Characterization of Acid-Soluble Collagen from Food Processing By-Products of Snakehead Fish (Channa striata)

The isolation of acid-soluble collagen (ASC) from by-products of snakehead fish (Channa striata), including skin and the mixture of skin and scale, has been investigated. The recovery yield of fish skin ASC (13.6%) was higher than ASC from fish skin and scale (12.09%). Both ASCs were identified as type I collagen and showed maximal solubility at pH 2. Collagen samples from the mixture of skin and scale had higher imino acid content (226 residues/1000 residues) and lower wavenumber in the amide I and amide III region (1642 and 1203 cm−1, respectively) than the fish skin ASC (the imino acid content was 220 residues/1000 residues and the wavenumber in the amide I and amide III were 1663 and 1206 cm−1, respectively. The difference scanning calorimeter (DSC) showed higher thermal stability in ASC from the mixture of skin and scale (Td of 35.78 °C) than fish skin ASC (34.21 °C). From the result, the denaturation temperature of ASC had a close relationship with the content of imino acid as well as with the degradation of α-helix in amide I and III. These results suggest that collagen could be obtained effectively from snakehead fish by-products and has potential as a realistic alternative to mammalian collagens.

Childhood in the Carpathians: An isotopic analysis of childhood diet and weaning in a medieval and Early Modern Transylvanian village

In this article, we explore aspects of daily life through the lens of non-adult diet in the High and Late Medieval (1000–1500 CE) and Early Modern (1500–1800 CE) periods using skeletons from a church cemetery located in Mugeni (Bögöz in Hungarian), Romania. Carbon and nitrogen isotope analysis was conducted on dentin collagen samples from 20 non-adults as well as bone collagen from 3 non-adults. The mean dentin δ13C (−18.4‰ ± 1.7‰) and δ15N (12.5‰ ± 1.2‰) measurements reveal a variable overall non-adult diet consisting primarily of terrestrial proteins and the use of both C3 and C4 grains to varying degrees. An average decrease in nitrogen values over the non-adult life course additionally provides context on the weaning process and other dietary changes.

A global carbon and nitrogen isotope perspective on modern and ancient human diet

Stable carbon and nitrogen isotope analyses are widely used to infer diet and mobility in ancient and modern human populations, potentially providing a means to situate humans in global food webs. We collated 13,666 globally distributed analyses of ancient and modern human collagen and keratin samples. We converted all data to a common "Modern Diet Equivalent" reference frame to enable direct comparison among modern human diets, human diets prior to the advent of industrial agriculture, and the natural environment. This approach reveals a broad diet prior to industrialized agriculture and continued in modern subsistence populations, consistent with the human ability to consume opportunistically as extreme omnivores within complex natural food webs and across multiple trophic levels in every terrestrial and many marine ecosystems on the planet. In stark contrast, isotope dietary breadth across modern nonsubsistence populations has compressed by two-thirds as a result of the rise of industrialized agriculture and animal husbandry practices and the globalization of food distribution networks.

Contribution of Quasifibrillar Properties of Collagen Hydrolysates Towards Lowering of Interface Tension in Emulsion-Based Food Leading to Shelf-Life Enhancement

This study was conducted to decipher the mechanism of emulsion-based food stabilization by fish-derived collagen hydrolysate. Collagen type I was isolated from seven fish processing by-products with yields ranging from 9.15 to 92.38%. The isolated samples had a mass of 110–120kDa and eluted at 30.44% NaCl in ion-exchange chromatography. The collagen samples were enzymatically digested to obtain collagen hydrolysate (CH) with mass <6kDa. The seven CH samples were subsequently screened for surface activity. CH obtained from Pacu skin and Tilapia bones exhibited more than 80% solubility over a wide pH range, zeta potential greater than +50mV, moisture retention up to 92–96%, emulsification activity of 53–70 m2g−1, and emulsion stability of 62–85min, and successfully increased the emulsion stability of a drug by 14 times. Both CH samples were able to retain the emulsion properties of butter and chocolate sauce for 25 weeks and did not show any cytotoxic effect on leukocytes and Vero cells. Structural studies revealed that the CH peptides existed in polyproline-II conformation that assembled to form a vast quasifibrillar network. Sequence analysis through tandem mass spectroscopy revealed that the peptides could be classified into multiple groups depending on the distribution of hydrophilic/lipophilic residues (H/L). The surface activity of the CH was found to be dependent on (1) small size and a wide array of H/L ratio, (2) abundance of hydroxyproline, and (3) assembly of the peptides in the emulsion interface to form a mimic-helix–based quasifibrillar network which ensured optimal orientation and subsequent interaction with multiple phases.

Initial Studies of Electron Beams as a Means of Modifying Collagen

We present the initial design studies and specifications for an accelerator and conveyor system to irradiate collagen samples, modifying properties such as the putrescibility and mechanical behaviours in a paradigm shift from existing, widely used technology. We show the integrated design requirements for a magnetic rastering scheme to move the beam position in order to ensure a uniform dose distribution over the full surface of the hide and discuss its dependence on factors such as the size of the hide, the beam current and conveyor speed. We also present initial energy deposition studies using beam particle interaction simulation program G4beamline, in order to determine the numerical beam parameters and angle of incidence needed to ensure a uniform depth-dose distribution throughout the hide thickness.

Human and hutia (Isolobodon portoricensis) interactions in pre-Columbian Hispaniola: The isotopic and morphological evidence

There are certain animal candidates for which researchers have long speculated as having been managed, or possibly domesticated, by indigenous peoples in the pre-Columbian Caribbean, the primary candidate being some members of a group of caviomorph rodents known as hutia (Capromyinae). This study comprises an isotopic and morphological investigation of the potential management of an extinct species endemic to Hispaniola, the Puerto Rican hutia (Isolobodon portoricensis). For comparisons, isotopic analysis was conducted of bone collagen samples examining carbon (∂13Cco) and nitrogen (∂15N) values of I. portoricensis with two other species of endemic hutia, guinea pig (Cavia porcellus), Antillean slider (Trachemys stejnegeri), rhinoceros iguana (Cyclura cornuta) and edible rat (Brotomys sp.) from four sites in the Dominican Republic: El Flaco, El Carril, El Cabo and La Entrada. This data was compared to human and dog collagen values available from El Flaco to assess similarities between these groups. Osteometric data was recorded for hutia mandibles, allowing for body mass estimations (n = 230). The findings suggest that some Isolobodon portoricensis specimens possessed carbon (δ13Cco) values similar to humans and dogs at El Flaco, possibly associated with the consumption of agriculturally produced maize. This research does not indicate whether domestication or management of this species was occurring, at least not in a systematic way. Concurrence with palaeoenvironmental and zooarchaeological data from the region in which El Flaco and El Carril are located indicate that indigenous agricultural practices may have affected populations of hutia, perhaps by attracting them and supporting them within anthropogenic mosaic landscapes. The data also suggests that some degree of either purposeful feeding or scavenging from human agricultural plots was occurring and supports some degree of commensalism between humans and Puerto Rican hutia at these sites.

Reevaluating the timing of Neanderthal disappearance in Northwest Europe

Elucidating when Neanderthal populations disappeared from Eurasia is a key question in paleoanthropology, and Belgium is one of the key regions for studying the Middle to Upper Paleolithic transition. Previous radiocarbon dating placed the Spy Neanderthals among the latest surviving Neanderthals in Northwest Europe with reported dates as young as 23,880 ± 240 B.P. (OxA-8912). Questions were raised, however, regarding the reliability of these dates. Soil contamination and carbon-based conservation products are known to cause problems during the radiocarbon dating of bulk collagen samples. Employing a compound-specific approach that is today the most efficient in removing contamination and ancient genomic analysis, we demonstrate here that previous dates produced on Neanderthal specimens from Spy were inaccurately young by up to 10,000 y due to the presence of unremoved contamination. Our compound-specific radiocarbon dates on the Neanderthals from Spy and those from Engis and Fonds-de-Forêt demonstrate that they disappeared from Northwest Europe at 44,200 to 40,600 cal B.P. (at 95.4% probability), much earlier than previously suggested. Our data contribute significantly to refining models for Neanderthal disappearance in Europe and, more broadly, show that chronometric models regarding the appearance or disappearance of animal or hominin groups should be based only on radiocarbon dates obtained using robust pretreatment methods.

Determination of glycosaminoglycans in biological matrices using a simple and sensitive reversed-phase HPLC method with fluorescent detection

This paper suggests a sensitive reversed-phase gradient HPLC method combined with fluorescence detection that has been developed, optimized and tested via the quantitative analysis of authentic biological material in an effort to determine and subsequently compare the total content of glycosaminoglycans (GAGs) in various collagen-based biomaterials intended for medical application.The proposed analytical method enabled the identification and separation of the GAGs present from the other components in the samples using commonly-available laboratory equipment; moreover, the very low detection limit of the method permits the determination of GAGs even for very small samples.This study describes the development of the method, including the isolation and processing of the collagen samples prior to HPLC analysis and the optimal parameters applied during the chromatographic analysis. The application of the method in laboratory practice is documented by means of several examples of the determination of GAGs employing both commercial standards and real collagen samples isolated from various animal tissues.

Bioactivation of Bovine Bone Matrix and Collagen Scaffold Using Argon Plasma: In Vitro Study.

Bone graft materials and soft tissue allografts are widely used in clinical practice to counteract physiologic postextraction site tridimensional shrinkage. The aim of this study was to test if plasma of argon treatment could have a bioactivation effect on hard and soft tissue scaffolds in clinical usage. Forty-eight bovine bone matrix and porcine collagen samples were subdivided into two groups (test and control) of 12 samples each. The test group was treated with argon plasma (10 W, 1 bar for 12 minutes), while the control group was left untreated. Immediate cell adhesion and a proliferation assay at 72 hours were performed in the perfusion chamber of a bioreactor. Additionally, micro-CT analysis was performed on the treated and untreated scaffolds, before and after soaking in cell culture medium (four samples). Osteoblasts seeded on plasma-treated bone matrix significantly increased the adhesion level compared with the untreated sample (43,144.3 ± 12,442.9 vs 21,736 ± 77,27.1; P = .0083). However, 3-day proliferation tests could not achieve significant differences between groups (105,715.5 ± 21,751.5 vs 107,108.6 ± 19,343.4; P = .998). No differences were measured on fibroblast adhesion on the collagen matrix in both conditions. Plasma of argon treatment and soaking in cell culture medium did not affect the bone matrix samples. The structure of collagen matrix samples was unaltered after plasma treatment, but became enlarged after soaking. Plasma of argon may be useful to biofunctionalize bone grafts, although benefits seemed to disappear after 3 days. No biologic response was detected on collagen matrix scaffolds. In vivo studies are needed to draw final clinical conclusions.

New Record of Terminal Pleistocene Elk/Wapiti (Cervus canadensis) from Ohio, USA

The earliest appearance of elk/wapiti (Cervus canadensis) in eastern North America is not thoroughly documented due to the small number of directly dated remains. Until recently, no absolute dates on elk bone older than 10,000 14C yr BP (11,621 to 11,306 calibrated years (cal yr) BP) were known from this region. The partial skeleton of the Tope Elk was discovered in 2017 during commercial excavation of peat deposits from a small bog in southeastern Medina County, Ohio, United States. Subsequent examination of the remains revealed the individual to be a robust male approximately 8.5 years old at death. The large size of this individual is compared with late Holocene specimens and suggests diminution of elk since the late Pleistocene. Two accelerator mass spectrometry (AMS) radiocarbon assays on bone collagen samples taken from the scapula and metacarpal of this individual returned ages of 10,270 ± 30 14C yr BP (Beta-477478) (12,154 to 11,835 cal yr BP) and 10,260 ± 30 14C yr BP (Beta-521748) (12,144 to 11,830 cal yr BP), respectively. These results place Cervus canadensis in the terminal Pleistocene of the eastern woodlands and near the establishment of the mixed deciduous forest biome over much of the region. This early temporal placement also situates this early elk as closely following the last representatives of now-extinct megafauna such as the American mastodon (Mammut americanum) and contemporary with the Late Paleoindian inhabitants of the region.

A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes.

To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at3:1 and 9:1 in which the structural and mechanical behavior of theresident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:ColIII was tested. Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters ofthe gels made with varying ratios of Col I:ColIII. SecondHarmonic Generation (SHG) was performed to observe the 3D characterization of thecollagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of thelyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay andthe applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample. Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases. This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.

Coronavirus disease 2019 and coagulopathy: other prothrombotic coagulation factors.

There is an increasing evidence supporting the existence of coagulopathy in coronavirus disease 2019 (COVID-19) patients. Most of reports are mainly focused on d-dimer. Our objective is to describe coagulation parameters in these patients that could be involved in a hypercoagulate state and to test platelet function to see if there are short closure times. We analyzed coagulation samples from 80 patients admitted with COVID-19 in our hospital. We also tested platelet function by closure times in a small subgroup of patients. Most of samples had increased d-dimer (96.2%) (median of d-dimer: 1158 ng/ml FEU), increased fibrinogen (75.2%) (median: 5.23 g/l), increased factor VIII (86%) (median: 264.8 U/dl), decreased protein S (22.5% of women, 62.5% of men) (median: 62.8 and 68.5 U/dl, respectively), decreased protein C (7.6%) (median: 100 U/dl), decreased factor XII (25.3%) (median: 90.3 U/dl) and decreased antithrombin activity (21%) (median: 86 U/dl). International normalized ratio was higher than normal in 24 patients (30%) (median: 1.13). The activated partial thromboplastin time ratio was below the normal range in nine patients (11.2%) and above normal in three (3.75%) (median: 0.93). The closure times were short in the 20% and 40% of samples of collagen and ADP and collagen and epinephrine, respectively. Twelve of the 80 patients (15%) had a thrombotic event and all had several abnormal coagulation parameters related with increased thrombotic risk. The results of this study support a hypercoagulability state in COVID-19 patients and it may help to explain the microvascular thrombosis caused by the inflammatory response.

In depth investigation of collagen non-enzymatic glycation by Raman spectroscopy

Non-enzymatic glycation is a post-translational modification of long-lived matrix proteins such as type I collagen. It occurs during aging and leads to the formation of advanced glycation end-products (AGEs). AGE accumulation is associated with severe complications in chronic and age-related diseases. The assessment of modifications induced by this (patho)physiological process represents an interest in biology and medicine for a better patient care. The objective of our work was to position the interest of Raman spectroscopy in the quantification of collagen glycation. Two types of in vitro glycation were used by incubating collagen samples, at different durations, with ribose or glyoxylic acid; these reducing agents acting on the chemical specificity of the glycation reaction. Glycation efficiency was evaluated by the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) quantification of carboxymethyllysine (CML) and pentosidine, which are among the most studied AGEs. Raman data were processed by PCA coupled to validity indices and Lasso regression as multivariate analysis tools. Regression models were constructed by considering the LC-MS/MS results as reference values. A marked variability was observed within the Raman datasets making difficult the identification of spectral differences between control and ribose-treated collagen samples. By taking advantage of the chemical specificity of the glyoxylic acid treatment leading to CML formation, on one hand, and the feature selection included in the Lasso algorithm, on the other hand, Raman markers associated with glycation were identified. The assigned vibrations corresponded to modifications of side chains of collagen. In addition, a threshold of CML concentration was determined as quantitative indicator of the applicability of Raman spectroscopy for potential patient follow-up purposes. Although lacking in sensitivity to directly detect AGEs in collagen, Raman spectroscopy allows to highlight the molecular modifications of collagen induced by glycation.

Evidence of a significant marine plant diet in a Pleistocene caribou from Haida Gwaii, British Columbia, through compound-specific stable isotope analysis

The Northwest Coast of Canada was likely an important location for glacial refugia in the Late Pleistocene. Recently, a caribou (Rangifer tarandus) antler fragment was reported from Graham Island dating prior to the Fraser Glaciation (MIS 3; 48,200–45,200 cal yr BP). The high carbon and nitrogen stable isotope values found in the bulk collagen samples of this caribou prompted further investigation into its diet and palaeoecological context. Today, caribou in British Columbia face rapid population and range decline, and have been placed on threatened and endangered species lists by both the U.S. and Canadian governments. Further insights into this species and its responses to changing environmental conditions can both improve our understanding of the Late Pleistocene ecosystems of the Northwest Coast and add relevant data to modern conservation issues. We employed compound-specific carbon and nitrogen stable isotope analysis to the individual amino acids of antler collagen to better understand the isotopic signal of this specimen. Our results suggest a significant marine plant contribution to the caribou's spring–summer diet, potentially reflecting an intensive foraging behavior observed in some modern-day Rangifer tarandus populations under conditions of terrestrial resource restriction.

Self-assembly study of type I collagen extracted from male Wistar Hannover rat tail tendons

BackgroundCollagen, the most abundant protein in the animal kingdom, represents a promising biomaterial for regenerative medicine applications due to its structural diversity and self-assembling complexity. Despite collagen’s widely known structural and functional features, the thermodynamics behind its fibrillogenic self-assembling process is still to be fully understood. In this work we report on a series of spectroscopic, mechanical, morphological and thermodynamic characterizations of high purity type I collagen (with a D-pattern of 65 nm) extracted from Wistar Hannover rat tail. Our herein reported results can be of help to elucidate differences in self-assembly states of proteins using ITC to improve the design of energy responsive and dynamic materials for applications in tissue engineering and regenerative medicine.MethodsHerein we report the systematic study on the self-assembling fibrillogenesis mechanism of type I collagen, we provide morphological and thermodynamic evidence associated to different self-assembly events using ITC titrations. We provide thorough characterization of the effect of pH, effect of salts and protein conformation on self-assembled collagen samples via several complementary biophysical techniques, including circular dichroism (CD), Fourier Transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), atomic force microscopy (AFM), scanning electron microscopy (SEM), dynamic mechanical thermal analysis (DMTA) and thermogravimetric analysis (TGA).ResultsEmphasis was made on the use of isothermal titration calorimetry (ITC) for the thermodynamic monitoring of fibrillogenesis stages of the protein. An overall self-assembly enthalpy value of 3.27 ± 0.85 J/mol was found. Different stages of the self-assembly mechanism were identified, initial stages take place at pH values lower than the protein isoelectric point (pI), however, higher energy release events were recorded at collagen’s pI. Denatured collagen employed as a control exhibited higher energy absorption at its pI, suggesting different energy exchange mechanisms as a consequence of different aggregation routes.Graphical abstract