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Zoll J.,RadboudUMC | Rahamat-Langendoen J.,RadboudUMC | Ahout I.,Laboratory of Pediatric Infectious Diseases | Jonge M.I.,Laboratory of Pediatric Infectious Diseases | And 5 more authors.
Journal of Clinical Virology | Year: 2015

Background: Acute respiratory tract infections (RTI) cause substantial morbidity during childhood, and are responsible for the majority of pediatric infectious diseases. Although most acute RTI are thought to be of viral origin, viral etiology is still unknown in a significant number of cases. Objectives: Multiplexed whole genome sequencing (WGS) was used for virome determination directly on clinical samples as proof of principle for the use of deep sequencing techniques in clinical diagnosis of viral infections. Study design: WGS was performed with nucleic acids from sputum and nasopharyngeal aspirates from four pediatric patients with known respiratory tract infections (two patients with human rhinovirus, one patient with human metapneumovirus and one patient with respiratory syncytial virus), and from four pediatric patients with PCR-negative RTI, and two control samples. Results: Viral infections detected by routine molecular diagnostic methods were confirmed by WGS; in addition, typing information of the different viruses was generated. In three out of four samples from pediatric patients with PCR-negative respiratory tract infections and the two control samples, no causative viral pathogens could be detected. In one sample from a patient with PCR-negative RTI, rhinovirus type-C was detected. Almost complete viral genomes could be assembled and in all cases virus species could be determined. Conclusions: Our study shows that, in a single run, viral pathogens can be detected and characterized, providing information for clinical assessment and epidemiological studies. We conclude that WGS is a powerful tool in clinical virology that delivers comprehensive information on the viral content of clinical samples. © 2015 Elsevier B.V. Source

Smeulders M.J.,Radboud University Nijmegen | Pol A.,Radboud University Nijmegen | Venselaar H.,Center for Molecular and Biomolecular Informatics | Barends T.R.M.,Max Planck Institute for Medical Research | And 3 more authors.
Journal of Bacteriology | Year: 2013

Carbon disulfide (CS2) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS2 is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO2) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS2-polluted airstreams. We report on the mechanism of bacterial CS2 conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS2 hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS2 hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of β-carbonic anhydrase (β-CA) homologues that may comprise a subclass of CS2 hydrolases within the β-CA family. Unlike CAs, the CS2 hydrolases did not hydrate CO2 but converted CS2 and COS with H2O toH2S and CO2. The CS2 hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS2 hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS2 hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS2 hydrolases based on the structure of Acidianus strain A1-3 CS2 hydrolase suggest that the A. thiooxidans strain G8 CS2 hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation. © 2013, American Society for Microbiology. Source

Soysa R.,Oregon Health And Science University | Venselaar H.,Center for Molecular and Biomolecular Informatics | Poston J.,Oregon Health And Science University | Ullman B.,Oregon Health And Science University | Hasne M.-P.,Oregon Health And Science University
Biochemical Journal | Year: 2013

The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by thisAPC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket. © The Authors Journal compilation © 2013 Biochemical Society. Source

Szalowska E.,RIKILT Institute of Food Safety | Tesfay H.A.,RIKILT Institute of Food Safety | Tesfay H.A.,Wageningen University | van Hijum S.A.F.T.,Center for Molecular and Biomolecular Informatics | Kersten S.,RIKILT Institute of Food Safety
BMC Genomics | Year: 2014

Background: The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor that regulates lipid catabolism and inflammation and is hepatocarcinogenic in rodents. It is presumed that the functions of PPARα in liver depend on cross-talk between parenchymal (hepatocytes) and non-parenchymal (Kupffer and endothelial cells) fractions as well as inter-organ interactions. In order to determine how cellular composition and inter-organ interactions influence gene expression upon pharmacological activation of PPARα, we performed a meta-analysis of transcriptomics data obtained from mouse hepatocytes (containing only the parenchymal fraction), mouse liver slices (containing both fractions), and mouse livers exposed to a PPARα agonist. The aim was to obtain a comprehensive view of common and model-specific PPARα-dependent genes and biological processes to understand the impact of cross-talk between parenchymal and non-parenchymal fractions as well as the effect of inter-organ interactions on the hepatic PPARα transcriptome. Results: In all models, activation of PPARα significantly altered processes related to various aspects of lipid metabolism. In ex vivo and in vivo models, PPARα activation significantly regulated processes involved in inflammation; these processes were unaffected in hepatocytes. Only in vivo models showed significant regulation of genes involved in coagulation, carcinogenesis, as well as vesicular trafficking and extracellular matrix. Conclusions: PPARα-dependent regulation of genes/processes involved in lipid metabolism is mostly independent of the presence of non-parenchymal cells or systemic factors, as it was observed in all liver models. PPARα-dependent regulation of inflammatory genes requires the presence of non-parenchymal cells, as it was observed only ex vivo and in vivo. However, the full spectrum of PPARα biology at the level of lipid metabolism, immunity, carcinogenesis, as well as novel aspects of PPARα signaling such as coagulation, vesicular trafficking and the extracellular matrix, seems to require systemic factors, as it was observed exclusively in vivo. © Szalowska et al.; licensee BioMed Central. 2014This article is published under license to BioMed Central Ltd. Source

Snelders E.,Radboud University Nijmegen | Camps S.M.T.,Radboud University Nijmegen | Karawajczyk A.,Center for Molecular and Biomolecular Informatics | Karawajczyk A.,LeadPharma Medicine B.V | And 6 more authors.
PLoS ONE | Year: 2012

Background: Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR 34/L98H). We investigated if TR 34/L98H could have developed through exposure to DMIs. Methods and Findings: Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in the Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR 34/L98H isolate in 1998. Through microsatellite genotyping of TR 34/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR 34/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. Conclusions: Our findings support a fungicide-driven route of TR 34/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs. © 2012 Snelders et al. Source

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