GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease

No positive effect of autologous platelet gel after total knee arthroplasty: A double-blind randomized controlled trial: 102 patients with a 3-month follow-up

Background and purpose Activated platelets release a cocktail of growth factors, some of which are thought to stimulate repair. We investigated whether the use of autologous platelet gel (PG) in total knee arthroplasty (TKA) would improve wound healing and knee function, and reduce blood loss and the use of analgesics. Patients and methods 102 patients undergoing TKA were randomly assigned to a PG group (n 50) or to a control (C) group (n 52). The primary analysis was based on 73 participants (PG: 32; C: 41) with comparison of postoperative wound scores, VAS, WOMAC, knee function, use of analgesics, and the pre- and postoperative hemoglobin values after a follow-up of 3 months. 29 participants were excluded due to insufficient data. Results The characteristics of the protocol-compliant patients were similar to those of the patients who w

N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells

The cellular prion protein (PrPC) plays a fundamental role in prion disease. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrPC is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrPC and also replaced the GPI-anchor of PrPC by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrPC in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrPC. Exchange of the PrPC GPI-anchor for the one of Thy-1 redirects PrPC to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrPC, with the GPI-anchor being dominant over N-glycans

Irradiation of wound blood from tumor surgery for retransfusion (transl.)

Intraoperative autotransfusion is contraindicated in tumor surgery because of the danger of tumor cell dissemination. We have tested the elimination of tumor cells in blood by irradiation for safe retransfusion. Tumor cells of various origin were mixed to washed red blood cells from volunteer blood donations. The blood was irradiated with 50 Gy. After isolation of the tumor cells by density gradient centrifugation they were tested for colony formation. While with different tumor cell lines (n = 12) 10 cells were sufficient to yield several colonies, as many as 10(10) cells did not result in any colony after irradiation of the blood. Similar results were obtained with cells cultured from blood salvaged during tumor surgery (n = 3), and with tumor cells prepared from various carcinomas (n = 10). Flow cytometric DNA analysis showed the irradiated cells in mitotic arrest. None of these cells had residual DNA metabolism expressed as incorporation of BrdUrd. We were able to demonstrate a rate of reduction in dividing cells of up to 10(9). With the typical irradiation sensitivity of tumor cells, with D0 values between 1 and 2 Gy, a dose of 50 Gy results in an effective log 12 reduction, sufficient for safe elimination of tumor cells found in shed blood. No adverse effects of the gamma-irradiation on the blood cells are to be expected, especially since the blood is retransfused without storage

Numerical analysis of particle deposition in an aircraft engine compressor cascade

Solid particles dispersed in the air represent a real hazard for gas turbines in both heavy-duty and aero-propulsion applications. Particles impacting the inner surfaces of the machine can stick to such surfaces or erode them. The geometry modifications related to such occurrences entail aerodynamic surface degradation. As the severity of the problem increases, the performance reduction can increase, demanding engine shut-down and off-line washing or refurbishing. Numerical modeling is one of the techniques employed for understanding and predicting the particle deposition problem. Multiple numerical studies investigated the influences of these phenomena. However, the basic challenge of modeling the particle wall-interaction with sufficient accuracy remains. In this work, a cascade exposed to a particle-laden flow is numerically investigated. The numerical analysis is devoted to investigate a test rig designed to be representative of aircraft engine compressor blading and exposed to an accelerated deposition process. Firstly, the relation between flow structures and particle trajectory is investigated. Then, a computational analysis is carried out considering different particle-wall interaction models of varying complexity levels (e.g. pure adhesion, presence of humidity or the influence of surface roughness) in order to identify advantages and disadvantages of each model and their ability to include different phenomena. The results are discussed by taking into account measurement data from a cascade test rig. The deviation between experimental data and the investigated model is evaluated, showing the increasing reliability that derives from successive model refinement. This work is proposed to be a test case for the numerical analyses of compressor fouling applications and a first step towards a general physical based particle-wall interaction model