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Preface xiii
List of Contributors xv
Diagnosis of Candida Infection in Tissue by Immunohistochemistry Malcolm D. Richardson Riina Rautemaa Jarkko Hietanen 1
Introduction 1
Specificity of monoclonal antibody 3H8 for C. albicans 3
Testing of specificity of monoclonal antibody 3H8 4
Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6
Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6
Application of immunohistochemistry in the diagnosis of Candida periodontal disease 7
Use of monoclonal antibody 3H8 in the detection of C. Albicans in tissue 8
References 11
Transmission Electron Microscopy of Pathogenic Fungi Guy Tronchin Jean-Philippe Bouchara 13
Introduction 13
Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis 16
Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis 17
Identification of the different compartments of the secretory pathway in yeasts 18
Identification of the different compartments of thesecretory pathway in yeasts 19
Cytochemical localization of acid phosphatase in yeasts 20
Cytochemical localization of acid phosphatase in yeasts 21
Detection of anionic sites 23
Detection of anionic sites 23
Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 25
Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 26
Enzyme-gold approach for the detection of polysaccharides in the cell wall 28
Enzyme-gold approach for the detection of polysaccharides in the cell wall 29
Detection of glycoconjugates by the lectin-gold technique 30
Detection of glycoconjugates by the lectin-gold technique 31
Immunogold detection of antigens on ultrathin sections of acrylic resin 33
Immunogold detection of antigens on ultrathin sections of acrylic resin 34
Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 36
Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 37
Overview 38
References 39
Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi Maria Simitsopoulou Emmanuel Roilides 43
Introduction 43
Preparation of conidia and hyphae of opportunistic fungi 45
Preparation of conidia and hyphae of opportunistic fungi 45
Preparation of hyphal fragments 47
Isolation of human monocytes from whole blood 48
Isolation of human MNCs from whole blood 48
Analysis of human MNC function in response to fungal infection 51
XTT microassay 52
Superoxide anion assay in 96-well plate 53
Hydrogen peroxide-rhodamine assay 55
Phagocytosis assay 55
Evaluation of immunomodulators in response to fungal infection 57
Analysis of gene expression by RT-PCR 58
Analysis of pathway-specific gene expression by microarray technology 63
Assessment of cytokines and chemokines by ELISA 66
Overview 67
References 68
Determination of the Virulence Factors of Candida Albicans and Related Yeast Species Khaled H. Abu-Elteen Mawieh Hamad 69
Introduction 69
Measurement of Candida species adhesion in vitro 70
The visual assessment of candidal adhesion to BECs 70
The radiometric measurement of candidal adhesion 73
Adhesion to inanimate surfaces 75
Assessment of candidal adhesion to denture acrylic material 75
Adherence of Candida to plastic catheter surfaces 76
C. albicans strain differentiation 77
Resistogram typing 77
The slide agglutination technique 79
Serotyping of C. albicans by flow cytomerty 80
Phenotypic switching in C. albicans 81
Evaluation of phenotype switching in C. albicans 82
Extracellular enzymes secreted by C. albicans 83
Measurement of extracellular proteinase production by C. albicans 85
Measurement of extracellular proteinase produced by C. albicans (staib method) 86
Measurement of extracellular phospholipases of C. albicans 87
Germ-tube formation in C. albicans 88
Germ-tube formation assay 88
References 89
Analysis of Drug Resistance in Pathogenic Fungi Gary P. Moran Emmanuelle Pinjon David C. Coleman Derek J. Sullivan 93
Introduction 93
Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 96
Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 97
Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters 102
Measurement of rhodamine 6G uptake and glucose-induced efflux 102
Analysis of expression of multidrug transporters in pathogenic fungi 105
Analysis of point mutations in genes encoding cytochrome P-450 lanosterol demethylase 106
Qualitative detection of alterations in membrane sterol contents 108
Qualitative detection of alterations in membrane sterol contents 109
Overview 110
References 110
Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi Eric Dannaoui 115
Introduction 115
Disseminated zygomycosis in non-immunosuppressed mice 118
Disseminated zygomycosis in non-immunosuppressed mice 118
Animal model of disseminated aspergillosis 125
Disseminated aspergillosis in neutropoenic mice 126
Study design for evaluation of antifungal combinations therapy in animal models 130
Study design for the evaluation of combination therapy in animal models 131
References 133
Proteomic Analysis of Pathogenic Fungi Alan Murphy 137
Introduction 137
2D SDS-PAGE of protein samples 139
Protein digestion in preparation for mass spectrometry by MALDI-TOF 140
Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry 141
In-gel digestion 142
In-solution digestion 143
MALDI-TOF mass spectrometry 145
Preparation of matrix for MALDI-TOF 147
Peptide mass fingerprinting (PMF) 149
Post-source decay (PSD) and chemically assisted fragmentation (CAF) 150
Interpreting MALDI-TOF result spectra 152
Overview 156
References 157
Extraction and Detection of DNA and RNA from Yeast Patrick Geraghty Kevin Kavanagh 159
Introduction 159
The extraction of yeast DNA with the aid of phenol: chloroform 161
Whole-cell DNA extraction from C. albicans using phenol: chloroform 161
Rapid extraction of DNA from C. albicans colonies for PCR 164
Detection of yeast DNA using radio-labelled probes 165
DNA detection by Southern blotting 165
Extraction of whole-cell RNA using two different protocols 169
The extraction of whole-cell RNA from yeast using phenol: chloroform 170
Rapid extraction of whole-yeast-cell RNA 172
Detection and expression levels of specific genes by the examination of mRNA in yeast 174
Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis 175
Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis 176
References 179
Microarrays for Studying Pathogenicity in Candida Albicans Andre Nantel Tracey Rigby Herve Hogues Malcolm Whiteway 181
Introduction 181
DNA microarrays 182
Building a second-generation 2-colour long oligonucleotide microarray for C. albicans 183
Isolation of C. albicans RNA 185
Isolation of total RNA using the hot phenol method 186
Isolation of Poly-A+ mRNA 187
Determination of the efficiency of incorporation of the probe 192
Hybridization to DNA microarrays 194
Experiment design 196
Microarray-based studies in C. albicans 200
Conclusion 205
References 205
Molecular Techniques for Application with Aspergillus Fumigatus Nir Osherov Jacob Romano 211
Introduction 211
Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 212
Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 213
Transformation of A. fumigatus 217
Chemical transformation of A. fumigatus 218
Molecular verification of correct single integration (PCR-based) 220
Molecular verification of correct integration by PCR 221
General strategies for the phenotypic characterization of A. fumigatus mutant strains 223
General strategies for the phenotypic characterization of mutants 223
References 229
Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus Matthias Brock Alexander Gehrke Venelina Sugareva Axel A. Brakhage 231
Introduction 231
Site-directed mutagenesis of promoter elements 233
Site-directed mutation of promoter elements 233
lacZ as a reporter gene 236
lacZ as a reporter gene: discontinuous determination of [Beta]-galactosidase activity 237
lacZ as a reporter gene: continuous determination of [Beta]-galactosidase activity 239
Transformation of A. fumigatus 241
Transformation of A. fumigatus 242
Hygromycin B as a selection marker for transformation 246
Hygromycin B as a selection marker for transformation 247
pyrG as a selection marker for transformation 249
pyrG as a selection marker for transformation 249
URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 251
URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 253
References 255
Microarray Technology for Studying the Virulence of Aspergillus Fumigatus Darius Armstrong-James Thomas Rogers 257
Introduction 257
Isolation of RNA from A. fumigatus 259
Isolation of total RNA from A. fumigatus 260
Reverse transcription of RNA and fluorescent labelling of cDNA 263
Indirect labelling of cDNA with fluorescent dyes 264
Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 266
Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 267
Image acquisition from hybridized microarrays 269
Image acquisition from hybridized microarrays 269
Microarray image analysis 270
Microarray image analysis 271
References 272
Techniques and Strategies for Studying Virulence Factors Cryptococcus Neoformans Nancy Lee Guilhem Janbon 275
Introduction 275
Construction of C. neoformans gene-disruption cassettes 278
Construction of C. neoformans gene-disruption cassettes 278
Genetic transformation of C. neoformans 283
Biolistic transformation of C. neoformans 283
Transformation via electroporation 286
Extraction of genomic DNA from C. neoformans 287
DNA for use in library construction and hybridization analysis 288
DNA for use in PCR 291
Screening and identification of deletion strains 292
Screening and identification of deletion strains 292
Measuring capsule size in C. neoformans 297
Measuring capsule size in C. neoformans 298
Purification of glucuronoxylomannan (GXM) 298
Purification of glucuronoxylomannan (GXM) 299
References 301
Genetic Manipulation of Zygomycetes Ashraf S. Ibrahim Christopher D. Skory 305
Introduction 305
Genetic tools to manipulate mucorales 306
Selectable markers used with mucorales fungi 307
Introduction of DNA used for transformation 308
Protoplasting of R. oryzae 309
Biolistic delivery system transformation of R. oryzae 313
A. tumefaciens-mediated transformation 316
Molecular analysis of transformants 319
Overview 322
References 323
Index 327
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