Allocating biocide sources and flow paths to surface waters using passive samplers and flood wave chemographs.

Biocide emissions have been shown to pose a comparable risk to aquatic life as pesticides in urbanized catchments. Sources of biocides have been reported to be steady flows from wastewater treatment plants as well as direct building wash off during rain events. A simple methodology to separate wastewater from surface runoff contribution during flood waves had been missing until now. This study introduces an elegant passive sampler method used to derive source allocation during flood waves based on the recalcitrant wastewater tracer carbamazepine. Field calibration of sampling rates during low- and high-flow with continuous autosampler monitoring indicated that uptake rates of polar compounds in caged POCIS are very close in both hydrological situations, allowing for a direct comparison. The passive sampler regressions showed that on a regional level carbendazim originated essentially from wastewater flows, while surface runoff contributed 31% and 74% respectively to terbutryn and diuron mass flows during flood-waves. A local autosampler campaign on a 38 km2 catchment using the same wastewater marker approach found increasing surface runoff allocations with event flow in the order terbutryn < carbendazim < diuron in accordance with results from a nearby combined sewer overflow. Both source allocation methods can be used to establish pertinent regional and local biocide mitigation plans. The passive sampler approach is by far the more cost efficient method.

Multiphotonic staging of chronic wounds and evaluation of sterile, optical transparent bacterial nanocellulose covering: A diagnostic window into human skin.

BACKGROUND: Diagnostics of healing, infection, and inflammation in chronic wounds in comparison with physiological wound healing in acute wounds may help for therapy decisions toward individualized therapy management. With emerging new optical techniques the coupling of optical diagnostic devices with tissue provides a great challenge. Traditional coupling with cover slips is used since the early days of microscopy. In modern health care, hygienic covering of surfaces is necessary to avoid infections and cross-contaminations. METHODS: Measurements in chronic wounds were performed at three different areas including the center of the wound, the border of the wound and healthy skin as comparison area. For each measurement area, three vertical stacks were taken by MPT. Additionally, three different optical measuring procedures (MPT, OCT, CLSM) were used for the examination of BNC foil. Examinations of BNC foil were carried out at two different areas of healthy skin compared to a standard setup as control. RESULTS: The MPT evaluation revealed a distinct difference in the second harmonic generation-to-autofluorescence aging index of dermis (SAAID) behavior between the vertical stacks taken at central wound areas and wound margins as well as unaffected skin. Through BNC foil covers, MPT CLSM and OCT images were captured with good quantitative and qualitative results. CONCLUSIONS: Phases in chronic wounds could be matched with physiologically healing in acute wounds according to SAAID and MPT imaging. BNC provided an alternative covering for MPT, OCT, and CLSM with clear morphological images.

Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine.

BACKGROUND: Mesenchymal stromal cells (MSCs) are promising cell therapy candidates. Clinical application is considered safe. However, minor side effects have included thromboembolism and instant blood-mediated inflammatory reactions suggesting an effect of MSC infusion on hemostasis. Previous studies focusing on plasmatic coagulation as a secondary hemostasis step detected both procoagulatory and anticoagulatory activities of MSCs. We now focus on primary hemostasis and analyzed whether MSCs can promote or inhibit platelet activation. METHODS: Effects of MSCs and MSC supernatant on platelet activation and function were studied using flow cytometry and further platelet function analyses. MSCs from bone marrow (BM), lipoaspirate (LA) and cord blood (CB) were compared to human umbilical vein endothelial cells or HeLa tumor cells as inhibitory or activating cells, respectively. RESULTS: BM-MSCs and LA-MSCs inhibited activation and aggregation of stimulated platelets independent of the agonist used. This inhibitory effect was confirmed in diagnostic point-of-care platelet function analyses in platelet-rich plasma and whole blood. Using inhibitors of the CD39-CD73-adenosine axis, we showed that adenosine produced by CD73 ectonucleotidase activity was largely responsible for the LA-MSC and BM-MSC platelet inhibitory action. With CB-MSCs, batch-dependent responses were obvious, with some batches exerting inhibition and others lacking this effect. CONCLUSIONS: Studies focusing on plasmatic coagulation suggested both procoagulatory and anticoagulatory activities of MSCs. We now show that MSCs can, dependent on their tissue origin, inhibit platelet activation involving adenosine converted from adenosine monophosphate by CD73 ectonucleotidase activity. These data may have strong implications for safety and risk/benefit assessment regarding MSCs from different tissue sources and may help to explain the tissue protective mode of action of MSCs. The adenosinergic pathway emerges as a key mechanism by which MSCs exert hemostatic and immunomodulatory functions.

von Willebrand disease type 2A phenotypes IIC, IID and IIE: A day in the life of shear-stressed mutant von Willebrand factor.

The bleeding disorder von Willebrand disease (VWD) is caused by mutations of von Willebrand factor (VWF), a multimeric glycoprotein essential for platelet-dependent primary haemostasis. VWD type 2A-associated mutations each disrupt VWF biosynthesis and function at different stages, depending on the VWF domain altered by the mutation. These effects cause considerable heterogeneity in phenotypes and symptoms. To characterise the molecular mechanisms underlying the specific VWF deficiencies in VWD 2A/IIC, IID and IIE, we investigated VWF variants with patient-derived mutations either in the VWF pro-peptide or in domains D3 or CK. Additionally to static assays and molecular dynamics (MD) simulations we used microfluidic approaches to perform a detailed investigation of the shear-dependent function of VWD 2A mutants. For each group, we found distinct characteristics in their intracellular localisation visualising specific defects in biosynthesis which are correlated to respective multimer patterns. Using microfluidic assays we further determined shear flow-dependent characteristics in polymer-platelet-aggregate formation, platelet binding and string formation for all mutants. The phenotypes observed under flow conditions were not related to the mutated VWF domain. By MD simulations we further investigated how VWD 2A/IID mutations might alter the ability of VWF to form carboxy-terminal dimers. In conclusion, our study offers a comprehensive picture of shear-dependent and shear-independent dysfunction of VWD type 2A mutants. Furthermore, our microfluidic assay might open new possibilities for diagnosis of new VWD phenotypes and treatment choice for VWD patients with shear-dependent VWF dysfunctions that are currently not detectable by static tests.

Circulating but not immobilized N-deglycosylated von Willebrand factor increases platelet adhesion under flow conditions.

The role of von Willebrand factor (VWF) as a shear stress activated platelet adhesive has been related to a coiled-elongated shape conformation. The forces dominating this transition have been suggested to be controlled by the proteins polymeric architecture. However, the fact that 20% of VWF molecular weight originates from glycan moieties has so far been neglected in these calculations. In this study, we present a systematic experimental investigation on the role of N-glycosylation for VWF mediated platelet adhesion under flow. A microfluidic flow chamber with a stenotic compartment that allows one to mimic various physiological flow conditions was designed for the efficient analysis of the adhesion spectrum. Surprisingly, we found an increase in platelet adhesion with elevated shear rate, both qualitatively and quantitatively fully conserved when N-deglycosylated VWF (N-deg-VWF) instead of VWF was immobilized in the microfluidic channel. This has been demonstrated consistently over four orders of magnitude in shear rate. In contrast, when N-deg-VWF was added to the supernatant, an increase in adhesion rate by a factor of two was detected compared to the addition of wild-type VWF. It appears that once immobilized, the role of glycans is at least modified if not-as found here for the case of adhesion-negated. These findings strengthen the physical impact of the circulating polymer on shear dependent platelet adhesion events. At present, there is no theoretical explanation for an increase in platelet adhesion to VWF in the absence of its N-glycans. However, our data indicate that the effective solubility of the protein and hence its shape or conformation may be altered by the degree of glycosylation and is therefore a good candidate for modifying the forces required to uncoil this biopolymer.