Approaches to detect alternative mechanisms of resistance to systemic antifungals

From the publisher\u27s website: Resistance to antifungals is a major concern in the management of fungal infections, especially while the incidence of pathogens with a poor susceptibility to current treatments is rising. New therapeutic strategies could be developed through the discovery of completely new fungal-specific targets or through the identification of new effectors of resistance to existing antifungals. In addition to basic molecular resistance mechanisms that are well understood, there are also numerous additional effectors able to modulate fungi susceptibility to the four main classes of antifungals. These effectors are unable to drive resistance alone, but they are now believed to be crucial for the establishment and maintenance of drug resistance, as they constitute key modulators allowing the phenotypic expression of resistance acquired by basic mechanisms. Formerly limited, the approaches to detect such alternative resistance mechanisms to antifungals were profoundly renewed with the "omics" era, allowing the study of whole organism\u27s response. This chapter will focus on the main strategies implemented in the last two decades, with a particular emphasis on high throughput technologies such as whole genome sequencing, transcriptomics, proteomics, and large-scale mutant collections screening

A modified hyperbolic tangent equation to determine equilibrium shape of headland bay beaches

When designing any artificial beach, it’s desirable to avoid (or minimise) future maintenance commitments by arranging the initial beach planshape so that it remains in equilibrium given the incident wave climate. Headlands bays, or embayments, where a sandy beach is held between two erosion resistant headlands, tend to evolve to a stable beach planshape with little movement of the beach contours over time. Several empirical bay shape equations have been derived to fit curves to the shoreline of headland bay beaches. One of the most widely adopted empirical equations is the parabolic bay shape equation, as it is the only equation that directly links the shoreline positions to the predominant wave direction and the point of diffraction. However, the main limitation with the application of the parabolic bay shape equation is locating the downcoast control point. As a result of research presented in this paper a new equation, based on the hyperbolic tangent shape equation was developed, which eliminates the requirement of placing the down coast control point and relies on defining a minimum beach width instead. This modified equation was incorporated into a new ArcGIS tool

Antifungals: from genomics to resistance and the development of novel agents

From the publisher\u27s website: Infections caused by pathogenic fungi are a significant global problem: a situation exacerbated by the limited availability of good antifungal options. Being eukaryotic organisms, these pathogens are phylogenetically much closer to the human host than bacterial pathogens. This sets serious limits to the range of exploitable fungal-specific drug targets. The advent of \u27omics\u27 and other high throughput technologies in recent years has revolutionised the field of antifungal research permitting researchers to quickly identify novel compounds and gain greater insights into drug resistance mechanisms. Researchers can analyse the whole organism\u27s response to any particular condition or compound thereby providing a deeper understanding of fungal biology and the host-fungus interaction. In this book a panel of high-profile authors provides an overview of current antifungal research. Chapters are written from a molecular and genomic perspective and contain speculative models upon which to base future research efforts. Topics include: the molecular mechanisms responsible for antifungal resistance to the classical molecules, azoles, polyenes, and echinocandins; fungal biofilms; fungal-specific biological pathways that constitute potential new targets; strategy to potentiate existing antifungal agents; Impact of high throughput screenings of chemical compound collections; modulating the host response; antifungal vaccines; and animals models

Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum.

A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized

Impact of cyclosporine dosing regimen and infection on voriconazole pharmacodynamics in an experimental model of cerebral scedosporiosis

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Impact of infection status and cyclosporine on voriconazole pharmacokinetics in an experimental model of cerebral scedosporiosis

Cerebral Scedosporium infections usually occur in lung transplant recipients as well as in immunocompetent patients in the context of near-drowning. Voriconazole is the first-line treatment. The diffusion of voriconazole through the blood-brain barriers in the context of cerebral infection and cyclosporine administration is crucial and remains a matter of debate. To address this issue, the pharmacokinetics of voriconazole were assessed in the plasma, cerebrospinal fluid (CSF), and brain, in an experimental model of cerebral scedosporiosis in rats receiving or not cyclosporine. A single dose of voriconazole (30 mg/kg, i.v.) was administrated to six groups of rats randomized according to the infection status and the cyclosporine dosing regimen (no cyclosporine, a single dose or three doses 15 mg/kg each). Voriconazole concentrations in plasma, CSF, and brain samples were quantified using UPLC-MS/MS and HPLC-UV methods and documented up to 48 hours after administration. Pharmacokinetic parameters were estimated using a non-compartmental approach. Voriconazole pharmacokinetic profiles were similar for plasma, CSF, and the brain in all groups studied. Voriconazole Cmax and AUC0=>48h were significantly higher in the plasma than in the CSF (CSF/plasma ratio, median [range] = 0.5 [0.39-0.55] for AUC0=>48h and 0.47 [0.35 and 0.75] for Cmax). Cyclosporine administration was significantly associated with an increase in voriconazole exposure in the plasma, CSF, and brain. In the plasma but not in the brain, an interaction between the infection and cyclosporine administration reduced the positive impact of cyclosporine on voriconazole exposure. Together these results emphasize the impact of cyclosporine on the brain voriconazole exposure

Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species