Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

An investigation into factors affecting electron density calibration for a megavoltage cone-beam CT system

There is a growing interest in the use of megavoltage cone-beam computed tomography (MV CBCT) data for radiotherapy treatment planning. To calculate accurate dose distributions, knowledge of the electron density (ED) of the tissues being irradiated is required. In the case of MV CBCT, it is necessary to determine a calibration-relating CT number to ED, utilizing the photon beam produced for MV CBCT. A number of different parameters can affect this calibration. This study was undertaken on the Siemens MV CBCT system, MVision, to evaluate the effect of the following parameters on the reconstructed CT pixel value to ED calibration: the number of monitor units (MUs) used (5, 8, 15 and 60 MUs), the image reconstruction filter (head and neck, and pelvis), reconstruction matrix size (256 by 256 and 512 by 512), and the addition of extra solid water surrounding the ED phantom. A Gammex electron density CT phantom containing EDs from 0.292 to 1.707 was imaged under each of these conditions. The linear relationship between MV CBCT pixel value and ED was demonstrated for all MU settings and over the range of EDs. Changes in MU number did not dramatically alter the MV CBCT ED calibration. The use of different reconstruction filters was found to affect the MV CBCT ED calibration, as was the addition of solid water surrounding the phantom. Dose distributions from treatment plans calculated with simulated image data from a 15 MU head and neck reconstruction filter MV CBCT image and a MV CBCT ED calibration curve from the image data parameters and a 15 MU pelvis reconstruction filter showed small and clinically insignificant differences. Thus, the use of a single MV CBCT ED calibration curve is unlikely to result in any clinical differences. However, to ensure minimal uncertainties in dose reporting, MV CBCT ED calibration measurements could be carried out using parameter-specific calibration measurements.

A simulation-based training system for surgical wound debridement

A simulation-based training system for surgical wound debridement was developed and comprises a multimedia introduction, a surgical simulator (tutorial component), and an assessment component. The simulator includes two PCs, a haptic device, and mirrored display. Debridement is performed on a virtual leg model with a shallow laceration wound superimposed. Trainees are instructed to remove debris with forceps, scrub with a brush, and rinse with saline solution to maintain sterility. Research and development issues currently under investigation include tissue deformation models using mass-spring system and finite element methods; tissue cutting using a high-resolution volumetric mesh and dynamic topology; and accurate collision detection, cutting, and soft-body haptic rendering for two devices within the same haptic space.

Emergence and the art system ‘plus minus now’

Emergence is discussed in the context of a practice-based study of interactive art and a new taxonomy of emergence is proposed. The interactive art system ‘plus minus now’ is described and its relationship to emergence is discussed. ‘Plus minus now’ uses a novel method for instantiating emergent shapes. A preliminary investigation of this art system has been conducted and reveals the creation of temporal compositions by a participant. These temporal compositions and the emergent shapes are described using the taxonomy of emergence. Characteristics of emergent interactions and the implications of designing for them are discussed.

Effects of various power system stabilizers on improving power system dynamic performance

To ensure the small-signal stability of a power system, power system stabilizers (PSSs) are extensively applied for damping low frequency power oscillations through modulating the excitation supplied to synchronous machines, and increasing interest has been focused on developing different PSS schemes to tackle the threat of damping oscillations to power system stability. This paper examines four different PSS models and investigates their performances on damping power system dynamics using both small-signal eigenvalue analysis and large-signal dynamic simulations. The four kinds of PSSs examined include the Conventional PSS (CPSS), Single Neuron based PSS (SNPSS), Adaptive PSS (APSS) and Multi-band PSS (MBPSS). A steep descent parameter optimization algorithm is employed to seek the optimal PSS design parameters. To evaluate the effects of these PSSs on improving power system dynamic behaviors, case studies are carried out on an 8-unit 24-bus power system through both small-signal eigenvalue analysis and large-signal time-domain simulations.

An intelligent network control system for plug-in electric vehicles and the associate communication mechanism

The development of an intelligent plug-in electric vehicle (PEV) network is an important research topic in the smart grid environment. An intelligent PEV network enables a flexible control of PEV charging and discharging activities and hence PEVs can be utilized as ancillary service providers in the power system concerned. Given this background, an intelligent PEV network architecture is first developed, and followed by detailed designs of its application layers, including the charging and discharging controlling system, mobility and roaming management, as well as communication mechanisms associated. The presented architecture leverages the philosophy in mobile communication network buildup

An ingenuous Decision Support System (iDSS) approach for remote area pregnant women

This paper addresses development of an ingenious decision support system (iDSS) based on the methodology of survey instruments and identification of significant variables to be used in iDSS using statistical analysis. A survey was undertaken with pregnant women and factorial experimental design was chosen to acquire sample size. Variables with good reliability in any one of the statistical techniques such as Chi-square, Cronbach’s α and Classification Tree were incorporated in the iDSS. The ingenious decision support system was implemented with Visual Basic as front end and Microsoft SQL server management as backend. Outcome of the ingenious decision support system include advice on Symptoms, Diet and Exercise to pregnant women.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.