Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Ball cattle (Bos javanicus). which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5 '- and 3 ' -long terminal repeats (LTRs), 0.4 kb of truncated gag and 1.1 kb of 3 ' -env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences. including gag, pol. vif, tar and rev: but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped. disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2) x 10(6) CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as wall. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus. (C) 2001 Elsevier Science B.V. All rights reserved.

Using the World Wide Web to revolutionise technology transfer and training in the water and wastewater industries

Industry professionals of the near future will be supported by an IT infrastructure that enables them to complete a task by drawing on resources and people with expertise anywhere in the world, and access to knowledge through specific training programs that address the task requirements. The increasing uptake of new technologies enables information to reach a diverse population and to provide flexible learning environments 24 hours a day, 7 days a week. This paper examines one of the key areas where the World Wide Web will impact on the water and wastewater industries, namely technology transfer and training. The authors will present their experiences of developing online training courses for wastewater industry professionals over the last two years. The perspective is that of two people working at the coalface.

A portable load cell for in-situ ore impact breakage testing

This paper discusses the design and characterisation of a short, and hence portable impact load cell for in-situ quantification of ore breakage properties under impact loading conditions. Much literature has been published in the past two decades about impact load cells for ore breakage testing. It has been conclusively shown that such machines yield significant quantitative energy-fragmentation information about industrial ores. However, documented load cells are all laboratory systems that are not adapted for in-situ testing due to their dimensions and operating requirements. The authors report on a new portable impact load cell designed specifically for in-situ testing. The load cell is 1.5 m in height and weighs 30 kg. Its physical and operating characteristics are detailed in the paper. This includes physical dimensions, calibration and signal deconvolution. Emphasis is placed on the deconvolution issue, which is significant for such a short load cell. Finally, it is conclusively shown that the short load cell is quantitatively as accurate as its larger laboratory analogues. (C) 2062 Elsevier Science B.V. All rights reserved.

Intramolecular electronic energy transfer in bichromophoric macrocyclic complexes

Efficient intramolecular electronic energy transfer (EET) has been demonstrated for three novel bichromophoric compounds utilizing a macrocyclic spacer as the bridge between the electronic energy donor and acceptor fragments. As their free base forms, emission from the electronically excited donor is absent and the acceptor emission is reductively quenched via photoinduced oxidation of proximate amine lone pairs. As their Zn(II) complexes, excitation of the donor results in sensitization of the electronic acceptor emission.

Wolbachia density and virulence attenuation after transfer into a novel host

The factors that control replication rate of the intracellular bacterium Wolbachia pipientis in its insect hosts are unknown and difficult to explore, given the complex interaction of symbiont and host genotypes. Using a strain of Wolbachia that is known to over-replicate and shorten the lifespan of its Drosophila melanogaster host, we have tracked the evolution of replication control in both somatic and reproductive tissues in a novel host/Wolbachia association. After transinfection (the transfer of a Wolbachia strain into a different species) of the over-replicating Wolbachia popcorn strain from D. metanogaster to Drosophila simulans, we demonstrated that initial high densities in the ovaries were in excess of what was required for perfect maternal transmission, and were likely causing reductions in reproductive fitness. Both densities and fitness costs associated with ovary infection rapidly declined in the generations after transinfection. The early death effect in D. simulans attenuated only slightly and was comparable to that induced in D. metanogaster. This study reveals a strong host involvement in Wolbachia replication rates, the independence of density control responses in different tissues, and the strength of natural selection acting on reproductive fitness.

Techniques for detailed heat transfer measurements in cold supersonic blowdown tunnels using thermochromic liquid crystals

The paper presents methods for measurement of convective heat transfer distributions in a cold flow, supersonic blowdown wind tunnel. The techniques involve use of the difference between model surface temperature and adiabatic wall temperature as the driving temperature difference for heat transfer and no active heating or cooling of the test gas or model is required. Thermochromic liquid crystals are used for surface temperature indication and results presented from experiments in a Mach 3 flow indicate that measurements of the surface heat transfer distribution under swept shock wave boundary layer interactions can be made. (C) 2002 Elsevier Science Ltd. All rights reserved.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

The effect of latent heat of fusion on heat transfer in fluidized-bed coating of thin plates

This paper presents a numerical study of fluidized-bed coating on thin plates using an orthogonal collocation technique. Inclusion of the latent heat of fusion term in the boundary conditions of the mathematical model accounts for the fact that some polymer powders used in coating may be partially crystalline. Predictions of coating thickness on flat plates were made with actual polymers used in fluidized-bed coating. Reasonably good agreement between numerical predictions of the coating thickness and experimental coating data of Richart was obtained for steel panels preheated to 316 degreesC. A good agreement was also obtained between numerical predictions and our coating thickness data for nylon-11 and polyethylene powders. Predicted coating thickness for polyethylene powder on flat plates were obtained with values of heat transfer coefficient closer to those obtained from our experiments. (C) 2002 Elsevier Science B.V. All rights reserved.

Mass transfer correlations for rotating drum bioreactors

Evaporative cooling is extremely important for large-scale operation of rotating drum bioreactors (RDBs). Outlet water vapour concentrations were measured for a RDB containing wet wheat bran with the aim of determining the mass transfer coefficient for evaporation from the bran bed to the headspace. Mass transfer was expressed as the mass transfer coefficient times the area for transfer per unit volume of void space in the drum. Values of ka' were determined under combinations of aeration superficial velocities ranging from 0.006 to 0.017 ms(-1) and rotation rates ranging from 0 to 9 rpm. Mass transfer coefficients were evaluated using a variety of residence time distributions (RTDs) for flow in the gas phase including plug flow and well-mixed and a Central Jet RTD based on RTD studies. If plug flow is assumed, the degree of holdup at low effective Peclet (Pe(eff)) numbers gives an apparent under-estimate of ka' compared with empirical correlations. Values of ka' calculated using the Central Jet RTD agree well with values of ka' from literature correlations. There was a linear relationship between ka' and effective Peclet number: ka' = 2.32 x 10(-3) Pe(eff). (C) 2002 Elsevier Science B.V. All rights reserved.

Simulation of the load-unload response ratio and critical sensitivity in the lattice solid model

The Load-Unload Response Ratio (LURR) method is an intermediate-term earthquake prediction approach that has shown considerable promise. It involves calculating the ratio of a specified energy release measure during loading and unloading where loading and unloading periods are determined from the earth tide induced perturbations in the Coulomb Failure Stress on optimally oriented faults. In the lead-up to large earthquakes, high LURR values are frequently observed a few months or years prior to the event. These signals may have a similar origin to the observed accelerating seismic moment release (AMR) prior to many large earthquakes or may be due to critical sensitivity of the crust when a large earthquake is imminent. As a first step towards studying the underlying physical mechanism for the LURR observations, numerical studies are conducted using the particle based lattice solid model (LSM) to determine whether LURR observations can be reproduced. The model is initialized as a heterogeneous 2-D block made up of random-sized particles bonded by elastic-brittle links. The system is subjected to uniaxial compression from rigid driving plates on the upper and lower edges of the model. Experiments are conducted using both strain and stress control to load the plates. A sinusoidal stress perturbation is added to the gradual compressional loading to simulate loading and unloading cycles and LURR is calculated. The results reproduce signals similar to those observed in earthquake prediction practice with a high LURR value followed by a sudden drop prior to macroscopic failure of the sample. The results suggest that LURR provides a good predictor for catastrophic failure in elastic-brittle systems and motivate further research to study the underlying physical mechanisms and statistical properties of high LURR values. The results provide encouragement for earthquake prediction research and the use of advanced simulation models to probe the physics of earthquakes.

Load-unload response ratio and accelerating moment/energy release critical region scaling and earthquake prediction

The main idea of the Load-Unload Response Ratio (LURR) is that when a system is stable, its response to loading corresponds to its response to unloading, whereas when the system is approaching an unstable state, the response to loading and unloading becomes quite different. High LURR values and observations of Accelerating Moment/Energy Release (AMR/AER) prior to large earthquakes have led different research groups to suggest intermediate-term earthquake prediction is possible and imply that the LURR and AMR/AER observations may have a similar physical origin. To study this possibility, we conducted a retrospective examination of several Australian and Chinese earthquakes with magnitudes ranging from 5.0 to 7.9, including Australia's deadly Newcastle earthquake and the devastating Tangshan earthquake. Both LURR values and best-fit power-law time-to-failure functions were computed using data within a range of distances from the epicenter. Like the best-fit power-law fits in AMR/AER, the LURR value was optimal using data within a certain epicentral distance implying a critical region for LURR. Furthermore, LURR critical region size scales with mainshock magnitude and is similar to the AMR/AER critical region size. These results suggest a common physical origin for both the AMR/AER and LURR observations. Further research may provide clues that yield an understanding of this mechanism and help lead to a solid foundation for intermediate-term earthquake prediction.

The effect of heat transfer mode on heart rate responses and hysteresis during heating and cooling in the estuarine crocodile Crocodylus porosus

The effect of heating and cooling on heart rate in the estuarine crocodile Crocodylus porosus was studied in response to different heat transfer mechanisms and heat loads. Three heating treatments were investigated. C. porosus were: (1) exposed to a radiant heat source under dry conditions; (2) heated via radiant energy while half-submerged in flowing water at 23degreesC and (3) heated via convective transfer by increasing water temperature from 23degreesC to 35degreesC. Cooling was achieved in all treatments by removing the heat source and with C. porosus half-submerged in flowing water at 23degreesC. In all treatments, the heart rate of C. porosus increased markedly in response to heating and decreased rapidly with the removal of the heat source. Heart rate during heating was significantly faster than during cooling at any given body temperature, i.e. there was a significant heart rate hysteresis. There were two identifiable responses to heating and cooling. During the initial stages of applying or removing the heat source, there was a dramatic increase or decrease in heart rate ('rapid response'), respectively, indicating a possible cardiac reflex. This rapid change in heart rate with only a small change or no change in body temperature (

Heat transfer and flow behind a step in high enthalpy superorbital flow

Heat transfer levels have been investigated behind a rearward-facing step in a superorbital expansion tube. The heat transfer was measured along a flat plate and behind 2 and 3mm steps with the same length to step height ratio. Results were obtained with air as the test gas at speeds of 6.76kms(-1) and 9-60kms(-1) corresponding to stagnation enthalpies of 26MJ/kg and 48MJ/kg respectively. A laminar boundary layer was established on the flat plate and measured heat transfer levels were consistent with classical empirical correlations. In the case of flow behind a step, the measurements showed a gradual rise in heat transfer from the rear of the step to a plateau several step heights downstream for both flow conditions. Reattachment distance was estimated to be approximately 1.6 step heights downstream of the 2mm step at the low enthalpy condition through the use of flow visualisation.