实验基础

Clostridium difficile is a gram-positive, spore-forming, toxin-producing anaerobic bacillus that is being increasingly implicated as the leading cause of diarrhea and colitis, particularly in hospitalized, elderly patients. Studies to date suggest that C. difficile toxins A a ...

The bacterial cell surface is an important structure as it mediates interactions with the external environment. In the case of pathogens like Clostridium difficile, the cell wall and its components also have to mediate interactions with the host cells and their products. In this chapter we di ...

Toxin A (TcdA) and Toxin B (TcdB) are the major virulence factors that contribute to the pathogenesis of Clostridium difficile-associated diarrhoea (CDAD). These enterotoxins act by glucosylation of members of the Rho protein family of small GTP-binding proteins. This leads to the disor ...

Multilocus sequence typing (MLST), a nucleotide sequence-based characterization of allelic polymorphism of housekeeping genes, has been proposed as a new approach for population and evolutionary genetics and global epidemiology of bacterial pathogens. MLST provides unam ...

Clostridium difficile shows considerable variability in the PaLoc region encoding two main virulence factors, toxins TcdA and TcdB. Strains with changes in PaLoc are defined as variant toxinotypes and currently 27 such groups are recognized (I to XXVII). Toxinotype 0 includes strains w ...

Molecular typing methods for Clostridium difficile are based on gel electrophoresis of restriction fragments (endonuclease restriction analysis, REA; pulsed field gel electrophoresis PFGE; toxinotyping), PCR amplification (PCR ribotyping, arbitrarily primed PCR, mu ...

Mouse models have been developed to study the pathogenic process of Clostridium difficile infections, first the intestinal colonization and second the toxin production. These models have also been used to test the role of environmental conditions that modulate infection. Differe ...

The Golden Syrian hamster is widely regarded as the most relevant small animal model of Clostridium difficile disease as oral infection of animals pre-treated with antibiotics reproduces many of the symptoms observed in man. These include diarrhoea, histological damage, colonisat ...

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 ...