Oxidative stress induces caspase-independent retinal apoptosis in vitro

Apoptosis is the mode of cell death in retinitis pigmentosa (RP), a heterogeneous group of retinal degenerations. The activation of the caspase proteases forms a pivotal step in the initiation and execution phase of apoptosis in many cells. Inhibition of caspases has been reported to prevent apoptosis in many model systems. However, we demonstrate the absence of caspase activation during retinal cell apoptosis in vitro which involves phosphatidylserine (PS) externalisation, DNA nicking and cell shrinkage. In addition, zVAD-fmk, DEVD-CHO and BD-fmk, inhibitors of the caspases, were unable to alter the characteristics or kinetics of apoptosis, implying that retinal cell death in vitro follows a caspase-independent pathway. We have previously demonstrated the ability of reactive oxygen species (ROS) to act as mediators of retinal cell apoptosis in vitro as well as the ability of antioxidants to prevent retinal cell apoptosis. Here we demonstrate the oxidative inactivation of caspases in this model of retinal apoptosis and provide evidence for an oxidative stress driven cell death pathway that does not involve caspase activity and which retains key features of apoptotic cell death. Furthermore, our data indicates that apoptotic events such as PS exposure, DNA nicking and cell shrinkage may occur independently of caspase activity.     

The flat-plate plant-microbial fuel cell: the effect of a new design on internal resistances

Due to a growing world population and increasing welfare, energy demand worldwide is increasing. To meet the increasing energy demand in a sustainable way, new technologies are needed. The Plant-Microbial Fuel Cell (P-MFC) is a technology that could produce sustainable bio-electricity and help meeting the increasing energy demand. Power output of the P-MFC, however, needs to be increased to make it attractive as a renewable and sustainable energy source. To increase power output of the P-MFC internal resistances need to be reduced. With a flat-plate P-MFC design we tried to minimize internal resistances compared to the previously used tubular P-MFC design. With the flat-plate design current and power density per geometric planting area were increased (from 0.15 A/m² to 1.6 A/m² and from 0.22 W/m² to and 0.44 W/m²)as were current and power output per volume (from 7.5 A/m³ to 122 A/m³ and from 1.3 W/m³ to 5.8 W/m³). Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing to calculate internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present; in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material-reduction in new designs. Concurrent reduction in material use and increase in power output brings the P-MFC a step closer to usable energy density and economic feasibility.     

Impact of enhanced Staphylococcus DNA extraction on microbial community measures in cystic fibrosis sputum

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF.     

Immunoaffinity Chromatography of Human β-Hexosaminidase A

A highly specific method for the purification of human 8-hexos-aminidase A employing immunoaffinity chromatography is described. Using an antiserum against the unique antigenic determinant, a, of 8-hexosaminidase A, and elution with 8.0M urea, a 283-or 1+17-fold purification of the enzyme was obtained in a single step from crude human placental homogenate.