Floyd Wittink

GENE EXPRESSION PROFILE ANALYSIS OF EPILEPSY-ASSOCIATED GANGLIOGLIOMAS

Gangliogliomas (GG) constitute the most frequent tumor entity in young patients undergoing surgery for intractable epilepsy. The histological composition of GG, with the presence of dysplastic neurons, corroborates their maldevelopmental origin. However, their histogenesis, the pathogenetic relationship with other developmental lesions, and the molecular alterations underlying the epileptogenicity of these tumors remain largely unknown. We performed gene expression analysis using the Affymetrix Gene Chip System (U133 plus 2.0 array). We used GENMAPP and the Gene Ontology database to identify global trends in gene expression data. Our analysis has identified various interesting genes and processes that are differentially expressed in GG when compared with normal tissue. The immune and inflammatory responses were the most prominent processes expressed in GG. Several genes involved in the complement pathway displayed a high level of expression compared with control expression levels. Higher expression was also observed for genes involved in cell adhesion, extracellular matrix and proliferation processes. We observed differential expression of genes as cyclin D1 and cyclin-dependent kinases, essential for neuronal cell cycle regulation and differentiation. Synaptic transmission, including GABA receptor signaling was an under-expressed process compared with control tissue. These data provide some suggestions for the molecular pathogenesis of GG. Furthermore, they indicate possible targets that may be investigated in order to dissect the mechanisms of epileptogenesis and possibly counteract the epileptogenic process in these developmental lesions.

Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm

INTRODUCTION Our previous studies showed that Streptococcus mutans and Veillonella parvula dual-species biofilms have a different acid production profile and a higher resistance to chlorhexidine than their single-species counterparts. The aim of the current study was to test whether the susceptibility of S. mutans grown in the presence of V. parvula is also decreased when it is exposed to various other antimicrobials. Furthermore, the aim was to identify other changes in the physiology of S. mutans when V. parvula was present using transcriptomics. METHODS Susceptibility to antimicrobials was assessed in killing experiments. Transcript levels in S. mutans were measured with the help of S. mutans microarrays. RESULTS When V. parvula was present, S. mutans showed an increase in survival after exposure to various antimicrobials. Furthermore, this co-existence altered the physiology of S. mutans. The expression of genes coding for proteins involved in amino acid synthesis, the signal recognition particle-translocation pathway, purine metabolism, intracellular polysaccharide synthesis, and protein synthesis all changed. CONCLUSION Growing in a biofilm together with a non-pathogenic bacterium like V. parvula changes the physiology of S. mutans, and gives this bacterium an advantage in surviving antimicrobial treatment. Thus, the study of pathogens implicated in polymicrobial diseases, such as caries and periodontitis, should be focused more on multispecies biofilms. In addition, the testing of susceptibility to currently used and new antimicrobials should be performed on a multispecies microbial community rather than with single pathogens.

Photorespiratory 2-phosphoglycolate metabolism and photoreduction of O2 cooperate in high-light acclimation of Synechocystis sp. strain PCC 6803.

In cyanobacteria, photorespiratory 2-phosphoglycolate (2PG) metabolism is mediated by three different routes, including one route involving the glycine decarboxylase complex (Gcv). It has been suggested that, in addition to conversion of 2PG into non-toxic intermediates, this pathway is important for acclimation to high-light. The photoreduction of O2 (Mehler reaction), which is mediated by two flavoproteins Flv1 and Flv3 in cyanobacteria, dissipates excess reductants under high-light by the four electron-reduction of oxygen to water. Single and double mutants defective in these processes were constructed to investigate the relation between photorespiratory 2PG-metabolism and the photoreduction of O2 in the cyanobacterium Synechocystis sp. PCC 6803. The single mutants Δflv1, Δflv3, and ΔgcvT, as well as the double mutant Δflv1/ΔgcvT, were completely segregated but not the double mutant Δflv3/ΔgcvT, suggesting that the T-protein subunit of the Gcv (GcvT) and Flv3 proteins cooperate in an essential process. This assumption is supported by the following results: (1) The mutant Δflv3/ΔgcvT showed a considerable longer lag phase and sometimes bleached after shifts from slow (low light, air CO2) to rapid (standard light, 5% CO2) growing conditions. (2) Photoinhibition experiments indicated a decreased ability of the mutant Δflv3/ΔgcvT to cope with high-light. (3) Fluorescence measurements showed that the photosynthetic electron chain is reduced in this mutant. Our data suggest that the photorespiratory 2PG-metabolism and the photoreduction of O2, particularly that catalyzed by Flv3, cooperate during acclimation to high-light stress in cyanobacteria.

Transcriptome analysis of Traf6 function in the innate immune response of zebrafish embryos

TRAF6 is a key player at the cross-roads of development and immunity. The analysis of its in vivo molecular function is a great challenge since severe developmental defects and early lethality caused by Traf6 deficiency in knock-out mice interfere with analyses of the immune response. In this study we have used a new strategy to analyze the function of Traf6 in a zebrafish-Salmonella infectious disease model. In our approach the effect of a Traf6 translation-blocking morpholino was titrated down to avoid developmental defects and the response to infection under these conditions was studied using the combination of microarray analysis and whole transcriptome deep sequencing. Transcriptome profiling of the traf6 knock-down allowed the identification of a gene set whose responsiveness during infection is highly dependent on Traf6. Expression trend analysis based on the resulting datasets identified nine clusters of genes with characteristic transcription response profiles, demonstrating Traf6 has a dynamic role as a positive and negative regulator. Among the Traf6-dependent genes was a large set of well known anti-microbial and inflammatory genes. Additionally, we identified several genes which were not previously linked to a response to microbial infection, such as the fertility hormone gene gnrh2 and the DNA-damage regulated autophagy modulator 1 gene dram1. With the use of the zebrafish embryo model we have now analyzed the in vivo function of Traf6 in the innate immune response without interference of adaptive immunity.

Operon structure of Staphylococcus aureus

In bacteria, gene regulation is one of the fundamental characteristics of survival, colonization and pathogenesis. Operons play a key role in regulating expression of diverse genes involved in metabolism and virulence. However, operon structures in pathogenic bacteria have been determined only by in silico approaches that are dependent on factors such as intergenic distances and terminator/promoter sequences. Knowledge of operon structures is crucial to fully understand the pathophysiology of infections. Presently, transcriptome data obtained from growth curves in a defined medium were used to predict operons in Staphylococcus aureus. This unbiased approach and the use of five highly reproducible biological replicates resulted in 93.5% significantly regulated genes. These data, combined with Pearson’s correlation coefficients of the transcriptional profiles, enabled us to accurately compile 93% of the genome in operon structures. A total of 1640 genes of different functional classes were identified in operons. Interestingly, we found several operons containing virulence genes and showed synergistic effects for two complement convertase inhibitors transcribed in one operon. This is the first experimental approach to fully identify operon structures in S. aureus. It forms the basis for further in vitro regulation studies that will profoundly advance the understanding of bacterial pathophysiology in vivo.

Interorgan Coordination of the Murine Adaptive Response to Fasting

Starvation elicits a complex adaptive response in an organism. No information on transcriptional regulation of metabolic adaptations is available. We, therefore, studied the gene expression profiles of brain, small intestine, kidney, liver, and skeletal muscle in mice that were subjected to 0–72 h of fasting. Functional-category enrichment, text mining, and network analyses were employed to scrutinize the overall adaptation, aiming to identify responsive pathways, processes, and networks, and their regulation. The observed transcriptomics response did not follow the accepted “carbohydrate-lipid-protein” succession of expenditure of energy substrates. Instead, these processes were activated simultaneously in different organs during the entire period. The most prominent changes occurred in lipid and steroid metabolism, especially in the liver and kidney. They were accompanied by suppression of the immune response and cell turnover, particularly in the small intestine, and by increased proteolysis in the muscle. The brain was extremely well protected from the sequels of starvation. 60% of the identified overconnected transcription factors were organ-specific, 6% were common for 4 organs, with nuclear receptors as protagonists, accounting for almost 40% of all transcriptional regulators during fasting. The common transcription factors were PPARα, HNF4α, GCRα, AR (androgen receptor), SREBP1 and -2, FOXOs, EGR1, c-JUN, c-MYC, SP1, YY1, and ETS1. Our data strongly suggest that the control of metabolism in four metabolically active organs is exerted by transcription factors that are activated by nutrient signals and serves, at least partly, to prevent irreversible brain damage.

Low-carbon acclimation in carboxysome-less and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803

Using metabolic and transcriptomic phenotyping, we studied acclimation of cyanobacteria to low inorganic carbon (LC) conditions and the requirements for coordinated alteration of metabolism and gene expression. To analyse possible metabolic signals for LC sensing and compensating reactions, the carboxysome-less mutant ΔccmM and the photorespiratory mutant ΔglcD1/D2 were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed accumulation of 2-phosphoglycolate (2PG) in ΔccmM and of glycolate in ΔglcD1/D2 in LC- but also in high inorganic carbon (HC)-grown mutant cells. The accumulation of photorespiratory metabolites provided evidence for the oxygenase activity of RubisCO at HC. The global gene expression patterns of HC-grown ΔccmM and ΔglcD1/D2 showed differential expression of many genes involved in photosynthesis, high-light stress and N assimilation. In contrast, the transcripts of LC-specific genes, such as those for inorganic carbon transporters and components of the carbon-concentrating mechanism (CCM), remained unchanged in HC cells. After a shift to LC, ΔglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas ΔccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in ΔccmM without CCM induction and of glycolate in ΔglcD1/D2 with CCM induction, we regard a direct role for 2PG as a metabolic signal for the induction of CCM during LC acclimation as less likely. Instead, our data suggest a potential role for glycolate as a signal molecule for enhanced expression of CCM genes.

The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis

BackgroundThe Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.ResultsOur analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others.ConclusionAnalyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized.

The effect of calcium on the transcriptome of sporulating B. subtilis cells.

Bacterial spores formed in the presence of high concentrations of minerals are a major problem in the food industry because of their extreme heat resistance. In order to enhance our insight in the molecular mechanisms underlying this phenomenon we have performed a detailed time-resolved analysis of the genome-wide transcriptome pattern of Bacillus subtilis sporulated both in the absence and presence of high calcium concentrations. The data was analysed in two ways. First, we determined the influence of the presence of high calcium levels during sporulation on the expression of gene groups as defined in Subtilist and KEGG pathways database. Second, we assessed the differential expression at the level of individual genes. When analysing groups and pathways, we found that those annotated as being involved in sporulation were significantly affected. Also, groups and pathways involved in flagella formation and biofilm matrix production were affected by the presence of calcium in the sporulation medium. When we analysed the behaviour of individual genes we found 305 genes influenced by calcium, including all known spore coat polysaccharide biosynthesis genes (10 induced and 1 repressed). A number of the calcium affected genes were also involved in biofilm formation. Minimal overlap with other stress outputs like sigma B activation and weak acid stress response was noted. Those genes that did overlap were unique to that combination which corroborates the notion that the cells sense these conditions differently.

Serious complications in gene-expression studies with stress perturbation: An example of UV-exposed p53-mutant mouse embryonic fibroblasts.

Reanalysis of our UV study of p53-mutant mouse embryonic fibroblasts revealed an intriguing orchestration of massive transcriptome responses. However, close scrutiny of the data uncovered an affected mRNA/rRNA ratio, effectively inhibiting valid data analysis. UV-dose range-finding showed low-dose UV specific- and high-dose stress-related responses, which represent a plea for UV dose range-finding in experimental design.