Genetic manipulation of Clostridium difficile is notoriously difficult, currently there is only one reliable method for generating random mutations in the organism and that is to use the conjugative transposon Tn916. Tn916 enters the genome of most strains of C. difficile with no obvious ...

Clostridium difficile is the causative agent of a range of intestinal diseases, collectively referred to as Clostridium difficile-associated disease (CDAD). The recent emergence of “hypervirulent” strains associated with increased rates of mortality and severity of disease ...

Members of the genus Clostridium have long been recognised as important to humankind and its animals, both in terms of the diseases they cause and the useful biological processes they undertake. This has led to increasing efforts directed at deriving greater information on their basic biolo ...

The use of shRNA for knockdown of gene expression is a powerful method. In addition to transient transfection of RNA oligonucleotides, various DNA-based vectors that express short hairpin RNAs have been successfully used for efficient depletion of gene products. Replication-defec ...

The adenovirus major late transcription unit (MLTU) encodes the main structural capsid proteins. Expression from the MLTU is accomplished through alternative mRNA processing and use of a terminal exon coding strategy. The capsid proteins hexon, penton, and fiber contribute to effic ...

Adenoviruses have become a popular vehicle for gene transfer into animal and human cells. However, wide prevalence of preexisting immunity to human adenovirus (HAdV) and the promiscuous nature of the virus have made the use of nonhuman adenoviruses an attractive alternative. Moreover, ...

Gene-directed enzyme prodrug therapy (GDEPT) is an emerging approach for the treatment of cancers. A variety of viral vectors have been used to deliver genes that encode the relevant enzymes, and some have been tested in clinical trials. To ensure the potency and efficacy of such vectors and to obta ...

One of the most time-consuming steps in the generation of adenoviral vectors is the construction of recombinant plasmids. This chapter describes a detailed method for the rapid construction of adenoviral vectors. The method described here uses homologous recombination machinery ...

This chapter describes a novel strategy that simplifies the generation and production of adenovirus type 5 (Ad5) mutants carrying defined mutations in early transcription units 1 (E1) and 4 (E4). The strategy involves three recombinant plasmids containing E1 (pE1-1235), E4 (pE4-1155), or ...

Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mut ...

A capture enzyme-linked immunosorbent assay (ELISA) was optimized for identification of mouse adenovirus type 1 in infected brain homogenates. The ELISA method allows for a much faster quantitation of virus in infected organs than plaque assays. Methods for organ homogenization and t ...

Precise and simple assay of purified and crude preparations of human adenoviruses is essential for basic and gene therapy research. Previous bioassays used to quantitate adenoviruses (such as the plaque assay or fluorescent focus assay) are time-consuming and subjective in their int ...

Advances in amplification techniques have revolutionized the ability to detect viruses both quantitatively and qualitatively and to study viral load. Real-time polymerase chain reaction (PCR) amplification depends on the ability to detect and quantify a fluorescent reporter ...

Pseudomonas aeruginosa produces a quorum sensing molecule termed the Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone; PQS) that regulates an array of genes involved in virulence. This chapter addresses four related techniques useful for detecting and quanti ...

The formation of biofilms in conjunction with quorum sensing (QS)-regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to de ...

Synthetic quorum-sensing systems in mammalian cells has enabled the implementation of time- and distance-dependent bioprocesses, as well as the design of synthetic ecosystems emulating clinically important host–parasite interactions. In this chapter, we provide a detailed ...

Quorum-quenching enzymes are useful as biochemical tools and possible therapeutic proteins. One of the best-characterized families of these catalysts is the N-acyl-l-homoserine lactone (AHL) lactonases, which rely on a dinuclear metal ion active site to hydrolytically cleave the ...

Bacterial quorum sensing (QS) system is a unique target for the development of a new class of drugs that potentially control pathogenicity and attenuate virulence. Thus, it has been of significant interest to discover small organic molecules that modulate QS circuits by competing with the s ...

The exchange of information within and among bacterial populations using small diffusible molecules has been termed “quorum sensing” (QS). Due to the extracellular distribution of the QS autoinducer molecules and the evolutionary highly conserved nature of signaling componen ...

Mammalian paraoxonases (PONs) are a unique, highly conserved family of calcium-dependent esterases consisting of PON1, PON2, and PON3. The PONs can hydrolyze the lactone ring of a range of N-acyl-l-homoserine lactone (AHL) quorum sensing signaling molecules, rendering them inactive. ...