Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5–10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.

We here present a detailed study of the ligand-receptor interactions between single and triple-helical strands of collagen and the α2A domain of integrin (α2A), providing valuable new insights into the mechanisms and dynamics of collagen-integrin binding at a sub-molecular level. The occurrence of single and triple-helical strands of the collagen fragments was scrutinized with atom force microscopy (AFM) techniques. Strong interactions of the triple-stranded fragments comparable to those of collagen can only be detected for the 42mer triple-helical collagen-like peptide under study (which contains 42 amino acid residues per strand) by solid phase assays as well as by surface plasmon resonance (SPR) measurements. However, changes in NMR signals during titration and characteristic saturation transfer difference (STD) NMR signals are also detectable when α2A is added to a solution of the 21mer single-stranded collagen fragment. Molecular dynamics (MD) simulations employing different sets of force field parameters were applied to study the interaction between triple-helical or single-stranded collagen fragments with α2A. It is remarkable that even single-stranded collagen fragments can form various complexes with α2A showing significant differences in the complex stability with identical ligands. The results of MD simulations are in agreement with the signal alterations in our NMR experiments, which are indicative of the formation of weak complexes between single-stranded collagen and α2A in solution. These results provide useful information concerning possible interactions of α2A with small collagen fragments that are of relevance to the design of novel therapeutic A-domain inhibitors.

Heterogeneous mixtures of collagen fragments can be used as nutrition supplement or as key ingredients for ointments with therapeutic relevance in wound healing. Some mixtures of collagen fragments are referred to as collagen hydrolysates owing to the production process with hydrolytic enzymes. Since the precise composition of collagen hydrolysates is generally unknown, it is of interest to analyze samples containing various collagen fragments with appropriate biophysical methods. Any product optimization without a profound knowledge concerning the size and the molecular weight distribution of its components is nearly impossible. It turned out that a combination of AFM methods with NMR techniques is exceptionally suited to examine the size range and the aggregation behavior of the collagen fragments in the hydrolysates of fish, jellyfish, chicken, porcine and bovine collagen. Supported by molecular modeling calculations, the AFM and NMR experiments provide a detailed knowledge about the composition of collagen hydrolysates and collagen ointments. Furthermore, the data allow a correlation between the size of the fragments and their potential bioactivity.

Proteoglycans and collagen molecules are interacting with each other thereby forming various connective tissues. The sulfation pattern of proteoglycans differs depending on the kind of tissue and/or the degree of maturation. Tissues from <i>Cnidaria</i> are suitable examples for exploration of the effects in relation to the presence and the absence of sulfate groups, when studying characteristic fragments of the long proteoglycan carbohydrate chains <i>in silico</i>. It has been described that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra. On the other hand, sulfated glucosaminoglycans play an important role in various developmental processes of <i>Cnidaria</i>. In order to understand this biological phenomenon on a sub-molecular level we have analysed the structures of sulfated and non-sulfated proteoglycan carbohydrate chains as well as the structure of diverse collagen molecules with computational methods including quantum chemical calculations. The strong interactions between the sulfate groups of the carbohydrates moieties in proteoglycans and positively charged regions of collagen are essential in stabilizing various <i>Cnidaria</i> tissues but could hinder the nematocyst formation and its proper function. The results of our quantum chemical calculations show that the sulfation pattern has a significant effect on the conformation of chondroitin structures under study

Today, substantial attention is given to biomaterial strategies for bone regeneration, and among them, there is a growing interest in using immunomodulatory biomaterials. The ability of a biomaterial to induce neo vascularization and macrophage polarization is a major factor in defining its success. Magnesium (Mg)-based degradable alloys have attracted significant attention for bone regeneration owing to their biodegradability and potential for avoiding secondary removal surgeries. However, there is insufficient evidence in the literature regarding the early inflammatory responses to these alloys in vivo. In this study, we investigated the early body responses to Mg-0.45wt%Zn-0.45wt%Ca pin-shaped alloy (known as ZX00 alloy) in rat femora 2, 5, and 10 days after implantation. We used 3D micro computed tomography (µCT), histological, immunohistochemical, histomorphometrical, and small angle X-ray scattering (SAXS) analyses to study new bone formation, early macrophage polarization, neo vascularization, and bone quality at the implant bone interface. The expression of macrophage type 2 biological markers increased significantly after 10 days of Mg alloy implantation, indicating its potential in stimulating macrophage polarization. Our biomineralization results using µCT as well as histological stained sections did not indicate any statistically significant differences between different time points for both groups. The activity of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx 2) biological markers decreased significantly for Mg group, indicating less osteoblast activity. Generally, our results supported the potential of ZX00 alloy to enhance the expression of macrophage polarization in vivo; however, we could not observe any statistically significant changes regarding biomineralization.