A high efficient and consistent method for harvesting large volumes of high-titre lentiviral vectors

Lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) are emerging as the vectors of choice for in vitro and in vivo gene therapy studies. However, the current method for harvesting lentivectors relies upon ultracentrifugation at 50 000 g for 2 h. At this ultra-high speed, rotors currently in use generally have small volume capacity. Therefore, preparations of large volumes of high-titre vectors are time-consuming and laborious to perform. In the present study, viral vector supernatant harvests from vector-producing cells (VPCs) were pre-treated with various amounts of poly-L-lysine (PLL) and concentrated by low speed centrifugation. Optimal conditions were established when 0.005% of PLL (w/v) was added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10 000 g for 2 h at 4 degreesC. Direct comparison with ultracentrifugation demonstrated that the new method consistently produced larger volumes (6 ml) of high-titre viral vector at 1 x 10(8) transduction unit (TU)/ml (from about 3000 ml of supernatant) in one round of concentration. Electron microscopic analysis showed that PLL/viral vector formed complexes, which probably facilitated easy precipitation at low-speed concentration (10 000 g), a speed which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of toxicity. These results suggest that the new method provides a convenient means for harvesting large volumes of high-titre lentivectors, facilitate gene therapy experiments in large animal or human gene therapy trials, in which large amounts of lentiviral vectors are a prerequisite.

A histone deacetylase inhibitor, azelaic bishydroxamic acid, shows cytotoxicity on Epstein-Barr virus-transformed B-cell lines - A potential therapy for posttransplant lymphoproliferative disease

Background. Posttransplant lymphoproliferative disease (PTLD), driven by the presence of Epstein-Barr virus (EBV), is becoming an increasingly important clinical problem after solid organ transplantation. The use of immunosuppressive therapy leads to the inhibition of the cytotoxic T cells that normally control the EBV latently infected B cells. The prognosis for many patients with PTLD is poor, and the optimal treatment strategy is not well defined. Method. This study investigates the use of a histone deacetylase inhibitor, azelaic bishydroxamic acid (ABRA), for its ability to effectively kill EBV-transformed lymphoblastoid cell lines. Results. In vitro treatment of lymphoblastoid cell lines with ABRA showed that they were effectively killed by low doses of the drug (ID50 2-5 mug/ml) within 48 hr. As well as being effective against polyclonal B-cell lines, ABHA was also shown to be toxic to seven of eight clonal Burkitt's lymphoma cell lines, indicating that the drug may also be useful in the treatment of late-occurring clonal PTLD. In addition, ABHA treatment did not induce EBV replication or affect EBV latent gene expression. Conclusion. These studies suggest that ABHA effectively kills both polyclonal and clonal B-cell lines and has potential in the treatment of PTLD.

The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.

A formal model of real-time program compilation

Program compilation can be formally defined as a sequence of equivalence-preserving transformations, or refinements, from high-level language programs to assembler code, Recent models also incorporate timing properties, but the resulting formalisms are intimidatingly complex. Here we take advantage of a new, simple model of real-time refinement, based on predicate transformer semantics, to present a straightforward compilation formalism that incorporates real-time constraints. (C) 2002 Elsevier Science B.V. All rights reserved.

A four-stage index 2 Diagonally Implicit Runge-Kutta method

High index Differential Algebraic Equations (DAEs) force standard numerical methods to lower order. Implicit Runge-Kutta methods such as RADAU5 handle high index problems but their fully implicit structure creates significant overhead costs for large problems. Singly Diagonally Implicit Runge-Kutta (SDIRK) methods offer lower costs for integration. This paper derives a four-stage, index 2 Explicit Singly Diagonally Implicit Runge-Kutta (ESDIRK) method. By introducing an explicit first stage, the method achieves second order stage calculations. After deriving and solving appropriate order conditions., numerical examples are used to test the proposed method using fixed and variable step size implementations. (C) 2001 IMACS. Published by Elsevier Science B.V. All rights reserved.

Application of a simplified method based on regular regime approach to determine the effective moisture diffusivity of mixture of low molecular weight sugars and maltodextrin during desorption

The binary diffusivities of water in low molecular weight sugars; fructose, sucrose and a high molecular weight carbohydrate; maltodextrin (DE 11) and the effective diffusivities of water in mixtures of these sugars (sucrose, glucose, fructose) and maltodextrin (DE 11) were determined using a simplified procedure based on the Regular Regime Approach. The effective diffusivity of these mixtures exhibited both the concentration and molecular weight dependence. Surface stickiness was observed in all samples during desorption, with fructose exhibiting the highest and maltodextrin the lowest. (C) 2002 Elsevier Science Ltd. All rights reserved.

Application of the maximum entropy method to the (2 + 1)D four-fermion model

We investigate spectral functions extracted using the maximum entropy method from correlators measured in lattice simulations of the (2+1)-dimensional four-fermion model. This model is particularly interesting because it has both a chirally broken phase with a rich spectrum of mesonic bound states and a symmetric phase where there are only resonances. In the broken phase we study the elementary fermion, pion, sigma, and massive pseudoscalar meson; our results confirm the Goldstone nature of the π and permit an estimate of the meson binding energy. We have, however, seen no signal of σ→ππ decay as the chiral limit is approached. In the symmetric phase we observe a resonance of nonzero width in qualitative agreement with analytic expectations; in addition the ultraviolet behavior of the spectral functions is consistent with the large nonperturbative anomalous dimension for fermion composite operators expected in this model.

Efficacy, timing and method of application of fungicides for management of sorghum ergot caused by Claviceps africana

Trials conducted in Queensland, Australia between 1997 and 2002 demonstrated that fungicides belonging to the triazole group were the most effective in minimising the severity of infection of sorghum by Claviceps africana, the causal agent of sorghum ergot. Triadimenol ( as Bayfidan 250EC) at 0.125 kg a. i./ha was the most effective fungicide. A combination of the systemic activated resistance compound acibenzolar-S-methyl ( as Bion 50WG) at 0.05 kg a. i./ha and mancozeb ( as Penncozeb 750DF) at 1.5 kg a. i./ha has the potential to provide protection against the pathogen, should triazole-resistant isolates be detected. Timing and method of fungicide application are important. Our results suggest that the triazole fungicides have no systemic activity in sorghum panicles, necessitating the need for multiple applications from first anthesis to the end of flowering, whereas acibenzolar-S-methyl is most effective when applied 4 days before flowering. The flat fan nozzles tested in the trials provided higher levels of protection against C. africana and greater droplet deposition on panicles than the tested hollow cone nozzles. Application of triadimenol by a fixed wing aircraft was as efficacious as application through a tractor-mounted boom spray.

Asymmetric blasting: a rock mass dependent blast design method

Blasting has been the most frequently used method for rock breakage since black powder was first used to fragment rocks, more than two hundred years ago. This paper is an attempt to reassess standard design techniques used in blasting by providing an alternative approach to blast design. The new approach has been termed asymmetric blasting. Based on providing real time rock recognition through the capacity of measurement while drilling (MWD) techniques, asymmetric blasting is an approach to deal with rock properties as they occur in nature, i.e., randomly and asymmetrically spatially distributed. It is well accepted that performance of basic mining operations, such as excavation and crushing rely on a broken rock mass which has been pre conditioned by the blast. By pre-conditioned we mean well fragmented, sufficiently loose and with adequate muckpile profile. These muckpile characteristics affect loading and hauling [1]. The influence of blasting does not end there. Under the Mine to Mill paradigm, blasting has a significant leverage on downstream operations such as crushing and milling. There is a body of evidence that blasting affects mineral liberation [2]. Thus, the importance of blasting has increased from simply fragmenting and loosing the rock mass, to a broader role that encompasses many aspects of mining, which affects the cost of the end product. A new approach is proposed in this paper which facilitates this trend 'to treat non-homogeneous media (rock mass) in a non-homogeneous manner (an asymmetrical pattern) in order to achieve an optimal result (in terms of muckpile size distribution).' It is postulated there are no logical reasons (besides the current lack of means to infer rock mass properties in the blind zones of the bench and onsite precedents) for drilling a regular blast pattern over a rock mass that is inherently heterogeneous. Real and theoretical examples of such a method are presented.

A rapid method for determining recent sunscreen use in field studies

The role of sunscreens in preventing skin cancer and melanoma is the focus of ongoing research. Currently, there is no objective measure which can be used in field studies to determine whether a person has applied sunscreen to their skin, and researchers must use indirect assessments such as questionnaires. We sought to develop a rapid, non-invasive method for identifying sunscreen on the skin for use in epidemiological studies. Our basic method is to swab the skin, elute any residues which have been adsorbed onto the swab by rinsing in ethanol, and submit the eluted washings for spectrophotometric analysis. In a controlled study, we applied 0.1 ml of sunscreen to a 50 cm(2) grid on both forearms of 21 volunteers. Each forearm was allocated one of 10 different sunscreen brands. The skin was swabbed after intervals of 20 min, 1 h, 2 h and 4 h. In a field study conducted among 12 children aged 2-4 years attending a child care centre, sunscreen was applied to the faces of half the children. Swabs were then taken from the face and back of all children without knowledge of sunscreen status. In the controlled study, sunscreen was clearly detectable up to 2 h after application for all brands containing organic sunscreen, and marginally detectable at 4 h. In the field study, this method correctly identified all children with and without sunscreen. We conclude that spectrophotometric analysis of skin swabs can reliably detect the presence of sunscreen on the skin for up to 2 It after application. (C) 2002 Elsevier Science B.V. All rights reserved.

An advanced method for eliminating impacts from frequency deviations in power system signal processing

Frequency deviation is a common problem for power system signal processing. Many power system measurements are carried out in a fixed sampling rate assuming the system operates in its nominal frequency (50 or 60 Hz). However, the actual frequency may deviate from the normal value from time to time due to various reasons such as disturbances and subsequent system transients. Measurement of signals based on a fixed sampling rate may introduce errors under such situations. In order to achieve high precision signal measurement appropriate algorithms need to be employed to reduce the impact from frequency deviation in the power system data acquisition process. This paper proposes an advanced algorithm to enhance Fourier transform for power system signal processing. The algorithm is able to effectively correct frequency deviation under fixed sampling rate. Accurate measurement of power system signals is essential for the secure and reliable operation of power systems. The algorithm is readily applicable to such occasions where signal processing is affected by frequency deviation. Both mathematical proof and numerical simulation are given in this paper to illustrate robustness and effectiveness of the proposed algorithm. Crown Copyright (C) 2003 Published by Elsevier Science B.V. All rights reserved.

A novel target-field method for magnetic resonance shim coils: III. Shielded zonal and tesseral coils

This paper continues the development of a new approach for the design of shim and gradient coils, used in magnetic resonance imaging (MRI) applications. A cylindrical primary coil of radius a and length 2L is placed inside a co-axial shield cylinder of radius b. An active shielding strategy is used to create a desired target field at an arbitrarily specified (cylindrical) location within the primary coil, and to annul the field at a certain radius outside the shield. The form of the interior target field may be chosen arbitrarily by the designer, although zonal and tesseral harmonics are typically used in MRI applications. The method presented here designs coil windings on both the primary and shielding cylinders, to produce fields that conform to the specified interior target field and the annulled field exterior to the shield. An additional feature of the method presented here is that the target field inside the primary coil is matched at two different radii, to improve overall accuracy. The method is illustrated by designing several shielded shim coils, for creating higher order tesseral fields located asymmetrically within the coil. The simpler case of pure zonal fields is discussed separately and applied to the design of some higher order shielded coils.

A practical method of estimating energy expenditure during tennis play

This study aimed to develop a practical method of estimating energy expenditure (EE) during tennis. Twenty-four elite female tennis players first completed a tennis-specific graded test in which five different intensity levels were applied randomly. Each intensity level was intended to simulate a game of singles tennis and comprised six 14 s periods of activity alternated with 20 s of active rest. Oxygen consumption (VO2) and heart rate (HR) were measured continuously and each player's rate of perceived exertion (RPE) was recorded at the end of each intensity level. Rate of energy expenditure (EEVO2) during the test was calculated using the sum of VO2 during play and the 'O-2 debt' during recovery, divided by the duration of the activity. There were significant individual linear relationships between EEVO2 and RPE, EEVO2 and HR, (rgreater than or equal to0.89 rgreater than or equal to0.93; p

Surface diffusion of strongly adsorbing vapors in activated carbon by a differential permeation method

Conventional methods to determine surface diffusion of adsorbed molecules are proven to be inadequate for strongly adsorbing vapors on activated carbon. Knudsen diffusion permeability (B-k) for strongly adsorbing vapors cannot be directly estimated from that of inert gases such as helium. In this paper three models are considered to elucidate the mechanism of surface diffusion in activated carbon. The transport mechanism in all three models is a combination of Knudsen diffusion, viscous flow and surface diffusion. The collision reflection factor f (which is the fraction of molecules undergoing collision to the solid surface over reflection from the surface) of the Knudsen diffusivity is assumed to be a function of loading. It was found to be 1.79 in the limit of zero loading, and decreases as loading increases. The surface diffusion permeability increases sharply at very low pressures and then starts to decrease after it has reached a maximum (B(mum)s) at a threshold pressure. The initial rapid increase in the total permeability is mainly attributed to surface diffusion. Interestingly the B(mum)s for all adsorbates appear at the same volumetric adsorbed phase concentration, suggesting that the volume of adsorbed molecules may play an important role in the surface diffusion mechanism in activated carbon. (C) 2003 Elsevier Ltd. All rights reserved.