Rendering Banana Plant Residues into a Potentially Commercial Byproduct by Doping Cellulose Films with Phenolic Compounds.

This study seeks to render residues from banana plants into a useful byproduct with possible applications in wound dressings and food packaging. Films based on cellulose extracted from banana plant pseudostem and doped with phenolic compounds extracted from banana plant leaves were developed. The phenolic compounds were extracted using batch solid-liquid and Soxhlet methods, with different drying temperatures and periods of time. The total phenolic content and antioxidant activity were quantified. The optimum values were obtained using a three-day period batch-solid extraction at 40 °C (791.74 ± 43.75 mg/L). SEM analysis indicates that the pseudostem (PS) films have a porous structure, as opposed to hydroxyethyl cellulose (HEC) films which presented a homogeneous and dense surface. Mechanical properties confirmed the poor robustness of PS films. By contrast HEC films manifested improved tensile strength at low levels of water activity. FTIR spectroscopy reinforced the need to improve the cellulose extraction process, the success of lignin and hemicellulose removal, and the presence of phenolic compounds. XRD, TGA and contact angle analysis showed similar results for both films, with an amorphous structure, thermal stability and hydrophilic behavior.

Expression levels of the receptor activator of NF-κB ligand and osteoprotegerin and the number of gram-negative bacteria in symptomatic and asymptomatic periapical lesions.

OBJECTIVE: The study aimed to verify the potential correlation between the detected amount of gram-negative bacteria and the radiographic sizes of the lesions in patients with symptomatic and asymptomatic apical periodontitis. Furthermore, to evaluate whether the expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) and the RANKL/OPG ratio are differentially regulated in both groups. DESIGN: Twenty patients with periapical lesions were divided into two groups: symptomatic (SYM) n=10 and asymptomatic (ASYM) n=10. After periapical surgery, the lesions were collected and processed for histological examination, and immunohistochemistry. The percentage of RANKL- and OPG-immunopositive areas relative to the total area of the microscopic field was calculated. For gram staining, the number of gram-negative cells per microscopic field was assessed. The radiographs of each patient were processed and measured. The Student's t-test and the Pearson correlation coefficient were performed. RESULTS: The SYM group showed a significantly higher number of gram-negative cells (p=0.007) when compared to the ASYM group. A higher number of gram-negative bacteria occurred more frequently in larger periapical lesions and the SYM group (p=0.03). The expression for RANKL and OPG and the RANKL/OPG ratio were not significantly different between the groups. There was a significant positive correlation between the number of bacteria and OPG levels in the SYM group (p=0.01). CONCLUSION: The number of bacteria seems to influence the symptoms and the radiographic size of a periapical lesion. Gram-negative bacteria may play an important role in OPG activity in the SYM group.

Evaluation of the potential of squash pumpkin by-products (seeds and shell) as sources of antioxidant and bioactive compounds.

The transformation of byproducts and wastes generated by agro-food companies is of high importance since only a small portion of plant material is utilized directly for human consumption. Squash pumpkin is greatly used in Portugal and as by-products of its processing are generated tons of shell and seeds. In this study we aim to evaluate the potential of these wastes as sources of beneficial and bioactive compounds (antioxidants and antimicrobials), studying the effect of different extraction solvents and drying methods. The samples (fresh and cooked) were freeze-dried and oven-dried followed by extraction with different solvents that revealed the following decreasing order of efficiency: 70 % ethanol, 70 % methanol, 70 % acetone, ultra-pure water and 100 % dichloromethane. The oven-dried samples showed higher values of antioxidant activity and phenolic content, with exception of the values of phenolics for the seeds material. The shell samples presented higher values (1.47 - 70.96 % inhibition) of antioxidant activity and total phenolic content (2.00 - 10.69 mg GAE/g DW). A positive correlation was found between these two parameters on the shell samples, however the squash seeds revealed a negative correlation between the phenolic content and the antioxidant activity. The results show that these industrial agro-food residues are potentially good sources of bioactive compounds with health benefits.

Screening of reducing agents for anaerobic growth of Candida albicans SC5314.

This study aimed to evaluate the influence of different redox potentials (Eh) on cell growth, whole-cell protein profile and cell surface hydrophobicity (CSH) of Candida albicans SC5314. The yeast was grown in YNB broth enriched with reducing (158mM sodium sulfite, 4mM sodium sulfite, 2.5mM sodium metabisulfite, 1.3mM 2-mercaptoethanol, 5.5mM thioglycolic acid, and 3.2mM l-cysteine hydrochloride) and oxidizing agents (15mM ammonium persulfate and 80mM potassium ferricyanide) and incubated in normoxic and anoxic atmospheres at 37°C, for 48h. Pre- and post-incubation Eh values were determined and cytoplasm proteins were extracted. Proteins were parted by SDS-PAGE and their profiles were compared. 3.2mM l-cysteine and 1.3mM 2-mercaptoethanol promoted and maintained negative Eh values during incubation. No differences were detected among SDS-PAGE profiles. CSH differences only were observed with 4mM sodium sulfite and 3.2mM l-cysteine. Results showed that 3.2mM l-cysteine is a reducing agent that allows maintenance of negative Eh in both anoxic and normoxic conditions and it seems not to interfere in the global expression of plasmatic proteins.

Proposal of protocols using D-glutamine to optimize the 2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay for indirect estimation of microbial loads in biofilms of medical importance.

Due to technical problems, biofilm biomasses are difficult to be precisely determined. One reliable strategy is based on the colorimetry of formazan compounds derived from tetrazolium salt reduction. XTT presents some desirable properties that make the biofilm measurements easier. However, cells entrapped within the extracellular matrixes normally do not metabolize the tetrazolium equally, leading to underestimation of cell contents. This study evaluated the effectiveness of D-glutamine, a plerotic substrate of tricarboxilic acid cycle (TAC), as inducer of XTT reduction. The metabolic activities of aerobic and anaerobic 48 h-old monospecific biofilms of Pseudomonas aeruginosa ATCC®27853™, Klebsiella pneumoniae ATCC®13883™, Staphylococcus epidermidis ATCC®12228™, Streptococcus mutans ATCC®25175™, and Candida albicans SC5314 were evaluated. Results showed that D-glutamine 50 mM (for P. aeruginosa, K. pneumoniae, and S. epidermidis) and 25 mM (for S. mutans and C. albicans) may enhance the detection of soluble formazan in a significant manner, what becomes the XTT reduction assay more robust.

Initial in vitro evaluations of the antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria.

AIMS: The aim of the study was to evaluate the in vitro antibacterial effects of glucosinolate hydrolysis products (GHP) against plant pathogenic micro-organisms namely Agrobacterium tumefaciens, Erwinia chrysanthemi, Pseudomonas cichorii, Pseudomonas tomato, Xanthomonas campestris and Xanthomonas juglandis. METHODS AND RESULTS: Using a disc diffusion assay, seven different doses of 10 GHP were tested against each bacteria. The results showed that the isothiocyanates were potent antibacterials, whilst the other GHP were much less efficient. Moreover, the antibacterial effects were dose-dependent, increasing with the dose applied; 2-phenylethylisothiocyanate and sulforaphane showed the strongest inhibitory effects. The overall results show a great potential for using the isothiocyanates as an alternative tool to control undesired bacterial growth in plants. CONCLUSIONS: Glucosinolate hydrolysis products and more specifically the isothiocyanates: benzylisothiocyanate, 2-phenylethylisothiocyanate, the isothiocyanate Mix and sulforaphane, were effective phytochemicals against the in vitro growth of the phytopathogenic bacteria. The antibacterial activity exhibited by these phytochemicals reinforces their potential as alternatives to the traditional chemical control of phytopathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This current in vitro study is the first providing comparative data on GHP as potential control agents for plant pathogenic bacteria. However, more studies are needed to determine their possible allelopathic impacts e.g. inhibition of plant growth and negative effects on beneficial soil bacteria and fungi (mycorrhizae).

The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract.

AIMS: The aim of the study was to evaluate the in vitro antibacterial activity of glucosinolates and their enzymatic hydrolysis product against bacteria isolated from the human intestinal tract. METHODS AND RESULTS: Using a disc diffusion bioassay, different doses of intact glucosinolates and their corresponding hydrolysis products were tested. There were clear structure-activity and concentration differences with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The most effective glucosinolate hydrolysis products were the isothiocyanates; sulforaphane and benzyl isothiocyanate were the best inhibitors of growth. Indole-3-carbinol had some inhibitory effects against the Gram-positive bacteria but had no effect, even at the highest dose, against the Gram-negative bacteria. Indole-3-acetonitrile had some inhibitory activity against the Gram-negative bacteria. Glucosinolates, nitriles and amines were ineffective at all the doses used. CONCLUSIONS: Glucosinolate hydrolysis products and specifically the isothiocyanates SFN and BITC have significant antimicrobial activity against Gram-positive and Gram-negative bacteria, and might be useful in controlling human pathogens through the diet. SIGNIFICANCE AND IMPACT OF THE STUDY: This the first major in vitro study demonstrating the potential of these natural dietary chemicals as an alternative to, or in combination with, current antibiotic-based therapies for treating infectious diseases.

Effect of chlorhexidine mouth rinse on Streptococci counts of tooth-tissue-borne palatal expander biofilm.

OBJECTIVES: To assess total Streptococci (TS) counts and biofilm mass over tooth-tissue-borne palatal expander (TTBPE), as well as the effect of chlorhexidine (CHX) mouth rinse on these variables. DESIGN: A cross-sectional study design employed clinical procedures and laboratorial techniques. SETTING AND SAMPLE POPULATION: Patients who had TTBPE removal indicated were divided into two groups: a CHX group (n = 26) in which three times a day of 0.2% CHX digluconate mouth rinses were prescribed 7 days before TTBPE removal; and a control (CON) group (n = 25) in which no antimicrobial treatment was applied. EXPERIMENTAL VARIABLE: 'Gender', 'Age', and 'TTBPE wear time' were recorded. After TTBPE removal, biofilm mass was determined by the difference between (TTBPE + biofilm) and (TTBPE only) masses. TS counts were determined by biofilm suspension followed by progressive dilutions and culture on Mitis Salivarius agar with incubation at 37 degrees C for 72 h. OUTCOME MEASURE: Biofilm mass (mg) and Colony Forming Units of TS / mg of biofilm (CFU-TS / mg) were calculated. Results - Total Streptococci mean values in CHX (6.77 x 10(6)CFU-TS / mg) were statistically lower (p < 0.01) than those in CON (3.82 x 10(7)CFU-TS / mg), but there was no statistical difference (p > 0.05) between CHX (168.88 mg) and CON (182.04 mg) masses nor statistical correlation (p > 0.05) between biofilm mass and CFU-TS / mg in the two groups. CONCLUSION: Chlorhexidine reduces the TS counts in TTBPE, but has no effect on biofilm mass.

Synthesis, anti-Toxoplasma gondii and antimicrobial activities of benzaldehyde 4-phenyl-3-thiosemicarbazones and 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids.

In the present communication, a new series of 2-[(phenylmethylene)hydrazono]-4-oxo-3-phenyl-5-thiazolidineacetic acids (2a-p) have been synthesized. Benzaldehyde 4-phenyl-3-thiosemicarbazones substituted (1a-p) were also obtained and used as intermediate to give the title compounds. All synthesized compounds were characterized by IR, (1)H and (13)C NMR. The in vitro anti-Toxoplasma gondii activity of 1a-p and 2a-p was evaluated. The 4-thiazolidinones (2a-p) were screened for their in vitro antimicrobial activity. For anti-Toxoplasma gondii activity, in general, all compounds promoted decreases in the percentage of infected cells leading to parasite elimination. These effects on intracellular parasites also caused a decrease in the mean number of tachyzoites. In addition, most of the 4-thiazolidinones showed more effective toxicity against intracellular parasites, with IC(50) values ranging from 0.05 to 1 mM. According to results of antimicrobial activity, compounds 2f, 2l, and 2p showed best activity against Mycobacterium luteus, 2c was more active against Mycobacterium tuberculosis, and 2g, 2l, and 2n showed same activity as nistatin (standard drug) against Candida sp. (4249).

Oral Candida albicans and Candida dubliniensis differentiation by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Five oral strains of Candida albicans and five C. dubliniensis, as well as their respective type-strains, were analyzed by multilocus enzyme electrophoresis (MLEE) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoreses and numerical analyses, we obtained two distinct species-specific taxa, which may justify the use of MLEE and SDS-PAGE as reliable methods for differentiation and complementary identification of C. dubliniensis.

DiferenciaciOn de Candida albicans y Candida dubliniensis por técnicas de electroforesis en geles de almidón y de poliacrilamida / Oral Candida albicans and Candida dubliniensis differentiatioin by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis

Cinco cepas de Candida albicans y otras cinco de C. dubliniensis así como sus cepas-patrón fueron evaluadas mediante el empleo de técnicas de electroforesis en geles de almidón y de poliacrilamida. Después de las electroforesis y evaluación numérica, se obtuvieron dos taxa muy distintos, que pueden justificar el uso de MLEE y SDS-PAGE como métodos confiables para la diferenciación e identificación complementaria de C.dubliniensis. (AU)

DiferenciaciÓn de Candida albicans y Candida dubliniensis por técnicas de electroforesis en geles de almidón y de poliacrilamida / Oral Candida albicans and Candida dubliniensis differentiatioin by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis

Cinco cepas de Candida albicans y otras cinco de C. dubliniensis así como sus cepas-patrón fueron evaluadas mediante el empleo de técnicas de electroforesis en geles de almidón y de poliacrilamida. Después de las electroforesis y evaluación numérica, se obtuvieron dos taxa muy distintos, que pueden justificar el uso de MLEE y SDS-PAGE como métodos confiables para la diferenciación e identificación complementaria de C.dubliniensis.

Oral Candida albicans and Candida dubliniensis differentiation by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Five oral strains of Candida albicans and five C. dubliniensis, as well as their respective type-strains, were analyzed by multilocus enzyme electrophoresis (MLEE) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoreses and numerical analyses, we obtained two distinct species-specific taxa, which may justify the use of MLEE and SDS-PAGE as reliable methods for differentiation and complementary identification of C. dubliniensis.

Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts.

The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

Candida spp. biotypes in the oral cavity of school children from different socioeconomic categories in Piracicaba-SP, Brazil.

Two hundred and thirty-nine (239) Brazilian children, distributed into five distinct socioeconomic categories (A to E) were studied. Saliva samples were analyzed as to flow rate, pH, buffer capacity and microbial parameters. The results revealed the presence of Candida spp. in 47.3% of the samples. The most commonly isolated species was C. albicans, in all socioeconomic categories, followed by C. tropicalis, C. krusei and C. parapsilosis. There was no statistical correlation between secretion rate, buffer capacity and Candida spp. CFU/ml. The prevalence of Candida spp. did not differ substantially among the groups; however the microorganisms were more detected in categories B and C. Among all species, C. albicans was the most prevalent. Only 5% of the sample presented more than one species--C. albicans associated with C. tropicalis, C. parapsilosis or C. krusei. It was possible to detect a significant correlation between caries indices and the socioeconomic categories. All categories presented increased caries indices; however the studied population was considered of low caries risk. There was no positive correlation between the presence of Candida and caries risk in the analyzed population.

Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts

The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly

Inter and intra-specific genetic variability of oral Candida species.

In this report, strains of five different Candida species (Candida albicans, Candida guilliermondii, Candida tropicalis, Candida krusei, and Candida parapsilosis) isolated from healthy human oral cavities as well as their respective type-strains were used in order to establish the genetic diversity existing among the different species and within a certain species, by the analysis of their electrophoretic alloenzyme patterns. These profiles were analyzed for their band positions in the gels, which allowed to group the strains of the same species in species-specific clusters and to treat them as conspecific populations. A total of thirteen enzymatic loci were obtained (ACO, ADH1, ADH2, CAT, G6PDH, GDH, GOT, IDH1, IDH2, LAP, LDH, PER, and SOD). The allelic frequencies (p) and the heterozygosity (h) for all the thirteen loci were determined by diversity index formulas. The GST index is the estimated proportion of genetic diversity that was applied in order to establish inter and intra populational diversity, which, for our results, indicated that 37.75% of total genetic diversity was attributable to differences among the species and the remaining 62.25% was attributable to differences within these populations. An Euclidian distance dendrogram for the different conspecific populations was built, showing that C. guilliermondii grouped first with C. tropicalis and thus formed a expanded cluster with C. albicans. This cluster combined later with another one composed by C. parapsilosis and C. krusei. Comparing our results to the others that were obtained by different molecular techniques, we have observed that the clustering hierarchies follow different paths of organization, varying according to the methodology employed.

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved

Analysis of parity between protein-based electrophoretic methods for the characterization of oral Candida species.

Electrophoretic studies of multilocus-enzymes (MLEE) and whole-cell protein (SDS-PAGE) were carried out in order to evaluate the parity between different methods for the characterization of five Candida species commonly isolated from oral cavity of humans by numerical taxonomy methods. The obtained data revealed that sodium dodecyl sulfate polyacrylamide gel electrophoresis is more efficient in grouping strains in their respective species while MLEE has much limited resolution in organizing all strains in their respective species-specific clusters. MLEE technique must be regarded for surveys in which just one species of Candida is involved.

Criteria for Candida albicans numerical analysis based on electrophoretic protein patterns.

Even in normalized gels, some problems with the choice and position of protein bands always have hindered the processing of electrophoretic data. We have developed a way to establish which criterion best fits the necessities in order to maximize the similarity indexes for numerical analysis. Some repetitions of a Candida albicans strain were carried out in eleven different gels. After staining, the bands were scored in numbers within ranges of +/- n values with increases of one quarter steps (+/- 0.25 kDa, +/- 0.5 kDa, +/- 0.75 kDa, +/- 1.0 kDa, +/- 1.25 kDa) that will limit the possibility of variation for the same bands that will appear on the repetitions. Using this criterion, we have determined that values scored within +/- 1.25 kDa could optimize the minimum limiting value of similarity in different dendrograms built.