Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Polyphyly of the hawk genera Leucopternis and Buteogallus (Aves, Accipitridae): multiple habitat shifts during the Neotropical buteonine diversification

Abstract Background The family Accipitridae (hawks, eagles and Old World vultures) represents a large radiation of predatory birds with an almost global distribution, although most species of this family occur in the Neotropics. Despite great morphological and ecological diversity, the evolutionary relationships in the family have been poorly explored at all taxonomic levels. Using sequences from four mitochondrial genes (12S, ATP8, ATP6, and ND6), we reconstructed the phylogeny of the Neotropical forest hawk genus Leucopternis and most of the allied genera of Neotropical buteonines. Our goals were to infer the evolutionary relationships among species of Leucopternis, estimate their relationships to other buteonine genera, evaluate the phylogenetic significance of the white and black plumage patterns common to most Leucopternis species, and assess general patterns of diversification of the group with respect to species' affiliations with Neotropical regions and habitats. Results Our molecular phylogeny for the genus Leucopternis and its allies disagrees sharply with traditional taxonomic arrangements for the group, and we present new hypotheses of relationships for a number of species. The mtDNA phylogenetic trees derived from analysis of the combined data posit a polyphyletic relationship among species of Leucopternis, Buteogallus and Buteo. Three highly supported clades containing Leucopternis species were recovered in our phylogenetic reconstructions. The first clade consisted of the sister pairs L. lacernulatus and Buteogallus meridionalis, and Buteogallus urubitinga and Harpyhaliaetus coronatus, in addition to L. schistaceus and L. plumbeus. The second clade included the sister pair Leucopternis albicollis and L. occidentalis as well as L. polionotus. The third lineage comprised the sister pair L. melanops and L. kuhli, in addition to L. semiplumbeus and Buteo buteo. According to our results, the white and black plumage patterns have evolved at least twice in the group. Furthermore, species found to the east and west of the Andes (cis-Andean and trans-Andean, respectively) are not reciprocally monophyletic, nor are forest and non-forest species. Conclusion The polyphyly of Leucopternis, Buteogallus and Buteo establishes a lack of concordance of current Accipitridae taxonomy with the mtDNA phylogeny for the group, and points to the need for further phylogenetic analysis at all taxonomic levels in the family as also suggested by other recent analyses. Habitat shifts, as well as cis- and trans-Andean disjunctions, took place more than once during buteonine diversification in the Neotropical region. Overemphasis of the black and white plumage patterns has led to questionable conclusions regarding the relationships of Leucopternis species, and suggests more generally that plumage characters should be used with considerable caution in the taxonomic evaluation of the Accipitridae.

The efficacy of chemical agents in cleaning and disinfection programs

Abstract Background Due to the growing number of outbreaks of infection in hospital nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Method For the purpose of evaluating the efficacy of a chemical agent, the minimum inhibitory concentration (MIC) was reached by the classic method of successive broth dilutions. The reference bacteria utilized were Bacillus subtilis var. globigii ATCC 9372, Bacillus stearothermophilus ATCC 7953, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923. The strains of Enterobacter cloacae IAL 1976 (Adolfo Lutz Institute), Serratia marcescens IAL 1478 and Acinetobactev calcoaceticus IAL 124 (ATCC 19606), were isolated from material collected from babies involved in outbreaks of infection in hospital nurseries. Results The MIC intervals, which reduced bacteria populations over 08 log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine digluconate; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of isopropanol or ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Conclusions Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus, was observed to show resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens.

Chemical resistance of the gram-negative bacteria to different sanitizers in a water purification system

Abstract Background Purified water for pharmaceutical purposes must be free of microbial contamination and pyrogens. Even with the additional sanitary and disinfecting treatments applied to the system (sequential operational stages), Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were isolated and identified from a thirteen-stage purification system. To evaluate the efficacy of the chemical agents used in the disinfecting process along with those used to adjust chemical characteristics of the system, over the identified bacteria, the kinetic parameter of killing time (D-value) necessary to inactivate 90% of the initial bioburden (decimal reduction time) was experimentally determined. Methods Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas alcaligenes, Pseudomonas picketti, Flavobacterium aureum, Acinetobacter lowffi and Pseudomonas diminuta were called in house (wild) bacteria. Pseudomonas diminuta ATCC 11568, Pseudomonas alcaligenes INCQS , Pseudomonas aeruginosa ATCC 15442, Pseudomonas fluorescens ATCC 3178, Pseudomonas picketti ATCC 5031, Bacillus subtilis ATCC 937 and Escherichia coli ATCC 25922 were used as 'standard' bacteria to evaluate resistance at 25°C against either 0.5% citric acid, 0.5% hydrochloric acid, 70% ethanol, 0.5% sodium bisulfite, 0.4% sodium hydroxide, 0.5% sodium hypochlorite, or a mixture of 2.2% hydrogen peroxide (H2O2) and 0.45% peracetic acid. Results The efficacy of the sanitizers varied with concentration and contact time to reduce decimal logarithmic (log10) population (n cycles). To kill 90% of the initial population (or one log10 cycle), the necessary time (D-value) was for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. Conclusion The contact time of 180 min of the system with the mixture of H2O2+ peracetic acid, a total theoretical reduction of 6 log10 cycles was attained in the water purified storage tank and distribution loop. The contact time between the water purification system (WPS) and the sanitary agents should be reviewed to reach sufficient bioburden reduction (over 6 log10).

Bioassay guided purification of the antimicrobial fraction of a Brazilian propolis from Bahia state

Abstract Background Brazilian propolis type 6 (Atlantic forest, Bahia) is distinct from the other types of propolis especially due to absence of flavonoids and presence of other non-polar, long chain compounds, but presenting good in vitro and in vivo antimicrobial activity. Several authors have suggested that fatty acids found in this propolis might be responsible for its antimicrobial activity; however, so far no evidence concerning this finding has been reported in the literature. The goals of this study were to evaluate the antibacterial activity of the main pure fatty acids in the ethanolic extract and fractions and elucidate the chemical nature of the bioactive compounds isolated from Brazilian propolis type 6. Methods Brazilian propolis type 6 ethanolic extract (EEP), hexane fraction (H-Fr), major fatty acids, and isolated sub-fractions were analyzed using high performance liquid chromatography (HPLC), high resolution gas chromatography with flame ionization detection (HRGC-FID), and gas chromatography-mass spectrometry (GC-MS). Three sub-fractions of H-Fr were obtained through preparative HPLC. Antimicrobial activity of EEP, H-Fr, sub-fractions, and fatty acids were tested against Staphyloccus aureus ATCC 25923 and Streptococcus mutans Ingbritt 1600 using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results EEP and H-Fr inhibited the growth of the microorganisms tested; nevertheless, no antimicrobial activity was found for the major fatty acids. The three sub-fractions (1, 2, and 3) were isolated from H-Fr by preparative HPLC and only sub-fraction 1 showed antimicrobial activity. Conclusion a) The major fatty acids tested were not responsible for the antimicrobial activity of propolis type 6; b) Sub-fraction 1, belonging to the benzophenone class, was responsible for the antimicrobial activity observed in the present study. The identification of the bioactive compound will improve the development of more efficient uses of this natural product.

Comparative analysis of Enterococcus Faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37 C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (mm3), live bacteria biovolume (mm3), and substrate coverage (%) were quantified by using the BioImage_L software. Results obtained were analyzed by nonparametric tests (P = .05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P < .05) and hydroxyapatite the highest at 21 days (P < .05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P < .05). The percentages of coverage were similar among all substrates at 21 days (P > .05), but at 14 days, bovine bone presented the highest coverage (P < .05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated

Differential arthritogenicity of Staphylococcus aureus strains isolated from biological samples

Background Staphylococcus aureus is the most common agent of septic arthritis that is a severe, rapidly progressive and destructive joint disease. Superantigens produced by S. aureus are considered the major arthritogenic factors. In this study, we compared the arthritogenic potential of five superantigen-producing staphylococcal strains. Methods Male C57BL/6 mice were intravenously infected with ATCC 19095 SEC+, N315 ST5 TSST-1+, S-70 TSST-1+, ATCC 51650 TSST-1+ and ATCC 13565 SEA+ strains. Clinical parameters as body weight, arthritis incidence and clinical score were daily evaluated. Joint histopathological analysis and spleen cytokine production were evaluated at the 14th day after infection. Results Weight loss was observed in all infected mice. ATCC 19095 SEC+, N315 ST5 TSST-1+ and S-70 TSST-1+ were arthritogenic, being the highest scores observed in ATCC 19095 SEC+ infected mice. Intermediate and lower clinical scores were observed in N315 ST5 TSST-1+ and S-70 TSST-1+ infected mice, respectively. The ATCC 13565 SEA+ strain caused death of 85% of the animals after 48 h. Arthritis triggered by the ATCC 19095 SEC+ strain was characterized by accentuated synovial hyperplasia, inflammation, pannus formation, cartilage destruction and bone erosion. Similar joint alterations were found in N315 ST5 TSST-1+ infected mice, however they were strikingly more discrete. Only minor synovial proliferation and inflammation were triggered by the S-70 TSST-1+ strain. The lowest levels of TNF-α, IL-6 and IL-17 production in response to S. aureus stimulation were found in cultures from mice infected with the less arthritogenic strains (S-70 TSST-1+ and ATCC 51650 TSST-1+). The highest production of IL-17 was detected in mice infected with the most arthritogenic strains (ATCC 19095 SEC+ and N315 ST5 TSST-1+). Conclusions Together these results demonstrated that S. aureus strains, isolated from biological samples, were able to induce a typical septic arthritis in mice. These results also suggest that the variable arthritogenicity of these strains was, at least in part, related to their differential ability to induce IL-17 production.

Characterization of the mechanisms underlying the crosstalk between galectins and notch in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Galectins form a family of β-galactosides binding proteins that recognize a variety of glycan-containing proteins at the cell surface and are overexpressed in various tumors, including gastric cancer. Galectins overexpression as well as changes in their subcellular distribution has been associated with gastric cancer progression and poor prognosis. It is not well understood, however, how the interaction between galectins and glycosylated receptors modulates tumor development and growth. Since Notch receptors and ligands contain glycan structures known to bind galectins, we aim to demonstrate that galectins expression in the tumor microenvironment may interfere with Notch signaling activation during tumor development and progression. Materials and methods Immunoprecipitation procedures with gastric cancer cell line AGS (ATCC CRL-1739) and MKN45 (ACC 409) were used to test for association between galectin-1/-3 and Notch-1 receptor. Furthermore, we transfected AGS cell line with siRNA against galectin-1/-3 or scramble using standard protocols (IDT DNA technologies), stimulate them with immobilized human recombinant delta-4 or Jagged-1 and assessed Notch-1 receptor activation. Results Galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling pathway upon activation by Delta/Jagged ligands. Galectin-1 knockdown alters Notch-1 activation induced by Delta-4 whereas galectin-3 knockdown alters jagged-1-mediated Notch-1 activation. Furthermore, we found that exogenously added galectin-3 can enhance Notch-1 activation by Jagged-1. Conclusion Our results suggest that galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling activation induced by Jagged-1 and Delta-4.

Invasion of Ureaplasma diversum in bovine spermatozoids

Background Ureaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay. Findings U. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids. Conclusions The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.

Influence of radiopacifying agents on the solubility, pH and antimicrobial activity of portland cement

The aim of this study was to evaluate the interference of the radiopacifiers bismuth oxide (BO), bismuth carbonate (BC), bismuth subnitrate (BS), and zirconiun oxide (ZO) on the solubility, alkalinity and antimicrobial properties of white Portland cement (WPC). The substances were incorporated to PC, at a ratio of 1:4 (v/v) and subjected to a solubility test. To evaluate the pH, the cements were inserted into retrograde cavities prepared in simulated acrylic teeth and immediately immersed in deionized water. The pH of the solution was measured at 3, 24, 72 and 168 h. The antimicrobial activity was evaluated by a radial diffusion method against the microorganisms S. aureus (ATCC 25923), P. aeruginosa (ATCC 27853), E. faecalis (ATCC 29212) and C. albicans (ATCC 10231). The zone of microbial growth inhibition was measured after 24 h. The addition of BS and BC increased the solubility of the cement. The pH values demonstrated that all materials produced alkaline levels. At 3 h, BS showed lower pH than WPC (p<0.05). At 168 h, all materials showed similar pHs (p>0.05). The materials did not present antimicrobial activity for S. aureus, P. aeruginosas and E. faecalis (p>0.05). With regards to C. albicans, all materials formed an inhibition zone, mainly the mixture of WPC with ZO (p<0.05). The type of radiopacifier incorporated into WPC interfered with its physical and antimicrobial properties. ZO was found to be a viable radiopacifier that can be used with WPC.

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.

Fermentative production of ribonucleotides from whey by Kluyveromyces marxianus: effect of temperature and pH

Ribonucleotides have shown many promising applications in food and pharmaceutical industries. The aim of the present study was to produce ribonucleotides (RNA) by Kluyveromyces marxianus ATCC 8,554 utilizing cheese whey, a dairy industry waste, as a main substrate under batch fermentation conditions. The effects of temperature, pH, aeration rate, agitation and initial cellular concentration were studied simultaneously through factorial design for RNA, biomass production and lactose consumption. The maximum RNA production (28.66 mg/g of dry biomass) was observed at temperature 30°C, pH 5.0 and 1 g/l of initial cellular concentration after 2 h of fermentation. Agitation and aeration rate did not influence on RNA concentration (p >0.05). Maximum lactose consumption (98.7%) and biomass production (6.0 g/l) was observed after 12 h of incubation. This study proves that cheese whey can be used as an adequate medium for RNA production by K. marxianus under the optimized conditions at industrial scale.

Differential transcription of virulence genes in Aggregatibacter actinomycetemcomitans serotypes

Background: Aggregatibacter actinomycetemcomitans serotypes are clearly associated with periodontitis or health, which suggests distinct strategies for survival within the host. Objective: We investigated the transcription profile of virulence-associated genes in A. actinomycetemcomitans serotype b (JP2 and SUNY 465) strains associated with disease and serotype a (ATCC 29523) strain associated with health. Design: Bacteria were co-cultured with immortalized gingival epithelial cells (OBA-9). The adhesion efficiency after 2 hours and the relative transcription of 13 genes were evaluated after 2 and 24 hours of interaction. Results: All strains were able to adhere to OBA-9, and this contact induced transcription of pgA for polysaccharide biosynthesis in all tested strains. Genes encoding virulence factors as Omp29, Omp100, leukotoxin, and CagE (apoptotic protein) were more transcribed by serotype b strains than by serotype a. ltxA and omp29, encoding the leukotoxin and the highly antigenic Omp29, were induced in serotype b by interaction with epithelial cells. Factors related to colonization (aae, flp, apaH, and pgA) and cdtB were upregulated in serotype a strain after prolonged interaction with OBA-9. Conclusion: Genes relevant for surface colonization and interaction with the immune system are regulated differently among the strains, which may help explaining their differences in association with disease.

Subcutaneous Tissue Thickness is an Independent Predictor of Image Noise in Cardiac CT

Background: Few data on the definition of simple robust parameters to predict image noise in cardiac computed tomography (CT) exist. Objectives: To evaluate the value of a simple measure of subcutaneous tissue as a predictor of image noise in cardiac CT. Methods: 86 patients underwent prospective ECG-gated coronary computed tomographic angiography (CTA) and coronary calcium scoring (CAC) with 120 kV and 150 mA. The image quality was objectively measured by the image noise in the aorta in the cardiac CTA, and low noise was defined as noise < 30HU. The chest anteroposterior diameter and lateral width, the image noise in the aorta and the skin-sternum (SS) thickness were measured as predictors of cardiac CTA noise. The association of the predictors and image noise was performed by using Pearson correlation. Results: The mean radiation dose was 3.5 ± 1.5 mSv. The mean image noise in CT was 36.3 ± 8.5 HU, and the mean image noise in non-contrast scan was 17.7 ± 4.4 HU. All predictors were independently associated with cardiac CTA noise. The best predictors were SS thickness, with a correlation of 0.70 (p < 0.001), and noise in the non-contrast images, with a correlation of 0.73 (p < 0.001). When evaluating the ability to predict low image noise, the areas under the ROC curve for the non-contrast noise and for the SS thickness were 0.837 and 0.864, respectively. Conclusion: Both SS thickness and CAC noise are simple accurate predictors of cardiac CTA image noise. Those parameters can be incorporated in standard CT protocols to adequately adjust radiation exposure.

Taxonomy, phylogeny and reproductive ecology of Gentiana lutea L.

This research focuses on taxonomy, phylogeny and reproductive ecology of Gentiana lutea. L.. Taxonomic analysis is a critical step in botanical studies, as it is necessary to recognize taxonomical unit. Herbarium specimens were observed to assess the reliability of several subspecies-diagnostic characters. The analysis of G. lutea genetic variability and the comparison with that of the other species of sect. Gentiana were performed to elucidate phylogenetic relationships among G. lutea subspecies and to propose a phylogenetic hypothesis for the evolution and the colonization dynamics of the section. Appropriate scientific information is critical for the assessment of species conservation status and for effective management plans. I carried out field work on five natural populations and performed laboratory analyses on specific critical aspects, with special regard to G. lutea breeding system and type and efficiency of plant-pollinator system. Bracts length is a reliable character to identify subsp. vardjanii, however it is not exclusive, hence to clearly identify subsp. vardjanii, other traits have to be considered. The phylogenetic hypotheses obtained from nuclear and chloroplast data are not congruent. Nuclear markers show a monophyly of sect. Gentiana, a strongly species identity of G. lutea and clear genetic identity of subsp. vardjanii. The little information emerging from plastid markers indicate a weak signal of hybridization and incomplete sorting of ancestral lineages. G. lutea shows a striking variation in intra-floral dichogamy probably evolved to reduce pollen-stigma interference. Although the species is partially self-compatible, pollen vectors are necessary for a successful reproduction, and moreover it shows a strong inbreeding depression. G. lutea is a generalist species: within its spectrum of visitors is possible to recognize "nectar thieves" and pollinators with sedentary or dynamic behaviour. Pollen limitation is frequent and it could be mainly explained by poor pollen quality.