Engineering a mammalian super producer

Mammalian cells are the preferred host for the manufacture of a wide range of biopharmaceuticals, but production costs are high owing to low productivity. A range of rational engineering strategies have been pursued in order to increase volumetric product titres from mammalian cells, such as delaying apoptosis, manipulation of the cell cycle, and improving metabolism and protein processing. Unfortunately, outcomes from these strategies have been mixed, with few instances where significant improvements in product yield have been achieved. This article reviews and contrasts many of the engineering strategies attempted to date, highlighting the variability and context specificity in outcome. The paper argues that this is a reflection of the complexity of mammalian cells, and that a deeper understanding of the biology underpinning protein production for biotechnological purposes is required. Copyright © 2011 Society of Chemical Industry     

Hydrogenation of dinitriles on Raney-type Ni catalysts: kinetic and mechanistic aspects

The mechanism of the hydrogenation of aliphatic C₄–C₆ dinitriles (succinonitrile, glutaronitrile, and adiponitrile) over Raney-type Ni catalysts was investigated in a fed-batch autoclave at 350 K and 5.0 MPa. The results are interpreted based on the strength of adsorption, the interaction with the solvent and intramolecular interactions. The kinetics of the hydrogenation of aliphatic dinitriles is highly dependent on the hydrocarbon chain length. Short dinitriles like succinonitrile adsorb stronger on the catalyst surface than longer dinitriles like adiponitrile. The yield of the intermediate aminonitriles decreases with increasing hydrocarbon chain length, due to the enhanced competitiveness of dinitrile and aminonitrile for the same active sites. It is proposed that the reactivity of dinitriles and aminonitriles is caused by difference in solvent interaction. It is remarkable that the stronger the adsorption, the higher the reactivity. The reactivity decreases in the order: succinonitrile > glutaronitrile > adiponitrile. In contrast, the reaction rate of the aminonitriles is fairly independent of the hydrocarbon chain length. The formation of undesired secondary amines in the form of cyclic compounds is under kinetic control and increases with decreasing chain length. It is found that adiponitrile can be very selectively hydrogenated to primary diamines. Promoting Raney-type Ni catalysts with traces of Mo, Cr or Fe enhances the performance in the hydrogenation of dinitriles. This gives the best opportunity for process improvement towards the desired primary amines.     

Molecular Heterojunctions of Oligo(phenylene ethynylene)s with Linear to Cruciform Framework

Electrical transport properties of molecular junctions are fundamentally affected by the energy alignment between molecular frontier orbitals (highest occupied molecular orbital (HOMO) or lowest unoccupied molecular orbital (LUMO)) and Fermi level (or work function) of electrode metals. Dithiafulvene (DTF) is used as substituent group to the oligo(phenylene ethynylene) (OPE) molecular wires and different molecular structures based on OPE3 backbone (with linear to cruciform framework) are achieved, with viable molecular orbitals and HOMO–LUMO energy gaps. OPE3, OPE3–DTF, and OPE3–tetrathiafulvalene (TTF) can form good self‐assembled monolayers (SAMs) on Au substrates. Molecular heterojunctions based on these SAMs are investigated using conducting probe–atomic force microscopy with different tips (Ag, Au, and Pt) and Fermi levels. The calibrated conductance values follow the sequence OPE3–TTF > OPE3–DTF > OPE3 irrespective of the tip metal. Rectification properties (or diode behavior) are observed in case of the Ag tip for which the work function is furthest from the HOMO levels of the OPE3s. Quantum chemical calculations of the transmission qualitatively agree with the experimental data and reproduce the substituent effect of DTF. Zero‐bias conductance, and symmetric or asymmetric couplings to the electrodes are investigated. The results indicate that improved fidelity of molecular transport measurements may be achieved by systematic studies of homologues series of molecular wires applying several different metal electrodes.     

Metabolic versatility in full-scale wastewater treatment plants performing enhanced biological phosphorus removal

This study analysed the enhanced biological phosphorus removal (EBPR) microbial community and metabolic performance of five full-scale EBPR systems by using fluorescence in situ hybridisation combined with off-line batch tests fed with acetate under anaerobic–aerobic conditions. The phosphorus accumulating organisms (PAOs) in all systems were stable and showed little variability between each plant, while glycogen accumulating organisms (GAOs) were present in two of the plants. The metabolic activity of each sludge showed the frequent involvement of the anaerobic tricarboxylic acid cycle (TCA) in PAO metabolism for the anaerobic generation of reducing equivalents, in addition to the more frequently reported glycolysis pathway. Metabolic variability in the use of the two pathways was also observed, between different systems and in the same system over time. The metabolic dynamics was linked to the availability of glycogen, where a higher utilisation of the glycolysis pathway was observed in the two systems employing side-stream hydrolysis, and the TCA cycle was more active in the A²O systems. Full-scale plants that showed higher glycolysis activity also exhibited superior P removal performance, suggesting that promotion of the glycolysis pathway over the TCA cycle could be beneficial towards the optimisation of EBPR systems.     

Are Perceived Sensory Dimensions a Reliable Tool for Urban Green Space Assessment and Planning?

Environmental psychologists have developed varying methods for landscape assessment according to how it is perceived and experienced. In the Nordic countries, eight perceived sensory dimensions are increasingly being used as a methodological framework for expert assessments. These are serene, nature, rich in species, space, prospect, refuge, social and culture. Using an onsite questionnaire distributed to green space visitors in Helsingborg, Sweden, this study is the first to examine the representation of the eight sensory dimensions in different types of urban green spaces as experienced by lay people and their relationships with green space attributes. The results showed that (1) prospect then serene and space were the most common, and culture and social the least; (2) the experience of the sensory dimensions varied across the six study sites and was related to their diversity of biotopes and overall size; and (3) respondent experience of the sensory dimensions was consistent across gender, age and frequency and type of recreational use. These results grant legitimacy to the method in assessment and mapping of recreational experience of urban green space.     

Influence of Microstructure and Surface Activation of Dual‐Phase Membrane Ce0.8Gd0.2O2−δ–FeCo2O4 on Oxygen Permeation

Dual‐phase oxygen transport membranes are fast‐growing research interest for application in oxyfuel combustion process. One such potential candidate is CGO‐FCO (60 wt% Ce_0.8Gd_0.2O_2?δ–40 wt% FeCo₂O₄) identified to provide good oxygen permeation flux with substantial stability in harsh atmosphere. Dense CGO‐FCO membranes of 1 mm thickness were fabricated by sintering dry pellets pressed from powders synthesized by one‐pot method (modified Pechini process) at 1200°C for 10 h. Microstructure analysis indicates presence of a third orthorhombic perovskite phase in the sintered composite. It was also identified that the spinel phase tends to form an oxygen deficient phase at the grain boundary of spinel and CGO phases. Surface exchange limitation of the membranes was overcome by La_0.6Sr_0.4Co_0.2Fe_0.8O_3?δ (LSCF) porous layer coating over the composite. The oxygen permeation flux of the CGO‐FCO screen printed with a porous layer of 10 μm thick LSCF is 0.11 mL/cm² per minute at 850°C with argon as sweep and air as feed gas at the rates of 50 and 250 mL/min.     

The Physical Properties and Self‐Assembly Potential of the RFFFR Peptide

The self‐assembly of fibers from peptides has attracted a tremendous amount of attention due to its many applications, such as in drug‐delivery systems, in tissue engineering, and in electronic devices. Recently, the self‐assembly potential of the designer peptide RFFFR has been reported. Here it is experimentally verified that the peptide forms fibers that are entangled and form solid spheres without water inside. Upon dilution below the critical fiber concentration, the fibers untangle and become totally separated prior to dissolution. These structures readily bind thioflavin T, resulting in a characteristic change in fluorescent properties consistent with β‐sheet‐rich amyloid structures with aromatic/hydrophobic grooves. The circular dichroism spectroscopy data are dominated by a π→π* transition, thus indicating that the fibers are stabilized by π‐stacking. Contrary to what was expected, the dissolution of the spheres/fibers results in increasing fluorescence anisotropy over time. This is explained in terms of HomoFRET between phenylalanine residues with a T‐shaped π‐stacking mode, which was determined in another study to be the dominant mode through atomistic simulations and semiempirical calculations. Kelvin probe force microscopy measurements indicate that the spheres and fibers have a conductivity comparable to that of gold. Hence, these self‐assembled structures might be applicable in organic solid‐state electronic devices. The dissolution properties of the spheres further suggest that they might be used as drug‐delivery systems.