Plasmin-Induced Activation of Human Platelets Is Modulated by Thrombospondin-1, Bona Fide Misfolded Proteins and Thiol Isomerases

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-β-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN⁻-denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.     

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d -ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.     

Inflammation in the Human Periodontium Induces Downregulation of the α1- and β1-Subunits of the sGC in Cementoclasts

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α₁- and β₁-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α₁- and β₁-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α₁- and β₁-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.     

Spatio-temporal patterns of birch regrowth in a Western Norwegian treeline ecotone

The study investigates spatio-temporal patterns of birch regrowth in a mountain valley in which intensive livestock grazing was formerly practised. Vegetation changes were identified through analysis of three sets of aerial photographs. The results of GIS analyses, one-way ANOVA and PCA showed that regrowth patterns are complex. In the early phase of succession, grazing history has a strong impact on where new forest establishes, both at a distance from the seasonal farmsteads where grazing intensity prior to abandonment was highest, and close to previously existing forest. Sprouting is most likely to be the dominating regeneration strategy in this early phase. In a later phase of succession, the impact of grazing history on forest distribution patterns decreases, as exemplified by the increasing distance from existing forest. The largest amounts of birch forest then establish in warm locations with moderate moisture. Protection against wind appears to be important for birch establishment.     

Chemikalienfreie Bekämpfung von Holzschädlingen durch dielektrische Erwärmung mit Radiowellen und Mikrowellen

Thermal pest control requires long treatment times due to the low thermal conductivity of wood and may lead to the formation of cracks. Here, the thermal treatment with radio waves as well as microwaves has been studied. The direct dielectric heating has the advantage of a good homogeneity. The obtained temperature profiles for radio waves were more homogeneous compared to microwaves. Detailed studies showed that elimination of pests was not related to the application of the electromagnetic field itself, but due to the temperature increase.     

Treatment of Critical Size Femoral Bone Defects with Biomimetic Hybrid Scaffolds of 3D Plotted Calcium Phosphate Cement and Mineralized Collagen Matrix

To treat critical-size bone defects, composite materials and tissue-engineered bone grafts play important roles in bone repair materials. The purpose of this study was to investigate the bone regenerative potential of hybrid scaffolds consisting of macroporous calcium phosphate cement (CPC) and microporous mineralized collagen matrix (MCM). Hybrid scaffolds were synthetized by 3D plotting CPC and then filling with MCM (MCM-CPC group) and implanted into a 5 mm critical size femoral defect in rats. Defects left empty (control group) as well as defects treated with scaffolds made of CPC only (CPC group) and MCM only (MCM group) served as controls. Eight weeks after surgery, micro-computed tomography scans and histological analysis were performed to analyze the newly formed bone, the degree of defect healing and the activity of osteoclasts. Mechanical stability was tested by 3-point-bending of the explanted femora. Compared with the other groups, more newly formed bone was found within MCM-CPC scaffolds. The new bone tissue had a clamp-like structure which was fully connected to the hybrid scaffolds and thereby enhanced the biomechanical strength. Together, the biomimetic hybrid MCM-CPC scaffolds enhanced bone defect healing by improved osseointegration and their differentiated degradation provides spatial effects in the process of critical-bone defect healing.     

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.     

Influence of vacuum skin packaging on color stability of beef longissimus lumborum compared with vacuum and high-oxygen modified atmosphere packaging

The objective of this study was to investigate how color stability of beef is affected by vacuum skin packaging (VSP) compared with vacuum packaging (VP) and high-oxygen modified atmosphere packaging (MAP; 80% O(2) and 20% CO(2)). Longissimus lumborum muscles were aged in vacuum for 7days and then cut into 2-cm-thick slices and repacked using VSP, VP and MAP for another 7days. Color stability was measured during the next 5days in air and samples for α-tocopherol and NADH analyses were obtained at the beginning and end of aerobic storage. Color stability, α-tocopherol and NADH of steaks were affected by packaging methods and storage time in air (P<0.05). Higher a* value was obtained in VSP on day 5 compared with VP. Steaks packed in VSP had better color stability than in VP and their color was similar to MAP at the end (day 5) of storage.     

Surface Acoustic Wave (SAW) Biosensor for Rapid and Label-Free Detection of Penicillin G in Milk

Surface acoustic wave (SAW) biosensors were introduced for rapid and label-free detection of penicillin G in milk. This is the first time that the use of SAW biosensors for antibiotic detection is reported. Penicillin G belongs to the β-lactam antibiotics which are commonly used in human and veterinary medicine. Particularly because of the latter, the detection of antibiotics in foodstuffs of animal origin is essential. Current methods for specific antibiotic detection often require complex laboratory equipment and procedures. SAW biosensors, however, offer rapid detection of analyte concentrations with a minimum of experimental effort. They use label-free acoustic (gravimetric) detection. Owing to the small mass of antibiotics, detection via binding inhibition assay was preferred to direct detection in this work. Samples containing penicillin G were preincubated with the corresponding antibody, and SAW biosensor surfaces were coated with penicillin G epitopes. The antibody in the sample bound to the biosensor surface, unless it was inhibited by penicillin G. The assay allowed the detection of 2 ng/ml penicillin G in buffer and 2.2 ng/ml in low-fat milk. This is below the maximum residue limit of 4 ng/ml given by the European Commission for penicillin G and other β-lactam residues in food.