Cytotoxic effects of white-MTA and MTA-Bio cements on odontoblast-like cells (MDPC-23)

This study evaluated the cytotoxic effects of 2 mineral trioxide aggregate (MTA) cements - White-MTA-Angelus and a new formulation, MTA-Bio - on odontoblast-like cell (MDPC-23) cultures. Twenty-four disc-shaped (2 mm diameter x 2 mm thick) specimens were fabricated from each material and immersed individually in wells containing 1 mL of DMEM culture medium for either 24 h or 7 days to obtain extracts, giving rise to 4 groups of 12 specimens each: G1 - White-MTA/24 h; G2 - White-MTA/7 days; G3 - MTA-Bio/24 h; and G4 - MTA-Bio/7 days. Plain culture medium (DMEM) was used as a negative control (G5). Cells at 30,000 cells/cm 2 concentration were seeded in the wells of 24-well plates and incubated in a humidified incubator with 5% CO 2 and 95% air at 37°C for 72 h. After this period, the culture medium of each well was replaced by 1 mL of extract (or plain DMEM in the control group) and the cells were incubated for additional 2 h. Cell metabolism was evaluated by the MTT assay and the data were analyzed statistically by ANOVA and Tukey's test (α=0.05). Cell morphology and the surface of representative MTA specimens of each group were examined by scanning electron microscopy. There was no statistically significant difference (p>0.05) between G1 and G2 or between G3 and G4. No significant difference (p>0.05) was found between the experimental and control groups either. Similar cell organization and morphology were observed in all groups, regardless of the storage periods. However, the number of cells observed in the experimental groups decreased compared to the control group. MTA-Bio presented irregular surface with more porosities than White-MTA. In conclusion, White-MTA and MTA-Bio presented low cytotoxic effects on odontoblast-like cell (MDPC-23) cultures.

Isolation and identification of black yeasts by enrichment on atmospheres of monoaromatic hydrocarbons

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species. © 2010 The Author(s).

Algebraic approach to optimal clone selection applied in metagenomic projects

Due to the wide diversity of unknown organisms in the environment, 99% of them cannot be grown in traditional culture medium in laboratories. Therefore, metagenomics projects are proposed to study microbial communities present in the environment, from molecular techniques, especially the sequencing. Thereby, for the coming years it is expected an accumulation of sequences produced by these projects. Thus, the sequences produced by genomics and metagenomics projects present several challenges for the treatment, storing and analysis such as: the search for clones containing genes of interest. This work presents the OCI Metagenomics, which allows defines and manages dynamically the rules of clone selection in metagenomic libraries, thought an algebraic approach based on process algebra. Furthermore, a web interface was developed to allow researchers to easily create and execute their own rules to select clones in genomic sequence database. This software has been tested in metagenomic cosmid library and it was able to select clones containing genes of interest. Copyright 2010 ACM.

Solubilization of iron phosphate by free or immobilized spores and pellets of Aspergillus niger

The production of pellets by Aspergillus niger and the FePO 4 (FeP) solubilization through the use of free or immobilized spores and pellets were studied. The media Sabouraud, MS-FeP, MS-K 2HPO 4, Czapek and Malt were used for pellets yield. Enhanced growth and pellets production were found in the Sabouraud and MS-FeP than those produced by the other media. Pellets produced in Sabouraud were larger in size and formed well-defined spheres than those produced in MS-FeP. Pellets amounts varying from 2 to 8 mg mL, -1 were inoculated in the MS-FeP medium. The greatest quantity of solubilized FeP was found when 6 mg of pellets mL, -1 were used. While the free spores were the worst form used for FeP solubilization in culture medium, free pellets allowed for the production of the greatest quantities of soluble phosphate, even after repeated use of the pellets. In the soil, pellets solubilized similar quantities of FeP compared to the immobilized A. niger spores and can be used with advantage since they are easily produced. © 2010 Academic Journals Inc.

Valor preditivo do potencial de implantação embrionário pela análise do perfil químico de meios de cultivo por espectrometria de massas (ESI-Q-ToF)

Objective: This case-control study analyzed mass spectrometry fingerprinting patterns of culture media samples used for embryo culture to predict embryo implantation. Methods: The culture medium harvested after embryo transfer of 22 embryos from 13 patients was used for the experiments. After embryo transfer, the remaining culture media were collected and samples were split in positive (n=8) and negative (n=14) implantation groups according to implantation outcomes (100% or 0% of implantation). Samples were individually diluted and injected directly to the Electrospray ionization (ESI) MS coupled to a Quadrupole Time-of-flight MS (Q-ToF-MS).Ions relative intensities of each spectrum were considered. Data analysis was conducted in MatLab 7.0 version using Partial Least Squares - Discriminant Analysis toolbox. Results: There were 3027 observed ions at 100% and 0% implantation groups by ESI-Q-ToF-MS. The statistical model could categorize the samples in two clusters, based on their positive and negative implantation outcomes. Less intense ions present in the mass spectra with statistical significance have contributed to the major differences to group distinction. Conclusions: Positive and negative implantation embryos showed a specific biochemical pattern present in culture media, which could be detected as a fast, simple and non-invasive way. This biochemical profile could help the selection of the most viable embryo, improving single embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Influence of TNF-α blockers on the oral prevalence of opportunistic microorganisms in ankylosing spondylitis patients

Objectives: To compare the oral prevalence and antimicrobial susceptibility of Candida spp., staphylococci, enterobacteriaceae, and pseudomonas spp.from ankylosing spondylitis (AS) patients receiving conventional and anti-TNF-α therapy. Methods: The study included 70 AS patients, diagnosed according to the modified New York criteria (1984). The volunteers were divided into 2 groups: a biological group (AS BioG) (n=35) (on anti-TNF-α therapy) and a conventional group (AS ConvG) (n=35). The control group (ContG) (n=70) was made up of healthy individuals matched for age, gender, and oral conditions. After clinical examination, oral rinse samples were collected and plated in specific culture media. The number of colony-forming units per milliliter (cfu/ml) was obtained, and isolates were identified using the API system. Antimicrobial susceptibility tests were performed according to the NCCLS guidelines. Prevalence and counts of microorganisms were statistically compared between the 3 groups, using the Mann-Whitney and Chi-square tests. Significance level was set at 5%. Results: In both the AS BioG and the AS ConvG, staphylococci counts were higher than that in the ContG (p<0.0001). Candida albicans and staphylococcus epidermidis were the most commonly found species in all the groups. Serratia marcescens and klebsiella oxytoca were more prevalent in the AS BioG and the AS ConvG, respectively. Two Candida isolates (2.8%) from the AS BioG and 5 (10.8%) from the AS ConvG were resistant to amphotericin B and 5-fluorocytosine. A low percentage of staphylococci isolates was resistant to amoxicillin, ciprofloxacin, and doxycycline. Conclusion: Higher counts of staphylococci were observed in both AS groups, regardless of the current therapy, age, sex, and oral conditions. Anti-TNF-α therapy could not be correlated with increased counts of microorganisms. © Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2012.

Wine aroma improvement using a β-glucosidase preparation from aureobasidium pullulans

Microbial β-glucosidases have been used for the enhancement of wine aroma. Nevertheless, few enzymes are active in the conditions of winemaking. In this work, the production of a β-glucosidase by an Aureobasidium pullulans strain (Ap-β-gl) isolated from grape ecosystems was evaluated. The maximum enzymatic synthesis using submerged fermentation was after 96 h of growth in complex media containing 20 g/L of cellobiose as the sole carbon source. The crude enzyme (Ap-β-gl) showed optimal pH at 5.5 and two peaks of optimum temperature (at 45 and 70 C). It showed a wide range of pH stability, stability at low temperatures, and tolerance to ethanol, showing suitable characteristics for winemaking conditions. The hydrolysis of glycosidic terpenes by Ap-β-gl was studied, and its ability to efficiently release free terpenols was demonstrated by gas chromatography/mass spectrometry. The enzymatic treatment notably increased the amount of monoterpenes, showing good prospects for its potential application for the development of aroma in wines. © 2012 Springer Science+Business Media New York.

Characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis

Tannases have attracted wider attention because of their biotechnological potential, especially enzymes from filamentous fungi and other microorganisms. However, the biodiversity of these microorganisms has been poorly explored, and few strains were identified for tannase production and characterization. This article describes the production, purification and characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis. High enzymatic levels were obtained in Khanna medium containing tannic acid up to 72 h at 30 °C under 100 rpm. The purified enzyme with 65% of carbohydrate content had an apparent native molecular mass of 218 kDa with subunits of 120 kDa and 93 kDa and was stable at 50 °C for 1 h. Optima of temperature and pH were 60 °C and 5.0-6.5, respectively. The enzyme was not affected significantly by most ions, detergents and organic solvents. While glucose did not affect the tannase activity, the addition of a high concentration of gallic acid did. The Km values were 1.7 mM (tannic acid), 14.3 mM (methyl-gallate) and 0.6 mM (propyl-gallate). The enzyme was able to catalyze the transesterification reaction to produce propyl-gallate. All biochemical properties suggest the biotechnological potential of the glucose- and solvent-tolerant tannase from A. phoenicis. © 2012 Elsevier B.V. All rights reserved.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Recovery of mutans streptococci on MSB, SB-20 and SB-20M agar media

Objective: The recovery of mutans streptococci in saliva and dental biofilm samples depends, in part, on the culture medium used. In this study, we compared (i) the culture media Sucrose-Bacitracin agar (SB-20), Modified SB-20 (SB-20M) and Mitis Salivarius Bacitracin agar (MSB) in the count of colony forming units (cfu) of mutans streptococci and (ii) in the morphological and biochemical differentiation between Streptococcus mutans and Streptococcus sobrinus. Design: Samples of non-stimulated saliva from 20 children were plated on SB-20, SB-20M and MSB, and incubated in microaerophilia at 37 °C for 72 h. Identification of microorganisms was based on analysis of colony morphology under stereomicroscopy. The biochemical identification of colonies was done by biochemical tests using sugar fermentation, resistance to bacitracin and hydrogen peroxide production. Results: There was no significant difference (p > 0.05) in the number of cfu of mutans streptococci recovered on SB-20 and SB-20M agar. Comparing the media, SB-20 and SB-20M yielded a larger number of mutans streptococci colonies (p < 0.05) and were more effective than MSB in the identification of S. sobrinus (p < 0.05), but not of S. mutans (p > 0.05). Conclusion: There was no significant difference between SB-20 and SB-20M culture media in the count of mutans streptococci, demonstrating that the replacement of sucrose by coarse granular cane sugar did not alter the efficacy of the medium. Compared with MSB, SB-20 and SB-20M allowed counting a larger number of mutans streptococci colonies and a more effective morphological identification of S. sobrinus. © 2012 Elsevier Ltd.

Effect of fluoride-treated enamel on indirect cytotoxicity of a16% carbamide peroxide bleaching gel to pulp cells

The aim of this study was to evaluate the possibility of fluoride solutions applied to enamel to protect pulp cells against the trans-enamel and transdentinal cytotoxicity of a 16% carbamide peroxide (CP) bleaching gel. The CP gel was applied to enamel/ dentin discs adapted to artificial pulp chambers (8 h/day) during 1, 7 or 14 days, followed by fluoride (0.05% or 0.2%) application for 1 min. The extracts (culture medium in contact with dentin) were applied to MDPC-23 cells for 1 h, and cell metabolism (MTT assay), alkaline phosphatase (ALP) activity and cell membrane damage (flow cytometry) were analyzed. Knoop microhardness of enamel was also evaluated. Data were analyzed statistically by ANOVA and Kruskal-Wallis tests (a=0.05). For the MTT assay and ALP activity, significant reductions between the control and the bleached groups were observed (p<0.05). No statistically significant difference occurred among bleached groups (p>0.05), regardless of fluoride application or treatment days. Flow cytometry analysis demonstrated 30% of cell membrane damage in all bleached groups. After 14 days of treatment, the fluoride-treated enamel presented significantly higher microhardness values than the bleached-only group (p<0.05). It was concluded that, regardless of the increase in enamel hardness due to the application of fluoride solutions, the treated enamel surface did not prevent the toxic effects caused by the 16% CP gel to odontoblast-like cells.

In vitro antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecalis in root canals

Objective: The aim of this study was to evaluate the antimicrobial activity of auxiliary chemical substances and natural extracts on Candida albicans and Enterococcus faecali's inoculated in root canals. Material and Methods: Seventy-two human tooth roots were contaminated with C. albicans and E. faecalis for 21 days. The groups were divided according to the auxiliary chemical substance into: G1) 2.5% sodium hypochlorite (NaOCl), G2) 2% chlorhexidine gel (CHX), G3) castor oil, G4) glycolic Aloe vera extract, g5) glycolic ginger extract, and G6) sterile saline (control). The samples of the root canal were collected at different intervals: confirmation collection, at 21 days after contamination; 1st collection, after instrumentation; and 2nd collection, seven days after instrumentation. Microbiological samples were grown in culture medium and incubated at 37°C for 48 hours. Results: The results were submitted to the Kruskal-Wallis and Dunn (5%) statistical tests. NaOCl and CHX completely eliminated the microorganisms of the root canals. Castor oil and ginger significantly reduced the number of CFU of the tested bacteria. Reduction of CFU/mL at the 1st and 2nd collections for groups G1, G2, G3 and G4 was greater in comparison to groups G5 and G6. Conclusion: It was concluded that 2.5% sodium hypochlorite and 2% chlorhexidine gel were more effective in eliminating C. albicans and E. faecalis, followed by the castor oil and glycolic ginger extract. The Aloe vera extract showed no antimicrobial activity.

Concentrações de sacarose no desenvolvimento in vitro e na aclimatização de Cattleya loddigesii Lindley

This work aimed to study the influence of sucrose in the culture medium for in vitro growth and acclimatization of the epiphytic orchid Cattleya loddigesii. Five sucrose treatments (absence, 10, 20, 30 and 40g L-1) were used in a randomic experimental design. Mature seeds were sowed in 1/2 MS culture medium and after 90 days the plantlets (1.0 +/-0.2 cm) were inoculated between the treatments, whereby they were remained more 90 days. After 180 days of the beginning of the experiment the plantlets were removed from the flasks and evaluated the number of roots, shoot length, number of leafs, total dry weight and photosynthetic pigments. Survival percentage was evaluated after 75 days of acclimatization. The data of biometric variables were analyzed by Anovaand polynomial regression (p<0.05). Theothers data were submitted to the Anova and the means compared by the Tukey test (p<0.05). The sucrose concentration of 20g L-1 favored the in vitro growth in all evaluated parameters, showed higher production of chlorophyll a, total chlorophyll and carotenoids, in addition to increased survival under ex vitro condition. The sucrose concentration of 20g L-1 in the culture medium was the most efficient among the tested concentrations both for in vitro growth and ex vitro establishment of Cattleya loddigesii.

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth.

Effect of low-level laser therapy on odontoblast-like cells exposed to bleaching agent

The aim of the present study was to evaluate the effect of low-level laser therapy (LLLT) on odontoblast-like MDPC-23 cells exposed to carbamide peroxide (CP 0.01 %-2.21 μg/mL of H2O2). The cells were seeded in sterile 24-well plates for 72 h. Eight groups were established according to the exposure or not to the bleaching agents and the laser energy doses tested (0, 4, 10, and 15 J/cm2). After exposing the cells to 0.01 % CP for 1 h, this bleaching solution was replaced by fresh culture medium. The cells were then irradiated (three sections) with a near-infrared diode laser (InGaAsP-780 ± 3 nm, 40 mW), with intervals of 24 h. The 0.01 % CP solution caused statistically significant reductions in cell metabolism and alkaline phosphate (ALP) activity when compared with those of the groups not exposed to the bleaching agent. The LLLT did not modulate cell metabolism; however, the dose of 4 J/cm2 increased the ALP activity. It was concluded that 0.01 % CP reduces the MDPC-23 cell metabolism and ALP activity. The LLLT in the parameters tested did not influence the cell metabolism of the cultured cells; nevertheless, the laser dose of 4 J/cm2 increases the ALP activity in groups both with and without exposure to the bleaching agent. © 2013 Springer-Verlag London.