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.

Graduate attributes in relation to curriculum design and delivery in a Bachelor of Materials Engineering Programme

This paper presents a critical analysis of the Bachelor of Materials Engineering programme compared with the expectations of the Institution of Engineers Australia (IEAust) and of UQ. To set the scene, the graduate attributes are listed, the programme framework is presented and the educational culture and available facilities are described Then, the programme delivery is described; this includes an analysis of the learning opportunities that allow students to develop the graduate attributes. Finally, an assessment is made of programme outcomes relating to graduate attributes.

Engineering education research - the UK perspective at a time of change

Engineering education in the United Kingdom is at the point of embarking upon an interesting journey into uncharted waters. At no point in the past have there been so many drivers for change and so many opportunities for the development of engineering pedagogy. This paper will look at how Engineering Education Research (EER) has developed within the UK and what differentiates it from the many small scale practitioner interventions, perhaps without a clear research question or with little evaluation, which are presented at numerous staff development sessions, workshops and conferences. From this position some examples of current projects will be described, outcomes of funding opportunities will be summarised and the benefits of collaboration with other disciplines illustrated. In this study, I will account for how the design of task structure according to variation theory, as well as the probe-ware technology, make the laws of force and motion visible and learnable and, especially, in the lab studied make Newton's third law visible and learnable. I will also, as a comparison, include data from a mechanics lab that use the same probe-ware technology and deal with the same topics in mechanics, but uses a differently designed task structure. I will argue that the lower achievements on the FMCE-test in this latter case can be attributed to these differences in task structure in the lab instructions. According to my analysis, the necessary pattern of variation is not included in the design. I will also present a microanalysis of 15 hours collected from engineering students' activities in a lab about impulse and collisions based on video recordings of student's activities in a lab about impulse and collisions. The important object of learning in this lab is the development of an understanding of Newton's third law. The approach analysing students interaction using video data is inspired by ethnomethodology and conversation analysis, i.e. I will focus on students practical, contingent and embodied inquiry in the setting of the lab. I argue that my result corroborates variation theory and show this theory can be used as a 'tool' for designing labs as well as for analysing labs and lab instructions. Thus my results have implications outside the domain of this study and have implications for understanding critical features for student learning in labs. Engineering higher education is well used to change. As technology develops the abilities expected by employers of graduates expand, yet our understanding of how to make informed decisions about learning and teaching strategies does not without a conscious effort to do so. With the numerous demands of academic life, we often fail to acknowledge our incomplete understanding of how our students learn within our discipline. The journey facing engineering education in the UK is being driven by two classes of driver. Firstly there are those which we have been working to expand our understanding of, such as retention and employability, and secondly the new challenges such as substantial changes to funding systems allied with an increase in student expectations. Only through continued research can priorities be identified, addressed and a coherent and strong voice for informed change be heard within the wider engineering education community. This new position makes it even more important that through EER we acquire the knowledge and understanding needed to make informed decisions regarding approaches to teaching, curriculum design and measures to promote effective student learning. This then raises the question 'how does EER function within a diverse academic community?' Within an existing community of academics interested in taking meaningful steps towards understanding the ongoing challenges of engineering education a Special Interest Group (SIG) has formed in the UK. The formation of this group has itself been part of the rapidly changing environment through its facilitation by the Higher Education Academy's Engineering Subject Centre, an entity which through the Academy's current restructuring will no longer exist as a discrete Centre dedicated to supporting engineering academics. The aims of this group, the activities it is currently undertaking and how it expects to network and collaborate with the global EER community will be reported in this paper. This will include explanation of how the group has identified barriers to the progress of EER and how it is seeking, through a series of activities, to facilitate recognition and growth of EER both within the UK and with our valued international colleagues.

Quality Assurance and Assessment in Technical Education System: A Web Based Approach

Engineering education quality embraces the activities through which a technical institution satisfies itself that the quality of education it provides and standards it has set are appropriate and are being maintained. There is a need to develop a standardised approach to most aspects of quality assurance for engineering programmes which is sufficiently well defined to be accepted for all assessments.We have designed a Technical Educational Quality Assurance and Assessment (TEQ-AA) System, which makes use of the information on the web and analyzes the standards of the institution. With the standards as anchors for definition, the institution is clearer about its present in order to plan better for its future and enhancing the level of educational quality.The system has been tested and implemented on the technical educational Institutions in the Karnataka State which usually host their web pages for commercially advertising their technical education programs and their Institution objectives, policies, etc., for commercialization and for better reach-out to the students and faculty. This helps in assisting the students in selecting an institution for study and to assist in employment.

Charge-based quantum computing using single donors in semiconductors

Solid-state quantum computer architectures with qubits encoded using single atoms are now feasible given recent advances in the atomic doping of semiconductors. Here we present a charge qubit consisting of two dopant atoms in a semiconductor crystal, one of which is singly ionized. Surface electrodes control the qubit and a radio-frequency single-electron transistor provides fast readout. The calculated single gate times, of order 50 ps or less, are much shorter than the expected decoherence time. We propose universal one- and two-qubit gate operations for this system and discuss prospects for fabrication and scale up.

Experimental and theoretical investigation of the structural, chemical, electronic, and high frequency dielectric properties of barium cadmium tantalate-based ceramics

Single-phase Ba(Cd1/3Ta2/3)O-3 powder was produced using conventional solid state reaction methods. Ba(Cd1/3Ta2/3)O-3 ceramics with 2 wt % ZnO as sintering additive sintered at 1550 degreesC exhibited a dielectric constant of similar to32 and loss tangent of 5x10(-5) at 2 GHz. X-ray diffraction and thermogravimetric measurements were used to characterize the structural and thermodynamic properties of the material. Ab initio electronic structure calculations were used to give insight into the unusual properties of Ba(Cd1/3Ta2/3)O-3, as well as a similar and more widely used material Ba(Zn1/3Ta2/3)O-3. While both compounds have a hexagonal Bravais lattice, the P321 space group of Ba(Cd1/3Ta2/3)O-3 is reduced from P (3) under bar m1 of Ba(Zn1/3Ta2/3)O-3 as a result of a distortion of oxygen away from the symmetric position between the Ta and Cd ions. Both of the compounds have a conduction band minimum and valence band maximum composed of mostly weakly itinerant Ta 5d and Zn 3d/Cd 4d levels, respectively. The covalent nature of the directional d-electron bonding in these high-Z oxides plays an important role in producing a more rigid lattice with higher melting points and enhanced phonon energies, and is suggested to play an important role in producing materials with a high dielectric constant and low microwave loss. (C) 2005 American Institute of Physics.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells. (c) 2005 Wiley Periodicals, Inc.

Fluorinated-plasma modification of polyetherimide films

Ultem 1000 polyetherimide films prepared by cast-evaporating technique were covered with a 1H,1H,2H-tridecafluoro-oct-1-ene (PFO) plasma-polymerized layer. The effects of the plasma exposure time on the surface composition were studied by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and surface energy analysis. The surface topography of the plasma layer was deduced from scanning electron microscopy. The F/C ratio for plasma-polymerized PFO under the input RF power of 50 W can be as high as 1.30 for 480 s and similar to 0.4-2 at % of oxygen was detected, resulting from the reaction of long-lived radicals in the plasma polymer with atmospheric oxygen. The plasma deposition of fluorocarbon coating from plasma PFO reduces the surface energy from 46 to 18.3 mJ m(-2). (c) 2006 Wiley Periodicals, Inc.

Structural evolution in a hydrothermal reaction between Nb2O5 and NaOH solution: From Nb2O5 grains to microporous Na2Nb2O6 center dot(2)/3H2O fibers and NaNbO3 cubes

Niobium pentoxide reacts actively with concentrate NaOH solution under hydrothermal conditions at as low as 120 degrees C. The reaction ruptures the corner-sharing of NbO7 decahedra and NbO6 octahedra in the reactant Nb2O5, yielding various niobates, and the structure and composition of the niobates depend on the reaction temperature and time. The morphological evolution of the solid products in the reaction at 180 degrees C is monitored via SEM: the fine Nb2O5 powder aggregates first to irregular bars, and then niobate fibers with an aspect ratio of hundreds form. The fibers are microporous molecular sieve with a monoclinic lattice, Na2Nb2O6 center dot(2)/3H2O. The fibers are a metastable intermediate of this reaction, and they completely convert to the final product NaNbO3 Cubes in the prolonged reaction of 1 h. This study demonstrates that by carefully optimizing the reaction condition, we can selectively fabricate niobate structures of high purity, including the delicate microporous fibers, through a direct reaction between concentrated NaOH solution and Nb2O5. This synthesis route is simple and suitable for the large-scale production of the fibers. The reaction first yields poorly crystallized niobates consisting of edge-sharing NbO6 octahedra, and then the microporous fibers crystallize and grow by assembling NbO6 octahedra or clusters of NbO6 octahedra and NaO6 units. Thus, the selection of the fibril or cubic product is achieved by control of reaction kinetics. Finally, niobates with different structures exhibit remarkable differences in light absorption and photoluminescence properties. Therefore, this study is of importance for developing new functional materials by the wet-chemistry process.

Beach face and berm morphodynamics fronting a coastal lagoon

This study documents two different modes of berm development: (I) vertical growth at spring tides or following significant beach cut due to substantial swash overtopping, and (2) horizontal progradation at neap tides through the formation of a proto-berm located lower and further seaward of the principal berm. Concurrent high-frequency measurements of bed elevation and the associated wave runup distribution reveal the details of each of these berm growth modes. In mode I sediment is eroded from the inner surf and lower swash zone where swash interactions are prevalent. The net transport of this sediment is landward only, resulting in accretion onto the upper beach face and over the berm crest. The final outcome is a steepening of the beach face gradient, a change in the profile shape towards concave and rapid vertical and horizontal growth of the berm. In mode 2 sediment is eroded from the lower two-thirds of the active swash zone during the rising tide and is transported both landward and seaward. On the falling tide sediment is eroded from the inner surf and transported landward to backfill the zone eroded on the rising tide. The net result is relatively slow steepening of the beach face, a change of the profile shape towards convex, and horizontal progradation through the formation of a neap berm. The primary factor determining which mode of berm growth occurs is the presence or absence of swash overtopping at the time of sediment accumulation on the beach face. This depends on the current phase of the spring-neap tide cycle, the wave runup height (and indirectly offshore wave conditions) and the height of the pre-existing berm. A conceptual model for berm morphodynamics is presented, based on sediment transport shape functions measured during the two modes of berm growth. (c) 2006 Elsevier B.V. All rights reserved.

Experimentation in the adaptation of the overhead projector utilizing 200 transparencies and 800 overlays in teaching engineering descriptive geometry curricula, June 1, 1959 to August 31, 1960. Final report, grant no. 741023.09, U. S. Office of Education, Dept. of Health, Education, and Welfare.

"Corrected graph iv" leaf inserted.