Feasibility of global gene expression analysis in testicular biopsies from infertile men

Numerous studies have documented the use of microarray analysis to identify patterns of global gene expression that distinguish normal development from that of the diseased state. Yet, there are no reports that compare global gene expression in the fertile and infertile human testis. Here, we report an initial study of global gene expression in testicular biopsies from several men with different infertility phenotypes. We found that microarray analysis of small biopsy samples was suitable for profiling expression of genes known to function in germ cell development and also identified expression of novel genes. Since it is now common for infertile men with spermatogenic failure to use intracytoplasmic sperm injection (ICSI) to achieve biological paternity, we hypothesize that molecular screening of testicular biopsies with microarrays may be suitable: (1) to categorize the molecular phenoytpes of infertile testes in a manner similar to standard morphologic analysis and (2) to initiate larger studies of gene expression in the infertile testes that may identify genetic signatures from biopsies that allow prediction of outcomes.     

Global synthetic-lethality analysis and yeast functional profiling

The Saccharomyces genome-deletion project created >5900 'molecularly barcoded' yeast knockout mutants (YKO mutants). The YKO mutant collections have facilitated large-scale analyses of a multitude of mutant phenotypes. For example, both synthetic genetic array (SGA) and synthetic-lethality analysis by microarray (SLAM) methods have been used for synthetic-lethality screens. Global analysis of synthetic lethality promises to identify cellular pathways that 'buffer' each other biologically. The combination of global synthetic-lethality analysis, together with global protein-protein interaction analyses, mRNA expression profiling and functional profiling will, in principle, enable construction of a cellular 'wiring diagram' that will help frame a deeper understanding of human biology and disease.     

Gene profile of replicative senescence is different from progeria or elderly donor

In vitro cellular senescence of human diploid fibroblast has been a good model for aging research, which shows similar phenotypes to in vivo aging. Gene expression profiling would provide an insight to understand the mechanism of senescence. Using cDNA microarray containing 384 known genes, we compared the expression profiles of three different types of aging models: replicative senescence, fibroblasts from progeria or from elderly donor. Although all of them showed senescence phenotypes, distinct sets of genes were altered in each group. Pairwise plots or cluster analysis of activation fold of gene expression revealed closer relationships between fibroblasts from progeria or from old individual, but not between replicative senescence fibroblasts and either models. Differential expression pattern of several genes were confirmed by RT-PCR. We suggest that the replicative senescence model might behave differently to other types of aging models due to the distinct gene expression.     

Cell protection, resistance and invasiveness of two malignant mesotheliomas as assessed by 10K-microarray

Malignant pleural mesothelioma (MPM) is an aggressive serosal tumor, strongly associated with former exposure to asbestos fibers and for which there is currently no effective treatment available. In human, MPM is characterized by a high local invasiveness, poor prognosis and therapeutic outcomes. In order to assess molecular changes that specify this phenotype, we performed a global gene expression profiling of human MPM. Using a 10,000-element microarray, we analyzed mRNA relative gene expression levels by comparing a mesothelioma cell line to either a pleural cell line or tumor specimens. To analyze these gene expression data, we used various bioinformatics softwares. Hierarchical clustering methods were used to group genes and samples with similar expression in an unsupervised mode. Genes of known function were further sorted by enzyme, function and pathway clusters using a supervised software (IncyteGenomics). Taken together, these data defined a molecular fingerprint of human MPM with more than 700 up- or down-regulated genes related to several traits of the malignant phenotype, specially associated with MPM invasiveness, protection and resistance to anticancer defenses. This portrait is meaningful in disease classification and management, and relevant in finding new specific markers of MPM. These molecular markers should improve the accuracy of mesothelioma diagnosis, prognosis and therapy.     

Characterization of the OxyR regulon of Neisseria gonorrhoeae

OxyR regulates the expression of the majority of H(2)O(2) responses in Gram-negative organisms. In a previous study we reported the OxyR-dependent derepression of catalase expression in the human pathogen Neisseria gonorrhoeae. In the present study we used microarray expression profiling of N. gonorrhoeae wild-type strain 1291 and an oxyR mutant strain to define the OxyR regulon. In addition to katA (encoding catalase), only one other locus displayed a greater than two-fold difference in expression in the wild type : oxyR comparison. This locus encodes an operon of two genes, a putative peroxiredoxin/glutaredoxin (Prx) and a putative glutathione oxidoreductase (Gor). Mutant strains were constructed in which each of these genes was inactivated. A previous biochemical study in Neisseria meningitidis had confirmed function of the glutaredoxin/peroxiredoxin. Assay of the wild-type 1291 cell free extract confirmed Gor activity, which was lost in the gor mutant strain. Phenotypic analysis of the prx mutant strain in H(2)O(2) killing assays revealed increased resistance, presumably due to upregulation of alternative defence mechanisms. The oxyR, prx and gor mutant strains were deficient in biofilm formation, and the oxyR and prx strains had decreased survival in cervical epithelial cells, indicating a key role for the OxyR regulon in these processes.     

3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3′-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (∼9.6K genes) and FFPET (∼8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (∼4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.     

Engineering HlyA hypersecretion in Escherichia coli based on proteomic and microarray analyses

Escherichia coli is a common host for recombinant protein production for biotechnology applications. Secretion to the extracellular media has the potential to reduce protein aggregation and to simplify downstream purification. However, the complexity of the mechanisms of protein secretion has confounded prior attempts to engineer enhanced secretion phenotypes. Here, mutagenesis was used to perturb E. coli W3110 cells secreting HlyA via a Type I pathway. An activity assay identified a mutant secreting fourfold more active alpha-hemolysin than the parent strain. The mutant was characterized using both high-density microarrays for mRNA profiling and a proteomics strategy for protein expression. The relative mRNA and protein expression levels of tRNA-synthetases were decreased in the mutant compared to the parent. A mathematical model of prokaryotic translation was used to design a variant of the hlyA gene that encodes the same amino acid sequence but uses rare codons to slow the rate of translation by altering five bases. Analysis of the parent strain transformed with a plasmid containing this variant gene resulted in the recovery of, and further improvement upon, the selected hypersecretion phenotype. These results present one of the first successful metabolic engineering attempts based on molecular information provided by mRNA and protein expression profiling approaches and resulting in a phenotype useful to the biotechnology community.