Collagen Amino Acid Research Articles

Stable isotopes of H, C and N in mice bone collagen as a reflection of isotopically controlled food and water intake

ABSTRACT2H/1H ratios in animal biomass reflect isotopic input from food and water. A 10-week controlled laboratory study raised 48 mice divided in two generations (8 mothers Mus musculus and their offspring). The mice were divided into four groups based on the combination of 2H, 13C, 15N-enriched and non-enriched food and water. Glycine, the most common amino acid in bone collagen, carried the 2H, 13C, 15N-isotopic spike in food. ANOVA data analysis indicated that hydrogen in food accounted for ∼81 % of the hydrogen isotope inventory in collagen whereas drinking water hydrogen contributed ∼17 %. Air humidity contributed an unspecified amount. Additionally, we monitored 13C and 15N-enrichment in bone collagen and found strong linear correlations with the 2H-enrichment. The experiments with food and water indicate two biosynthetic pathways, namely (i) de novo creation of non-essential amino acids using hydrogen from water, and (ii) the integration of essential and non-essential amino acids from food. The lower rate of isotope uptake in mothers’ collagen relative to their offspring indicates incomplete bone collagen turnover after ten weeks. The variance of hydrogen stable isotope ratios within the same cohort may limit its usefulness as a single sample proxy for archaeological or palaeoenvironmental research.

Exceptionally high δ15N values in collagen single amino acids confirm Neandertals as high-trophic level carnivores

Isotope and archeological analyses of Paleolithic food webs have suggested that Neandertal subsistence relied mainly on the consumption of large herbivores. This conclusion was primarily based on elevated nitrogen isotope ratios in Neandertal bone collagen and has been significantly debated. This discussion relies on the observation that similar high nitrogen isotopes values could also be the result of the consumption of mammoths, young animals, putrid meat, cooked food, freshwater fish, carnivores, or mushrooms. Recently, compound-specific C and N isotope analyses of bone collagen amino acids have been demonstrated to add significantly more information about trophic levels and aquatic food consumption. We undertook single amino acid C and N isotope analysis on two Neandertals, which were characterized by exceptionally high N isotope ratios in their bulk bone or tooth collagen. We report here both C and N isotope ratios on single amino acids of collagen samples for these two Neandertals and associated fauna. The samples come from two sites dating to the Middle to Upper Paleolithic transition period (Les Cottés and Grotte du Renne, France). Our results reinforce the interpretation of Neandertal dietary adaptations as successful top-level carnivores, even after the arrival of modern humans in Europe. They also demonstrate that high δ15N values of bone collagen can solely be explained by mammal meat consumption, as supported by archeological and zooarcheological evidence, without necessarily invoking explanations including the processing of food (cooking, fermenting), the consumption of mammoths or young mammals, or additional (freshwater fish, mushrooms) dietary protein sources.

Fish Collagen – Molecular Structure After Thermal Treatment

Modern medicine widely uses exogenous collagen as a good material for tissue regeneration, also as a natural substrate for cell attachment and proliferation, used to create dressings and to support the treatment of burn and diabetic wounds, or finally as a source of amino acids in the form of a dietary supplement. Collagen is a safe material that has high biocompatibility and biodegradability as well as good cell adhesion. Due to the possibility of transferring Creutzfeld-Jacob’s disease (Bovine Spongiform Encephalopathy) from animals to the human body, interest in collagen from fish is currently increasing. The collagen we examined was derived from the skin of the silver carp fish (Hypophtalmichthys molitrix) and was obtained by the method of hydration in an aqueous lactic acid solution. The topography of the test sample was performed with the AFM method, showing its fibrillar structure with dimensions equivalent to those given in iterature. Raman spectroscopy was used to study fish collagen using a Renishaw Ramanscope with a helium-neon laser at a wavelength of 633 nm. Analysis of Raman spectra allowed to determine the content of amino acids in collagen, as well as glycine, proline and hydroxyproline. It also showed the native nature of the material at 20 °C. The partial renaturation of the secondary structure of this material heated to about 85 °C and cooled was also proved. Raman spectroscopy has been presented as an effective method for testing biopolymers.

Comparative evaluation between two nutritional supplements in the improvement of telogen effluvium.

PurposeTelogen effluvium (TE) is defined as a diffuse hair loss characterized by shortening of the anagen phase and precipitation of the telogen phase, with a consequent reduction of total hair volume. Nutritional supplementation is indicated under conditions in which TE is related to dietary disorders. The aim of this clinical study was to compare the efficacy of two different supplements in monotherapy for TE.Patients and methodsFemale adult patients were randomized to receive two oral nutritional supplements (group 1: a supplement composed of zinc, biotin, iron, vitamins A, C, E, and B complex, folic acid, magnesium, and amino acids of keratin and collagen and group 2: calcium pantothenate cystine, thiamine nitrate, medicinal yeast, keratin, and aminobenzoic acid) to treat hair loss for 180 days. They were evaluated clinically and by digital trichoscopy.ResultsClinical evaluation showed significant clinical improvement (P<0.05) for the evaluated parameters: hair loss, hair volume, density of hair (scalp cover), hair shine, hair strength, in 180 days. At 90 days evaluation, group 1 showed significant improvement for all parameters, while group 2 did not show any significant improvement for hair shine and hair strength. In the digital trichoscopy, there was a significant improvement only in group 1 (11.09%×7.76%) after 180 days.ConclusionIn idiopathic TE, the nutritional component should be suspected; the supplementation of an association of nutrients in recommended daily intake can lead to significant improvement of the condition from the first trimester of use. The use of an association with proven efficacy and a safety profile and posologic convenience facilitate its indication and patient adherence.

Cyclodextrin Modulated Type I Collagen Self-Assembly to Engineer Biomimetic Cornea Implants.

Collagen-rich tissues in the cornea exhibit unique and highly organized extracellular matrix ultrastructures, which contribute to its high load-bearing capacity and light transmittance. Corneal collagen fibrils are controlled during development by small leucine-rich proteoglycans (SLRPs) that regulate the fibril diameter and spacing in order to achieve the unique optical transparency. Cyclodextrins (CDs) of varying size and chemical functionality for their ability to regulate collagen assembly during vitrification process are screened in order to create biosynthetic materials that mimic the native cornea structure. Addition of βCD to collagen vitrigels produces materials with aligned fibers and lamellae similar to native cornea, resulting in mechanically robust and transparent materials. Biochemistry analysis revealed that CD interacts with hydrophobic amino acids in collagen to influence assembly and fibril organization. To translate the self-assembled collagen materials for cornea reconstruction, custom molds for gelation and vitrification are engineered to create βCD/Col implants with curvature matching that of the cornea. Acellular βCD/Col materials are implanted in a rabbit partial keratoplasty model with interrupted sutures. The implants demonstrate tissue integration and support re-epithelialization. Therefore, the addition of CD molecules regulates collagen self-assembly and provides a simple process to engineer corneal mimetic substitutes with advanced structural and functional properties.

Isotopic insights on cave bear palaeodiet

More than 300 cave bear bones from all over Europe have carbon and nitrogen isotopic composition that match overwhelmingly a diet based on plants, except for samples from two caves in Romania, for which high nitrogen-15 amounts have been interpreted as reflecting an omnivorous diet. This paper aims at deciphering the various factors influencing the carbon and nitrogen isotopic composition of a potential omnivorous species like cave bear, those linked to trophic levels and variations among plants and those caused by physiological factors. The comparison of European cave bears with coeval Late Pleistocene large mammals with different diets clearly shows that all the cave bear populations, including those from Romania, present isotopic values overlapping with herbivores, not with carnivores. Therefore omnivory is very unlikely for cave bears. Consumption of plants with high δ15N values, such as graminoids, forbs and possibly fungi, could explain in part the observed isotopic pattern. In addition, the variations in δ15N values through ontogeny support the hypothesis of a different hibernation pattern for the Romanian cave bears with high δ15N values. Future investigations using new isotopic approaches, especially nitrogen isotopic composition of collagen amino acids, should contribute to decipher the paleoecology of these Romanian cave bears.

Surface treatment with amino acids of porous collagen based scaffolds to improve cell adhesion and proliferation

AbstractThe purpose of this study was to improve the biocompatibility of glutaraldehyde (GA) cross‐linked chitosan coated collagen scaffold for cartilage tissue regeneration. In order to prevent the potential toxicity of GA, we treated the designed scaffold with either glutamic acid or glycine. Amino acid treated scaffolds were characterized by scanning electron microscopy (SEM) techniques. Afterward, chondrocyte interaction with the composite scaffold was investigated assessing cell adhesion and proliferation using Hoechst staining and MTT cell proliferation assay, respectively. The SEM analyses of the scaffolds’ surface and cross‐section confirmed the adhesion of amino acids on the surface of the scaffolds. We also observed that scaffolds’ porosity was reduced due to the coverage of the pores by chitosan and amino acids, leading to low porosity. The use of amino acid improved the chondrocyte adhesion and proliferation inside the scaffolds’ pores when cells were cultured onto the chitosan‐coated collagen scaffolds. Overall, our in vitro results suggest the use of amino acid to improve the biocompatibility of natural polymer composite scaffold being crosslinked with glutaraldehyde. Such scaffold has improved mechanical properties; biocompatibility thus may be useful for tissue regeneration such as cartilage.

Hydroxylysine deficiency, conspicuous skeletal lesions and a strange burial practice in a historical German island population

AbstractEighty‐two complete and partial human skeletons were excavated from a burial site on the island of Borkum in the North Sea. The site covers a period of 400 years from the middle of the 14th century AD until 1720 AD. The position of the skeletons in situ suggests rapid disposal of the bodies. About 75% of the skeletons were male and of a quite robust constitution, and about 60% of all individuals died rather young between 20 and 40 years of age. All male skeletons exhibited particular pathological alterations as a result of the typical daily activities of seafaring men, including severe degenerative joint disease and pronounced osteochondropathies. In juvenile skeletons, these pathologies were accompanied by inflammatory processes without exception. Palaeodietary reconstruction by stable isotope analysis resulted in a protein rich diet, confirming fishing as an important part of the subsistence economy. The juveniles derived substantially more protein from plant food than the adults. Bone collagen amino acid profiles were short of hydroxylysine, suggesting that the conspicuous pathological lesions could also have been caused by a systemic connective tissue disease. Possible etiology of the disease is discussed and related to both the uneven sex ratio of the inhumations and the burial context.

The Effects of Local and Systemic Administration of Proline on Wound Healing in Rats

ABSTRACTPurpose: Wound healing consists of a sequence of complex molecular and cellular events. Collagen is composed mainly of proline and hydroxyproline. Proline and hydroxyproline constitute 1/3 of the amino acids in collagen, which makes up approximately 30% of the proteins within the body. The hydroxylation of proline found in collagen determines the stability of the triple helical structure of collagen. In this study, we examined the effects of local and systemic administration of proline on wound healing. Materials and Methods: 24 female Sprague-Dawley rats were used in the study and divided into three groups. Group 1: The defect created in the backs of the subjects was left to secondary healing. Group 2: 200 µl proline per day was administered topically for 30 days on the defect in the backs of the subjects. Group 3: 200 µl per day was administered intraperitoneally for 30 days on the defect in the backs of the subjects. Results: On day 21, there was a statistically significant difference between the groups in terms of the mean re-epithelialization score. On days 7 and 14, there was a statistically significant difference between the groups in terms of the mean granulation score. On days 7, 14, and 21, there was a statistically significant difference between the groups in terms of the mean collagen accumulation score. On day 30, there was a statistically significant difference between Groups 1 and 3 in terms of the mean E-mode score on mechanical tensile test. Conclusion: Our study confirmed that proline has positive effects on wound healing. However, it revealed that systemic administration of proline is more effective than local administration of proline.

Calling all archaeologists: guidelines for terminology, methodology, data handling, and reporting when undertaking and reviewing stable isotope applications in archaeology.

Stable isotope analysis has been utilized in archaeology since the 1970s, yet standardized protocols for terminology, sampling, pretreatment evaluation, calibration, quality assurance and control, data presentation, and graphical or statistical treatment still remain lacking in archaeological applications. Here, we present recommendations and requirements for each of these in the archaeological context of: bulk stable carbon and nitrogen isotope analysis of organics; bulk stable carbon and oxygen isotope analysis of carbonates; single compound stable carbon and nitrogen isotope analysis on amino acids in collagen and keratin; and single compound stable carbon and hydrogen isotope analysis on fatty acids. The protocols are based on recommendations from the Commission on Isotopic Abundances and Atomic Weights of the International Union of Pure and Applied Chemistry (IUPAC) as well as an expanding geochemical and archaeological science experimental literature. We hope that this will provide a useful future reference for authors and reviewers engaging with the growing number of stable isotope applications and datasets in archaeology.

Reply to “Comment on “Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen.” [J. Hum. Evol. 93 (2016) 82–90]” [J. Hum. Evol. 117 (2018) 53–55

Evidence for plants rarely survives on Paleolithic sites, while animal bones and biomolecular analyses suggest animal produce was important to hominin populations, leading to the perspective that Neanderthals had a very-high-protein diet. But although individual and short-term survival is possible on a relatively low-carbohydrate diet, populations are unlikely to have thrived and reproduced without plants and the carbohydrates they provide. Today, nutritional guidelines recommend that around half the diet should be carbohydrate, while low intake is considered to compromise physical performance and successful reproduction. This is likely to have been the same for Paleolithic populations, highlighting an anomaly in that the basic physiological recommendations do not match the extensive archaeological evidence. Neanderthals had large, energy-expensive brains and led physically active lifestyles, suggesting that for optimal health they would have required high amounts of carbohydrates. To address this anomaly, we begin by outlining the essential role of carbohydrates in the human reproduction cycle and the brain and the effects on physical performance. We then evaluate the evidence for resource availability and the archaeological evidence for Neanderthal diet and investigate three ways that the anomaly between the archaeological evidence and the hypothetical dietary requirements might be explained. First, Neanderthals may have had an as yet unidentified genetic adaptation to an alternative physiological method to spare blood glucose and glycogen reserves for essential purposes. Second, they may have existed on a less-than-optimum diet and survived rather than thrived. Third, the methods used in dietary reconstruction could mask a complex combination of dietary plant and animal proportions. We end by proposing that analyses of Paleolithic diet and subsistence strategies need to be grounded in the minimum recommendations throughout the life course and that this provides a context for interpretation of the archaeological evidence from the behavioral and environmental perspectives.

Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth.

Glycine, proline, and hydroxyproline (Hyp) contribute to 57% of total amino acids (AAs) in collagen, which accounts for one-third of proteins in animals. As the most abundant protein in the body, collagen is essential to maintain the normal structure and strength of connective tissue, such as bones, skin, cartilage, and blood vessels. Mammals, birds, and fish can synthesize: (1) glycine from threonine, serine, choline, and Hyp; (2) proline from arginine; and (3) Hyp from proline residues in collagen, in a cell- and tissue-specific manner. In addition, livestock (e.g., pigs, cattle, and sheep) produces proline from glutamine and glutamate in the small intestine, but this pathway is absent from birds and possibly most fish species. Results of the recent studies indicate that endogenous synthesis of glycine, proline, and Hyp is inadequate for maximal growth, collagen production, or feed efficiency in pigs, chickens, and fish. Although glycine, proline and Hyp, and gelatin can be used as feed additives in animal diets, these ingredients except for glycine are relatively expensive, which precludes their inclusion in practical rations. Alternatively, hydrolyzed feather meal (HFM), which contains 9% glycine, 5% Hyp, and 12% proline, holds great promise as a low cost but abundant dietary source of glycine, Hyp, and proline for ruminants and nonruminants. Because HFM is deficient in most AAs, future research efforts should be directed at improving the bioavailability of its AAs and the balance of AAs in HFM-supplemented diets. Finally, HFM may be used as a feed additive to prevent or ameliorate connective tissue disorders in domestic and aquatic animals.

Comment on “Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen” [J. Hum. Evol. 93 (2016) 82–90

Comment on “Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen” [J. Hum. Evol. 93 (2016) 82–90

Collagen from the Marine Sponges Axinella cannabina and Suberites carnosus: Isolation and Morphological, Biochemical, and Biophysical Characterization.

In search of alternative and safer sources of collagen for biomedical applications, the marine demosponges Axinella cannabina and Suberites carnosus, collected from the Aegean and the Ionian Seas, respectively, were comparatively studied for their insoluble collagen, intercellular collagen, and spongin-like collagen content. The isolated collagenous materials were morphologically, physicochemically, and biophysically characterized. Using scanning electron microscopy and transmission electron microscopy the fibrous morphology of the isolated collagens was confirmed, whereas the amino acid analysis, in conjunction with infrared spectroscopy studies, verified the characteristic for the collagen amino acid profile and its secondary structure. Furthermore, the isoelectric point and thermal behavior were determined by titration and differential scanning calorimetry, in combination with circular dichroism spectroscopic studies, respectively.

Broad-scale trophic shift in the pelagic North Pacific revealed by an oceanic seabird.

Human-induced ecological change in the open oceans appears to be accelerating. Fisheries, climate change and elevated nutrient inputs are variously blamed, at least in part, for altering oceanic ecosystems. Yet it is challenging to assess the extent of anthropogenic change in the open oceans, where historical records of ecological conditions are sparse, and the geographical scale is immense. We developed millennial-scale amino acid nitrogen isotope records preserved in ancient animal remains to understand changes in food web structure and nutrient regimes in the oceanic realm of the North Pacific Ocean (NPO). Our millennial-scale isotope records of amino acids in bone collagen in a wide-ranging oceanic seabird, the Hawaiian petrel (Pterodroma sandwichensis), showed that trophic level declined over time. The amino acid records do not support a broad-scale increase in nitrogen fixation in the North Pacific subtropical gyre, rejecting an earlier interpretation based on bulk and amino acid specific δ15N chronologies for Hawaiian deep-sea corals and bulk δ15N chronologies for the Hawaiian petrel. Rather, our work suggests that the food web structure in the NPO has shifted at a broad geographical scale, a phenomenon potentially related to industrial fishing.

Dietary protein content and tissue type control 13 C discrimination in mammals: an analytical approach.

RationaleCarbon isotope measurements on collagen and other tissues are used in modern and paleodiet studies, and relating these patterns to the dietary protein fraction (fdp) and to δ13C values of dietary protein and energy (lipids and carbohydrates) remains important for determining diets.MethodsCarbon fluxes from dietary protein to tissues in feeding studies of mice, rats, and pigs were estimated to test how tissue δ13C values related to the δ13C values of dietary protein, dietary energy, fdp, or loge fdp. Values of fdp, protein δ13C, and energy δ13C were used in stepwise regressions that estimated the metabolic routing of dietary protein and the 13C enrichment of tissues relative to dietary protein.ResultsThe 13C enrichment of rat collagen and mouse muscle relative to dietary protein depended on loge fdp and the 13C enrichment of dietary protein on energy: urn:x-wiley:09514198:media:rcm7830:rcm7830-math-0001 The collagen (fcp) or muscle (fmp) fractions derived from dietary protein were estimated as: urn:x-wiley:09514198:media:rcm7830:rcm7830-math-0002 The coefficients of loge fdp reflected the higher proportion of non‐essential amino acids in collagen (79%) than in muscle (61%). In rats, dietary protein contributed from 66% (liver) to 85% (hair) whereas, in mice, dietary protein contributed from 76% (liver) to 85% (body protein). In pigs, collagen derived primarily (83 ± 5%) from dietary protein.ConclusionsThese equations provided analytical frameworks to interpret dietary patterns from isotopic measurements. Metabolic routing and 13C enrichment varied with fdp and with the tissue amino acid composition. Thus, fdp and tissue amino acid compositions were key parameters to assess dietary sources from isotopic values. Incorporating these insights will improve our ability to interpret the isotopic paleorecord and modern diet studies. Copyright © 2017 John Wiley &amp; Sons, Ltd.

Dietary 2-oxoglutarate prevents bone loss caused by neonatal treatment with maximal dexamethasone dose.

Synthetic glucocorticoids (GCs) are widely used in the variety of dosages for treatment of premature infants with chronic lung disease, respiratory distress syndrome, allergies, asthma, and other inflammatory and autoimmune conditions. Yet, adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Conversely, 2-oxoglutarate (2-Ox), a precursor of glutamine, glutamate, and collagen amino acids, exerts protective effects on bone development. Our aim was to elucidate the effect of dietary administered 2-Ox on bone loss caused by neonatal treatment with clinically relevant maximal therapeutic dexamethasone (Dex) dose. Long bones of neonatal female piglets receiving Dex, Dex+2-Ox, or untreated were examined through measurements of mechanical properties, density, mineralization, geometry, histomorphometry, and histology. Selected hormones, bone turnover, and growth markers were also analyzed. Neonatal administration of clinically relevant maximal dose of Dex alone led to over 30% decrease in bone mass and the ultimate strength ( P < 0.001 for all). The length (13 and 7% for femur and humerus, respectively) and other geometrical parameters (13-45%) decreased compared to the control ( P < 0.001 for all). Dex impaired bone growth and caused hormonal imbalance. Dietary 2-Ox prevented Dex influence and vast majority of assessed bone parameters were restored almost to the control level. Piglets receiving 2-Ox had heavier, denser, and stronger bones; higher levels of growth hormone and osteocalcin concentration; and preserved microarchitecture of trabecular bone compared to the Dex group. 2-Ox administered postnatally had a potential to maintain bone structure of animals simultaneously treated with maximal therapeutic doses of Dex, which, in our opinion, may open up a new opportunity in developing combined treatment for children treated with GCs. Impact statement The present study has showed, for the first time, that dietary 2-oxoglutarate (2-Ox) administered postnatally has a potential to improve/maintain bone structure of animals simultaneously treated with maximal therapeutic doses of dexamethasone (Dex). It may open the new direction in searching and developing combined treatment for children treated with glucocorticoids (GCs) since growing group of children is exposed to synthetic GCs and adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Currently proposed combined therapies have numerous side effects. Thus, this study proposed a new direction in combined therapies utilizing dietary supplementation with glutamine derivative. Impairment caused by Dex in presented long bones animal model was prevented by dietary supplementation with 2-Ox and vast majority of assessed bone parameters were restored almost to the control level. These results support previous thesis on the regulatory mechanism of nutrient utilization regulated by glutamine derivatives and enrich the nutritional science.

An exceptionally well-preserved skeleton of Thomashuxleya externa (Mammalia, Notoungulata), from the Eocene of Patagonia, Argentina

We describe one of the oldest notoungulate skeletons with associated craniodental and postcranial elements: Thomashuxleya externa (Isotemnidae) from Canadon Vaca in Patagonia, Argentina (Vacan subage of the Casamayoran SALMA, middle Eocene). We provide body mass estimates given by different elements of the skeleton, describe the bone histology, and study its phylogenetic position. We note differences in the scapulae, humerii, ulnae, and radii of the new specimen in comparison with other specimens previously referred to this taxon. We estimate a body mass of 84 ± 24.2 kg, showing that notoungulates had acquired a large body mass by the middle Eocene. Bone histology shows that the new specimen was skeletally mature. The new material supports the placement of Thomashuxleya as an early, divergent member of Toxodontia. Among placentals, our phylogenetic analysis of a combined DNA, collagen, and morphology matrix favor only a limited number of possible phylogenetic relationships, but cannot yet arbitrate between potential affinities with Afrotheria or Laurasiatheria. With no constraint, maximum parsimony supports Thomashuxleya and Carodnia with Afrotheria. With Notoungulata and Litopterna constrained as monophyletic (including Macrauchenia and Toxodon known for collagens), these clades are reconstructed on the stem to Euungulata (i.e., Perissodactyla and Artiodactyla). Unconstrained, Bayesian analysis weakly supports the possibility that Thomashuxleya is a stem xenarthran; with Notoungulata and Litopterna constrained as monophyletic, the two clades are recovered as sister to Perissodactyla. Anatomical data sampled thus far for Thomashuxleya, combined with collagen amino acids for Pleistocene meridiungulates, substantially limit the number of possible affinities for endemic South American species among mammals, although ambiguity still remains.

Molecular imaging of fibrosis using a novel collagen-binding peptide labelled with 99mTc on SPECT/CT.

Fibrosis, closely related to chronic various diseases, is a pathological process characterised by the accumulation of collagen (largely collagen type I). Non-invasive methods are necessary for the diagnosis and follow-up of fibrosis. This study aimed to develop a collagen-targeted probe for the molecular imaging of fibrosis. We identified CPKESCNLFVLKD (CBP1495) as an original collagen-binding peptide using isothermal titration calorimetry and enzyme-linked immunosorbent assay. CBP1495 effectively bound to collagen type I (K d=861nM) and (GPO)9 (K d=633nM), a collagen mimetic peptide. Western blot and histochemistry validated CBP1495 targeting collagen in vitro and ex vivo. (Gly-(D)-Ala-Gly-Gly) was introduced to CBP1495 for coupling 99mTc. Labelling efficiency of 99mTc-CBP1495 was 95.06±1.08%. The physico-chemical properties, tracer kinetics and biodistribution of 99mTc-CBP1495 were carried out, and showed that the peptide stably chelated 99mTc in vitro and in vivo. SPECT/CT imaging with 99mTc-CBP1495 was performed in rat fibrosis models, and revealed that 99mTc-CBP1495 significantly accumulated in fibrotic lungs or livers of rats. Finally, 99mTc-CBP1495 uptake and hydroxyproline (Hyp), a specific amino acid of collagen, were quantitatively analysed. The results demonstrated that 99mTc-CBP1495 uptake was positvely correlated with Hyp content in lungs (P<0.0001, r 2=0.8266) or livers (P<0.0001, r 2=0.7581). Therefore, CBP1495 is a novel collagen-binding peptide, and 99mTc-labelled CBP1495 may be a promising radiotracer for the molecular imaging of fibrosis.

Evidence for herbivorous cave bears (Ursus spelaeus) in Goyet Cave, Belgium: implications for palaeodietary reconstruction of fossil bears using amino acid δ15N approaches

ABSTRACTInsights into causes of extinction in fossil animals can contribute to an understanding of how environmental or anthropogenic processes may affect extant animals. Cave bears that went extinct in the late Pleistocene in Europe have been considered largely herbivorous based on tooth, skull and jaw morphology. Nitrogen and carbon isotopic composition (δ15N, δ13C) of bone collagen of many cave bears having values similar to or lower than those of coeval herbivores support an exclusive plant diet and their occurrence in habitats with denser vegetation. A complicating aspect of this hypothesis is that isotopic compositions of bulk collagen, especially those of nitrogen, could reflect environmental fluctuation as well as behavioural and physiological traits, which are not related to trophic position and so may lead to uncertainty in palaeodietary reconstruction. Here we show that δ15N analysis of individual collagen amino acids of fossil bears from Goyet Cave (Belgium) indicates that cave bears had a constant trophic position of 1.9–2.1, indicating purely herbivorous diets, while brown bears had a trophic position of 2.0–2.4, indicating a slightly more omnivorous diet. Results might support the hypothesis of the extinction of cave bear due to the inflexibility in feeding habits.