Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Evaluation of experimental cleanser solution of Ricinus communis: effect on soft denture liner properties

Objective: This study evaluated colour stability, hardness and roughness of soft denture liners after immersion in various cleansers. Materials and methods: Thirty specimens (14 mm x 4 mm) of Elite Soft Relining (ES) and Mucopren Soft (MS) were randomly immersed in distilled water at 37 degrees C, sodium hypochlorite 1%, and an experimental Ricinus communis solution (RC) for 7, 15 and 183 continuous days. Results: ANOVA (p < 0.05) and Tukey's test indicated that after T7 (mu =8.79 +/- 7.36); T15 (mu = 4.23 +/- 2.62) and T183 (mu = 8.78 +/- 3.16), MS presented a higher increase in hardness than ES. After T7, MS underwent an increase in roughness (mu = 0.09 +/- 0.80); ES underwent a decrease (mu = -0.08 +/- 0.16). RC caused the smallest variation in roughness. After T15, both materials presented an increase in roughness. After T183, ES (mu = -0.30 +/- 0.48) presented a higher roughness variation than MS (mu = -0.07 +/- 0.32). Hypochlorite caused an increase in roughness (mu = 0.02 +/- 0.19). Conclusion: After all periods ES presented higher colour alteration than MS; highest colour alteration was caused by hypochlorite. Both materials were more stable after immersion in RC.

Spray drying microencapsulation of Lippia sidoides extracts in carbohydrate blends

The microencapsulation of Lippia sidoides extracts in blends of carbohydrates was investigated. The extraction conditions were determined through a 2(2) factorial design. The effects of the plant:solvent ratio (A - 7.5:100 and 15:100 m/m) and the extraction time (B - 30 and 90 min) on thymol content of extractive solutions were evaluated, using a 2:1 (v/v) of ethanol:water at a temperature of 50 degrees C, as a solvent system. The selected extract was subjected to spray drying. Blends of maltodextrin and gum arabic at different proportions (4:1; 3:2; 2:3; 0:1) (m/m) were used as encapsulating material. The protective effects of the maltodextrin and gum arabic blends were evaluated by determination of the thymol retention in the dried product, which ranged from 70.2 to 84.2% (related to the content in the extractive solution). An increase in the gum arabic to maltodextrin (DE10) ratio has positive effect on thymol retention. L. sidoides extracts and spray-dried products showed antifungal activity against tested fungal strains (Candida albicans - ATCC 64548, Candida glabrata - ATCC 90030, Candida krusei - ATCC 6258, and Candida parapsilosis - ATCC 22019), evidencing their potential as a natural antifungal agent for medicinal, food, and cosmeceutical purposes. (C) 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Activation pattern of neutrophils from blood of elderly individuals with -related denture stomatitis

We have identified impaired neutrophils in elderly individuals which could be involved with -related denture stomatitis (DS), an oral infection predominantly caused by , affecting especially elderly individuals using dental prosthesis. However, specific mechanisms performed by neutrophil contributing to the susceptibility of the elderly to DS are not fully understood. This study evaluated activation features of blood neutrophils from elderly and young individuals with DS. Blood neutrophils cultured with . from elderly subjects secreted decreased levels of CXCL8. However, . challenged-neutrophils from DS patients produced high IL-4 and IL-10, and low GM-CSF levels, regardless of age. Additional elastase activity of neutrophils from both elderly groups was detected after incubation with . , but only neutrophils from elderly DS demonstrated high myeloperoxidase activity. Therefore, DS patients have affected neutrophils, and the advance of age intensifies these damages. In sumamry, individuals with -related denture stomatitis presented variation in the neutrophil phenotype and activation. Such alterations were more intense in neutrophils from infected elderly individuals.

COMPARISON OF PRIMERS FOR RAPD-PCR FROM ENVIRONMENTAL ISOLATES OF CRYPTOCOCCUS NEOFORMANS, CRYPTOCOCCUS ALBIDUS AND CRYPTOCOCCUS LAURENTII COMPLEX

Various organisms have been characterized by molecular methods, including fungi of the genus Cryptococcus. The purposes of this study were: to determine the discriminatory potential of the RAPD (Random Amplified Polymorphic DNA) primers, the pattern of similarity of the Cryptococcus species, and discuss their useful application in epidemiological studies. We analyzed 10 isolates of each specie/group: C. albidus, C. laurentii complex, C. neoformans var. grubii, all from environmental source, and two ATCC strains, C. neoformans var. grubii ATCC 90112, and C. neoformans var. neoformans ATCC 28957 by RAPD-PCR using the primers CAV1, CAV2, ZAP19, ZAP20, OPB11 and SEQ6. The primers showed a good discriminatory power, revealing important differences between them and between species; the SEQ6 primer discriminated a larger number of isolates of three species. Isolates of C. laurentii showed greater genetic diversity than other species revealed by all six primers. Isolates of C. neoformans were more homogeneous. Only the primer CAV2 showed no amplification of DNA bands for C. albidus. It was concluded that the use of limited number of carefully selected primers allowed the discrimination of different isolates, and some primers (e. g., CAV2 for C. albidus) may not to be applied to some species.

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis

Background: Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. Candida albicans is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution. Results: Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-alpha, IFN-gamma and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mice Conclusions: Gomesin proved to be effective against experimental Candida albicans infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin's mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.

Microbiological quality assessment of sand and water from three selected beaches of South Coast, Sao Paulo State, Brazil

This study aimed to assess the sanitary quality of water, and wet and dry sand from three beaches located in the South Coast region of Sao Paulo State, Brazil, selected taking into account the frequency of tourists and the water quality (good, fair and poor). Thirty-six water samples each of wet and dry sand and seawater were collected monthly over a period of one year and analyzed for fecal indicator bacteria (FIB: thermotolerant coliforms, Escherichia coli, and enterococci), presumptive Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and dermatophytes. The results revealed FIB concentrations more elevated in dry sand followed by wet sand and water. P. aeruginosa and presumptive S. aureus were detected with a similar frequency in water and sand samples, but maximum concentrations and geometric means were higher in dry sand. C. albicans was detected only in water samples whereas the dermatophyte Microsporum sp. was isolated exclusively from dry and wet sand samples. This evaluation showed also that the environment had a significant influence on P. aeruginosa but not on presumptive S. aureus concentrations. According to threshold values proposed in the literature for E. coli and enterococci dry sand densities, none of the beaches would be considered of sufficient quality for recreational activities.

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T>G and c.476 G>C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

Epidemiological and clinical characteristics of nosocomial candidiasis in university hospitals in Cuiaba - Mato Grosso, Brazil

Background: Fungal infections are emerging as an important cause of human disease, especially among hospitalized patients with serious underlying disease and several risk factors. Aims: To evaluate epidemiological and clinical characteristics of patients with nosocomial candidiasis in university hospitals in Cuiaba - MT, Brazil. Methods: A descriptive study of 91 patients admitted to university hospitals in Cuiaba - MT, with clinical and laboratory diagnosis of nosocomial candidiasis, over a 20-month period. Results: A rate for nosocomial infections by Candida spp. of 5 per 1000 admissions, proportional mortality of 14.4% and lethality of 53.8% were determined. The patient age ranged from 29 days to 82 years-old, among which, 74.7% were adults and 25.3% children. The intensive care units contributed with the highest number of cases of infection by Candida spp. (69.2%). The most important underlying disease was gastrointestinal tract disease (11%). Prematurity and low birth weight were the most important risk factors among newborns. The use of antibiotics, invasive procedures, H-2 blockers, multiple blood transfusions and stay length of >= 21 days were the most frequent risk factors among adults. Candida albicans was the most common species in all cases. Conclusions: In this study, C. albicans was the most frequently detected species in candidiasis and risk factors increased the susceptibility of hospitalized patients to acquiring a nosocomial infection by Candida spp. (C) 2011 Revista Iberoamericana de Micologia. Published by Elsevier Espana, S.L. All rights reserved.

Antimicrobial Properties and Cytotoxicity of an Antimicrobial Monomer for Application in Prosthodontics

Purpose: This study aimed to investigate the antimicrobial properties and cytotoxicity of the monomer methacryloyloxyundecylpyridinium bromide (MUPB), an antiseptic agent capable of copolymerizing with denture base acrylic resins. Materials and Methods: The antimicrobial activity of MUPB was tested against the species Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus, and Streptococcus mutans. The minimum inhibitory and fungicidal/bactericidal concentrations (MIC, MFC/MBC) of MUPB were determined by serial dilutions in comparison with cetylpyridinium chloride (CPC). The cytotoxic effects of MUPB at concentrations ranging from 0.01 to 1 g/L were assessed by MTT test on L929 cells and compared with methyl methacrylate (MMA). The antimicrobial activity of copolymerized MUPB was tested by means of acrylic resin specimens containing three concentrations of the monomer (0, 0.3, 0.6% w/w). Activity was quantified by means of a disc diffusion test and a quantification of adhered planktonic cells. Statistical analysis employed the Mann-Whitney test for MIC and MFC/MBC, and ANOVA for the microbial adherence test (a= 0.05). Results: MUBP presented lower MIC values when compared with CPC, although differences were significant for C. dubliniensis and S. mutans only (p= 0.046 and 0.043, respectively). MFC/MBC values were similar for all species except C. albicans; in that case, MUPB presented significantly higher values (p= 0.046). MUPB presented higher cytotoxicity than MMA for all tested concentrations (p < 0.001) except at 0.01 g/L. Irrespective of the concentration incorporated and species, there was no inhibition halo around the specimens. The incorporation of MUPB influenced the adhesion of C. albicans only (p= 0.003), with lower CFU counts for the 0.6% group. Conclusions: It was concluded that non-polymerized MUPB has an antimicrobial capacity close to that of CPC and high cytotoxicity when compared with MMA. The antimicrobial activity of MUPB after incorporation within a denture base acrylic resin did not depend on its elution, but was shown to be restricted to C. albicans.

Effect of Chlorhexidine on Denture Biofilm Accumulation

Purpose: Adequate denture hygiene can prevent and treat infection in edentulous patients, who are frequently elderly and have difficulty brushing their teeth. This study evaluated the efficacy of complete denture biofilm removal using a chlorhexidine solution in two concentrations: 0.12% and 2.0%. Materials and Methods: Sixty complete denture wearers participated in a trial for 21 days after receiving brushing instructions. They were distributed into three groups, according to the tested solution and regimen (n = 20): (G1) Control (daily overnight soaking in water); (G2) daily immersion at home in 0.12% chlorhexidine for 20 minutes after dinner; and (G3) a single immersion in 2.0% chlorhexidine for 5 minutes at the end of the experimental period, performed by a professional. Biofilm coverage area (%) was quantified on the internal surface of maxillary dentures at baseline and after 21 days. Afterward, the differences between initial and posttreatment results were compared by means of the Kruskal-Wallis test (a = 0.05). Results: Median values for biofilm coverage area after treatment were: (G1) 36.0%; (G2) 5.3%; and (G3) 1.4%. Differences were significant (KW = 35.25; p < 0.001), although G2 and G3 presented similar efficacy in terms of biofilm removal. Conclusions: Both chlorhexidine-based treatments had a similar ability to remove denture biofilm. Immersion in 0.12% or 2.0% chlorhexidine solutions can be used as an auxiliary method for cleaning complete dentures.