An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

Background: Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods: In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results: The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions: Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.

The enzymatic nature of fungal bioluminescence

The uncertainty about the possible involvement of a luciferase in fungal bioluminescence has not only hindered the understanding of its biochemistry but also delayed the characterization of its constituents. The present report describes how in vitro light emission can be obtained enzymatically from the cold and hot extracts assay using different species of fungi, which also indicates a common mechanism for all these organisms. Kinetic data suggest a consecutive two-step enzymatic mechanism and corroborate the enzymatic proposal of Airth and Foerster. Finally, overlapping of light emission spectra from the fungal bioluminescence and the in vitro assay confirm that this reaction is the same one that occurs in live fungi.

Interaction between polymorphisms of the Human Leukocyte Antigen and HPV-16 Variants on the risk of invasive cervical cancer

Background: Persistent infection with oncogenic types of human papillomavirus (HPV) is the major risk factor for invasive cervical cancer (ICC), and non-European variants of HPV-16 are associated with an increased risk of persistence and ICC. HLA class II polymorphisms are also associated with genetic susceptibility to ICC. Our aim is to verify if these associations are influenced by HPV-16 variability. Methods: We characterized HPV-16 variants by PCR in 107 ICC cases, which were typed for HLA-DQA1, DRB1 and DQB1 genes and compared to 257 controls. We measured the magnitude of associations by logistic regression analysis. Results: European ( E), Asian-American ( AA) and African (Af) variants were identified. Here we show that inverse association between DQB1*05 ( adjusted odds ratio [ OR] = 0.66; 95% confidence interval [CI]: 0.39-1.12]) and HPV-16 positive ICC in our previous report was mostly attributable to AA variant carriers ( OR = 0.27; 95% CI: 0.10-0.75). We observed similar proportions of HLA DRB1*1302 carriers in E-P positive cases and controls, but interestingly, this allele was not found in AA cases ( p = 0.03, Fisher exact test). A positive association with DRB1*15 was observed in both groups of women harboring either E ( OR = 2.99; 95% CI: 1.13-7.86) or AA variants ( OR = 2.34; 95% CI: 1.00-5.46). There was an inverse association between DRB1*04 and ICC among women with HPV-16 carrying the 350T [83L] single nucleotide polymorphism in the E6 gene ( OR = 0.27; 95% CI: 0.08-0.96). An inverse association between DQB1*05 and cases carrying 350G (83V) variants was also found ( OR = 0.37; 95% CI: 0.15-0.89). Conclusion: Our results suggest that the association between HLA polymorphism and risk of ICC might be influenced by the distribution of HPV-16 variants.

Combined photocatalytic and fungal processes for the treatment of nitrocellulose industry wastewater

The objective of this work was to characterize the delignification effluent originating from the delignification industry and evaluate the combination of the fungus and photocatalytic process (TiO(2)/UV system) for the treatment of this effluent. The delignification effluent has proven harmful to the environment because it presents high color (3516 CU), total phenol (876 mg/L and TOC (1599 mg/L) and is also highly toxic even in a low concentration. The results of photocatalysis were 11%, 25% and 13% higher for reductions in color, total phenol and TOC, respectively. The combined treatments presented benefits when compared to the non-combined treatments. Fungus and photocatalysis in combination proved to be the best treatment, reducing the color, total phenol, toxicity (inhibition of Escherichia coli growth) and TOC by 94.2%, 92.6%, 4.9% and 62%, respectively. (C) 2008 Elsevier B.V. All rights reserved.

Technological advances and mechanistic basis for fungal biopulping

Biopulping fundamentals, technology and mechanisms are reviewed in this article. Mill evaluation of Eucalyptus grandis wood chips biotreated by Ceriporiopsis subvermispora on a 50-tonne pilot-plant demonstrated that equivalent energy savings can be obtained in lab- and mill-scale biopulping. Some drawbacks concerning limited improvements in pulp strength and contamination of the chip pile with opportunist fungi have been observed. The use of pre-cultured wood chips as inoculum seed for the biotreatment process minimized contamination problems related to the use of blended mycelium and corn-steep liquor in the inoculation step. Alkaline wash restored part of the brightness in biopulps and marketable brightness values were obtained by one-stage bleaching with 5% H2O2 when bio-TMP pulps were under evaluation. Considering the current scenario, the understanding of biopulping mechanisms has gained renewed attention because more resistant and competitive fungal species could be selected with basis on a function-directed screening project. A series of studies aimed to elucidate structural changes in lignin during wood biodegradation by C. subvermispora had indicated that lignin depolymerization occurs during initial stages of wood biotreatment. Aromatic hydroxyls did not increase with the split of aryl-ether linkages, suggesting that the ether-cleavage-products remain as quitione-type structures. On the other hand, cellulose is more resistant to the attack by C subvermispora. MnP-initiated lipid peroxidation reactions have been proposed to explain degradation of non-phenolic lignin substructures by C subvermispora, while the lack of cellobiohydrolases and the occurrence of systems able to suppress Fenton`s reaction in the cultures have explained non-efficient cellulose degradation by this biopulping fungus. (C) 2007 Elsevier Inc. All rights reserved.

Biomimetic oxidative treatment of spruce wood studied by pyrolysis-molecular beam mass spectrometry coupled with multivariate analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis: implications for fungal degradation of wood

In this work, pyrolysis-molecular beam mass spectrometry analysis coupled with principal components analysis and (13)C-labeled tetramethylammonium hydroxide thermochemolysis were used to study lignin oxidation, depolymerization, and demethylation of spruce wood treated by biomimetic oxidative systems. Neat Fenton and chelator-mediated Fenton reaction (CMFR) systems as well as cellulosic enzyme treatments were used to mimic the nonenzymatic process involved in wood brown-rot biodegradation. The results suggest that compared with enzymatic processes, Fenton-based treatment more readily opens the structure of the lignocellulosic matrix, freeing cellulose fibrils from the matrix. The results demonstrate that, under the current treatment conditions, Fenton and CMFR treatment cause limited demethoxylation of lignin in the insoluble wood residue. However, analysis of a water-extractable fraction revealed considerable soluble lignin residue structures that had undergone side chain oxidation as well as demethoxylation upon CMFR treatment. This research has implications for our understanding of nonenzymatic degradation of wood and the diffusion of CMFR agents in the wood cell wall during fungal degradation processes.

Discoloration and fungal growth on three fiber cement formulations exposed in urban, rural and coastal zones

Three formulations of fiber cement were evaluated for fungal colonization and color change after five years of exposure in aging stations located in urban (Sao Paulo), rural (Pirassununga) and coastal (Rio Grande) zones in Brazil. The lowest color change and fungal colonization were registered in Rio Grande, which has a temperate climate, as opposed to Sao Paulo and Pirassununga, which are tropical. The highest fungal colonization was recorded in Sao Paulo, one of the most air polluted cities in Brazil. Pirassununga samples had an intermediate fungal colonization, in spite of showing the highest color change with visible dark spots on the surfaces. These spots were identified as cyanobacteria, which significantly contributed to the darkening of the specimens. The fiber cement formulation, varying in proportion of organic fibers such as poly (vinyl alcohol) and cellulose, was less significant for fungal bioreceptivity than the characteristics of the exposure site. The most frequent fungal genus found in the tropical climate, in both urban and rural zones, and the main one responsible for the higher records in Sao Paulo, was Scytalidiurn sp. which was registered for the first time on this building material in Brazil. (C) 2010 Elsevier Ltd. All rights reserved.

Climate as the most important factor determining anti-fungal biocide performance in paint films

The effect of Pigment Volume Content (PVC) on fungal growth on acrylic paint formulations with and without biocide, exposed to weathering in three different climatic regions in Brazil for four years, was studied Latex paints. with PVC of 30%, 35% and 50%, were applied to autoclaved aerated concrete blocks pre-covered with acrylic sealer and acrylic plaster They were exposed to equatorial, tropical and temperate climates in north, south-east, and south Brazil Cladosporium was the most abundant fungal genus detected in the biofilm on the surfaces of all paint formulations at all sites after four years Heaviest fungal colonization occurred in the tropical south-east and lightest in the temperate south of the country, but more phototrophs, principally cyanobacteria, were detected in the equatorial region PVC and presence of biocides were shown to be of less importance than environmental conditions (irradiance, humidity and temperature) for biofilm formation and consequent discolouration These results have important implications for testing of paint formulations (C) 2010 Elsevier B V All rights reserved

Fungal pathogens as classical biological control agents against arthropods

Fungal entomopathogens have been used more frequently than other types of pathogens for classical biological control. Among 136 programs using different groups of arthropod pathogens, 49.3% have introduced fungal pathogens (including both the traditional fungi and microsporidia). The most commonly introduced species was Metarhizium anisopliae (Metschnikoff) Sorokin, with 13 introductions, followed by Entomophaga maimaiga Humber, Shimazu & Soper, which was released seven times. The majority of introduction programs have focused on controlling invasive species of insects or mites (70.7%) rather than on native hosts (29.4%). Almost half of the introductions of traditional fungi targeted species of Hemiptera and 75% of the microsporidia introduced have been introduced against lepidopteran species. The United States was the country where most introductions of fungi took place (n = 24). From 1993 to 2007, no arthropod pathogens were released in the US due to the rigorous regulatory structure, but in 2008 two species of microsporidia were introduced against the gypsy moth, Lymantria dispar (L.). Establishment of entomopathogenic fungi in programs introducing traditional fungi was 32.1% and establishment was 50.0% for programs introducing microsporidia. In some programs, releases have resulted in permanent successful establishment with no non-target effects. In summary, classical biological control using fungal entomopathogens can provide a successful and environmentally friendly avenue for controlling arthropod pests, including the increasing numbers of invasive non-native species.

Impact of natural epizootics of the fungal pathogen Neozygites floridana (Zygomycetes: Entomophthorales) on population dynamics of Tetranychus evansi (Acari: Tetranychidae) in tomato and nightshade

The tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae) was recently introduced in Africa and Europe, where there is an increasing interest in using natural enemies to control this pest on solanaceous crops. Two promising candidates for the control of T. evansi were identified in South America, the fungal pathogen, Neozygites floridana and the predatory mite Phytoseiulus longipes. In this study, population dynamics of T. evansi and its natural enemies together with the influence of environmental conditions on these organisms were evaluated during four crop cycles in the field and in a protected environment on nightshade and tomato plants with and without application of chemical pesticides. N. floridana was the only natural enemy found associated with T. evansi in the four crop cycles under protected environment but only in the last crop cycle in the field. In the treatments where the fungus appeared, reduction of mite populations was drastic. N. floridana appeared in tomato plants even when the population density of T. evansi was relatively low (less than 10 mites/3.14 cm(2) of leaf area) and even at this low population density, the fungus maintained infection rates greater than 50%. The application of pesticides directly affected the fungus by delaying epizootic initiation and contributing to lower infection rates than unsprayed treatments. Rainfalls did not have an apparent impact on mite populations. These results indicate that the pathogenic fungus, N. floridana can play a significant role in the population dynamics of T. evansi, especially under protected environment, and has the potential to control this pest in classical biological control programs. (C) 2009 Elsevier Inc. All rights reserved.

The effect of host plants on Tetranychus evansi, Tetranychus urticae (Acari: Tetranychidae) and on their fungal pathogen Neozygites floridana (Entomophthorales: Neozygitaceae)

In a series of tritrophic-level interaction experiments, the effect of selected host plants of the spider mites, Tetranychus evansi and Tetranychus urticae, on Neozygites floridana was studied by evaluating the attachment of capilliconidia, presence of hyphal bodies in the infected mites, mortality from fungal infection, mummification and sporulation from fungus-killed mite cadavers. Host plants tested for T. evansi were tomato, cherry tomato, eggplant, nightshade, and pepper while host plants tested for T. urticae were strawberry, jack bean, cotton and Gerbera. Oviposition rate of the mites on each plant was determined to infer host plant suitability while host-switching determined antibiosis effect on fungal activity. T. evansi had a high oviposition on eggplant, tomato and nightshade but not on cherry tomato and pepper. T. urticae on jack bean resulted in a higher oviposition than on strawberry, cotton and Gerbera. Attachment of capilliconidia to the T. evansi body, presence of hyphal bodies in infected T. evansi and mortality from fungal infection were significantly higher on pepper, nightshade and tomato. The highest level of T. evansi mummification was observed on tomato. T. evansi cadavers from tomato and eggplant produced more primary conidia than those from cherry tomato, nightshade and pepper. Switching N. floridana infected T. evansi from one of five Solanaceous host plants to tomato had no prominent effect on N. floridana performance. For T. urticae, strawberry and jack bean provided the best N. floridana performance when considering all measured parameters. Strawberry also had the highest primary conidia production. This study shows that performance of N. floridana can vary with host plants and may be an important factor for the development of N. floridana epizootics. (C) 2011 Elsevier Inc. All rights reserved.

Postharvest diseases in citrus and characterization of the fungal population in Sao Paulo`s wholesale market

The purposes of this workwere to characterize postharvest injuries and to evaluate the physicochemical characteristics of`Nra` and `Lima`oranges and `Murcott` tangor at Ceagesp market, as well as to characterize the environmental mycoflora in retail points at Ceagesp in 2006. Fruits collected at retail points were stored for 14 days at 25 degrees C and 85-90% RH. The incidence of injuries was visually evaluated every three days. The physicochemical characteristics analyzed were titratable acidity and soluble solids amount. The environmental mycoflora was sampled according to the gravimetric method, using Petri dishes containing potato-dextrose-agar medium+pentabiotic opened for two minutes. The average rot incidences in `Pera` and `Lima` oranges and `Murcott` tangor were 12.8, 14.9 and 25.8%, respectively, at the end of the storage period, and green mold was the main postharvest disease. Associations between physicochemical parameters and rot incidence was, in general, not significant. The environmental fungal population varied significantly between the sampling months in retail points with an average of 25.3 cfu/plate. Penicillium and Cladosporium were the most recorded genera of fungi. Positive correlation (r=0.96) was observed between frequency of P digitatum found in the environment of retail points and the green mold in on-sale fruits of `Pera` orange. However, for `Lima` orange and `Murcott` tangor such a correlation was not verified.

Characterization of fungal soil communities by F-RISA and arbuscular mycorrhizal fungi from Araucaria angustifolia forest soils after replanting and wildfire disturbances

The Fungal Ribosomal Intergenic Spacer Analysis (F-RISA) was used to characterize soil fungal communities from three ecosystems of Araucaria angustifolia from Brazil: a native forest and two replanted forest ecosystems, one of them with a past history of wildfire. The arbuscular mycorrhizal fungi (AMF) infection was evaluated in Araucaria roots of 18-month-old axenic plants previously inoculated with soils collected from those areas in a greenhouse experiment. The principal component analysis of F-RISA profiles showed different soil fungal community between the three studied areas. Sixty three percent of F-RISA fragments amplified in the soil and the substrate samples presented lengths between 500 and 700 bp. The number of Operational Taxonomic Units (OTUs) was 34 for soil and 38 for substrate, however, more fragments were detected in soil (214) than in substrate (163). An in silico F-RISA analysis to compare our data with ITS1-5.8S-ITS2 sequences from NCBI database showed the presence of Ascomycota, Basidiomycota and Glomeromycota among the soil and substrate fungal communities. AMF infection was higher in plants inoculated with soil from the native forest and the replanted forest with wildfire, both presenting similar chemical characteristics but with different disturbance levels. These results indicate that soil chemical composition may influence the soil fungal community structures rather than the anthropogenic or fire disturbances.

Restoration of Pattern Recognition Receptor Costimulation to Treat Chromoblastomycosis, a Chronic Fungal Infection of the Skin

Chromoblastomycosis is a chronic skin infection caused by the fungus Fonsecaea pedrosoi. Exploring the reasons underlying the chronic nature of F. pedrosoi infection in a murine model of chromoblastomycosis, we find that chronicity develops due to a lack of pattern recognition receptor (PRR) costimulation. F. pedrosoi was recognized primarily by C-type lectin receptors (CLRs), but not by Toll-like receptors (TLRs), which resulted in the defective induction of proinflammatory cytokines. Inflammatory responses to F. pedrosoi could be reinstated by TLR costimulation, but also required the CLR Mincle and signaling via the Syk/CARD9 pathway. Importantly, exogenously administering TLR ligands helped clear F. pedrosoi infection in vivo. These results demonstrate how a failure in innate recognition can result in chronic infection, highlight the importance of coordinated PRR signaling, and provide proof of the principle that exogenously applied PRR agonists can be used therapeutically.