Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site-directed mutant of eIF2, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single-and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2 protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2 phosphorylation in cells transfected with the mutant eIF2 construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.

Impacts of morphological, physical and chemical properties of sludge flocs on dewaterability of activated sludge

This study examined how the floc characteristics affect dewaterability of activated sludge. The floc properties were characterized by morphological parameters (floc size distribution, fractal dimension and filament index), physical properties (flocculating ability, surface charge, relative hydrophobicity and viscosity), and chemical constituents in sludge and extracted extracellular polymeric substances (EPS), including the polymeric compounds protein, humic substances, carbohydrates and the ions Ca2+, Mg2+, Fe3+ and Al3+. The dewaterability was defined in terms of the bound water content and capillary suction time (CST). The bound water and CST corresponded to a similar indication with respect to dewaterability of activated sludge. The floc physical parameters were the most important factors which effect significantly on the water binding ability of the sludge flocs. The morphological characteristics had relatively weak impact on the dewaterability. The polymeric components protein and carbohydrate had a significant contribution to enhance the water binding ability of the sludge flocs. The effect of humic substances in the sludge on the dewaterability was, however, insignificant. The CST had good statistical correlations with the polymeric constituents measured in both sludge and the extracted EPS, and the bound water was only correlated well with the individual polymers measured in the sludge. High concentration of Ca2+, Mg2+, Fe3+ and Al3+ had significant improvement for dewaterability. (C) 2003 Elsevier B.V. All rights reserved.

The physical and surface chemical characteristics of activated carbons and the adsorption of methylene blue from wastewater

Adsorption of a basic dye, methylene blue, from aqueous solutions onto as-received activated carbons and acid-treated carbons was investigated. The physical and surface chemical properties of the activated carbons were characterized using BET-N-2 adsorption, X-ray photoelectron spectroscopy (XPS), and mass titration. It was found that acid treatment had little effect on carbon textural characteristics but significantly changed the surface chemical properties, resulting in an adverse effect on dye adsorption. The physical properties of activated carbon, such as surface area and pore volume, have little effect on dye adsorption, while the pore size distribution and the surface chemical characteristics play important roles in dye adsorption. The pH value of the solution also influences the adsorption capacity significantly. For methylene blue, a higher pH of solution favors the adsorption capacity. The kinetic adsorption of methylene blue on all carbons follows a pseudo-second-order equation. (c) 2004 Elsevier Inc. All rights reserved.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Biomolecular engineering at interfaces

A broad review of technologically focused work concerning biomolecules at interfaces is presented. The emphasis is on developments in interfacial biomolecular engineering that may have a practical impact in bioanalysis, tissue engineering, emulsion processing or bioseparations. We also review methods for fabrication in an attempt to draw out those approaches that may be useful for product manufacture, and briefly review methods for analysing the resulting interfacial nanostructures. From this review we conclude that the generation of knowledge and-innovation at the nanoscale far exceeds our ability to translate this innovation into practical outcomes addressing a market need, and that significant technological challenges exist. A particular challenge in this translation is to understand how the structural properties of biomolecules control the assembled architecture, which in turn defines product performance, and how this relationship is affected by the chosen manufacturing route. This structure-architecture-process-performance (SAPP) interaction problem is the familiar laboratory scale-up challenge in disguise. A further challenge will be to interpret biomolecular self- and directed-assembly reactions using tools of chemical reaction engineering, enabling rigorous manufacturing optimization of self-assembly laboratory techniques. We conclude that many of the technological problems facing this field are addressable using tools of modem chemical and biomolecular engineering, in conjunction with knowledge and skills from the underpinning sciences. (c) 2005 Elsevier Ltd. All rights reserved.

Systematic selection of solvents for the fabrication of 3D combined macro- and microporous polymeric scaffolds for soft tissue engineering

In this study, we investigate the fabrication of 3D porous poly(lactic-co-glycolic acid) (PLGA) scaffolds using the thermally-induced phase separation technique. The current study focuses on the selection of alternative solvents for this process using a number of criteria, including predicted solubility. toxicity, removability and processability. Solvents were removed via either vacuum freeze-drying or leaching, depending on their physical properties. The residual solvent was tested using gas chromatography-mass spectrometry. A large range of porous, highly interconnected scaffold architectures with tunable pore size and alignment was obtained, including combined macro- and microporous structures and an entirely novel 'porous-fibre' structure. The morphological features of the most promising poly(lactic-co-glycolic acid) scaffolds were analysed via scanning electron microscopy and X-ray micro-computed tomography in both two and three dimensions. The Young's moduli of the scaffolds under conditions of temperature, pH and ionic strength similar to those found in the body were tested and were found to be highly dependent on the architectures.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract ( Matrigel (TM)) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 mu L collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.