Diane Kelly

Swansea University | SWAN · Institute of Life Science "ILS"

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min⁻¹), palmitic (1.61 min⁻¹) and stearic acids (1.24 min⁻¹), at both the ω-1- and ω-2...

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivati...

Three types of biochar (microalgae, rice straw and sorghum straw) from biomass thermal conversion production were tested for producing biochar-derived carbonaceous nanomaterials (BCN). BCN were obtained after using chemical depolymerisation and solvent extraction, NanoRefinery process. Microalgae biochar-derived carbo-naceous nanomaterials (MAB-CN)...

Here we report the first evaluation of isavuconazole inhibition of Aspergillus fumigatus CYP51 and thus sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant A. fumigatus CYP51 isoenzymes A and B (AfCYP51A and AfCYP51B) isolated in Escherichia coli membranes. CYP51 reconstitution assays confirmed that A...

This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRe...

This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically...

Recombinant C. albicans CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild type enzyme were expressed in Escherichia coli and purified by Ni ²⁺ -NTA agarose chromatogrpahy. Catalytic tolerance to azole antifungals was assessed by IC 50 [concentration causing 50% enzyme inhibiti...

Cytochrome P450 (CYP) 27A1 is a key enzyme in both the acidic and neutral pathways of bile acid biosynthesis accepting cholesterol and ring-hydroxylated sterols as substrates introducing a (25R)26-hydroxy and ultimately a (25R)26-acid group to the sterol side-chain. In human, mutations in the CYP27A1 gene are the cause of the autosomal recessive di...

This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy...

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol...

The antifungal effects of the novel triazole, PC1244, designed for topical or inhaled administration, against A. fumigatus have been tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (N=96) with a MIC range of 0.016--0.25 μg/ml, whereas the MIC range for vorico...

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency cha...

Background Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticoste...

Prior to characterization of antifungal inhibitors that target this enzyme, Trichophyton rubrum CYP51 was expressed in Escherichia coli, purified and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol and eburicol with similar affinities (Kd values 22.7, 20.3 and 20.9 μM), but displayed substrate specificity insofar as only eburicol was...

The profile of PC945, a novel triazole antifungal, designed for administration via inhalation, has been assessed in a range of in vitro and in vivo studies. PC945 was characterized as a potent, tight-binding inhibitor of Aspergillus fumigatus sterol 14α-demethylase (CYP51A and CYP51B) activity (IC50, 0.23 μM and 0.22 μM, respectively), with charact...

Biochar is a carbon rich product composed of recalcitrant aromatic and heterocyclic structures. Biochar is produced as part of biomass transformation into energy or biofuels using pyrolysis or gasification processes. In this work, the production and purification of fluorescent carbon dots from biochar were evaluated. Manure biochar was used as init...

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 μM, respectively, catalyzing sterol 14α-demethylation...

Cryptococcosis is a life-threatening disease often associated with HIV. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (Kd 14 to 25 nM) with similar affinity as fluconazole, voriconazole,...

The incidence of triazole-resistant Aspergillus infections is increasing worldwide, often mediated through mutations in the CYP51A amino acid sequence. New classes of azole-based drugs are required to combat the increasing resistance to existing triazole therapeutics. In this study, a CYP51 reconstitution assay is described consisting of eburicol,...

In this study we investigate the amoebacidal activities of the pharmaceutical triazole CYP51 inhibitors fluconazole, itraconazole and voriconazole against Acanthamoeba castellanii and Acanthamoeba polyphaga and assess their potential as therapeutic agents against Acanthamoeba infections in humans. Amoebacidal activities of the triazoles were assess...

Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals are the treatment of choice, however, resistance has arisen against these compounds, which coul...

Mycosphaerella graminicola (Zymoseptoria tritici) is an ascomycete filamentous fungus that causes Septoria leaf blotch in wheat crops. In Europe the most widely used fungicides for this major disease are demethylation inhibitors (DMIs). Their target is the essential sterol 14α-demethylase (CYP51), which requires Cytochrome P450 reductase (CPR) as i...

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 pr...

Abstract Background: 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 usin...

The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, ch...

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 exist...

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 mecha-nisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of wi...

A candidate CYP51 gene encoding sterol 14α-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 sim...

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 alle...

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