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Harm van Bakel

Publications and source records attributed to Harm van Bakel.

11 recordsLinked to original sources
PubMed2024

No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Humans↗
PubMed2023

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Humans↗
PubMed2007

Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes.

Copper toxicity in the liver is mediated by free-radical generation, resulting in oxidative stress. To prevent toxic accumulation of copper, liver cells adapt to high copper levels. Here, we used microarray analysis to compare the adaptive responses on global gene expression in liver cells exposed to high copper levels in vitro and in vivo. In HepG2 cells we identified two clusters of upregulated genes over time, an "early" cluster that comprised metallothionein genes and a "late" cluster, highly enriched in genes involved in proteasomal degradation and in oxidative stress response. Concomitant with the "late" cluster, we detected a significant downregulation of several copper metabolism MURR1 domain (COMMD) genes that were recently implicated in copper metabolism and inhibition of nuclear transcription factor kappaB (NF-kappaB) signaling. As metal-induced oxidative stress increases NF-kappaB activity, our data suggest a role for reduced COMMD protein levels in prolonged activation of NF-kappaB, thus inducing cell survival. Mice exposed to a copper diet that highly exceeded normal daily intake accumulated only twofold more hepatic copper than control mice. Although a moderate, but significant upregulation of a set of 22 genes involved in immunity, iron and cholesterol metabolism was detected, these cannot account for direct mechanisms involved in copper excretion. In conclusion, we identified a novel set of genes that represent a delayed response to copper overload, thus providing insight into the adaptive transcriptional response to copper-induced oxidative stress.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Reconstruction of a functional human gene network, with an application for prioritizing positional candidate genes.

Most common genetic disorders have a complex inheritance and may result from variants in many genes, each contributing only weak effects to the disease. Pinpointing these disease genes within the myriad of susceptibility loci identified in linkage studies is difficult because these loci may contain hundreds of genes. However, in any disorder, most of the disease genes will be involved in only a few different molecular pathways. If we know something about the relationships between the genes, we can assess whether some genes (which may reside in different loci) functionally interact with each other, indicating a joint basis for the disease etiology. There are various repositories of information on pathway relationships. To consolidate this information, we developed a functional human gene network that integrates information on genes and the functional relationships between genes, based on data from the Kyoto Encyclopedia of Genes and Genomes, the Biomolecular Interaction Network Database, Reactome, the Human Protein Reference Database, the Gene Ontology database, predicted protein-protein interactions, human yeast two-hybrid interactions, and microarray co-expressions. We applied this network to interrelate positional candidate genes from different disease loci and then tested 96 heritable disorders for which the Online Mendelian Inheritance in Man database reported at least three disease genes. Artificial susceptibility loci, each containing 100 genes, were constructed around each disease gene, and we used the network to rank these genes on the basis of their functional interactions. By following up the top five genes per artificial locus, we were able to detect at least one known disease gene in 54% of the loci studied, representing a 2.8-fold increase over random selection. This suggests that our method can significantly reduce the cost and effort of pinpointing true disease genes in analyses of disorders for which numerous loci have been reported but for which most of the genes are unknown.

Computational Biology↗
PubMed2005

Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets.

Mediator is an evolutionarily conserved coregulator of RNA polymerase II transcription. Microarray structure-function analysis of S. cerevisiae Mediator reveals functional antagonism between the cyclin-dependent kinase (Cdk) submodule and components from the Tail (Med15, Med2, Med3), Head (Med20, Med18), and Middle (Med31). Certain genes exhibit increased or decreased expression, depending on which subunit is deleted. Epistasis analysis with expression-profile phenotypes shows that MED2 and MED18 are downstream of CDK8. Strikingly, Cdk8-mediated modification of a single amino acid within Mediator represses the regulon of a single transcription factor, Rcs1/Aft1. Highly specific gene regulation is thought to be determined by activators and combinatorial use of cofactors. Here, subtle modification of the general transcription machinery through one of its own components is shown to determine highly specific expression patterns. Expression profiling can therefore precisely map regulatory cascades, and our findings support a role for Mediator as a direct processor of signaling pathways for determining specificity.

Chromosomal Proteins, Non-Histone↗
PubMed2005

Gene expression profiling and phenotype analyses of S. cerevisiae in response to changing copper reveals six genes with new roles in copper and iron metabolism.

Exhaustive microarray time course analyses of Saccharomyces cerevisiae during copper starvation and copper excess reveal new aspects of metal-induced gene regulation. Aside from identifying targets of established copper- and iron-responsive transcription factors, we find that genes encoding mitochondrial proteins are downregulated and that copper-independent iron transport genes are preferentially upregulated, both during prolonged copper deprivation. The experiments also suggest the presence of a small regulatory iron pool that links copper and iron responses. One hundred twenty-eight genes with putative roles in metal metabolism were further investigated by several systematic phenotype screens. Of the novel phenotypes uncovered, hsp12-Delta and arn1-Delta display increased sensitivity to copper, cyc1-Delta and crr1-Delta show resistance to high copper, vma13-Delta exhibits increased sensitivity to iron deprivation, and pep12-Delta results in reduced growth in high copper and low iron. Besides revealing new components of eukaryotic metal trafficking pathways, the results underscore the previously determined intimate links between iron and copper metabolism and mitochondrial and vacuolar function in metal trafficking. The analyses further suggest that copper starvation can specifically lead to downregulation of respiratory function to preserve iron and copper for other cellular processes.

Amino Acid Motifs↗
PubMed2004

Prokaryotic diversity of the Saccharomyces cerevisiae Atx1p-mediated copper pathway.

MOTIVATION: Several genes involved in the cellular import of copper and its subsequent incorporation into the high-affinity iron transport complex in Saccharomyces cerevisiae are known to be conserved between eukaryotes and prokaryotes. However, the degree to which these genes share their functional context as members of the same pathway in the prokaryotic domain is less clear. RESULTS: The co-occurrence of gene families involved in Atx1p-mediated copper transport in the genomes and operon structures of 80 non-redundant prokaryotes was investigated. For this purpose, we developed a Web tool (SHOPS) to display the operon context for a given set of proteins. In total, a set of 43 putative operons was identified. These were found to be involved in a variety of pathways and indicate a large diversity in the functional context of the individual gene family members. AVAILABILITY: The SHOPS tool can be found at http://www.bioinformatics.med.uu.nl/shops supplemental data are available at http://humgen.med.uu.nl/publications/vanbakel/pathway/

Algorithms↗
PubMed2004

TEAM: a tool for the integration of expression, and linkage and association maps.

The identification of genes primarily responsible for complex genetic disorders is a daunting task. Despite the assignment of many susceptibility loci, there has only been limited success in identifying disease genes based solely on positional information from genome-wide screens. The incorporation of several complementary strategies in a single integrated approach should facilitate and further enhance the efficacy of this search for genes. To permit the integration of linkage, association and expression data, together with functional annotations, we have developed a Java-based software tool: TEAM (tool for the integration of expression, and linkage and association maps). TEAM includes a genome viewer, capable of overlaying karyobands, genes, markers, linkage graphs, association data, gene expression levels and functional annotations in one composite view. Data management, analysis and filtering functionality was implemented and extended with links to the Ensembl, Unigene and Gene Ontology databases to facilitate gene annotation. Filtering functionality can help prevent the exclusion of poorly annotated, but differentially expressed, genes that reside in candidate regions that show linkage or association. Here we demonstrate the program's functionality in our study on coeliac disease (OMIM 212750), a multifactorial gluten-sensitive enteropathy. We performed a combined data analysis of a genome-wide linkage screen in 82 Dutch families with affected siblings and the microarray expression profiles of 18,110 cDNAs in 22 intestinal biopsies.

Celiac Disease↗
PubMed2004

In control: systematic assessment of microarray performance.

Expression profiling using DNA microarrays is a powerful technique that is widely used in the life sciences. How reliable are microarray-derived measurements? The assessment of performance is challenging because of the complicated nature of microarray experiments and the many different technology platforms. There is a mounting call for standards to be introduced, and this review addresses some of the issues that are involved. Two important characteristics of performance are accuracy and precision. The assessment of these factors can be either for the purpose of technology optimization or for the evaluation of individual microarray hybridizations. Microarray performance has been evaluated by at least four approaches in the past. Here, we argue that external RNA controls offer the most versatile system for determining performance and describe how such standards could be implemented. Other uses of external controls are discussed, along with the importance of probe sequence availability and the quantification of labelled material.

Animals↗
PubMed2003

Monitoring global messenger RNA changes in externally controlled microarray experiments.

Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.

Bacillus subtilis↗