The ABC transporter gene in the sirodesmin biosynthetic gene cluster of Leptosphaeria maculans is not essential for sirodesmin production but facilitates self-protection

Epipolythiodioxopiperazine toxins are secreted by a range of fungi, including Leptosphaeria maculans, which produces sirodesmin, and Aspergillus fumigatus, which produces gliotoxin. The L. maculans biosynthetic gene cluster for sirodesmin includes an ABC transporter gene, sirA. Disruption of this gene led to increased secretion of sirodesmin into the medium and an altered ratio of sirodesmin to its immediate precursor. The transcription pattern of a peptide synthetase that catalyses an early step in sirodesmin biosynthesis was elevated in the sirA mutant by 47% over a 7-day period. This was consistent with the finding that the transporter mutant had elevated sirodesmin levels. Despite increased production of sirodesmin, the sit-A mutant was more sensitive to both sirodesmin and gliotoxin. The putative gliotoxin transporter gene, gliA, (a major facilitator superfamily transporter) from A.fumigatus complemented the tolerance of the L. maculans sirA mutant to gliotoxin, but not to sirodesmin. The results indicate that SirA contributes to self-protection against sirodesmin in L. maculans and suggest a transporter other than SirA is primarily responsible for efflux of endogenously produced sirodesmin. (C) 2004 Elsevier Inc. All rights reserved.

Successful treatment of post-influenza pseudomembranous necrotising bronchial aspergillosis with liposomal amphotericin, inhaled amphotericin B, gamma interferon and GM-CSF

A case of aspergillus tracheobronchitis following influenza A infection in an immunocompetent 35 year old woman is described that required prolonged mechanical ventilation for airways obstruction. Treatment included liposomal amphotericin, inhaled amphotericin, gamma interferon and GM-CSF. Liposomal amphotericin therapy was associated with reversible hepatosplenomegaly. Inhaled corticosteroids with continued antifungal therapy were used for the management of severe recurrent airway obstruction. After a prolonged course of treatment she survived with fixed airways obstruction unresponsive to corticosteroids.

Aflatoxins and their relationship with sugars in peanut (Arachis hypogaea L.)

Aspergillus spp. produce aflatoxins in peanut, which poses a health risk to humans and animals, as well as affecting the marketability of peanuts. The current research found that more aflatoxin is produced under rain fed (RF) compared to irrigated (IRR) conditions, and was more predominant in juvenile (R3-5 stage) compared to older (R6-8 stage) pods. No aflatoxin was recorded in marketable pods of the Streeton cultivar in either of the growing conditions, whereas the cultivar NC-7 produced aflatoxin under RF conditions only. Sugars such as glucose, fructose and sucrose were positively correlated with total aflatoxins. It appears that Aspergillus utilises these simple carbohydrates as substrates in biosynthesis of aflatoxins.

A D-optimal designed population pharmacokinetic study of itraconazole capsules and solution in adults with cystic fibrosis

Background: Oral itraconazole (ITRA) is used for the treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis (CF) because of its antifungal activity against Aspergillus species. ITRA has an active hydroxy-metabolite (OH-ITRA) which has similar antifungal activity. ITRA is a highly lipophilic drug which is available in two different oral formulations, a capsule and an oral solution. It is reported that the oral solution has a 60% higher relative bioavailability. The influence of altered gastric physiology associated with CF on the pharmacokinetics (PK) of ITRA and its metabolite has not been previously evaluated. Objectives: 1) To estimate the population (pop) PK parameters for ITRA and its active metabolite OH-ITRA including relative bioavailability of the parent after administration of the parent by both capsule and solution and 2) to assess the performance of the optimal design. Methods: The study was a cross-over design in which 30 patients received the capsule on the first occasion and 3 days later the solution formulation. The design was constrained to have a maximum of 4 blood samples per occasion for estimation of the popPK of both ITRA and OH-ITRA. The sampling times for the population model were optimized previously using POPT v.2.0.[1] POPT is a series of applications that run under MATLAB and provide an evaluation of the information matrix for a nonlinear mixed effects model given a particular design. In addition it can be used to optimize the design based on evaluation of the determinant of the information matrix. The model details for the design were based on prior information obtained from the literature, which suggested that ITRA may have either linear or non-linear elimination. The optimal sampling times were evaluated to provide information for both competing models for the parent and metabolite and for both capsule and solution simultaneously. Blood samples were assayed by validated HPLC.[2] PopPK modelling was performed using FOCE with interaction under NONMEM, version 5 (level 1.1; GloboMax LLC, Hanover, MD, USA). The PK of ITRA and OH‑ITRA was modelled simultaneously using ADVAN 5. Subsequently three methods were assessed for modelling concentrations less than the LOD (limit of detection). These methods (corresponding to methods 5, 6 & 4 from Beal[3], respectively) were (a) where all values less than LOD were assigned to half of LOD, (b) where the closest missing value that is less than LOD was assigned to half the LOD and all previous (if during absorption) or subsequent (if during elimination) missing samples were deleted, and (c) where the contribution of the expectation of each missing concentration to the likelihood is estimated. The LOD was 0.04 mg/L. The final model evaluation was performed via bootstrap with re-sampling and a visual predictive check. The optimal design and the sampling windows of the study were evaluated for execution errors and for agreement between the observed and predicted standard errors. Dosing regimens were simulated for the capsules and the oral solution to assess their ability to achieve ITRA target trough concentration (Cmin,ss of 0.5-2 mg/L) or a combined Cmin,ss for ITRA and OH-ITRA above 1.5mg/L. Results and Discussion: A total of 241 blood samples were collected and analysed, 94% of them were taken within the defined optimal sampling windows, of which 31% where taken within 5 min of the exact optimal times. Forty six per cent of the ITRA values and 28% of the OH-ITRA values were below LOD. The entire profile after administration of the capsule for five patients was below LOD and therefore the data from this occasion was omitted from estimation. A 2-compartment model with 1st order absorption and elimination best described ITRA PK, with 1st order metabolism of the parent to OH-ITRA. For ITRA the clearance (ClItra/F) was 31.5 L/h; apparent volumes of central and peripheral compartments were 56.7 L and 2090 L, respectively. Absorption rate constants for capsule (kacap) and solution (kasol) were 0.0315 h-1 and 0.125 h-1, respectively. Comparative bioavailability of the capsule was 0.82. There was no evidence of nonlinearity in the popPK of ITRA. No screened covariate significantly improved the fit to the data. The results of the parameter estimates from the final model were comparable between the different methods for accounting for missing data, (M4,5,6)[3] and provided similar parameter estimates. The prospective application of an optimal design was found to be successful. Due to the sampling windows, most of the samples could be collected within the daily hospital routine, but still at times that were near optimal for estimating the popPK parameters. The final model was one of the potential competing models considered in the original design. The asymptotic standard errors provided by NONMEM for the final model and empirical values from bootstrap were similar in magnitude to those predicted from the Fisher Information matrix associated with the D-optimal design. Simulations from the final model showed that the current dosing regimen of 200 mg twice daily (bd) would provide a target Cmin,ss (0.5-2 mg/L) for only 35% of patients when administered as the solution and 31% when administered as capsules. The optimal dosing schedule was 500mg bd for both formulations. The target success for this dosing regimen was 87% for the solution with an NNT=4 compared to capsules. This means, for every 4 patients treated with the solution one additional patient will achieve a target success compared to capsule but at an additional cost of AUD $220 per day. The therapeutic target however is still doubtful and potential risks of these dosing schedules need to be assessed on an individual basis. Conclusion: A model was developed which described the popPK of ITRA and its main active metabolite OH-ITRA in adult CF after administration of both capsule and solution. The relative bioavailability of ITRA from the capsule was 82% that of the solution, but considerably more variable. To incorporate missing data, using the simple Beal method 5 (using half LOD for all samples below LOD) provided comparable results to the more complex but theoretically better Beal method 4 (integration method). The optimal sparse design performed well for estimation of model parameters and provided a good fit to the data.

Mycotoxins and abnormal embryonic development

This chapter reviews studies on the effects of mycotoxins on embryonic and fetal development, especially those toxins that are global food and feed contaminants. The toxins discussed include aflatoxin produced by Aspergillus flavus and A. parasiticus, ochratoxin which is produced by Aspergillus species particularly A. ochraceus as well as Penicillium verrucosum, ergot alkaloids produced by Claviceps spp., and the Fusarium toxins (fumonisins, deoxynivalenol [vomitoxin], and zearalenone). These toxins have been shown to be teratogenic and/or embryotoxic in different animal bioassays. The implications of toxicity on embryogenesis, and the progress of research on these mycotoxins, are also examined.

Monitoring inflammation and airway remodeling by FMT in a chronic asthma model

Asthma is a multifactorial disease for which a variety of mouse models have been developed. A major drawback of these models is represented by the transient nature of the airway pathology peaking 24 to 72 hours after challenge and resolving in 1 to 2 weeks. The objective of this study is to characterize the temporal evolution of pulmonary inflammation and remodeling in a recently described mouse model of chronic asthma (8 week treatment with 3 allergens relevant for the human pathology: Dust mite, Ragweed, and Aspergillus; DRA). We studied the DRA model taking advantage of fluorescence molecular tomography (FMT) imaging using near-infrared probes to non-invasively evaluate lung inflammation and airway remodeling. At 4, 6, 8 or 11 weeks, cathepsin- and metalloproteinase-dependent fluorescence was evaluated in vivo. A subgroup of animals, after 4 weeks of DRA, was treated with Budesonide (100 µg/kg intranasally) daily for 4 weeks. Cathepsin-dependent fluorescence in DRA-sensitized mice resulted significantly increased at 6 and 8 weeks, and was markedly inhibited by budesonide. This fluorescent signal well correlated with ex vivo analysis such as bronchoalveolar lavage eosinophils and alveolar cell infiltration. Metalloproteinase-dependent fluorescence was significantly increased at 8 and 11 weeks, nicely correlated with collagen deposition, as evaluated histologically by Masson’s Trichrome staining, and airway epithelium hypertrophy, and was also partly inhibited by budesonide. In conclusion, FMT proved suitable for longitudinal study to evaluate asthma progression, both in terms of inflammatory cell infiltration and airway remodeling, allowing the determination of treatment efficacy in a chronic asthma model in mice.

The development of filamentous fungi in submerged culture

During the 24 hour period following inoculation, aggregation of spores and sporelings can have an important effect on the subsequent growth of filamentous fungi in submerged culture. This early phase of growth does not appear to have received much attention, and it was for this reason that the author's research was started. The aggregation, germination and early growth of the filamentous fungus Aspergillus niger have been followed in aerated tower fermenters, by microscopic examination. By studying many individual sporelings it has been possible to estimate the specific growth rate and germination times, and then to assess the branching characteristics of the fungus over a period of from 1 to 10 hours after germination. The results have been incorporated into computer models to simulate the development of the physical structure of individual and aggregated sporelings. Following germination, and an initial rapid growth phase, fungi were found to grow exponentially: in the case of A.niger the mean germination time was about 5 hours and the doubling time was as short as 1.5 hours. Branching also followed an exponential pattern and appeared to be related to hyphal length. Using a simple hypothesis for growth along with empirical parameters, typical fungal structures were generated using the computer models : these compared well with actual sporelings observed under the microscope. Preliminary work suggested that the techniques used in this research could be successfully applied to a range of filamentous fungi.

Ocular mycoses:infections of the eye caused by fungi

Fungi are ubiquitous organisms in nature and can be found in association with healthy eyes. The incidence of actual fungal infection of the eye, however, is relatively low compared with that attributable to viruses and bacteria. Nevertheless, fungal infection of the eye is increasing especially in immuno-compromised patients and a wide variety of fungal infections have now been described worldwide with species of Fusarium, Aspergillus, Candida, and dematiaceous fungi predominating. At present there are a limited number of compounds available to control ocular mycoses while resistance to anti-fungal agents has been growing in recent years, especially to azoles. Several mechanisms of resistance have been identified including modification of sterol synthesis pathways by the fungus, modification of enzymes to reduce the binding of azoles to fungal components and increased efficiency of removal of the azole within fungal cells. Although resistance to amphotericin-B has been reported, it continues to be the most important treatment for life-threatening conditions and more severe ophthalmic infections. Natamycin is often first choice for filamentous fungal keratitis and topical amphotericin-B for Candida keratitis. Continued monitoring of the behaviour of ocular fungi will be essential in future together with the development of new anti-fungal agents.

DNA Mutation of the progranulin (GRN) gene in familial frontotemporal lobar degeneration (FTLD):a study of the pathology of nine cases

Frontotemporal lobar degeneration (FTLD) with transactive response (TAR) DNA-binding protein of 43kDa (TDP-43) proteinopathy (FTLD-TDP) is a neurodegenerative disease characterized by variable neocortical and allocortical atrophy principally affecting the frontal and temporal lobes. Histologically, there is neuronal loss, microvacuolation in the superficial cortical laminae, and a reactive astrocytosis. A variety of TDP-43 immunoreactive changes are present in FTLD-TDP including neuronal cytoplasmic inclusions (NCI), neuronal intranuclear inclusions (NII), dystrophic neurites (DN) and, oligodendroglial inclusions (GI). Many cases of familial FTLD-TDP are caused by DNA mutations of the progranulin (GRN) gene. Hence, the density, spatial patterns, and laminar distribution of the pathological changes were studied in nine cases of FLTD-TDP with GRN mutation. The densities of NCI and DN were greater in cases caused by GRN mutation compared with sporadic cases. In cortical regions, the commonest spatial pattern exhibited by the TDP-43 immunoreactive lesions was the presence of clusters of inclusions regularly distributed parallel to the pia mater. In approximately 50% of cortical gyri, the NCI exhibited a peak of density in the upper cortical laminae while the GI were commonly distributed across all laminae. The distribution of the NII and DN was variable, the most common pattern being a peak of NII density in the lower cortical laminae and DN in the upper cortical laminae. These results suggest in FTLD-TDP caused by GRN mutation: 1) there are greater densities of NCI and DN than in sporadic cases of the disease, 2) there is degeneration of the cortico-cortical and cortico-hippocampal pathways, and 3) cortical degeneration occurs across the cortical laminae, the various TDP-43 immunoreactive inclusions often being distributed in different cortical laminae.

Neuropathological heterogeneity in frontotemporal lobar degeneration with TDP-43 proteinopathy: a quantitative study of 94 cases using principal components analysis

Studies suggest that frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein of 43kDa (TDP-43) proteinopathy (FTLD-TDP) is heterogeneous with division into four or five subtypes. To determine the degree of heterogeneity and the validity of the subtypes, we studied neuropathological variation within the frontal and temporal lobes of 94 cases of FTLD-TDP using quantitative estimates of density and principal components analysis (PCA). A PCA based on the density of TDP-43 immunoreactive neuronal cytoplasmic inclusions (NCI), oligodendroglial inclusions (GI), neuronal intranuclear inclusions (NII), and dystrophic neurites (DN), surviving neurons, enlarged neurons (EN), and vacuolation suggested that cases were not segregated into distinct subtypes. Variation in the density of the vacuoles was the greatest source of variation between cases. A PCA based on TDP-43 pathology alone suggested that cases of FTLD-TDP with progranulin (GRN) mutation segregated to some degree. The pathological phenotype of all four subtypes overlapped but subtypes 1 and 4 were the most distinctive. Cases with coexisting motor neuron disease (MND) or hippocampal sclerosis (HS) also appeared to segregate to some extent. We suggest: 1) pathological variation in FTLD-TDP is best described as a ‘continuum’ without clearly distinct subtypes, 2) vacuolation was the single greatest source of variation and reflects the ‘stage’ of the disease, and 3) within the FTLD-TDP ‘continuum’ cases with GRN mutation and with coexisting MND or HS may have a more distinctive pathology.

A morphometric study of the spatial patterns of TDP-43 immunoreactive neuronal inclusions in frontotemporal lobar degeneration (FTLD) with progranulin (GRN) mutation

Mutations of the progranulin (GRN) gene are a major cause of familial frontotemporal lobar degeneration with transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) proteinopathy (FTLD-TDP). We studied the spatial patterns of TDP-43 immunoreactive neuronal cytoplasmic inclusions (NCI) and neuronal intranuclear inclusions (NII) in histological sections of the frontal and temporal lobe in eight cases of FTLD-TDP with GRN mutation using morphometric methods and spatial pattern analysis. In neocortical regions, the NCI were clustered and the clusters were regularly distributed parallel to the pia mater; 58% of regions analysed exhibiting this pattern. The NII were present in regularly distributed clusters in 35% of regions but also randomly distributed in many areas. In neocortical regions, the sizes of the regular clusters of NCI and NII were 400-800 µm, approximating to the size of the modular columns of the cortico-cortical projections, in 31% and 36% of regions respectively. The NCI and NII also exhibited regularly spaced clustering in sectors CA1/2 of the hippocampus and in the dentate gyrus. The clusters of NCI and NII were not spatially correlated. The data suggest degeneration of the cortico-cortical and cortico-hippocampal pathways in FTLD-TDP with GRN mutation, the NCI and NII affecting different clusters of neurons.

The spectrum and severity of FUS-immunoreactive inclusions in the frontal and temporal lobes of ten cases of neuronal intermediate filament inclusion disease

Neuronal intermediate filament inclusion disease (NIFID), a rare form of frontotemporal lobar degeneration (FTLD), is characterized neuropathologically by focal atrophy of the frontal and temporal lobes, neuronal loss, gliosis, and neuronal cytoplasmic inclusions (NCI) containing epitopes of ubiquitin and neuronal intermediate filament proteins. Recently, the 'fused in sarcoma' (FUS) protein (encoded by the FUS gene) has been shown to be a component of the inclusions of familial amyotrophic lateral sclerosis with FUS mutation, NIFID, basophilic inclusion body disease, and atypical FTLD with ubiquitin-immunoreactive inclusions (aFTLD-U). To further characterize FUS proteinopathy in NIFID, and to determine whether the pathology revealed by FUS immunohistochemistry (IHC) is more extensive than a-internexin, we have undertaken a quantitative assessment of ten clinically and neuropathologically well-characterized cases using FUS IHC. The densities of NCI were greatest in the dentate gyrus (DG) and in sectors CA1/2 of the hippocampus. Anti-FUS antibodies also labeled glial inclusions (GI), neuronal intranuclear inclusions (NII), and dystrophic neurites (DN). Vacuolation was extensive across upper and lower cortical layers. Significantly greater densities of abnormally enlarged neurons and glial cell nuclei were present in the lower compared with the upper cortical laminae. FUS IHC revealed significantly greater numbers of NCI in all brain regions especially the DG. Our data suggest: (1) significant densities of FUS-immunoreactive NCI in NIFID especially in the DG and CA1/2; (2) infrequent FUS-immunoreactive GI, NII, and DN; (3) widely distributed vacuolation across the cortex, and (4) significantly more NCI revealed by FUS than a-internexin IHC.

The effects of substrate water availability on the isolation and growth of fungi

The work reported in this thesis was carried out to contribute to the knowledge of the effects of substrate water availability or water activity (a ) on fungal growth parameters and its implications in the preparationw of materials susceptible to biodeterioration. Fungi were isolated from soils of different ecological sites at a range of substrate aw levels controlled by sodium chloride (NaCl). Three groups of fungi were isolated : firstly, those isolated only at high a (aw about 0.997).secondly, those isolated at high and decreasing aw (aw 0.997 to 0.85) and finally, those isolated at only decreased aw (aw O.95 to 0.80). From these isolations, test fungi were selected to study the effects of pH, temperature, exo-enzyme production and biocide efficacy at decreased aw levels, with glycerol and NaCl as a controlling solutes. The linear extension rates of the fungi increased at all test pH values near optimum a of growth. Test fungi of the Aspergillus glaucus group were found to be most resistant to low aw. Growth and survival of vegetative and fruiting bodies at elevated temperatures were enhanced with the addition of a controlling solutes. A. flavus, A. fumigatus displayed high heat resistance and A. amstelodami, A. versicolor and Penicillium citrinum displayed low heat resistance at high aw levels and vice versa at low aw levels. Amylase, lipase and protease activities were studied at lowered aw , using modifications of the test tube method of Raute11a and Cowling. Amylase and protease production in most xerophilic fungi ceased around 0.80 aw , but lipase production in some xerophilic fungi, including A. glatlcus fungi, was up to and including 0.70 aw with g1ycero1.

TDP-43-Immunoreactive pathology in frontotemporal lobar degeneration with TDP proteinopathy (FTLD-TDP) with and without associated motor neuron disease

A proportion of patients with motor neuron disease (MND) exhibit frontotemporal dementia (FTD) and some patients with FTD develop the clinical features of MND. Frontotemporal lobar degeneration (FTLD) is the pathological substrate of FTD and some forms of this disease (referred to as FTLD-U) share with MND the common feature of ubiquitin-immunoreactive, tau-negative cellular inclusions in the cerebral cortex and hippocampus. Recently, the transactive response (TAR) DNA-binding protein of 43 kDa (TDP-43) has been found to be a major protein of the inclusions of FTLD-U with or without MND and these cases are referred to as FTLD with TDP-43 proteinopathy (FTLD-TDP). To clarify the relationship between MND and FTLD-TDP, TDP-43 pathology was studied in nine cases of FTLD-MND and compared with cases of familial and sporadic FTLD–TDP without associated MND. A principal components analysis (PCA) of the nine FTLD-MND cases suggested that variations in the density of surviving neurons in the frontal cortex and neuronal cytoplasmic inclusions (NCI) in the dentate gyrus (DG) were the major histological differences between cases. The density of surviving neurons in FTLD-MND was significantly less than in FTLD-TDP cases without MND, and there were greater densities of NCI but fewer neuronal intranuclear inclusions (NII) in some brain regions in FTLD-MND. A PCA of all FTLD-TDP cases, based on TDP-43 pathology alone, suggested that neuropathological heterogeneity was essentially continuously distributed. The FTLD-MND cases exhibited consistently high loadings on PC2 and overlapped with subtypes 2 and 3 of FTLD-TDP. The data suggest: (1) FTLD-MND cases have a consistent pathology, variations in the density of NCI in the DG being the major TDP-43-immunoreactive difference between cases, (2) there are considerable similarities in the neuropathology of FTLD-TDP with and without MND, but with greater neuronal loss in FTLD-MND, and (3) FTLD-MND cases are part of the FTLD-TDP ‘continuum’ overlapping with FTLD-TDP disease subtypes 2 and 3.

Modelling a biochemical reaction with computational fluid dynamics

Earlier investigations (Cartland Glover et al., 2004) into the use of computational fluid dynamics (CFD) for the modelling of gas-liquid and gas-liquid-solid flow allowed a simple biochemical reaction model to be implemented. A single plane mesh was used to represent the transport and reaction of molasses, the mould Aspergillus niger and citric acid in a bubble column with a height to diameter aspect ratio of 20:1. Two specific growth rates were used to examine the impact that biomass growth had on the local solids concentration and the effect this had on the local hydrodynamics of the bubble column.