The RNase activity of the envelope glycoprotein E rns of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E rns is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E rns . Here, we show that the activity of soluble E rns as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E rns protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E rns mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E rns remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E rns acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCEThe pestiviral RNase E rns was previously shown to inhibit viral ssRNA-and dsRNA-induced interferon (IFN) synthesis. However, the localization of E rns at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E rns is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E rns remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Of all our mechanosensitive tissues, skeletal muscle is the most developmentally responsive to physical activity. Conversely, restricted mobility due to injury or disease results in muscle atrophy. Gravitational force is another form of mechanical input with profound developmental consequences. The mechanical unloading resulting from the reduced gravitational force experienced during spaceflight results in oxidative muscle loss. We examined the early stages of myogenesis under conditions of simulated microgravity (SM). C2C12 mouse myoblasts in SM proliferated more slowly (2.23× less) as a result of their being retained longer within the G2/M phase of the cell cycle (2.10× more) relative to control myoblasts at terrestrial gravity. Blocking calcium entry via TRP channels with SKF-96365 (10-20 μM) accumulated myoblasts within the G2/M phase of the cell cycle and retarded their proliferation. On the genetic level, SM resulted in the reduced expression of TRPC1 and IGF-1 isoforms, transcriptional events regulated by calcium downstream of mechanical input. A decrease in TRPC1-mediated calcium entry thus appears to be a pivotal event in the muscle atrophy brought on by gravitational mechanical unloading. Hence, relieving the constant force of gravity on cells might prove one valid experimental approach to expose the underlying mechanisms modulating mechanically regulated developmental programs.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

The overall predictability of smoke exposure indicators and the importance of different influencing factors were assessed in a cross-sectional study (n = 144), using multiple linear regression and bivariate correlation analyses. Respiratory CO, and plasma nicotine and cotinine concentrations were measured before and after smoking, for lip or holder smoking, and natural or standardized (30 puffs) puffing. The prediction of smoke exposure measures varied considerably across sampling times, smoking conditions, and dependent variables. The variance of plasma cotinine and nicotine were predictable to a considerable extent (30%; 19-41%) by cigarette yield, consumption and self-reported inhalation, whereas respiratory CO was less predictable (15-27%). Generally, consumption was the most important predictor, surpassed by nicotine yield for post-smoking plasma nicotine. Smoke exposure from a single smoking period could be predicted to a variable degree (CO, 11-42%; nicotine, 33-54%) by a subset of smoker's sex, cigarette yield, self-reported inhalation and puffing characteristics. The highest prediction was found under standardized smoking conditions (30 puffs through a holder), the lowest under natural smoking conditions. The best subset of predictors, especially with respect to puffing parameters, was found to vary considerably across smoking conditions and dependent variables.