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.

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis of peptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin (TCP-resin) with 1,4-phenylenediamine, subsequent coupling with desired amino acids by the standard Fmoc protocol, and oxidation of the intermediate para-aminoanilides (pAA) with Oxone(R). This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.

A(2)((2)) parafermions: a new conformal field theory

A new parafermionic algebra associated with the homogeneous space A(2)((2))/U(1) and its corresponding Z-algebra have been recently proposed. In this paper, we give a free boson representation of the A(2)((2)) parafermion algebra in terms of seven free fields. Free field realizations of the parafermionic energy-momentum tensor and screening currents are also obtained. A new algebraic structure is discovered, which contains a W-algebra type primary field with spin two. (C) 2002 Published by Elsevier Science B.V.

Theory and applications of fuzzy Volterra integral equations

Formulations of fuzzy integral equations in terms of the Aumann integral do not reflect the behavior of corresponding crisp models. Consequently, they are ill-adapted to describe physical phenomena, even when vagueness and uncertainty are present. A similar situation for fuzzy ODEs has been obviated by interpretation in terms of families of differential inclusions. The paper extends this formalism to fuzzy integral equations and shows that the resulting solution sets and attainability sets are fuzzy and far better descriptions of uncertain models involving integral equations. The investigation is restricted to Volterra type equations with mildly restrictive conditions, but the methods are capable of extensive generalization to other types and more general assumptions. The results are illustrated by integral equations relating to control models with fuzzy uncertainties.

Efficient simulation of a Tandem Jackson Network

The two-node tandem Jackson network serves as a convenient reference model for the analysis and testing of different methodologies and techniques in rare event simulation. In this paper we consider a new approach to efficiently estimate the probability that the content of the second buffer exceeds some high level L before it becomes empty, starting from a given state. The approach is based on a Markov additive process representation of the buffer processes, leading to an exponential change of measure to be used in an importance sampling procedure. Unlike changes of measures proposed and studied in recent literature, the one derived here is a function of the content of the first buffer. We prove that when the first buffer is finite, this method yields asymptotically efficient simulation for any set of arrival and service rates. In fact, the relative error is bounded independent of the level L; a new result which is not established for any other known method. When the first buffer is infinite, we propose a natural extension of the exponential change of measure for the finite buffer case. In this case, the relative error is shown to be bounded (independent of L) only when the second server is the bottleneck; a result which is known to hold for some other methods derived through large deviations analysis. When the first server is the bottleneck, experimental results using our method seem to suggest that the relative error is bounded linearly in L.

The Role of Non-Market Valuation in Forest Management and Recreation Policy

Forests, and particularly those where native and mixed species are gown, provide a variety of non-wood values, important among which are recreation and environmental services. Substantial progress has been made in recent years in estimating economic values on these services. A considerable amount of research on forest values has been carried out recently in tropical and sub-tropical eastern Australia, some of which is reported in the following papers. The need for estimates of non-wood forest benefits is apparent, and it is clear that further development of techniques and a greater understanding of the way these values can be integrated into public-sector decision making is required.

Resolving the puzzle of productive and destructive conflict in culturally heterogeneous workgroups: A communication accommodation theory approach

This study presents an investigation of the communicative behaviors and strategies employed in the stimulation and management of productive and destructive conflict in culturally heterogeneous workgroups. Using communication accommodation theory (CAT), we argue that the type and course of conflict in culturally heterogeneous workgroups is impacted by the communicative behaviors and strategies employed by group members during interactions. Analysis of data from participant observations, non-participant observations, semi-structured interviews, and self-report questionnaires support CA T-based predictions and provide fresh insights into the triggers and management strategies associated with conflict in culturally heterogeneous workgroups. In particular, results indicated that the more groups used discourse management Strategies, the more they experienced productive conflict. In addition, the use of explanation and checking of own and others' understanding was a major feature of productive conflict, while speech interruptions emerged as a strategy leading to potential destructive conflict. Groups where leaders emerged and assisted in reversing communication breakdowns were better able to manage their discourse, and achieved consensus On task processes. Contributions to the understanding of the triggers and the management of productive conflict in culturally heterogeneous workgroups are discussed.