JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
Resistance to antifungals that target CYP51.
J Chem Biol
PUBLISHED: 10-01-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance.
Mol. Biol. Evol.
PUBLISHED: 04-14-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept.
AMB Express
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
Co-production of ethanol and squalene using a Saccharomyces cerevisiae ERG1 (squalene epoxidase) mutant and agro-industrial feedstock.
Biotechnol Biofuels
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
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.
Related JoVE Video
An algorithm for rapid computational construction of metabolic networks: a cholesterol biosynthesis example.
Comput. Biol. Med.
PUBLISHED: 02-16-2013
Show Abstract
Hide Abstract
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.
Related JoVE Video
The cytochrome P450 complement (CYPome) of Mycosphaerella graminicola.
Biotechnol. Appl. Biochem.
PUBLISHED: 01-25-2013
Show Abstract
Hide Abstract
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.
Related JoVE Video
Flux, questions, exclusion and compassion: collective learning in secondary care.
Med Educ
PUBLISHED: 01-07-2013
Show Abstract
Hide Abstract
Health care organisations are increasingly conceptualised as complex, indivisible entities made up of web-like networks of staff that connect to each other in changeable ways. This study draws on the theoretical framework of activity theory and the concept of knotworking to illustrate how health professionals improvise collaboratively to negotiate everyday challenges and contribute positively to patients health priorities.
Related JoVE Video
Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
We report the discovery of a novel dual inhibitor targeting fungal sterol 14?-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50?=?700 nM) and CYP51 (IC50?=?43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.
Related JoVE Video
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola.
PLoS ONE
PUBLISHED: 05-17-2011
Show Abstract
Hide Abstract
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.
Related JoVE Video
Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity.
Appl. Environ. Microbiol.
PUBLISHED: 04-08-2011
Show Abstract
Hide Abstract
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.
Related JoVE Video
Collective learning, change and improvement in health care: trialling a facilitated learning initiative with general practice teams.
J Eval Clin Pract
PUBLISHED: 02-17-2011
Show Abstract
Hide Abstract
Many patients, families, health care professionals and politicians desire for quality improvement within the UK National Health Service. One way to achieve this change is for health care teams to work and learn together more effectively. This research aimed to design and trial a facilitated learning programme with the aim of supporting general practice teams in fostering the characteristics of learning organizations.
Related JoVE Video
Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals.
Appl. Environ. Microbiol.
PUBLISHED: 12-17-2010
Show Abstract
Hide Abstract
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.
Related JoVE Video
Cultivation of a learning culture in general practice: an educational intervention.
Educ Prim Care
PUBLISHED: 09-28-2010
Show Abstract
Hide Abstract
Increasing challenges are faced in primary care, including the increase in chronic disease and its management in the community. This paper describes an educational initiative developed to help local general practitioners (GPs) and primary care teams manage chronic conditions and address referral behaviour. The purpose of this study was to evaluate the initiative through the exploration of the experiences of the participants and providers.
Related JoVE Video
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
Show Abstract
Hide Abstract
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).
Related JoVE Video
Identification and characterization of four azole-resistant erg3 mutants of Candida albicans.
Antimicrob. Agents Chemother.
PUBLISHED: 08-23-2010
Show Abstract
Hide Abstract
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.
Related JoVE Video
Expression, purification, and characterization of Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A and B.
Antimicrob. Agents Chemother.
PUBLISHED: 07-26-2010
Show Abstract
Hide Abstract
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.
Related JoVE Video
Azole binding properties of Candida albicans sterol 14-alpha demethylase (CaCYP51).
Antimicrob. Agents Chemother.
PUBLISHED: 07-12-2010
Show Abstract
Hide Abstract
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.
Related JoVE Video
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
Research paradigms in medical education research.
Med Educ
PUBLISHED: 05-07-2010
Show Abstract
Hide Abstract
The growing popularity of less familiar methodologies in medical education research, and the use of related data collection methods, has made it timely to revisit some basic assumptions regarding knowledge and evidence.
Related JoVE Video
Practice-based small group learning: how health professionals view their intention to change and the process of implementing change in practice.
Med Teach
PUBLISHED: 11-14-2009
Show Abstract
Hide Abstract
Commitment-to-change (CTC) strategies used as part of Continuing Professional Development (CPD) programmes have been found to influence changes in the clinical practice of health professionals. However, there is an unquestioned usage of the term commitment to describe the statements made by learners specifying the changes they intend to make following CPD programmes. The Practice-based Small Group Learning (PBSGL) programme is one approach to the CPD of health professionals that includes a CTC instrument. This study reports on a pilot PBSGL programme in Scotland, which composed of mixed groups of General Practitioners (GPs) and Practice Nurses (PNs), as well as PN-only groups. Using qualitative methods, the study examines the meaning that learners ascribe to their statements of intention to introduce changes in their practice. It reveals that for some participants commitment is too strong a word to describe their intention. Nevertheless, others did feel that they were committing to the changes that they identified at PBSGL meetings. This study also explores the factors that influenced the decision of PBSGL participants to introduce practice changes, and the process of implementing change in clinical practice.
Related JoVE Video
Social structure predicts genital morphology in African mole-rats.
PLoS ONE
PUBLISHED: 06-11-2009
Show Abstract
Hide Abstract
African mole-rats (Bathyergidae, Rodentia) exhibit a wide range of social structures, from solitary to eusocial. We previously found a lack of sex differences in the external genitalia and morphology of the perineal muscles associated with the phallus in the eusocial naked mole-rat. This was quite surprising, as the external genitalia and perineal muscles are sexually dimorphic in all other mammals examined. We hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure.
Related JoVE Video
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
Show Abstract
Hide Abstract
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.
Related JoVE Video
The Practice-based Small Group Learning programme: experiences of learners in multi-professional groups.
J Interprof Care
PUBLISHED: 05-06-2009
Show Abstract
Hide Abstract
This study describes the experiences of General Practitioners (GPs) and Practice Nurses (PNs) as they came together and engaged in the Practice-based Small Group Learning (PBSGL) programme. Based on principles of adult and small group learning, PBSGL, which was developed in Canada, was used for the first time for the Continuing Professional Development (CPD) of multi-professional groups in the UK. The findings detail the main reasons GPs and PNs participated in PBSGL, the nature of interaction and development of cohesion in the groups, factors influencing contribution to discussions, the learning process, and outcomes for learners. Respect shown for different roles and perspectives enabled participants to be open about gaps in their knowledge and to ask questions. A mutual keenness to understand the perspectives of and learn from the other profession emerges as a key ingredient for learners to feel that their learning needs were met. The learning process in the groups came close to transformative learning--there were changes in perspectives, acquisition of new knowledge and increased self-esteem. The appropriateness of the PBSGL approach for the CPD of mixed groups of GPs and PNs is discussed.
Related JoVE Video
The CYPome (Cytochrome P450 complement) of Aspergillus nidulans.
Fungal Genet. Biol.
PUBLISHED: 04-08-2009
Show Abstract
Hide Abstract
The cytochromes P450 (CYPs) are found in all biological kingdoms and genome sequencing projects continue to reveal an ever increasing number. The principle aim of this paper is to identify the complete CYPome of Aspergillus nidulans from the genome sequence version AN.3 deposited at the Broad institute, assign the appropriate CYP nomenclature and define function where possible. The completed analysis revealed a total of 111 CYP genes, 3 of which were previously unknown and 8 pseudogenes, representing 89CYP families, 21 of which are unique. We have identified 28 potential gene clusters associated with one or more CYP genes and discussed those with putative PKS and NRPS associated function. The chromosomal location of the genes, predicted cellular location of the proteins and possible function(s) are discussed.
Related JoVE Video
The practice-based small group learning approach: making evidence-based practice come alive for learners.
Nurse Educ Today
PUBLISHED: 02-15-2009
Show Abstract
Hide Abstract
This paper reports the study of a pilot learning programme for the Continuing Professional Development of Practice Nurses (PNs) in Scotland. Two PN groups used the practice-based small group (PBSG) approach to learning, which is designed to encourage learning relevant to patient problems faced in day-to-day practice. Learners kept a log of their experiences of engaging in PBSG learning for three consecutive meetings. Data was also collected through interviews. The findings detail the motivation for joining the PBSGs, group dynamics and the development of cohesion, factors influencing participation in group-discussions and the nature of learning that took place in the groups. PNs from both groups reported that discussions led to habitual practices being challenged but without hostility, and that various sources of information were considered. The suitability of PBSG learning to promote evidence-based nursing practice is discussed.
Related JoVE Video
The 2008 update of the Aspergillus nidulans genome annotation: a community effort.
Fungal Genet. Biol.
PUBLISHED: 01-17-2009
Show Abstract
Hide Abstract
The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.
Related JoVE Video
Prothioconazole and prothioconazole-desthio activities against Candida albicans sterol 14-?-demethylase.
Appl. Environ. Microbiol.
Show Abstract
Hide Abstract
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.
Related JoVE Video
Azole affinity of sterol 14?-demethylase (CYP51) enzymes from Candida albicans and Homo sapiens.
Antimicrob. Agents Chemother.
Show Abstract
Hide Abstract
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.
Related JoVE Video
Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2.
Antimicrob. Agents Chemother.
Show Abstract
Hide Abstract
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.
Related JoVE Video
Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi.
Mem. Inst. Oswaldo Cruz
Show Abstract
Hide Abstract
Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.
Related JoVE Video
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.
Show Abstract
Hide Abstract
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.
Related JoVE Video
S279 point mutations in Candida albicans Sterol 14-? demethylase (CYP51) reduce in vitro inhibition by fluconazole.
Antimicrob. Agents Chemother.
Show Abstract
Hide Abstract
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.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.