30 research outputs found
Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia
Background: Community-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. Objective: To obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. Methods: Monocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1β and IL-10. Results: 50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1β and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. Conclusion: These results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.peer-reviewe
A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
In 5–40% of respiratory infections in children, the diagnostics
remain negative, suggesting that the patients might be infected with a yet
unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery
method based on recognition of restriction enzyme cleavage sites, ligation of
adaptors and subsequent amplification by PCR. However, direct discovery of
unknown pathogens in nasopharyngeal swabs is difficult due to the high
concentration of ribosomal RNA (rRNA) that acts as competitor. In the current
study we optimized VIDISCA by adjusting the reverse transcription enzymes and
decreasing rRNA amplification in the reverse transcription, using hexamer
oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA
templates was further reduced with oligonucleotides that anneal to rRNA but can
not be extended due to 3′-dideoxy-C6-modification. With these
modifications >90% reduction of rRNA amplification was established.
Further improvement of the VIDISCA sensitivity was obtained by high throughput
sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all
containing known respiratory viruses. We could identify the proper virus in the
majority of samples tested (11/18). The median load in the VIDISCA-454 positive
samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6).
Our results show that optimization of VIDISCA and subsequent
high-throughput-sequencing enhances sensitivity drastically and provides the
opportunity to perform virus discovery directly in patient material
Ribonuclease H1 maps to chromosome 2 and has at least three pseudogene loci in the human genome
We have analyzed the genomic structure of ribonuclease H1 (RNase H1) loci in the human genome. Human PAC library screening combined with database searches indicated that several loci are present. The transcribed gene is localized on chromosome 2p25. This was confirmed by RNA analysis of a monochromosomal hybrid cell line that expressed human chromosome 2. These data contradict a previous report, as well as the current Human Genome Project (HGP) annotation, which had placed the gene on chromosome 17p11.2. This location represents a pseudogene. Another highly similar pseudogene is present at a separate locus located more distal on chromosome 17p, while a third pseudogene is localized on chromosome 1
Looking ultra deep: short identical sequences and transcriptional slippage
Studying transcriptomes by ultra deep sequencing provides an in-depth picture of transcriptional regulation and it facilitates the detection of rare transcriptional events. Using ultra deep sequencing of amplicons we identified known isoforms and also various new low frequency variants. Most of these variants likely involve the splicing machinery except for two events that we named variations affecting multiple exons, which are mainly deletions affecting parts of adjacent exons and intra-exonic deletions. Both events involve short identical sequences of 1 to 8 nucleotides at the junction and canonical splice sites are missing. They were identified in different genes and species at very low frequencies. We excluded that they are an artifact of PCR, sequencing, or reverse transcription. We propose that these variants represent intramolecular slippage events that require short identical sequences for reannealing of dissociated transcript
In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides
Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo
Amplified fragment length polymorphism and whole genome sequencing : A comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci
Background: Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods: Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-Analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results: Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4-5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion: AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management
Amplified fragment length polymorphism and whole genome sequencing: A comparison of methods in the investigation of a nosocomial outbreak with vancomycin resistant enterococci
Background: Recognition of nosocomial outbreaks with antimicrobial resistant (AMR) pathogens and appropriate infection prevention measures are essential to limit the consequences of AMR pathogens to patients in hospitals. Because unrelated, but genetically similar AMR pathogens may circulate simultaneously, rapid high-resolution molecular typing methods are needed for outbreak management. We compared amplified fragment length polymorphism (AFLP) and whole genome sequencing (WGS) during a nosocomial outbreak of vancomycin-resistant Enterococcus faecium (VRE) that spanned 5 months. Methods: Hierarchical clustering of AFLP profiles was performed using unweighted pair-grouping and similarity coefficients were calculated with Pearson correlation. For WGS-Analysis, core single nucleotide polymorphisms (SNPs) were used to calculate the pairwise distance between isolates, construct a maximum likelihood phylogeny and establish a cut-off for relatedness of epidemiologically linked VRE isolates. SNP-variations in the vanB gene cluster were compared to increase the comparative resolution. Technical replicates of 2 isolates were sequenced to determine the number of core-SNPs derived from random sequencing errors. Results: Of the 721 patients screened for VRE carriage, AFLP assigned isolates of 22 patients to the outbreak cluster. According to WGS, all 22 isolates belonged to ST117 but only 21 grouped in a tight phylogenetic cluster and carried vanB resistance gene clusters. Sequencing of technical replicates showed that 4-5 core-SNPs were derived by random sequencing errors. The cut-off for relatedness of epidemiologically linked VRE isolates was established at ≤7 core-SNPs. The discrepant isolate was separated from the index isolate by 61 core-SNPs and the vanB gene cluster was absent. In AFLP analysis this discrepant isolate was indistinguishable from the other outbreak isolates, forming a cluster with 92% similarity (cut-off for identical isolates ≥90%). The inclusion of the discrepant isolate in the outbreak resulted in the screening of 250 patients and quarantining of an entire ward. Conclusion: AFLP was a rapid and affordable screening tool for characterising hospital VRE outbreaks. For in-depth understanding of the outbreak WGS was needed. Compared to AFLP, WGS provided higher resolution typing of VRE isolates with implications for outbreak management
Targeted sequence capture and GS-FLX Titanium sequencing of 23 hypertrophic and dilated cardiomyopathy genes: implementation into diagnostics
Genetic evaluation of cardiomyopathies poses a challenge. Multiple genes are involved but no clear genotype-phenotype correlations have been found so far. In the past, genetic evaluation for hypertrophic (HCM) and dilated (DCM) cardiomyopathies was performed by sequential screening of a very limited number of genes. Recent developments in sequencing have increased the throughput, enabling simultaneous screening of multiple genes for multiple patients in a single sequencing run. Development and implementation of a next generation sequencing (NGS) based genetic test as replacement for Sanger sequencing. In order to increase the number of genes that can be screened in a shorter time period, we enriched all exons of 23 of the most relevant HCM and DCM related genes using on-array multiplexed sequence capture followed by massively parallel pyrosequencing on the GS-FLX Titanium. After optimisation of array based sequence capture it was feasible to reliably detect a large panel of known and unknown variants in HCM and DCM patients, whereby the unknown variants could be confirmed by Sanger sequencing. The rate of detection of (pathogenic) variants in both HCM and DCM patients was increased due to a larger number of genes studied. Array based target enrichment followed by NGS showed the same accuracy as Sanger sequencing. Therefore, NGS is ready for implementation in a diagnostic settin