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.

Denitrification, leaching and immobilisation of N-15-labelled nitrate in winter under windrowed harvesting residues in hoop pine plantations of 1-3 years old in subtropical Australia

A field study was carried out to investigate the impacts of windrowed harvesting residues on denitrification, immobilisation and leaching of N-15-labelled nitrate applied at 20 kg N ha(-1) to microplots in second-rotation hoop pine (Araucaria cunninghamii) plantations of 1-3 years old in southeast Queensland, Australia. The PVC microplots were 235 mm in diameter and 150 mm. long, and driven into the 100 mm soil. There were three replications of such microplots for each of the six treatments which were areas just under and between 1-, 2- and 3-year-old windrows of harvesting residues. Based on gaseous N losses estimated by the difference between the recoveries of bromide (Br) applied at 100 kg Br ha(-1) and N-15-labelled nitrate, denitrification was highest (23% based on N-15 loss) in the areas just under the 1-year-old windrows 25 days after a simulated 75 mm rainfall and following several natural rainfall events. There was no significant difference in N-15 losses (14-17%) among the other treatments. The N-15 immobilisation rate was highest for microplots in the areas between the 1-year-old windrows and generally higher for microplots in the areas just under the windrows (30-39%) than that (26-30%) between the windrows. Direct measurement of N-15 gas emissions (N-15(2) + (N2O)-N-15) confirmed that the highest denitrification rate occurred in the microplots under the 1-year-old windrows although the gaseous N-15 loss calculated by gas emission was only about one-quarter that estimated by the N-15 mass balance method. A significant, positive linear relationship (P < 0.05) existed between the gaseous N-15 losses measured by the two methods used. The research indicates that considerable mineral N could be lost via denitrification during the critical inter-rotation period and early phase of the second rotation. However, the impacts of windrowed harvesting residues on N losses via denitrification might only last for a period of about 2 years. Published by Elsevier Science B.V.

Mining wheat germplasm collections for yield enhancing traits

The material in genebanks includes valuable traditional varieties and landraces, non-domesticated species, advanced and obsolete cultivars, breeding lines and genetic stock. It is the wide variety of potentially useful genetic diversity that makes collections valuable. While most of the yield increases to date have resulted from manipulation of a few major traits (such as height, photoperiodism, and vernalization), meeting future demand for increased yields will require exploitation of novel genetic resources. Many traits have been reported to have potential to enhance yield, and high expression of these can be found in germplasm collections. To boost yield in irrigated situations, spike fertility must be improved simultaneously with photosynthetic capacity. CIMMYT's Wheat Genetic Resources program has identified a source of multi-ovary florets, with up to 6 kernels per floret. Lines from landrace collections have been identified that have very high chlorophyll concentration, which may increase leaf photosynthetic rate. High chlorophyll concentration and high stomatal conductance are associated with heat tolerance. Recent studies, through augmented use of seed multiplication nurseries, identified high expression of these traits in bank accessions, and both traits were heritable. Searches are underway for drought tolerance traits related to remobilization of stem fructans, awn photosynthesis, osmotic adjustment, and pubescence. Genetic diversity from wild relatives through the production of synthetic wheats has produced novel genetic diversity.

Relationships between crude protein and mineral concentrations in alfalfa and value of alfalfa silage as a mineral source for periparturient cows

One hundred and eight samples from three cultivars of alfalfa were obtained from three cuttings in 1996-1998 to evaluate the relationship between crude protein (CP) and mineral concentrations of alfalfa with cutting and maturation. The CP content drastically decreased from 27.9 to 11.4% on DM with maturity. Highly positive correlations were observed between CP and K in the first and the second cutting of alfalfa. The Ca content remained almost constant throughout the growth period. Four multiparous Holstein cows were assigned an alfalfa silage diet or an orchardgrass silage diet from 3 weeks prepartum to 1 week postpartum to examine the effect on the mineral balance. In the prepartum and postpartum diet, the roughage to concentrate ratio was 70:30 and 50:50, with alfalfa being 50 and 100% of the roughage, respectively. The alfalfa contained 1.93% of K. No metabolic disorders occurred, but the body weight decreased drastically from 1 to 6 days postpartum with each diet because of the high milk production immediately after the parturition. Positive retention of N, Ca, P, Mg, and K was observed prepartum, whereas the cows had negative N and mineral retention from 2 to 4 days postpartum. The Ca and P absorption, and Mg retention of cows with the alfalfa diet were higher than with the grass diet. The plasma Ca and inorganic P were not affected by diet, but the plasma PTH at parturition and plasma hydroxyproline from 1 week prepartum to 1 week postpartum were higher with the alfalfa diet. These results suggest that the low K alfalfa is suitable not only to prevent the incidence of milk fever but also to increase Ca, P and Mg utilization of periparturient cows, but the mineral supplementation is needed for the postpartum cows immediately after the parturition. (C) 2001 Elsevier Science B.V. All rights reserved.

An automated multi-unit composting facility for biodegradability evaluations

A system has been developed for studying the biodegradation of natural and synthetic polymeric material. The system is based on standard methods developed by the European Committee for Standardisation (CEN TC 261) (ISO/DIS 14855) and the American Society of Testing Materials, 'ASTM. Standard Test Method for Determining Aerobic. Biodegradation of Plastic Materials under Controlled Composting Conditions' (ASTM D 5338-92). A new low-cost compost facility has been used which satisfies the requirements of these standards. The system has been automated for data collection and has been run under the conditions specified by the standards. In the system, cellulose, newspaper and two starch-based polymers were treated with compost in a series of 3dm(3) vessels at 52 degreesC and under conditions of optimum moisture and pH. The degradation was followed over time by measuring the amount of carbon released as carbon dioxide. (C) 2001 Society of Chemical Industry.

A multi-purpose rainfall simulator for field infiltration and erosion studies

This paper describes a rainfall simulator developed for field and laboratory studies that gives great flexibility in plot size covered, that is highly portable and able to be used on steep slopes, and that is economical in its water use. The simulator uses Veejet 80100 nozzles mounted on a manifold, with the nozzles controlled to sweep to and from across a plot width of 1.5 m. Effective rainfall intensity is controlled by the frequency with which the nozzles sweep. Spatial uniformity of rainfall on the plots is high, with coefficients of variation (CV) on the body of the plot being 8-10%. Use of the simulator for erosion and infiltration measurements is discussed.

Impact of point source pollution on nitrogen isotope signatures (delta N-15) of vegetation in SE Brazil

This study presents novel evidence that N-15 natural abundance can be used as a robust indicator to detect pollutant nitrogen in natural plant communities. Vegetation from the heavily polluted industrial area of Cubatao in Sao Paulo State, SE Brazil, was strongly N-15 depleted compared to plants at remote sites. Historic herbarium samples from Cubatao were significantly less N-15 depleted than extant plants, indicating that N-15 depletion of vegetation is associated with present-day nitrogen pollution in Cubatao. The heavy load of nitrogenous atmospheric pollutants in Cubatao provides a nitrogen source for plants, and strongly N-15 depleted air NH3 is likely to contribute to plant and soil N-15 depletion. Epiphytic plants from Cubatao were extremely N-15 depleted (average -10.9parts per thousand) contrasting with epiphytes at remote sites (averages -1.0parts per thousand and -3.0parts per thousand). Nitrogen isotope composition of vegetation provides a tool to determine input of pollutant nitrogen into plant communities. The strong isotopic change of epiphytes suggests that epiphytes are particularly sensitive biomonitors for atmospheric pollutant nitrogen.

Establishment of Nilaparvata Iugens Stl in rice crop nurseries: a possible source of outbreaks

We tested the hypothesis that early-planted seedbeds of rioe are mere heavily infested with brown planthopper (BPH) than later seedbeds, and that transplanted plants with lBPH are a source of subsequent population increase and possible outbreaks. The experiments were conducted at CARDI and Takeo province in wet season 2000 and early wet 2 season 200 I. BPH at O. 25. 50, 100, 200 1m were infested onto plants with low and high fertilizer treatments. Rice seeds of varieties moderately and highly susceptible to BPH were sown 3 weeks early, 2 weeks early, at the normal time, and later than normal (5 weeks) and treated with low and high fertilizer rates. At Takeo, the 3< weeks early seedbeds were infested by BPH migration, and both varieties with high fertilizer caught more immigrant insects and subsequently had damaging outbreaks of BPH in the third generation. At CARDl, no seedbeds were infested with immigrant BPH. Seedbeds in areas with continuous cropping of rice have a high risk of BPH attack, Seedlings infested with 200, 100, and 50 BPI[/m2 resulted in death of the plant. Plants with 100 and 200 BPH/m'! were kj[Jed sooner. With 25 BPIVm2 plants were not kllled, but subsequent population increase caused yi eld reduction. Yield loss was high ill higlh fertilizer treated plants. Key words , ,

High resolution 3-D imaging via multi-pass SAR

Spaceborne/airborne synthetic aperture radar (SAR) systems provide high resolution two-dimensional terrain imagery. The paper proposes a technique for combining multiple SAR images, acquired on flight paths slightly separated in the elevation direction, to generate high resolution three-dimensional imagery. The technique could be viewed as an extension to interferometric SAR (InSAR) in that it generates topographic imagery with an additional dimension of resolution. The 3-D multi-pass SAR imaging system is typically characterised by a relatively short ambiguity length in the elevation direction. To minimise the associated ambiguities we exploit the relative phase information within the set of images to track the terrain landscape. The SAR images are then coherently combined, via a nonuniform DFT, over a narrow (in elevation) volume centred on the 'dominant' terrain ground plane. The paper includes a detailed description of the technique, background theory, including achievable resolution, and the results of an experimental study.

Agents as multi-threaded logical objects

In this paper we describe a distributed object oriented logic programming language in which an object is a collection of threads deductively accessing and updating a shared logic program. The key features of the language, such as static and dynamic object methods and multiple inheritance, are illustrated through a series of small examples. We show how we can implement object servers, allowing remote spawning of objects, which we can use as staging posts for mobile agents. We give as an example an information gathering mobile agent that can be queried about the information it has so far gathered whilst it is gathering new information. Finally we define a class of co-operative reasoning agents that can do resource bounded inference for full first order predicate logic, handling multiple queries and information updates concurrently. We believe that the combination of the concurrent OO and the LP programming paradigms produces a powerful tool for quickly implementing rational multi-agent applications on the internet.

Empirical evidence for ultrametric structure in multi-layer perceptron error surfaces

Combinatorial optimization problems share an interesting property with spin glass systems in that their state spaces can exhibit ultrametric structure. We use sampling methods to analyse the error surfaces of feedforward multi-layer perceptron neural networks learning encoder problems. The third order statistics of these points of attraction are examined and found to be arranged in a highly ultrametric way. This is a unique result for a finite, continuous parameter space. The implications of this result are discussed.

Applications of accurate, high-precision Pb isotope ratio measurement by multi-collector ICP-MS

The isotope composition of Ph is difficult to determine accurately due to the lack of a stable normalisation ratio. Double and triple-spike addition techniques provide one solution and presently yield the most accurate measurements. A number of recent studies have claimed that improved accuracy and precision could also be achieved by multi-collector ICP-MS (MC-ICP-MS) Pb-isotope analysis using the addition of Tl of known isotope composition to Pb samples. In this paper, we verify whether the known isotope composition of Tl can be used for correction of mass discrimination of Pb with an extensive dataset for the NIST standard SRM 981, comparison of MC-ICP-MS with TIMS data, and comparison with three isochrons from different geological environments. When all our NIST SRM 981 data are normalised with one constant Tl-205/Tl-203 of 2.38869, the following averages and reproducibilities were obtained: Pb-207/Pb-206=0.91461+/-18; Pb-208/Ph-206 = 2.1674+/-7; and (PbPh)-Pb-206-Ph-204 = 16.941+/-6. These two sigma standard deviations of the mean correspond to 149, 330, and 374 ppm, respectively. Accuracies relative to triple-spike values are 149, 157, and 52 ppm, respectively, and thus well within uncertainties. The largest component of the uncertainties stems from the Ph data alone and is not caused by differential mass discrimination behaviour of Ph and Tl. In routine operation, variation of sample introduction memory and production of isobaric molecular interferences in the spectrometer's collision cell currently appear to be the ultimate limitation to better reproducibility. Comparative study of five different datasets from actual samples (bullets, international rock standards, carbonates, metamorphic minerals, and sulphide minerals) demonstrates that in most cases geological scatter of the sample exceeds the achieved analytical reproducibility. We observe good agreement between TIMS and MC-ICP-MS data for international rock standards but find that such comparison does not constitute the ultimate. test for the validity of the MC-ICP-MS technique. Two attempted isochrons resulted in geological scatter (in one case small) in excess of analytical reproducibility. However, in one case (leached Great Dyke sulphides) we obtained a true isochron (MSWD = 0.63) age of 2578.3 +/- 0.9 Ma, which is identical to and more precise than a recently published U-Pb zircon age (2579 3 Ma) for a Great Dyke websterite [Earth Planet. Sci. Lett. 180 (2000) 1-12]. Reproducibility of this age by means of an isochron we regard as a robust test of accuracy over a wide dynamic range. We show that reliable and accurate Pb-isotope data can be obtained by careful operation of second-generation MC-ICP magnetic sector mass spectrometers. (C) 2002 Elsevier Science B.V. All rights reserved.

Polyethylene flow prediction with a differential multi-mode Pom-Pom model

We report the first steps of a collaborative project between the University of Queensland, Polyflow, Michelin, SK Chemicals, and RMIT University; on simulation, validation and application of a recently introduced constitutive model designed to describe branched polymers. Whereas much progress has been made on predicting the complex flow behaviour of many - in particular linear - polymers, it sometimes appears difficult to predict simultaneously shear thinning and extensional strain hardening behaviour using traditional constitutive models. Recently a new viscoelastic model based on molecular topology, was proposed by McLeish and Larson (1998). We explore the predictive power of a differential multi-mode version of the pom-pom model for the flow behaviour of two commercial polymer melts: a (long-chain branched) low-density polyethylene (LDPE) and a (linear) high-density polyethylene (HDPE). The model responses are compared to elongational recovery experiments published by Langouche and Debbaut (1999), and start-up of simple shear flow, stress relaxation after simple and reverse step strain experiments carried out in our laboratory.