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Microbiologists have realized that it is unlikely that all virulence determinants of a human pathogen could be identified simply by studying the pathogen in the laboratory since it is technically impossible to determine and mimic all of the complex and changing environmental stimuli that occur at the site of an infection. This shortcoming hampers our complete understanding of the virulence mechanisms employed by human pathogens, which is reflected in the relatively small number of effective vaccines that are currently available to combat the myriad of infections that afflict mankind. To overcome this problem, a number of investigators have designed methods to identify genes of pathogens that are specifically expressed during infection. The advent of in vivo expression technology (IVET) in 1993 triggered a concerted effort in various fields of microbiology to seek for differentially-expressed and/or in vivo induced genes. These genes were originally proposed as likely targets for the development of new vaccine, diagnostic and antibiotherapy strategies in the medical field. In counterpart, other non-human systems quickly followed the trend and included the development of methods particularly suited to study plant and animal pathogens in vivo. This Special Edition of Infectious Disorders-Drug Targets reviews some of the latest and outstanding developments accomplished using a number of methods that focus on differentially expressed genes to further our understanding of molecular microbial pathogenesis. A plethora of reviews have exhaustively covered many aspects of the novel techniques that led to the identification of numerous differentially expressed microbial genes. The present overview focuses on those methods that show substantial promise for-or have already led to-novel approaches for diagnosing, preventing or treating microbial diseases. In the first two papers, methods based on in vivo screens and selection will be presented. Jackson and Giddens (Development and Application of In Vivo Expression Technology (IVET) for Analysing Microbial Gene Expression in Complex Environments) will describe IVET and its various spin-offs as tools for analyzing microbial gene expression in complex environments and providing new targets for biotechnological development. Bossé et al. (High-Throughput Identification of Conditionally Essential Genes in Bacteria: From STM to TSM) will next depict the latest development obtained with signature-tagged mutagenesis (STM) and transposon screen by microarray (TSM), which combine the negative-selection principle of STM with the genome-wide screening strength of DNA microarrays. The next three papers will focus on transcriptomic approaches that have been used to dissect hostpathogen interactions. A review on the recent and ongoing developments obtained with bacterial microarrays will first be presented by Chen (DNA Microarrays - An Armory for Combating Infectious Diseases in the New Century). Besides describing exciting progress in microarray technology applied to the study of microbial pathogenesis, drug response, vaccine development and disease agent identification, Chen will address certain issues and challenges in the analysis, management and interpretation of microarray data. Kronstad (Serial Analysis of Gene Expression in Eukaryotic Pathogens) will then present SAGE as an alternative technique to microarrays to obtain information on transcript abundance and differential RNA expression, particularly with eukaryotic systems such as Saccharomyces cerevisiae and Caenorhabditis elegans. Finally, Mans et al. (Microarray Analysis of Human Epithelial Cell Responses to Bacterial Interaction) will present how the host transcriptional responses have been recently used to infer the function of virulence determinants of bacterial pathogens that are interacting with the epithelial mucosa during disease. 206 Infectious Disorders - Drug Targets 2006, Vol. 6, No. 3 Editorial In the last two papers, particular emphasis will be granted to the use of proteomic approaches leading to understanding of microbial pathogenesis and development of new vaccine, diagnostic and antibiotherapy tools. Lamont et al. (Mass Spectrometry-Based Proteomics and Its Application to Studies of Porphyromonas gingivalis Invasion and Pathogenicity) will depict the recent advances in proteomic methods based on multidimensional capillary HPLC and tandem mass spectrometry, which allow the acquisition of comprehensive protein expression datasets. These datasets are comparable with spotted cDNA arrays in terms of coverage and quantitative precision. Finally, Handfield and Hillman (In Vivo Induced Antigen Technology (IVIAT) and Change Mediated Antigen Technology (CMAT)) will review recent applications of IVIAT and CMAT to various human and plant pathogens.