Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen
Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins ...characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in
A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of
Leptosphaeria maculans. The physical limits of the cluster in
A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.
Summary
Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the ...epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two‐module non‐ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co‐regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.
We describe a case series of 35 Ebola virus disease (EVD) survivors during the epidemic in West Africa who had neurologic and accompanying psychiatric sequelae. Survivors meeting neurologic criteria ...were invited from a cohort of 361 EVD survivors to attend a preliminary clinic. Those whose severe neurologic features were documented in the preliminary clinic were referred for specialist neurologic evaluation, ophthalmologic examination, and psychiatric assessment. Of 35 survivors with neurologic sequelae, 13 had migraine headache, 2 stroke, 2 peripheral sensory neuropathy, and 2 peripheral nerve lesions. Of brain computed tomography scans of 17 patients, 3 showed cerebral and/or cerebellar atrophy and 2 confirmed strokes. Sixteen patients required mental health followup; psychiatric disorders were diagnosed in 5. The 10 patients who experienced greatest disability had co-existing physical and mental health conditions. EVD survivors may have ongoing central and peripheral nervous system disorders, including previously unrecognized migraine headaches and stroke.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human apolipoprotein C-II (apoC-II) slowly forms amyloid fibers in lipid-free solutions at physiological pH and salt concentrations (Hatters, D. M., MacPhee, C. E., Lawrence, L. J., Sawyer, W. H., ...and Howlett, G. J. (2000)Biochemistry 39, 8276–8283). Measurements of the time dependence of solution turbidity, thioflavin T reactivity, and the amount of sedimentable aggregate reveal that the rate and extent of amyloid formation are significantly increased by the addition of an inert polymer, dextran T10, at concentrations exceeding 20 g/liter. High dextran concentrations do not alter the secondary structure of the protein, fiber morphology, or the thioflavin T and Congo Red binding capacity of apoC-II amyloid. Analytical ultracentrifugation studies show that monomeric apoC-II does not associate significantly with dextran. The observed dependence of the overall rate of amyloid formation on dextran concentration may be accounted for quantitatively by a simple model for nonspecific volume exclusion. The model predicts that an increase in the fractional volume occupancy of macromolecules in a physiological fluid can nonspecifically accelerate the formation of amyloid fibers by any amyloidogenic protein.
Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made ...these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a ...class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.
Putative ETP gene clusters are present in 14 ascomycete taxa, but absent in numerous other ascomycetes examined. These clusters are discontinuously distributed in ascomycete lineages. Gene content is not absolutely fixed, however, common genes are identified and phylogenies of six of these are separately inferred. In each phylogeny almost all cluster genes form monophyletic clades with non-cluster fungal paralogues being the nearest outgroups. This relatedness of cluster genes suggests that a progenitor ETP gene cluster assembled within an ancestral taxon. Within each of the cluster clades, the cluster genes group together in consistent subclades, however, these relationships do not always reflect the phylogeny of ascomycetes. Micro-synteny of several of the genes within the clusters provides further support for these subclades.
ETP gene clusters appear to have a single origin and have been inherited relatively intact rather than assembling independently in the different ascomycete lineages. This progenitor cluster has given rise to a small number of distinct phylogenetic classes of clusters that are represented in a discontinuous pattern throughout ascomycetes. The disjunct heredity of these clusters is discussed with consideration to multiple instances of independent cluster loss and lateral transfer of gene clusters between lineages.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Gliotoxin, a major product of the gli non-ribosomal peptide synthetase gene cluster, is strongly associated with virulence of the opportunistic human pathogen Aspergillus fumigatus. Despite ...identification of the gli cluster, the pathway of gliotoxin biosynthesis has remained elusive, in part because few potential intermediates have been identified. In addition, previous studies suggest that knowledge of gli-dependent metabolites is incomplete. Here we use differential analysis by 2D NMR spectroscopy (DANS) of metabolite extracts derived from gli knock-out and wild-type (WT) strains to obtain a detailed inventory of gli-dependent metabolites. DANS-based comparison of the WT metabolome with that of ΔgliZ, a knock-out strain devoid of the gene encoding the transcriptional regulator of the gli cluster, revealed nine novel gliZ-dependent metabolites including unexpected structural motifs. Their identification provides insight into gliotoxin biosynthesis and may benefit studies of the role of the gli cluster in A. fumigatus virulence. Our study demonstrates the utility of DANS for correlating gene expression and metabolite biosynthesis in microorganisms.
The cytosolic C-terminal domain of the membrane copper transporter Ctr1 from the yeast Saccharomyces cerevisiae, Ctr1c, was expressed in E. coli as an oxygen-sensitive soluble protein with no ...significant secondary structure. Visible-UV spectroscopy demonstrated that Ctr1c bound four Cu(I) ions, structurally identified as a CuI 4(μ-S−Cys)6 cluster by Xray absorption spectroscopy. This was the only metalated form detected by electrospray ionization mass spectrometry. An average dissociation constant K D = (K 1 K 2 K 3 K 4)1/4 = 10-19 for binding of Cu(I) to Ctr1c was estimated via competition with the ligand bathocuproine disulfonate bcs (β2 = 1019.8). Equivalent experiments for the yeast chaperone Atx1 and an N-terminal domain of the yeast Golgi pump Ccc2, which both bind a single Cu(I) ion, provided similar K D values. The estimates of K D were supported by independent estimates of the equilibrium constants K ex for exchange of Cu(I) between pairs of these three proteins. It is apparent that, in vitro, the three proteins buffer “free” Cu(I) concentrations in a narrow range around 10-19 M. The results provide quantitative support for the proposals that, in yeast, (a) “free” copper concentrations are very low in the cytosol and (b) the Cu(I) trafficking gradient is shallow along the putative Ctrlc → Atx1 → Ccc2n metabolic pathway. In addition, both Ctr1c and its copper-responsive transcription factor Mac1 contain similar clusters which may be important in signaling copper status in yeast.
Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this ...parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by β-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native β-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.
Background: Successful percutaneous nephrolithotomy (PCNL) relies on a technically challenging, precise needle puncture of the renal collecting system. We aimed to compare, in an ex vivo model, the ...use of a real time image guidance system (the SabreSource™) and a mechanical stabilising device with conventional manual techniques for the accuracy of needle placement.Methods: The SabreSource™ system (Minrad International Inc.; New York, USA) is a real time image guidance system. The system platform is mounted on a C-arm fluoroscope. It employs targeting cross hairs on the fluoroscopic image that can be easily positioned to target the desired renal calyx. The system directs a visible laser beam onto the patient which is precisely aligned with the cross hairs on the fluoroscopic image. This provides the correct “bull’s-eye” angle of approach to the calyx, even after the x-ray source is turned off. The locator then stabilises the needle in the “bull’s-eye” position so that only screening for depth is required. Objective assessment using a simulated PCNL puncture was performed by 7 urologic trainees on a kidney phantom with and without using the SabreSource™. Fluoroscopy screening time (FST) and amount of radiation (mGy) used to achieve successful puncture were compared.Results: Simulated PCNL puncture was quicker and resulted in reduced radiation exposure when the apparatus was used. The mean FST for traditional “bull’s-eye” vs SabreSource™ puncture was 17 vs 5 seconds (p = 0.01), and the mean radiation exposure to puncture was 0.7 vs 0.2 mGy (p = 0.03), respectively.Conclusion: The SabreSource™ is a novel assistant to achieving successful PCNL puncture. In combination with “the locator” the preliminary in vitro testing suggests that the device reduces fluoroscopy exposure and is quicker. The device warrants further evaluation in the clinical setting.
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