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Find video protocols related to scientific articles indexed in Pubmed.
Use of 70% alcohol for the routine removal of microbial hard surface bioburden in life science cleanrooms.
Future Microbiol
PUBLISHED: 11-19-2014
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ABSTRACT? Alcohol-based disinfectants are used for the removal of microbial hard surface bioburden in Life science Cleanrooms. Evidence for using formulations containing 70% alcohol has been lost over time but probably originates from historical observations of the activity of 60-70% alcohol. Tradition is no longer adequate to inform contemporary cleaning practice. We evaluated the efficacy of ethanol, isopropanol and trade-specific denatured alcohol 7 against vegetative Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Enterococcus hirae using standardized European Suspension and Hard Surface tests. All three alcohols were effective at lower concentrations than the 70% standard. This constitutes the first evaluation of disinfectant formulations containing ?70% alcohol using standard methodology. The utility of trade-specific denatured alcohol #7 and evidence-based cleanroom practice warrant further validation.
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Resistance to antifungals that target CYP51.
J Chem Biol
PUBLISHED: 10-01-2014
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Fungal diseases are an increasing global burden. Fungi are now recognised to kill more people annually than malaria, whilst in agriculture, fungi threaten crop yields and food security. Azole resistance, mediated by several mechanisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of widespread use of triazole fungicides in agriculture and triazole antifungal drugs in the clinic. Mutations similar to those seen in clinical isolates as long ago as the 1990s in Candida albicans and later in Aspergillus fumigatus have been identified in agriculturally important fungal species and also wider combinations of point mutations. Recently, evidence that mutations originate in the field and now appear in clinical infections has been suggested. This situation is likely to increase in prevalence as triazole fungicide use continues to rise. Here, we review the progress made in understanding azole resistance found amongst clinically and agriculturally important fungal species focussing on resistance mechanisms associated with CYP51. Biochemical characterisation of wild-type and mutant CYP51 enzymes through ligand binding studies and azole IC50 determinations is an important tool for understanding azole susceptibility and can be used in conjunction with microbiological methods (MIC50 values), molecular biological studies (site-directed mutagenesis) and protein modelling studies to inform future antifungal development with increased specificity for the target enzyme over the host homologue.
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Design and construction of an urban runoff research facility.
J Vis Exp
PUBLISHED: 08-08-2014
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As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m(2) facility was constructed which consists of 24 individual 33.6 m(2) field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO??P, K(+), Mg(2+), and Ca(2+) had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.
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SLaP mapper: A webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes.
Mol. Biochem. Parasitol.
PUBLISHED: 08-08-2014
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The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ?39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website.
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Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14?-demethylase (CYP51).
Appl. Environ. Microbiol.
PUBLISHED: 08-01-2014
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A candidate CYP51 gene encoding sterol 14?-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 ?M). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ?3 nM) and prothioconazole-desthio the lowest (Kd, ?51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 ?M using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 ?g ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 ?g ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ?1 ?g ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ?1 ?M) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ?1 to 2 ?g ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.
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Touching from a distance: Evolution of interplay between the nuclear pore complex, nuclear basket, and the mitotic spindle.
Nucleus
PUBLISHED: 08-01-2014
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The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.
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Deep evolutionary comparison of gene expression identifies parallel recruitment of trans-factors in two independent origins of C4 photosynthesis.
PLoS Genet.
PUBLISHED: 06-01-2014
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With at least 60 independent origins spanning monocotyledons and dicotyledons, the C4 photosynthetic pathway represents one of the most remarkable examples of convergent evolution. The recurrent evolution of this highly complex trait involving alterations to leaf anatomy, cell biology and biochemistry allows an increase in productivity by ? 50% in tropical and subtropical areas. The extent to which separate lineages of C4 plants use the same genetic networks to maintain C4 photosynthesis is unknown. We developed a new informatics framework to enable deep evolutionary comparison of gene expression in species lacking reference genomes. We exploited this to compare gene expression in species representing two independent C4 lineages (Cleome gynandra and Zea mays) whose last common ancestor diverged ? 140 million years ago. We define a cohort of 3,335 genes that represent conserved components of leaf and photosynthetic development in these species. Furthermore, we show that genes encoding proteins of the C4 cycle are recruited into networks defined by photosynthesis-related genes. Despite the wide evolutionary separation and independent origins of the C4 phenotype, we report that these species use homologous transcription factors to both induce C4 photosynthesis and to maintain the cell specific gene expression required for the pathway to operate. We define a core molecular signature associated with leaf and photosynthetic maturation that is likely shared by angiosperm species derived from the last common ancestor of the monocotyledons and dicotyledons. We show that deep evolutionary comparisons of gene expression can reveal novel insight into the molecular convergence of highly complex phenotypes and that parallel evolution of trans-factors underpins the repeated appearance of C4 photosynthesis. Thus, exploitation of extant natural variation associated with complex traits can be used to identify regulators. Moreover, the transcription factors that are shared by independent C4 lineages are key targets for engineering the C4 pathway into C3 crops such as rice.
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The impact of widespread regulatory neofunctionalization on homeolog gene evolution following whole-genome duplication in maize.
Genome Res.
PUBLISHED: 04-30-2014
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Whole-genome duplications are a widespread feature of plant genome evolution, having been detected in all flowering plant lineages. Despite the prevalence of these events, the extent to which duplicated genes (homeolog gene pairs) functionally diverge (neofunctionalization) is unclear. We present a genome-wide analysis of molecular evolution and regulatory neofunctionalization in maize (Zea mays L.). We demonstrate that 13% of all homeolog gene pairs in maize are regulatory neofunctionalized in leaves, and that regulatory neofunctionalized genes experience enhanced purifying selection. We show that significantly more genes have been regulatory neofunctionalized in foliar leaves than in husk leaves and that both leaf types have experienced selection for distinct functional roles. Furthermore, we demonstrate that biased subgenome expression dominance occurs only in the presence of regulatory neofunctionalization and that in nonregulatory neofunctionalized genes subgenome dominance is progressively acquired during development. Taken together, our study reveals several novel insights into the evolution of maize, genes, and gene expression, and provides a general model for gene evolution following whole-genome duplication in plants.
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Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 04-14-2014
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Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.
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Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance.
Mol. Biol. Evol.
PUBLISHED: 04-14-2014
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Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.
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Shared origins of a key enzyme during the evolution of C4 and CAM metabolism.
J. Exp. Bot.
PUBLISHED: 03-17-2014
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CAM and C4 photosynthesis are two key plant adaptations that have evolved independently multiple times, and are especially prevalent in particular groups of plants, including the Caryophyllales. We investigate the origin of photosynthetic PEPC, a key enzyme of both the CAM and C4 pathways. We combine phylogenetic analyses of genes encoding PEPC with analyses of RNA sequence data of Portulaca, the only plants known to perform both CAM and C4 photosynthesis. Three distinct gene lineages encoding PEPC exist in eudicots (namely ppc-1E1, ppc-1E2 and ppc-2), one of which (ppc-1E1) was recurrently recruited for use in both CAM and C4 photosynthesis within the Caryophyllales. This gene is present in multiple copies in the cacti and relatives, including Portulaca. The PEPC involved in the CAM and C4 cycles of Portulaca are encoded by closely related yet distinct genes. The CAM-specific gene is similar to genes from related CAM taxa, suggesting that CAM has evolved before C4 in these species. The similar origin of PEPC and other genes involved in the CAM and C4 cycles highlights the shared early steps of evolutionary trajectories towards CAM and C4, which probably diverged irreversibly only during the optimization of CAM and C4 phenotypes.
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Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity.
Mol. Biol. Cell
PUBLISHED: 03-05-2014
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The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle-dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.
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Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept.
AMB Express
PUBLISHED: 01-01-2014
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Microbial biotechnology and biotransformations promise to diversify the scope of the biorefinery approach for the production of high-value products and biofuels from industrial, rural and municipal waste feedstocks. In addition to bio-based chemicals and metabolites, microbial biomass itself constitutes an obvious but overlooked by-product of existing biofermentation systems which warrants fuller attention. The probiotic yeast Saccharomyces boulardii is used to treat gastrointestinal disorders and marketed as a human health supplement. Despite its relatedness to S. cerevisiae that is employed widely in biotechnology, food and biofuel industries, the alternative applications of S. boulardii are not well studied. Using a biorefinery approach, we compared the bioethanol and biomass yields attainable from agriculturally-sourced grass juice using probiotic S. boulardii (strain MYA-769) and a commercial S. cerevisiae brewing strain (Turbo yeast). Maximum product yields for MYA-769 (39.18 [±2.42] mg ethanol mL(-1) and 4.96 [±0.15] g dry weight L(-1)) compared closely to those of Turbo (37.43 [±1.99] mg mL(-1) and 4.78 [±0.10] g L(-1), respectively). Co-production, marketing and/or on-site utilisation of probiotic yeast biomass as a direct-fed microbial to improve livestock health represents a novel and viable prospect for rural biorefineries. Given emergent evidence to suggest that dietary yeast supplementations might also mitigate ruminant enteric methane emissions, the administration of probiotic yeast biomass could also offer an economically feasible way of reducing atmospheric CH4.
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Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock.
Biotechnol Biofuels
PUBLISHED: 01-01-2014
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Genetically customised Saccharomyces cerevisiae that can produce ethanol and additional bio-based chemicals from sustainable agro-industrial feedstocks (for example, residual plant biomass) are of major interest to the biofuel industry. We investigated the microbial biorefinery concept of ethanol and squalene co-production using S. cerevisiae (strain YUG37-ERG1) wherein ERG1 (squalene epoxidase) transcription is under the control of a doxycycline-repressible tet0 7 -CYC1 promoter. The production of ethanol and squalene by YUG37-ERG1 grown using agriculturally sourced grass juice supplemented with doxycycline was assessed.
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Functional and structural characterisation of a viral cytochrome b5.
FEBS Lett.
PUBLISHED: 08-27-2013
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Cytochrome b5 is a ubiquitous electron transport protein. The sequenced viral OtV-2 genome, which infects Ostreococcus tauri, was predicted to encode a putative cytochrome b5 enzyme. Using purified OtV-2 cytochrome b5 we confirm this protein has identical spectral properties to purified human cytochrome b5 and additionally that the viral enzyme can substitute for yeast cytochrome b5 in yeast cytochrome P450 51 mediated sterol 14?-demethylation. The crystal structure of the OtV-2 cytochrome b5 enzyme reveals a single domain, comprising four ? sheets, four ? helices and a haem moiety, which is similar to that found in larger eukaryotic cytochrome proteins. As a product of a horizontal gene transfer event involving a subdomain of the host fumarate reductase-like protein, OtV-2 cytochrome b5 appears to have diverged in function and is likely to have evolved an entirely new role for the virus during infection. Indeed, lacking a hydrophobic C-terminal anchor, OtV-2 encodes the first cytosolic cytochrome b5 characterised. The lack of requirement for membrane attachment (in contrast to all other microsomal cytochrome b5s) may be a reflection of the small size of the host cell, further emphasizes the unique nature of this virus gene product and draws attention to the potential importance of cytochrome b5 metabolic activity at the extremes of cellular scale.
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Genome-wide transcript analysis of early maize leaf development reveals gene cohorts associated with the differentiation of C4 Kranz anatomy.
Plant J.
PUBLISHED: 03-26-2013
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Photosynthesis underpins the viability of most ecosystems, with C4 plants that exhibit Kranz anatomy being the most efficient primary producers. Kranz anatomy is characterized by closely spaced veins that are encircled by two morphologically distinct photosynthetic cell types. Although Kranz anatomy evolved multiple times, the underlying genetic mechanisms remain largely elusive, with only the maize scarecrow gene so far implicated in Kranz patterning. To provide a broader insight into the regulation of Kranz differentiation, we performed a genome-wide comparative analysis of developmental trajectories in Kranz (foliar leaf blade) and non-Kranz (husk leaf sheath) leaves of the C4 plant maize. Using profile classification of gene expression in early leaf primordia, we identified cohorts of genes associated with procambium initiation and vascular patterning. In addition, we used supervised classification criteria inferred from anatomical and developmental analyses of five developmental stages to identify candidate regulators of cell-type specification. Our analysis supports the suggestion that Kranz anatomy is patterned, at least in part, by a SCARECROW/SHORTROOT regulatory network, and suggests likely components of that network. Furthermore, the data imply a role for additional pathways in the development of Kranz leaves.
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An algorithm for rapid computational construction of metabolic networks: a cholesterol biosynthesis example.
Comput. Biol. Med.
PUBLISHED: 02-16-2013
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Alternative pathways of metabolic networks represent the escape routes that can reduce drug efficacy and can cause severe adverse effects. In this paper we introduce a mathematical algorithm and a coding system for rapid computational construction of metabolic networks. The initial data for the algorithm are the source substrate code and the enzyme/metabolite interaction tables. The major strength of the algorithm is the adaptive coding system of the enzyme-substrate interactions. A reverse application of the algorithm is also possible, when optimisation algorithm is used to compute the enzyme/metabolite rules from the reference network structure. The coding system is user-defined and must be adapted to the studied problem. The algorithm is most effective for computation of networks that consist of metabolites with similar molecular structures. The computation of the cholesterol biosynthesis metabolic network suggests that 89 intermediates can theoretically be formed between lanosterol and cholesterol, only 20 are presently considered as cholesterol intermediates. Alternative metabolites may represent links with other metabolic networks both as precursors and metabolites of cholesterol. A possible cholesterol-by-pass pathway to bile acids metabolism through cholestanol is suggested.
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DendroBLAST: approximate phylogenetic trees in the absence of multiple sequence alignments.
PLoS ONE
PUBLISHED: 02-07-2013
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The rapidly growing availability of genome information has created considerable demand for both fast and accurate phylogenetic inference algorithms. We present a novel method called DendroBLAST for reconstructing phylogenetic dendrograms/trees from protein sequences using BLAST. This method differs from other methods by incorporating a simple model of sequence evolution to test the effect of introducing sequence changes on the reliability of the bipartitions in the inferred tree. Using realistic simulated sequence data we demonstrate that this method produces phylogenetic trees that are more accurate than other commonly-used distance based methods though not as accurate as maximum likelihood methods from good quality multiple sequence alignments. In addition to tests on simulated data, we use DendroBLAST to generate input trees for a supertree reconstruction of the phylogeny of the Archaea. This independent analysis produces an approximate phylogeny of the Archaea that has both high precision and recall when compared to previously published analysis of the same dataset using conventional methods. Taken together these results demonstrate that approximate phylogenetic trees can be produced in the absence of multiple sequence alignments, and we propose that these trees will provide a platform for improving and informing downstream bioinformatic analysis. A web implementation of the DendroBLAST method is freely available for use at http://www.dendroblast.com/.
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The cytochrome P450 complement (CYPome) of Mycosphaerella graminicola.
Biotechnol. Appl. Biochem.
PUBLISHED: 01-25-2013
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Mycosphaerella graminicola is a key fungal pathogen of wheat and a major target for azole fungicides, many of whose central mode of action is through inhibition of cytochrome P450 51 (lanosterol 14?-demethylase) in the ergosterol biosynthetic pathway. The range of activities of other fungal CYPs is thought to be a reflection of the differences between different organisms and their range of secondary metabolic pathways as a response to their niche environments, for example, in the production of mycotoxins. The present study collates information from a range of databases, to classify the CYPs found in M. graminicola and assign them an internationally recognized nomenclature, which, when referenced to the recent publication of the JGI version 2.0 genome model, creates a current, robust model for the CYP complement (CYPome) of M. graminicola. These CYPome data, which examined 82 CYPs and one pseudo-gene, may be utilized not only to further characterize and describe the physiology of the organism but also to enhance our understanding of CYP function and diversity.
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Cyclosporine 0.05% ophthalmic emulsion for the treatment of radiation-associated dry eye in children.
Pediatr Blood Cancer
PUBLISHED: 01-17-2013
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Dry eye disease is a well-known late complication of radiation therapy and is often difficult to treat. We evaluated the usefulness of cyclosporine 0.05% ophthalmic emulsion for the treatment of radiation-associated dry eye in children. Eleven children received cyclosporine 0.05% emulsion twice daily after failure of conventional therapy. After 6 months, dry eye manifestations improved in three children (27.3%). The remaining eight children showed no improvement with cyclosporine 0.05% ophthalmic emulsion. These results suggest that twice-daily cyclosporine 0.05% ophthalmic emulsion has limited use in children with refractory radiation-associated chronic dry eye.
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Characterization of the sterol 14?-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function.
New Phytol.
PUBLISHED: 01-15-2013
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CYP51 encodes the cytochrome P450 sterol 14?-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ?FgCYP51B and ?FgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14?-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14?-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14?-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51.
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Adaptin evolution in kinetoplastids and emergence of the variant surface glycoprotein coat in African trypanosomatids.
Mol. Phylogenet. Evol.
PUBLISHED: 01-07-2013
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The kinetoplastids are an important group of protozoa from the Excavata supergroup, and cause numerous diseases with wide environmental, economic and ecological impact. Trypanosoma brucei, the causative agent of human African trypanosomiasis, expresses a dense variant surface glycoprotein (VSG) coat, facilitating immune evasion via rapid switching and antigenic variation. Coupled to VSG switching is efficient clathrin-mediated endocytosis (CME), which removes anti-VSG antibody from the parasite surface. While the precise molecular basis for an extreme CME flux is unknown, genes encoding the AP2 complex, central to CME in most organisms, are absent from T. brucei, suggesting a mechanistic divergence in trypanosome CME. Here we identify the AP complex gene cohorts of all available kinetoplastid genomes and a new Trypanosoma grayi genome. We find multiple secondary losses of AP complexes, but that loss of AP2 is restricted to T. brucei and closest relatives. Further, loss of AP2 correlates precisely with the presence of VSG genes, supporting a model whereby these two adaptations may function synergistically in immune evasion.
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Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans.
J. Antimicrob. Chemother.
PUBLISHED: 01-04-2013
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In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains.
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Targeted versus tailored multimedia patient engagement to enhance depression recognition and treatment in primary care: randomized controlled trial protocol for the AMEP2 study.
BMC Health Serv Res
PUBLISHED: 01-01-2013
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Depression in primary care is common, yet this costly and disabling condition remains underdiagnosed and undertreated. Persisting gaps in the primary care of depression are due in part to patients reluctance to bring depressive symptoms to the attention of their primary care clinician and, when depression is diagnosed, to accept initial treatment for the condition. Both targeted and tailored communication strategies offer promise for fomenting discussion and reducing barriers to appropriate initial treatment of depression.
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Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?
Philos. Trans. R. Soc. Lond., B, Biol. Sci.
PUBLISHED: 01-01-2013
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The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo- and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14?-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated.
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An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.
Antimicrob. Agents Chemother.
PUBLISHED: 10-28-2011
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CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 ?M), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 ?M). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.
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Multiple treatment comparison meta-analyses: a step forward into complexity.
Clin Epidemiol
PUBLISHED: 05-26-2011
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The use of meta-analysis has become increasingly useful for clinical and policy decision making. A recent development in meta-analysis, multiple treatment comparison (MTC) meta-analysis, provides inferences on the comparative effectiveness of interventions that may have never been directly evaluated in clinical trials. This new approach may be confusing for clinicians and methodologists and raises specific challenges relevant to certain areas of medicine. This article addresses the methodological concepts of MTC meta-analysis, including issues of heterogeneity, choice of model, and adequacy of sample sizes. We address domain-specific challenges relevant to disciplines of medicine, including baseline risks of patient populations. We conclude that MTC meta-analysis is a useful tool in the context of comparative effectiveness and requires further study, as its utility and transparency will likely predict its uptake by the research and clinical community.
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Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola.
PLoS ONE
PUBLISHED: 05-17-2011
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A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ?Y459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.
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Mixed treatment comparison of repeated measurements of a continuous endpoint: an example using topical treatments for primary open-angle glaucoma and ocular hypertension.
Stat Med
PUBLISHED: 05-07-2011
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Mixed treatment comparison (MTC) meta-analyses estimate relative treatment effects from networks of evidence while preserving randomisation. We extend the MTC framework to allow for repeated measurements of a continuous endpoint that varies over time. We used, as a case study, a systematic review and meta-analysis of intraocular pressure (IOP) measurements from randomised controlled trials evaluating topical ocular hypotensives in primary open-angle glaucoma or ocular hypertension because IOP varies over the day and over the treatment course, and repeated measurements are frequently reported. We adopted models for conducting MTC in WinBUGS (The BUGS Project, Cambridge, UK) to allow for repeated IOP measurements and to impute missing standard deviations of the raw data using the predictive distribution from observations with standard deviations. A flexible model with an unconstrained baseline for IOP variations over time and time-invariant random treatment effects fitted the data well. We also adopted repeated measures models to allow for class effects; assuming treatment effects to be exchangeable within classes slightly improved model fit but could bias estimated treatment effects if exchangeability assumptions were not valid. We enabled all timepoints to be included in the analysis, allowing for repeated measures to increase precision around treatment effects and avoid bias associated with selecting timepoints for meta-analysis.The methods we developed for modelling repeated measures and allowing for missing data may be adapted for use in other MTC meta-analyses. Copyright © 2011 John Wiley & Sons, Ltd.
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Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity.
Appl. Environ. Microbiol.
PUBLISHED: 04-08-2011
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The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14?-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
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Ab initio identification of novel regulatory elements in the genome of Trypanosoma brucei by Bayesian inference on sequence segmentation.
PLoS ONE
PUBLISHED: 02-11-2011
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The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes.
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Smoking cessation reduces postoperative complications: a systematic review and meta-analysis.
Am. J. Med.
PUBLISHED: 02-08-2011
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We aimed to review randomized trials and observational evidence to establish the effect of preoperative smoking cessation on postoperative complications and to determine if there is an optimal cessation period before surgery.
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Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals.
Appl. Environ. Microbiol.
PUBLISHED: 12-17-2010
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Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 ?g of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14?-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
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Cyclization of a cellular dipentaenone by Streptomyces coelicolor cytochrome P450 154A1 without oxidation/reduction.
J. Am. Chem. Soc.
PUBLISHED: 10-29-2010
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We report a comprehensive genetic, metabolomic, and biochemical study on the catalytic properties of Streptomyces coelicolor cytochrome P450 (P450) 154A1, known to have a unique heme orientation in its crystal structure. Deletion of the P450 154A1 gene compromised the long-term stability of the bacterial spores. A novel dipentaenone (1) with a high degree of conjugation was identified as an endogenous substrate of P450 154A1 using a metabolomics approach. The biotransformation of 1 by P450 154A1 was shown to be an unexpected intramolecular cyclization to a Paterno?-Bu?chi-like product, without oxidation/reduction.
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Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (sterol 14?-demethylase) doxycycline-regulated mutant and screening of the azole sensitivity of Aspergillus fumigatus isoenzymes CYP51A and CYP51B.
Antimicrob. Agents Chemother.
PUBLISHED: 08-23-2010
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Aspergillus fumigatus sterol 14?-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs showed identical sensitivity to itraconazole, posaconazole, clotrimazole, and voriconazole. Conversely, YUG37-pcyp51A withstood 16-fold-higher concentrations of fluconazole than YUG37-pcyp51B (8 and 0.5 ?g ml?¹, respectively).
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Identification and characterization of four azole-resistant erg3 mutants of Candida albicans.
Antimicrob. Agents Chemother.
PUBLISHED: 08-23-2010
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Sterol analysis identified four Candida albicans erg3 mutants in which ergosta 7,22-dienol, indicative of perturbations in sterol ?(5,6)-desaturase (Erg3p) activity, comprised >5% of the total sterol fraction. The erg3 mutants (CA12, CA488, CA490, and CA1008) were all resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under standard CLSI assay conditions (MIC values, ?256, 16, 16, 8, and 1 ?g ml?¹, respectively). Importantly, CA12 and CA1008 retained an azole-resistant phenotype even when assayed in the presence of FK506, a multidrug efflux inhibitor. Conversely, CA488, CA490, and three comparator isolates (CA6, CA14, and CA177, in which ergosterol comprised >80% of the total sterol fraction and ergosta 7,22-dienol was undetectable) all displayed azole-sensitive phenotypes under efflux-inhibited assay conditions. Owing to their ergosterol content, CA6, CA14, and CA177 were highly sensitive to amphotericin B (MIC values, <0.25 ?g ml?¹); CA1008, in which ergosterol comprised <2% of the total sterol fraction, was less sensitive (MIC, 1 ?g ml?¹). CA1008 harbored multiple amino acid substitutions in Erg3p but only a single conserved polymorphism (E266D) in sterol 14?-demethylase (Erg11p). CA12 harbored one substitution (W332R) in Erg3p and no residue changes in Erg11p. CA488 and CA490 were found to harbor multiple residue changes in both Erg3p and Erg11p. The results suggest that missense mutations in ERG3 might arise in C. albicans more frequently than currently supposed and that the clinical significance of erg3 mutants, including those in which additional mechanisms also contribute to resistance, should not be discounted.
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Expression, purification, and characterization of Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A and B.
Antimicrob. Agents Chemother.
PUBLISHED: 07-26-2010
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Aspergillus fumigatus sterol 14-? demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K(s), 8.6 ?M) and eburicol (K(s), 22.6 ?M). Membrane suspensions of AF51A bound to both lanosterol (K(s), 3.1 ?M) and eburicol (K(s), 4.1 ?M). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K(d) (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 ?M, respectively, in comparison with a K(d) value of 4 ?M for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K(d) values ranging from 1 ?M for itraconazole to 11.9 ?M for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.
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Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51).
Antimicrob. Agents Chemother.
PUBLISHED: 07-12-2010
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Purified Candida albicans sterol 14-? demethylase (CaCYP51) bound the CYP51 substrates lanosterol and eburicol, producing type I binding spectra with K(s) values of 11 and 25 ?M, respectively, and a K(m) value of 6 ?M for lanosterol. Azole binding to CaCYP51 was "tight" with both the type II spectral intensity (?A(max)) and the azole concentration required to obtain a half-?A(max) being proportional to the CaCYP51 concentration. Tight binding of fluconazole and itraconazole was confirmed by 50% inhibitory concentration determinations from CYP51 reconstitution assays. CaCYP51 had similar affinities for clotrimazole, econazole, itraconazole, ketoconazole, miconazole, and voriconazole, with K(d) values of 10 to 26 ?M under oxidative conditions, compared with 47 ?M for fluconazole. The affinities of CaCYP51 for fluconazole and itraconazole appeared to be 4- and 2-fold lower based on CO displacement studies than those when using direct ligand binding under oxidative conditions. Econazole and miconazole were most readily displaced by carbon monoxide, followed by clotrimazole, ketoconazole, and fluconazole, and then voriconazole (7.8 pmol min(-1)), but itraconzole could not be displaced by carbon monoxide. This work reports in depth the characterization of the azole binding properties of wild-type C. albicans CYP51, including that of voriconazole, and will contribute to effective screening of new therapeutic azole antifungal agents. Preliminary comparative studies with the I471T CaCYP51 protein suggested that fluconazole resistance conferred by this mutation was through a combination of increased turnover, increased affinity for substrate, and a reduced affinity for fluconazole in the presence of substrate, allowing the enzyme to remain functionally active, albeit at reduced velocity, at higher fluconazole concentrations.
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A clinical isolate of Candida albicans with mutations in ERG11 (encoding sterol 14alpha-demethylase) and ERG5 (encoding C22 desaturase) is cross resistant to azoles and amphotericin B.
Antimicrob. Agents Chemother.
PUBLISHED: 06-14-2010
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A clinical isolate of Candida albicans was identified as an erg5 (encoding sterol C22 desaturase) mutant in which ergosterol was not detectable and ergosta 5,7-dienol comprised >80% of the total sterol fraction. The mutant isolate (CA108) was resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole (MIC values, 64, 8, 2, 1, and 2 microg ml(-1), respectively); azole resistance could not be fully explained by the activity of multidrug resistance pumps. When susceptibility tests were performed in the presence of a multidrug efflux inhibitor (tacrolimus; FK506), CA108 remained resistant to azole concentrations higher than suggested clinical breakpoints for C. albicans (efflux-inhibited MIC values, 16 and 4 microg ml(-1) for fluconazole and voriconazole, respectively). Gene sequencing revealed that CA108 was an erg11 erg5 double mutant harboring a single amino acid substitution (A114S) in sterol 14alpha-demethylase (Erg11p) and sequence repetition (10 duplicated amino acids), which nullified C22 desaturase (Erg5p) function. Owing to a lack of ergosterol, CA108 was also resistant to amphotericin B (MIC, 2 microg ml(-1)). This constitutes the first report of a C. albicans erg5 mutant isolated from the clinic.
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Spontaneous pneumomediastinum (Hammans syndrome).
Surgeon
PUBLISHED: 02-04-2010
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Spontaneous pneumomediastinum is defined as the presence of free air in the mediastium in the absence of any obvious precipitating cause. The purpose of this study was to review our experience with this condition, discuss mechanisms and provide a management algorithm.
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Streptomyces coelicolor A3(2) CYP102 protein, a novel fatty acid hydroxylase encoded as a heme domain without an N-terminal redox partner.
Appl. Environ. Microbiol.
PUBLISHED: 01-22-2010
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The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.
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Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site.
J. Biol. Chem.
PUBLISHED: 10-26-2009
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Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 A) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 A). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 alpha-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an alpha-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.
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Efficacy and safety of prostaglandin analogues in patients with predominantly primary open-angle glaucoma or ocular hypertension: a meta-analysis.
Clin Ophthalmol
PUBLISHED: 08-03-2009
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First-line therapy for primary open-angle glaucoma and ocular hypertension generally involves prostaglandin analogue therapy. The relative efficacy of differing prostaglandin therapy is disputed.
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Identification of a crenarchaeal orthologue of Elf1: implications for chromatin and transcription in Archaea.
Biol. Direct
PUBLISHED: 07-04-2009
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The transcription machineries of Archaea and eukaryotes are similar in many aspects, but little is understood about archaeal chromatin and its role in transcription. Here, we describe the identification in hyperthermophilic Crenarchaeota and a Korarchaeon of an orthologue of the eukaryotic transcription elongation factor Elf1, which has been shown to function in chromatin structure maintenance of actively transcribed templates. Our discovery has implications for the relationship of chromatin and transcription in Archaea and the evolution of these processes in eukaryotes.
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The first virally encoded cytochrome p450.
J. Virol.
PUBLISHED: 06-10-2009
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The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes (YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.
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Identification, characterization, and azole-binding properties of Mycobacterium smegmatis CYP164A2, a homolog of ML2088, the sole cytochrome P450 gene of Mycobacterium leprae.
Antimicrob. Agents Chemother.
PUBLISHED: 05-20-2009
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The genome sequence of Mycobacterium leprae revealed a single open reading frame, ML2088 (CYP164A1), encoding a putative full-length cytochrome P450 monooxygenase and 12 pseudogenes. We have identified a homolog of ML2088 in Mycobacterium smegmatis and report here the cloning, expression, purification, and azole-binding characteristics of this cytochrome P450 (CYP164A2). CYP164A2 is 1,245 bp long and encodes a protein of 414 amino acids and molecular mass of 45 kDa. CYP164A2 has 60% identity with Mycobacterium leprae CYP161A1 and 66 to 69% identity with eight other mycobacterial CYP164A1 homologs, with three identified highly conserved motifs. Recombinant CYP164A2 has the typical spectral characteristics of a cytochrome P450 monooxygenase, predominantly in the ferric low-spin state. Unusually, the spin state was readily modulated by increasing ionic strength at pH 7.5, with 50% high-spin occupancy achieved with 0.14 M NaCl. CYP164A2 bound clotrimazole, econazole, and miconazole strongly (K(d), 1.2 to 2.5 muM); however, strong binding with itraconazole, ketoconazole, and voriconazole was only observed in the presence of 0.5 M NaCl. Fluconazole did not bind to CYP164A2 at pH 7.5 and no discernible type II binding spectrum was observed.
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Molecular characterization of a subgroup IE intron with wide distribution in the large subunit rRNA genes of dermatophyte fungi.
Med. Mycol.
PUBLISHED: 05-20-2009
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Group I introns have the ability to catalyse their own excision (self-splice) from pre-RNA, and are found in a wide range of eukaryotic organisms. In fungal nuclear genomes, they have been identified in the small subunit (SSU) and large subunit (LSU) of the ribosomal RNA gene. Sequencing of the 3 region of the LSU rRNA gene of the dermatophyte Trichophyton interdigitale revealed a 393 bp group I intron, Tin.2563, containing the four characteristic conserved motifs (P,Q,R and S) essential for self-splicing. The predicted secondary structure revealed nine sets of conserved paired regions (P1-P9), with most similarity to a subgroup IE intron of the entomopathogenic hyphomycete Beauveria bassiana. Tin.2563 was inserted at a site in the LSU rDNA corresponding to position 2563 of the Escherichia coli 23S rRNA. PCR and sequence analysis showed an intron to be present at an identical location in the LSU rDNA of many dermatophytes, although its distribution was erratic. In contrast, an intron was present at the same location in multiple isolates (n = 20) of the clinically important anthrophilic species Trichophyton rubrum and T. interdigitale. Conservation of intron insertion site, subgroup and P helix sequences showed intron genotyping to be unsuitable for strain identification in dermatophytes. Phylogenetic analysis of intron sequences from different dermatophyte species indicated that lateral transfer of the element was likely to be a rare event.
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Prothioconazole and prothioconazole-desthio activities against Candida albicans sterol 14-?-demethylase.
Appl. Environ. Microbiol.
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Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14?-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
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Azole affinity of sterol 14?-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens.
Antimicrob. Agents Chemother.
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Candida albicans CYP51 (CaCYP51) (Erg11), full-length Homo sapiens CYP51 (HsCYP51), and truncated ?60HsCYP51 were expressed in Escherichia coli and purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (K(s), 14 to 18 ?M) and catalyzed the 14?-demethylation of lanosterol using Homo sapiens cytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [K(d)s], 42 to 131 nM) but bound fluconazole (K(d), ~30,500 nM) and voriconazole (K(d), ~2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (K(d)s, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over ?60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and ?60HsCYP51, producing atypical type I UV-visible difference spectra (K(d)s, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and ?60HsCYP51 (K(d), ~40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC(50)) values, which were 9- to 2,000-fold higher for ?60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC(50), ~150 ?M) and did not significantly inhibit ?60HsCYP51.
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Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection.
New Phytol.
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Plant-derived nitric oxide (NO) triggers defence, priming the onset of the hypersensitive response and restricting pathogen ingress during incompatibility. However, little is known about the role of pathogen-produced NO during pre-infection development and infection. We sought evidence for NO production by the rice blast fungus during early infection. NO production was measured using fluorescence of DAR-4M and the role of NO assessed using NO scavengers. The synthesis of NO was investigated by targeted knockout of genes potentially involved in NO synthesis, including nitric oxide synthase-like genes (NOL2 and NOL3) and nitrate (NIA1) and nitrite reductase (NII1), generating single and double ?nia1?nii1, ?nia1?nol3, and ?nol2?nol3 mutants. We demonstrate that Magnaporthe oryzae generates NO during germination and in early development. Removal of NO delays germling development and reduces disease lesion numbers. NO is not generated by the candidate proteins tested, nor by other arginine-dependent NO systems, by polyamine oxidase activity or non-enzymatically by low pH. Furthermore, we show that, while NIA1 and NII1 are essential for nitrate assimilation, NIA1, NII1, NOL2 and NOL3 are all dispensable for pathogenicity. Development of M. oryzae and initiation of infection are critically dependent on fungal NO synthesis, but its mode of generation remains obscure.
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Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2.
Antimicrob. Agents Chemother.
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Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 ?g ml(-1)) were found to be ERG2 mutants, wherein ?(8)-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr(121) in ERG2; the corresponding residue (Thr(119)) in Saccharomyces cerevisiae is essential for sterol ?8-?7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.
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Evolution of GOLDEN2-LIKE gene function in C(3) and C (4) plants.
Planta
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A pair of GOLDEN2-LIKE transcription factors is required for normal chloroplast development in land plant species that encompass the range from bryophytes to angiosperms. In the C(4) plant maize, compartmentalized function of the two GLK genes in bundle sheath and mesophyll cells regulates dimorphic chloroplast differentiation, whereas in the C(3) plants Physcomitrella patens and Arabidopsis thaliana the genes act redundantly in all photosynthetic cells. To assess whether the cell-specific function of GLK genes is unique to maize, we analyzed gene expression patterns in the C(4) monocot Sorghum bicolor and C(4) eudicot Cleome gynandra. Compartmentalized expression was observed in S. bicolor, consistent with the development of dimorphic chloroplasts in this species, but not in C. gynandra where bundle sheath and mesophyll chloroplasts are morphologically similar. The generation of single and double mutants demonstrated that GLK genes function redundantly in rice, as in other C(3) plants, despite the fact that GLK gene duplication in monocots preceded the speciation of rice, maize and sorghum. Together with phylogenetic analyses of GLK gene sequences, these data have allowed speculation on the evolutionary trajectory of GLK function. Based on current evidence, most species that retain single GLK genes belong to orders that contain only C(3) species. We therefore propose that the ancestral state is a single GLK gene, and hypothesize that GLK gene duplication enabled sub-functionalization, which in turn enabled cell-specific function in C(4) plants with dimorphic chloroplasts. In this scenario, GLK gene duplication preconditioned the evolution of C(4) physiology that is associated with chloroplast dimorphism.
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Structural Analysis of Cytochrome P450 105N1 Involved in the Biosynthesis of the Zincophore, Coelibactin.
Int J Mol Sci
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Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the ?-helical domain and the ?-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation.
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Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei.
Cell Rep
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Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion.
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MergeAlign: improving multiple sequence alignment performance by dynamic reconstruction of consensus multiple sequence alignments.
BMC Bioinformatics
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The generation of multiple sequence alignments (MSAs) is a crucial step for many bioinformatic analyses. Thus improving MSA accuracy and identifying potential errors in MSAs is important for a wide range of post-genomic research. We present a novel method called MergeAlign which constructs consensus MSAs from multiple independent MSAs and assigns an alignment precision score to each column.
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Facultative sterol uptake in an ergosterol-deficient clinical isolate of Candida glabrata harboring a missense mutation in ERG11 and exhibiting cross-resistance to azoles and amphotericin B.
Antimicrob. Agents Chemother.
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We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 ?g ml(-1), respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14?-demethylase (Erg11p) mutant, wherein 14?-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14?-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ((glc)YM). However, when grown on sterol-supplemented (glc)YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, ?(7)-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented (glc)YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using (glc)YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using (glc)YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 ?g AMB ml(-1), respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.
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S279 point mutations in Candida albicans Sterol 14-? demethylase (CYP51) reduce in vitro inhibition by fluconazole.
Antimicrob. Agents Chemother.
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The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities (K(s), 7.1 and 8.0 ?M, respectively) compared to the wild-type CaCYP51 protein (K(s), 13.5 ?M). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC??s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher K(d) values observed.
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Alterations in predicted regulatory and coding regions of the sterol 14?-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae.
Mol. Plant Pathol.
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The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14?-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.
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