Evaluation of Primers OPF-01, P54, and 1253 to Identify A. fumigatus, A. flavus, and A. niger from Polymorphic Patterns Obtained by RAPD-PCR.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Peptide derived from plant defensins: A promising 68Ga radiolabelled agent for diagnostic of infection foci in PET.

The development of new radiopharmaceuticals for the detection of hidden infection foci has great relevance for early detection and the selection of the correct treatment, particularly in immunosuppressed patients. In that sense, the labelling of antimicrobial peptides (AMPs) that are capable of binding specifically to the pathogenic microorganism which causes the infection, should provide a sufficiently specific agent, able to distinguish an infection from a sterile inflammation. Defensins are particularly interesting molecules with antimicrobial activity, the EcgDf1 defensin was identified from the genome of a Uruguayan native plant, Erythrina crista-galli, the 'Ceibo' tree. Our group has previously reported a synthetic biologically active short analogue EcgDf21 (ERFTGGHCRGFRRRCFCTKHC) successfully labelled with 99mTc. Herein we present a shorter analogue which also preserves the γ-core domain, as a pharmacophore for a potential infection detection agent. This peptide was derivatized with the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) through a lysine linker in the amino-terminal group (NOTA-KGHCRGFRRRC) and radiolabelled with 68Ga ([68Ga]Ga-NOTA-K-EcgDf1(10)). The [68Ga]Ga-NOTA-K-EcgDf1(10) labelling procedure rendered a product with high radiochemical purity and stability in the labelling milieu. The Log P value indicated that the complex has a hydrophilic behaviour, confirmed by the biodistribution profile. The [68Ga]Ga-NOTA-K-EcgDf1(10) complex demonstrated specific binding to cultures of Candida albicans and Aspergillus niger. Its biodistribution showed renal elimination and low accumulation in the rest of the body. It was possible to successfully differentiate sterile inflammation from infection by PET images in nude mice with a target/non-target ratio of 3.3 for C. albicans and 3.7 for A. niger, respectively.

[99m Tc]Tc-HYNIC-EcgDf21: A defensin short analogue with potential application in infection foci imaging.

Opportunistic infections are a problem of great relevance in public health and the precise detection and localization of infection in the early stages of the disease is of great importance for patient management as well as cost containment. Our proposal seeks to contribute to developing a new agent that meets the needs of diagnosis and follow-up of fungal and bacterial infections, focused on the design of a radiotracer with the potential for recognition of hidden infection foci. Defensins are plant antimicrobial peptides that not only show activity against plant pathogens but also against human ones. A short analogue of EcgDf1 defensin, EcgDf21d (NH2 -ERFTGGHCRGFRRRCFCTKHC-COOH), was labelled through the formation of a 99m Tc-HYNIC complex which was assessed for physicochemical and biological behaviour both in vitro and in vivo. The [99m Tc]Tc-HYNIC-EcgDf21 labelling procedure rendered a single product with remarkably high RCP and stability in the labelling milieu. The Log p value indicated that [99m Tc]Tc-HYNIC-EcgDf21 has a hydrophilic behaviour, confirmed by the biodistribution profiles. The optimal uptake value was obtained for Candida albicans infection model reaching a lesion/muscle ratio of 3, this correlates with in vitro binding studies, and the lesion can be definitely observed in the scintigraphic images.

Assessing the intracellular primary metabolic profile of Trichoderma reesei and Aspergillus niger grown on different carbon sources.

Trichoderma reesei and Aspergillus niger are efficient biological platforms for the production of various industrial products, including cellulases and organic acids. Nevertheless, despite the extensive research on these fungi, integrated analyses of omics-driven approaches are still missing. In this study, the intracellular metabolic profile of T. reesei RUT-C30 and A. niger N402 strains grown on glucose, lactose, carboxymethylcellulose (CMC), and steam-exploded sugarcane bagasse (SEB) as carbon sources for 48 h was analysed by proton nuclear magnetic resonance. The aim was to verify the changes in the primary metabolism triggered by these substrates and use transcriptomics data from the literature to better understand the dynamics of the observed alterations. Glucose and CMC induced higher fungal growth whereas fungi grown on lactose showed the lowest dry weight. Metabolic profile analysis revealed that mannitol, trehalose, glutamate, glutamine, and alanine were the most abundant metabolites in both fungi regardless of the carbon source. These metabolites are of particular interest for the mobilization of carbon and nitrogen, and stress tolerance inside the cell. Their concomitant presence indicates conserved mechanisms adopted by both fungi to assimilate carbon sources of different levels of recalcitrance. Moreover, the higher levels of galactose intermediates in T. reesei suggest its better adaptation in lactose, whereas glycolate and malate in CMC might indicate activation of the glyoxylate shunt. Glycerol and 4-aminobutyrate accumulated in A. niger grown on CMC and lactose, suggesting their relevant role in these carbon sources. In SEB, a lower quantity and diversity of metabolites were identified compared to the other carbon sources, and the metabolic changes and higher xylanase and pNPGase activities indicated a better utilization of bagasse by A. niger. Transcriptomic analysis supported the observed metabolic changes and pathways identified in this work. Taken together, we have advanced the knowledge about how fungal primary metabolism is affected by different carbon sources, and have drawn attention to metabolites still unexplored. These findings might ultimately be considered for developing more robust and efficient microbial factories.

Aspergillus species from Brazilian dry beans and their toxigenic potential.

Aspergilli are common contaminants of food and feed and a major source of mycotoxins. In this study, 87 Aspergillus strains were isolated from beans from 14 different cities in Brazil and identified to the species level based on partial calmodulin and ß-tubulin sequence data. All green spored isolates belonged to section Flavi and were identified as A. flavus (n = 39) or A. pseudocaelatus (n = 1). All black spored isolates belonged to section Nigri and were identified as A. niger (n = 24) or A. luchuensis (n = 10), while the yellow spored strains were identified as A. westerdijkiae (n = 7), A. ostianus (n = 3), A. ochraceus (n = 1) or A. wentii (n = 2). The toxigenic potential of these Aspergillus strains from beans was studied by the prospection of genes in three of the major mycotoxin clusters: aflatoxin (seven genes checked), ochratoxin A (four genes) and fumonisin (ten genes and two intergenic regions). Genes involved in the biosynthesis of aflatoxin were only detected in A. flavus isolates: 17/39 A. flavus isolates proved to contain all the aflatoxin genes tested, the others missed one or more genes. The full complement of fumonisin biosynthesis genes was identified in all A. niger isolates. Finally, no genes for ochratoxin A were detected in any of the isolates. Our work suggests that aflatoxin production by some A. flavus strains and fumonisin production by A. niger isolates form the largest mycotoxin risks in Brazilian beans.

Prospecting for the incidence of genes involved in ochratoxin and fumonisin biosynthesis in Brazilian strains of Aspergillus niger and Aspergillus welwitschiae.

Aspergillus niger "aggregate" is an informal taxonomic rank that represents a group of species from the section Nigri. Among A. niger "aggregate" species Aspergillus niger sensu stricto and its cryptic species Aspergillus welwitschiae (=Aspergillus awamori sensu Perrone) are proven as ochratoxin A and fumonisin B2 producing species. A. niger has been frequently found in tropical and subtropical foods. A. welwitschiae is a new species, which was recently dismembered from the A. niger taxon. These species are morphologically very similar and molecular data are indispensable for their identification. A total of 175 Brazilian isolates previously identified as A. niger collected from dried fruits, Brazil nuts, coffee beans, grapes, cocoa and onions were investigated in this study. Based on partial calmodulin gene sequences about one-half of our isolates were identified as A. welwitschiae. This new species was the predominant species in onions analyzed in Brazil. A. niger and A. welwitschiae differ in their ability to produce ochratoxin A and fumonisin B2. Among A. niger isolates, approximately 32% were OTA producers, but in contrast only 1% of the A. welwitschiae isolates revealed the ability to produce ochratoxin A. Regarding fumonisin B2 production, there was a higher frequency of FB2 producing isolates in A. niger (74%) compared to A. welwitschiae (34%). Because not all A. niger and A. welwitschiae strains produce ochratoxin A and fumonisin B2, in this study a multiplex PCR was developed for detecting the presence of essential genes involved in ochratoxin (polyketide synthase and radHflavin-dependent halogenase) and fumonisin (α-oxoamine synthase) biosynthesis in the genome of A. niger and A. welwitschiae isolates. The frequency of strains harboring the mycotoxin genes was markedly different between A. niger and A. welwitschiae. All OTA producing isolates of A. niger and A. welwitschiae showed in their genome the pks and radH genes, and 95.2% of the nonproducing isolates did not contain these genes. The α-oxoamine synthase gene was detected in 100% and 36% of the A. niger and A. welwitschiae isolates, respectively. The loss of ochratoxin A production in A. niger and A. welwitschiae is highly associated with gene deletions within the ochratoxin biosynthetic gene cluster. The loss of fumonisin production in A. welwitschiae is associated with gene deletions within the fumonisin biosynthetic gene cluster, but this is not the case with A. niger.

Mechanisms of interaction of chromium with Aspergillus niger var tubingensis strain Ed8.

Experiments were conducted to determine the mechanisms of interaction with chromium of Aspergillus niger var tubingensis strain Ed8 in batch culture and in bioreactor experiments. Results obtained in this work showed that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process and also, secondly, in a sorption process. Using electron microscopy techniques the ultrathin sections obtained from the mycelium biomass produced by the fungus in batch cultures showed the ability to incorporate Cr intracellulary, into low electron-dense inclusions, but not extracellularly. On the other hand, cultures without Cr(VI) of A. niger var tubingensis Ed8, grown in a bubble column bioreactor, reduced Cr(VI) immediately after repeated addition of this oxyanion; after six loads, 460 mg Cr(VI) was reduced to Cr(III) in 60 h, corresponding to a reduction rate of 2.62 mg Cr(VI)g(-1) dry biomass h(-1).

Production of rabbit antibodies against purified Glucose oxidase

Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose) resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

Evaluación biológica de 99mTc-voriconazol como potencial agente de diagnóstico de infecciones fúngicas por centellografía gamma / Biological evaluation of voriconazole with 99mTc for the diagnosis of fungal infections

El avance del HIV ha favorecido el aumento de infecciones fúngicas como candidiasis y aspergilosis invasivas. Varias clases de antifúngicos son utilizados para el tratamiento de las mismas y éstos pueden ser radiomarcados con un agente emisor gamma que permita la detección mediante centellografia de focos de infección. El Voriconazol es un triazol adecuado para la marcación mediante la formación de un complejo unido al precursor [99mTc(H2O)3(CO)3]+. El objetivo fue marcar y determinar las características fisicoquímicas y biológicas del voriconazol con 99mTc para la detección temprana de infecciones. La pureza radioquímica se determinó por HPLC y permitió establecer que el complejo permanece estable durante al menos 120 min. Los estudios in vivo en modelos de inflamación estéril, infección con C. Albicans y A. Niger mostraron diferenciación de los procesos tanto en biodistribución como en imágenes centellográficas.

The spread of HIV has led to an increase of fungal infections such as candidiasis and invasive aspergillosis. Several types of antifungals are used to treat them and some of them can be radiolabeled with a gamma emitting agent to allow detection by scintigraphy of foci of infection. Voriconazole is a triazole agent, suitable for the synthesis of a complex linked with the precursor [99mTc(H2O)3(CO)3]+. The aim of his work was to label and determine the physicochemical and biological characteristics of voriconazole with 99mTc for the early detection of fungal infections. Radiochemical purity was determined by HPLC and the complex remained stable during at least 120 min. In vivo studies in rats bearing either sterile inflammation, infection with C. Albicans or A. Niger showed differentiation of the processes not only in biosdistribution but also in scintigraphic images.

Influences of phenolic compounds on citric acid productivity by Aspergillus niger in stirred fermentor

Influences of phenol, alpha-naphtol and beta-naphtol, which are toxic chemicals, on citric acid biosynthesis and biomass in the artificial culture setting of Aspergillus niger using batch fermenter are examined in the most favorable fermentation conditions, and a model is proposed. Addition of certain concentrations of phenol, alpha-naphtol and beta-naphtol to the culture increases the citric acid production. According to this model, maximum citric acid concentration is 48.3 g L-1 in a culture that does not contain any toxic chemicals, whereas the maximum concentrations obtained in cultures containing 25 mg L-1 phenol, 25 mg L-1 alpha-naphtol and 15 mg L-1 beta-naphtol are 62.5 g L-1, 78.1 g L-1 and 86.0 g L-1, respectively. Moreover, addition of toxic chemicals to the culture reduces fermentation time by 24 hrs.

Effect of antioxidant mixtures on growth and ochratoxin a production of Aspergillus section Nigri species under different water activity conditions on peanut meal extract agar.

The effect of mixtures of antioxidants butylated hydroxyanisol (BHA) and propyl paraben (PP) on lag phase, growth rate and ochratoxin A (OTA) production by four Aspergillus section Nigri strains was evaluated on peanut meal extract agar (PMEA) under different water activities (a(w)). The antioxidant mixtures used were: BHA + PP (mM), M1 (0.5 + 0.5), M2 (1.0 + 0.5), M3 (2.5 + 0.5), M4 (0.5 + 1.0), M5 (1.0 + 1.0), M6 (2.5 + 1.0), M7 (5.0 + 2.5) and M8 (10 + 2.5). The mixture M8 completely suppressed mycelial growth for all strains. A significant stimulation in OTA production was observed with mixtures M1 to M5 mainly at the highest a(w); whereas M6, M7 and M8 completely inhibited OTA production in all strains assayed; except M6 in A. carbonarius strain (RCP G). These results could enable a future intervention strategy to minimize OTA contamination.