SHG Fiberscopy Assessment of Collagen Morphology and Its Potential for Breast Cancer Optical Histology.

OBJECTIVE: This study is to investigate the feasibility of our recently developed nonlinear fiberscope for label-free in situ breast tumor detection and lymph node status assessment based on second harmonic generation (SHG) imaging of fibrillar collagen matrix with histological details. The long-term goal is to improve the current biopsy-based cancer paradigm with reduced sampling errors. METHODS: In this pilot study we undertook retrospective SHG imaging study of ex vivo invasive ductal carcinoma human biopsy tissue samples, and carried out quantitative image analysis to search for collagen structural signatures that are associated with the malignance of breast cancer. RESULTS: SHG fiberscopy image-based quantitative assessment of collagen fiber morphology reveals that: 1) cancerous tissues contain generally less extracellular collagen fibers compared with tumor-adjacent normal tissues, and 2) collagen fibers in lymph node positive biopsies are more aligned than lymph node negative counterparts. CONCLUSION/SIGNIFICANCE: The results demonstrate the promising potential of our SHG fiberscope for in situ breast tumor detection and lymph node involvement assessment and for offering real-time guidance during ongoing tissue biopsy.

In vitro assessment of Momordica charantia/Hypericum perforatum oils loaded PCL/Collagen fibers: Novel scaffold for tissue engineering.

The research on tissue engineering applications has been progressing to manufacture ideal tissue scaffold biomaterials. In this study, a double-layered electrospun biofiber scaffold biomaterial including Polycaprolactone (PCL)/Collagen (COL) fibrous inner layer and PCL/ Momordica charantia (MC) and Hypericum perforatum (HP) oils fibrous outer layer was developed to manufacture a functional, novel tissue scaffold with the advantageous mechanical and biological properties. The main approach was to combine the natural perspective using medicinal oils with an engineering point of view to fabricate a potential functional scaffold for tissue engineering. Medicinal plants MC and HP are rich in functional oils and incorporation of them in a tissue scaffold will unveil their potential to augment both new tissue formation and wound healing. In this study, a novel double-layered scaffold prototype was fabricated using electrospinning technique with two PCL fiber layers, first is composed of collagen, and second is composed of oils extracted from medicinal plants. Initially, the composition of plant oils was analyzed. Thereafter the biofiber scaffold layers were fabricated and were evaluated in terms of morphology, physicochemistry, thermal and mechanical features, wettability, in vitro bio-degradability. Double-layered scaffold prototype was further analyzed in terms of in vitro biocompatibility and antibacterial effect. The medicinal oils blend provided antioxidant and antibacterial properties to the novel PCL/Oils layer. The results signify that inner PCL/COL layer exhibited advanced biodegradability of 8.5% compared to PCL and enhanced wettability with 11.7° contact angle. Strength of scaffold prototype was 5.98 N/mm2 thanks to the elastic PCL fibrous matrix. The double-layered functional biofiber scaffold enabled 92% viability after 72 h contact with fibroblast cells and furthermore provided feasible attachment sites for the cells. The functional scaffold prototype's noteworthy mechanical, chemical, and biological features enable it to be suggested as a different novel biomaterial with the potential to be utilized in tissue engineering applications.

Assessment of collagen content in fish skin - development of a flow analysis method for hydroxyproline determination.

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L-1, with a limit of detection (LOD) of 2.6 mg L-1 and a limit of quantification (LOQ) of 23.8 mg L-1. Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

Stable isotope analyses of amino acids reveal the importance of aquatic resources to Mediterranean coastal hunter-gatherers.

Determining the degree to which humans relied on coastal resources in the past is key for understanding long-term social and economic development, as well as for assessing human health and anthropogenic impacts on the environment. Prehistoric hunter-gatherers are often assumed to have heavily exploited aquatic resources, especially those living in regions of high marine productivity. For the Mediterranean, this view has been challenged, partly by the application of stable isotope analysis of skeletal remains which has shown more varied coastal hunter-gatherer diets than in other regions, perhaps due to its lower productivity. By undertaking a more specific analysis of amino acids from bone collagen of 11 individuals from one of the oldest and best-known Mesolithic cemeteries in the Mediterranean, at El Collado, Valencia, we show that high levels of aquatic protein consumption were achieved. By measuring both carbon and nitrogen in amino acids, we conclude that some of the El Collado humans relied heavily on local lagoonal fish and possibly shellfish, rather than open marine species. By contrast to previous suggestions, this study demonstrates that the north-western coast of the Mediterranean basin could support maritime-oriented economies during the Early Holocene.

Chemical Characterization of a Collagen-Derived Protein Hydrolysate and Biostimulant Activity Assessment of Its Peptidic Components.

Protein hydrolysates (PHs) are plant biostimulants consisting of oligopeptides and free amino acids exploited in agriculture to increase crop productivity. This work aimed to fractionate a commercial collagen-derived protein hydrolysate (CDPH) according to the molecular mass of the peptides and evaluate the bioactivity of different components. First, the CDPH was dialyzed and/or filtrated and analyzed on maize, showing that smaller compounds were particularly active in stimulating lateral root growth. The CDPH was then fractionated through fast protein liquid chromatography and tested on in vitro grown tomatoes proving that all the fractions were bioactive. Furthermore, these fractions were characterized by liquid chromatography-electrospray ionization-tandem mass spectrometry revealing a consensus sequence shared among the identified peptides. Based on this sequence, a synthetic peptide was produced. We assessed its structural similarity with the CDPH, the collagen, and polyproline type II helix by comparing the respective circular dichroism spectra and for the first time, we proved that a signature peptide was as bioactive as the whole CDPH.

Assessment of gelatinolytic proteinases in chilled grass carp (Ctenopharyngodon idellus) fillets: characterization and contribution to texture softening.

BACKGROUND: Texture softening is always a problem during chilling of grass carp fillets. To solve this problem and provide for better quality of flesh, understanding the mechanism of softening is necessary. Gelatinolytic proteinases are suspected to play an essential role in the disintegration of collagen in softening of fish flesh. In the present study, the types and contribution of gelatinolytic proteinases in chilled fillets were investigated. RESULTS: Four active bands (G1, 250 kDa; G2, 68 kDa; G3, 66 kDa; G4, 29 kDa) of gelatinolytic proteinases were identified in grass carp fillets by gelatin zymography. The effect of inhibitors and metal ions revealed that G1 was possibly a serine proteinase, G2 and G3 were calcium-dependent metalloproteinases and G4 was a cysteine proteinase. The effect of the inhibitors phenylmethanesulfonyl fluoride (PMSF), l-3-carboxy-trans-2,3-epoxy-propionyl-l-leucine-4-guanidinobutylamide (E-64) and 1,10-phenanthroline (Phen) on chilled fillets revealed that gelatinolytic proteinase activities were significantly suppressed. Collagen solubility indicated that metalloproteinase and serine proteinase played critical roles in collagen breakdown during the first 3 days, and cysteine proteinase revealed its effect after 3 days. Meanwhile, during chilled storage for 11 days, the final values of shear force increased 19.68% and 24.33% in PMSF and E-64 treatments when compared to control fillets respectively, whereas the increase after Phen treatment was 49.89%. CONCLUSION: Our study concluded that the disintegration of collagen in post-mortem softening of grass carp fillets was mainly mediated by metalloproteinase and to a lesser extent by serine proteinase and cysteine proteinase. © 2021 Society of Chemical Industry.

Assessment of Bioavailability after In Vitro Digestion and First Pass Metabolism of Bioactive Peptides from Collagen Hydrolysates.

Collagen hydrolysates (CHs) are composed of bioactive peptides (BAPs), which possess health enhancing properties. There is a knowledge gap regarding the bioavailability of these BAPs that involves intestinal transport and hepatic first pass effects. A simulated gastrointestinal model was used to generate digesta from two CHs (CH-GL and CH-OPT), which were applied to a novel transwell co-culture of human intestinal epithelium cell line-6 (HIEC-6) and hepatic (HepG2) cells to simulate in vivo conditions of absorption and first pass metabolism. Peptide transport, hepatic first pass effects, and bioavailability were determined by measuring BAPs (Gly-Pro, Hyp-Gly, Ala-Hyp, Pro-Hyp, Gly-Pro-Hyp) using an innovative capillary electrophoresis method. All peptides were transported across the intestinal cell layer to varying degrees with both CHs; however, Gly-Pro-Hyp was transported only with CH-GL, but not CH-OPT. Notable hepatic production was observed for Ala-Hyp with both CH treatments, and for Pro-Hyp and Gly-Pro with CH-GL only. All peptides were bioavailable (>10%), except for Gly-Pro-Hyp after CH-OPT. Overall, a high degree of transport and hepatic first pass effects on CH-derived BAPs were observed. Further research is needed to explore the hepatic mechanisms related to the production of BAPs and the bifunctional effects of the bioavailable BAPs noted in this study.

Gastrointestinal Digestion Model Assessment of Peptide Diversity and Microbial Fermentation Products of Collagen Hydrolysates.

Osteoarthritis (OA), the most common form of arthritis, is associated with metabolic diseases and gut microbiome dysbiosis. OA patients often take supplements of collagen hydrolysates (CHs) with a high peptide content. Following digestion, some peptides escape absorption to induce prebiotic effects via their colonic fermentation to generate short-chain fatty acids (SCFAs), branched-chain fatty acids (BCFAs) and colonic gases (NH4 and H2S). The capacity of CHs to generate microbial metabolites is unknown. Proteomic analysis of two CHs (CH-GL and CH-OPT) demonstrated different native peptide profiles with increased peptide diversity after in vitro gastric and small intestinal digestion. Subsequent 24 h fermentation of the CH digests in a dynamic gastrointestinal (GI) digestion model containing human fecal matter showed that CH-OPT increased (p < 0.05) H2S, SCFAs (propionic, butyric and valeric acids), BCFAs, and decreased NH4 in the ascending colon reactor with no major changes seen with CH-GL. No major effects were observed in the transverse and descending vessels for either CH. These findings signify that CHs can induce prebiotic effects in the ascending colon that are CH dependent. More studies are needed to determine the physiological significance of CH-derived colonic metabolites, in view of emerging evidence connecting the gut to OA and metabolic diseases.

Distinguishing African bovids using Zooarchaeology by Mass Spectrometry (ZooMS): New peptide markers and insights into Iron Age economies in Zambia.

Assessing past foodways, subsistence strategies, and environments depends on the accurate identification of animals in the archaeological record. The high rates of fragmentation and often poor preservation of animal bones at many archaeological sites across sub-Saharan Africa have rendered archaeofaunal specimens unidentifiable beyond broad categories, such as "large mammal" or "medium bovid". Identification of archaeofaunal specimens through Zooarchaeology by Mass Spectrometry (ZooMS), or peptide mass fingerprinting of bone collagen, offers an avenue for identification of morphologically ambiguous or unidentifiable bone fragments from such assemblages. However, application of ZooMS analysis has been hindered by a lack of complete reference peptide markers for African taxa, particularly bovids. Here we present the complete set of confirmed ZooMS peptide markers for members of all African bovid tribes. We also identify two novel peptide markers that can be used to further distinguish between bovid groups. We demonstrate that nearly all African bovid subfamilies are distinguishable using ZooMS methods, and some differences exist between tribes or sub-tribes, as is the case for Bovina (cattle) vs. Bubalina (African buffalo) within the subfamily Bovinae. We use ZooMS analysis to identify specimens from extremely fragmented faunal assemblages from six Late Holocene archaeological sites in Zambia. ZooMS-based identifications reveal greater taxonomic richness than analyses based solely on morphology, and these new identifications illuminate Iron Age subsistence economies c. 2200-500 cal BP. While the Iron Age in Zambia is associated with the transition from hunting and foraging to the development of farming and herding, our results demonstrate the continued reliance on wild bovids among Iron Age communities in central and southwestern Zambia Iron Age and herding focused primarily on cattle. We also outline further potential applications of ZooMS in African archaeology.

Balancing Cost and Accuracy in Quantum Mechanical Simulations on Collagen Protein Models.

Collagen proteins are spread in almost every vertebrate's tissue with mechanical function. The defining feature of this fundamental family of proteins is its well-known collagen triple-helical domain. This helical domain can have different geometries, varying in helical elongation and interstrands contact, as a function of the amino acidic composition. The helical geometrical features play an important role in the interaction of the collagen protein with cell receptors, but for the vast majority of collagen compositions, these geometrical features are unknown. Quantum mechanical (QM) simulations based on the density functional theory (DFT) provide a robust approach to characterize the scenario on the collagen composition-structure relationships. In this work, we analyze the role of the adopted computational method in predicting the collagen structure for two purposes. First, we look for a cost-effective computational approach to apply to a large-scale composition-structure analysis. Second, we attempt to assess the robustness of the predictions by varying the QM methods. Therefore, we have run geometry optimization on periodic models of the collagen protein using a variety of approaches based on the most commonly used DFT functionals (PBE, HSE06, and B3LYP) with and without dispersion correction (D3ABC). We have coupled these methods with several different basis sets, looking for the highest accuracy/cost ratio. Furthermore, we have studied the performance of the composite HF-3c method and the semiempirical GFN1-xTB method. Our results identify a computational recipe that is potentially capable of predicting collagen structural features in line with DFT simulations, with orders of magnitude reduced computational cost, encouraging further investigations on the topic.

Fabrication and assessment of chondroitin sulfate-modified collagen nanofibers for small-diameter vascular tissue engineering applications.

Chondroitin sulfate (ChS) has shown promising results in promoting cell proliferation and antithrombogenic activity. To engineered develop a dual-function vascular scaffold with antithrombosis and endothelialization, ChS was tethered to collagen to accelerate the growth of endothelial cells and prevent platelet activation. First, ChS was used to conjugate with collagen to generate glycosylated products (ChS-COL) via reductive amination. Then, the fabricated ChS-COL conjugates were electrospun into nanofibers and their morphologies and physicochemical characteristics, cell-scaffold responses and platelet behaviors upon ChS-COL nanofibers were comprehensively characterized to evaluate their potential use for small-diameter vascular tissue-engineered scaffolds. The experimental results demonstrated that the ChS modified collagen electrospun nanofibers were stimulatory of endothelial cell behavior, alleviated thrombocyte activation and maintained an antithrombotic effect in vivo in 10-day post-transplantation. The ChS-COL scaffolds encouraged rapid endothelialization, thus probably ensuring the antithrombotic function in long-term implantation, suggesting their promise for small-diameter vascular tissue engineering applications.

Comparative assessment of the efficiency of various decellularization agents for bone tissue engineering.

Bone regeneration can be possible through grafts or engineered bone replacement when bone defects are larger than the critical size. Decellularized bone extracellular matrix (ECM) is an alternative that is able to accelerate tissue regeneration, while decellularization protocols influence engineered bone quality. The objective of this study was to compare the quality of decellularized bone produced through different methods. Four decellularization methods were employed using (a) sodium lauryl ether sulfate (SLES), (b) sodium dodecyl sulfate (SDS) 0.5%, (c) SDS 1% and (d) trypsin/EDTA. All samples were then washed in triton X-100. DNA quantification, hematoxylin and eosin, and Hoechst staining showed that although DNA was depleted in all scaffolds, treatment with SLES led to a significantly lower DNA content. Glycosaminoglycan quantification, Raman confocal microscopy, alcian blue and PAS staining exhibited higher carbohydrate retention in the scaffolds treated with SLES and SDS 0.5%. Raman spectra, scanning electron microscopy and trichrom Masson staining showed more collagen content in SLES and SDS-treated scaffolds compared to trypsin/EDTA-treated scaffolds. Therefore, although trypsin/EDTA could efficiently decellularize the scaffolds, it washed out the ECM contents. Also, both MTT and attachment tests showed a significantly higher cell viability in SLES-treated scaffolds. Raman spectra revealed that while the first washing procedure did not remove SLES traces in the scaffolds, excessive washing reduced ECM contents. In conclusion, SLES and, to a lesser degree, SDS 0.5% protocols could efficiently preserve ultrastructure and ECM constituents of decellularized bone tissue and can thus be suggested as nontoxic and safe protocols for bone regeneration.

Early specialized maritime and maize economies on the north coast of Peru.

We assess diet and economies of middle Holocene (∼7,500 to 4,000 calibrated [cal] B.P.) humans at coexisting mound sites (Huaca Prieta and Paredones) in north coastal Peru and document regular consumption of maize by ∼6,500 to 6,000 cal B.P. and its earliest use as a staple food in this area of the Andes between 5,000 and 4,500 cal B.P. Stable isotope data from enamel carbonates and dentin collagen (childhood diet) and dental microwear texture analysis (adult diet) demonstrate dietary and economic specialization. Previous studies revealed maize and mixed-food refuse at both sites, but this study documents actual food consumption, showing that these communities situated a few hundred meters apart had significantly distinct diets in childhood and adulthood. Huaca Prieta focused on marine resources, although there are some contributions from terrestrial meat. Paredones individuals primarily consumed maize during childhood (up to 70% of the juvenile diet), as shown by δ13C values, apatite-collagen spacing, and discriminant analysis of δ13Ccoll, δ13Ccarb, and δ15N values. Maize was likely used as a weaning food (e.g., gruel and/or chicha-a maize beverage), hinting at the significant role of breastfeeding mothers, weanling infants, and children in the development of maize as a staple crop. Additionally, dental microwear data show Paredones adult diets are high in abrasives, potentially from maize processing. The distinct foodways at these neighboring sites result from and also reflect their social and political distinctions. These differences in food production, distribution, and consumption generated opportunities for exchange, an interaction that bound them together in mutual benefit.

Decellularized matrices for tumor cell modeling.

Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.

Assessment of nutritional stress in famine burials using stable isotope analysis.

OBJECTIVES: We compared δ15 N and δ13 C values from bone and dentine collagen profiles of individuals interred in famine-related and attritional burials to evaluate whether individuals in medieval London who experienced nutritional stress exhibit enriched nitrogen in bone and tooth tissue. Dentine profiles were evaluated to identify patterns that may be indicative of famine during childhood and were compared with the age of enamel hypoplasia (EH) formation to assess whether isotopic patterns of undernutrition coincide with the timing of physiological stress. MATERIALS AND METHODS: δ15 N and δ13 C isotope ratios of bone collagen were obtained from individuals (n = 128) interred in attritional and famine burials from a medieval London cemetery (c. 1120-1539). Temporal sequences of δ15 N and δ13 C isotope profiles for incrementally forming dentine collagen were obtained from a subset of these individuals (n = 21). RESULTS: Results indicate that individuals from attritional graves exhibit significantly higher δ15 N values but no significant differences were found between burial types for the sexes. Analyses of dentine profiles reveal that a lower proportion of famine burials exhibit stable dentine profiles and that several exhibit a pattern of opposing covariance between δ15 N and δ13 C. EH were also observed to have formed during or after the opposing covariance pattern for some individuals. CONCLUSIONS: The results of this study may reflect differences in diet between burial types rather than nutritional stress. Though nutritional stress could not be definitively identified using bone and dentine collagen, the results from dentine analysis support previous observations of biochemical patterns associated with nutritional stress during childhood.

Comprehensive assessment of Nile tilapia skin collagen sponges as hemostatic dressings.

Nile tilapia skin collagen sponge was fabricated by lyophilization and cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide (EDC/NHS). The physicochemical properties were examined. The EDC/NHS cross-linked collagen sponge presented an enhanced water absorption capacity. In addition, biocompatibility and hemostatic efficiency were evaluated by acute systemic toxicity assay, dermal irritation test, intradermal reaction test, sensitization test, cytotoxicity, blood clotting assay in vitro, and liver and femoral artery hemorrhage models in vivo. Results showed that the produced collagen sponges before and after EDC/NHS cross-linking had excellent biocompatibility. Furthermore, EDC/NHS cross-linking promoted fibroblast cells viability and proliferation reflected by the MTT reduction assay. Meanwhile, EDC/NHS cross-linked collagen sponge exhibited the best blood clotting ability and hemostatic efficiency in rat femoral artery hemorrhage model in comparison with non-crosslinked and commercial collagen sponges. Our results demonstrated that the fabricated collagen sponges could be used as perfect hemostatic dressings.

Assessment of biological properties of recombinant collagen-hyaluronic acid composite scaffolds.

OBJECTIVE: Recombinant collagen (rCOL)-hyaluronic acid (HA) composite scaffolds were prepared to thoroughly investigate their biological properties. METHODS: The rCOL and HA composite scaffolds were formulated via lyophilization. The scaffolds were characterized for various materials properties, including porosity, surface modification, and degradation rates. Biological properties such as in vitro cytotoxicity, cell adhesion, proliferation and migration effects were also evaluated. RESULTS: The water absorption, mechanical strength, degradation resistance and thermal stability of the prepared rCOL-HA composites were improved over that of the control studied. Scanning electron microscopy (SEM) revealed that the composites formed a three-dimensional network structure with uniform pore distribution. The cytotoxicity of the composites was minimal (grade I) and the material showed strong adhesion and proliferation effects when grown with mouse fibroblasts, particularly the composite material of rCOL (5% HA) group (P < 0.05). CONCLUSION: The rCOL-HA composite prepared via lyophilization after cross-linking is characterized by high porosity, high water absorption, and good interaction between the material and cells, as well as good biodegradability. Compared with rCOL materials, rCOL-HA has increased mechanical strength, water absorption and thermal stability. The biocompatibility and fibroblast proliferation of rCOL-HA have excellent biological performance, providing a new material for wound healing applications.

Assessment of various crosslinking agents on collagen/chitosan scaffolds for myocardial tissue engineering.

Suitable material for scaffolds that support cell attachment, proliferation, vascularization and contraction has always been a challenge in myocardial tissue engineering. Much research effort has been focused on natural polymers including collagen, gelatin, chitosan, fibrin, alginate, etc. Among them, a collagen/chitosan composite scaffold was widely used for myocardial tissue engineering. Due to the non-proliferative and contractile characteristics of cardiomyocytes, the biocompatibility and mechanical properties of the scaffolds are extremely important for supporting intercellular connection and tissue function for myocardial tissue engineering. To the best of our knowledge, the three crosslinking agents (glutaraldehyde (GTA), genipin (GP), tripolyphosphate (TPP)) have not yet been comparatively studied in myocardial tissue engineering. Thus, the aim of this study is to compare and identify the crosslinking effect of GTA, GP and TPP for myocardial tissue engineering. The collagen/chitosan scaffolds prepared with various crosslinking agents were fabricated and evaluated for physical characteristics, biocompatibility and contractile performance. All the groups of scaffolds exhibited high porosity (>65%) and good swelling ratio suitable for myocardial tissue engineering. TPP crosslinked scaffolds showed excellent mechanical properties, with their elastic modulus (81.0 ± 8.1 kPa) in the physiological range of native myocardium (20∼100 kPa). Moreover, GP and TPP crosslinked scaffolds exhibited better biocompatibility than GTA crosslinked scaffolds, as demonstrated by the live/dead staining and proliferation assay. In addition, cardiomyocytes within TPP crosslinked scaffolds showed the highest expression of cardiac-specific marker protein and the best contractile performance. To conclude, of the three crosslinking agents, TPP was recommended as the most suitable crosslinking agent for collagen/chitosan scaffold in myocardial tissue engineering.

Hybrid Kinetic Monte Carlo/Molecular Dynamics Simulations of Bond Scissions in Proteins.

Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)materials under load and related chemistries.

Does faecal matter reflect location? An initial assessment of isotopic variability between consumed prey remains and faecal matter for wild jaguars.

Faecal isotopic analysis may complement other non-invasive wildlife survey tools for monitoring landscape use by carnivores, such as motion-detecting cameras and non-invasive genetic sampling. We analysed carbon, nitrogen, and strontium isotopes in faecal matter produced by jaguars (Panthera onca) as well as bones from consumed prey at the Mountain Pine Ridge Forest Reserve (MPR) in Belize, Central America. The MPR is ideally suited for a spatial isotope study as vegetation and geology both vary considerably. The isotopic composition of faecal matter should reflect the habitat and geology where consumed prey lived. We used bone from consumed prey recovered from jaguar scats as a proxy for diet. Faecal matter and bone showed comparable spatial isotopic trends, suggesting that the isotopic composition of jaguar faeces can be used to detect foraging in different habitats (pine forest versus broadleaf forest) or on different geologies (Mesozoic carbonates; Palaeozoic granite, contact metamorphics, and metasediments). This result is reassuring as bones are not always present in carnivore scats. Studying landscape use by cryptic and wide-ranging carnivore species like jaguars remains challenging. Isotopic analysis of faecal matter complements the existing array of non-invasive spatial monitoring tools.