Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P(BAD) promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

Preparation and Evaluation of Sustained-Release Matrix Tablets Based on Metoprolol and an Acrylic Carrier Using Injection Moulding

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading. © 2012 American Association of Pharmaceutical Scientists.

Production and evaluation of the utility of novel phage display-derived peptide ligands to Salmonella spp. for magnetic separation

Aims: The objectives of this study were to produce Salmonella-specific peptide ligands by phage display biopanning and evaluate their use for magnetic separation (MS).
Methods and Results: Four phage display biopanning rounds were performed and the peptides expressed by the two most Salmonella-specific (on the basis of phage binding ELISA results) phage clones, MSal020401 and MSal020417, were chemically synthesized and coupled to MyOne™ tosylactivated Dynabeads®. Peptide capture capability for whole Salmonella cells from non-enriched broth cultures was quantified by MS + plate counts and MS + Greenlight™ detection, and compared to capture capability of anti-Salmonella (antibody-coated) Dynabeads®. MS + Greenlight™ gave a more comprehensive picture of capture capability than MS + plate counts and showed that Peptide MSal020417-coated beads exhibited at least similar, if not better, capture capability to anti-Salmonella Dynabeads® (mean capture values of 36.0 ± 18.2 % and 31.2 ± 20.1 %, respectively, over Salmonella spp. concentration range 3 x 101 - 3 x 106 cfu ml-1) with minimal cross-reactivity (= 1.9 %) to three other foodborne bacteria.
Conclusions: One of the phage display-derived peptide ligands was demonstrated by MS + Greenlight™ to be a viable antibody-alternative for MS of Salmonella spp.
Significance and Impact of Study: This study demonstrates an antibody-free approach to Salmonella detection and opens substantial possibilities for more rapid tests for this bacterium.

Implementation and Evaluation of Measurement-Based Admission Control Schemes Within a Converged Networks QoS Management Framework

Policy-based network management (PBNM) paradigms provide an effective tool for end-to-end resource
management in converged next generation networks by enabling unified, adaptive and scalable solutions
that integrate and co-ordinate diverse resource management mechanisms associated with heterogeneous
access technologies. In our project, a PBNM framework for end-to-end QoS management in converged
networks is being developed. The framework consists of distributed functional entities managed within a
policy-based infrastructure to provide QoS and resource management in converged networks. Within any
QoS control framework, an effective admission control scheme is essential for maintaining the QoS of
flows present in the network. Measurement based admission control (MBAC) and parameter basedadmission control (PBAC) are two commonly used approaches. This paper presents the implementationand analysis of various measurement-based admission control schemes developed within a Java-based
prototype of our policy-based framework. The evaluation is made with real traffic flows on a Linux-based experimental testbed where the current prototype is deployed. Our results show that unlike with classic MBAC or PBAC only schemes, a hybrid approach that combines both methods can simultaneously result in improved admission control and network utilization efficiency