On the fit of statistical and the k-C* models to projecting treatment performance in a constructed wetland system.

The objective of this study was to assess the suitability of statistical and the k-C* models to projecting treatment performance of constructed wetlands by applying the models to predict the final effluent concentrations of a pilot field-scale constructed wetlands system (CWs) treating animal farm wastewater. The CWs achieved removal rates (in g/m(2).d) ranging from 7.1-149.8 for BOD(5), 49.8-253.8 for COD and 7.1-47.0 for NH(4)-N. Generally, it was found that the statistical models developed from multiple regression analyses (MRA) were stronger in predicting final effluent concentrations than the k-C* model. However, both models were inadequate in predicting the final effluent concentrations of NO(3)-N. The first-order area-based removal rate constants (k, m/yr) determined from the experimental data were 200.5 for BOD(5), 80.1 for TP and 173.8 for NH(4)-N and these indicate a high rate of pollutant removal within the CWs.

Performance evaluation and prediction for a pilot two-stage on-site constructed wetland system employing dewatered alum sludge as main substrate.

Dewatered alum sludge, a widely generated by-product of drinking water treatment plants using aluminium salts as coagulants was used as main substrate in a pilot on-site constructed wetland system treating agricultural wastewater for 11 months. Treatment performance was evaluated and spreadsheet analysis was used to establish correlations between water quality variables. Results showed that removal rates (in g/m(2)d) of 4.6-249.2 for 5 day biochemical oxygen demand (BOD(5)), 35.6-502.0 for chemical oxygen demand (COD), 2.5-14.3 for total phosphorus (TP) and 2.7-14.6 for phosphate (PO(4)P) were achieved. Multiple regression analysis showed that effluent BOD(5) and COD can be predicted to a reasonable accuracy (R(2)=0.665 and 0.588, respectively) by using input variables which can be easily monitored in real time as sole predictor variables. This could provide a rapid and cheap alternative to such laborious and time consuming analyses and also serve as management tools for day-to-day process control.

Multiplexed PCR genotyping of HPVs from plantaris verrucae.

BACKGROUND: Plantaris verrucae are a common diagnosis in childhood, consume a significant amount of health-care resources, have many painful treatment options and many recurrences. OBJECTIVES: The objective of this study was to design and test a single site-anchored, multiplexed and expandable PCR assay for common types of cutaneous HPVs. STUDY DESIGN: Common forward and unique reverse primers were selected from the E2 open reading frames of five cutaneous HPV genotypes. These were analyzed for sensitivity and selectivity using pHPV plasmids and several control DNAs in an optimized multiplexed assay. This standardized assay was used to analyze human verruca plantaris tissue for genome type and to evaluate the effect of a commonly used treatment protocol. RESULTS: A sensitive, multiplexed PCR assay for human cutaneous HPV genotypes 1a, 2a and 4 was developed. Specific-unique primers and a consensus anchor primer were selected within the HPV E2 region to produce amplicons varying by greater than 100bp. In analytical sensitivity studies, fewer than 100 genome copies of HPV1a and 2a were detected, and fewer than 1000 copies of HPV4 were detected. The multiplexed assay did not amplify regions of human placenta, calf thymus, CaSki or SiHa DNA and E. coli, pBR322 or non-HPV virus DNAs. In combination with a forensic DNA extraction procedure, the multiplexed HPV assay detected and identified HPV types in 23 of 51 (45%) deep plantaris verrucae. Two patients were found with two different genotypes in single deep plantaris verruca. Detection of the HPV genome was followed as a function of tissue ablation and Mediplast treatment in one patient. In healing tissue, the genome content was reduced but had not totally disappeared. CONCLUSIONS: The multiplexed HPV assay can be used to determine genotype prevalence that may correlate with treatment efficacy.

Virulence factors of Porphyromonas gingivalis are modified by polyphenol oxidase and asparaginase.

Porphyromonas gingivalis is a well-adapted pathogen of the periodontal pocket distinguished by its wide array of proteolytic activities and its ability to adhere to multiple substrata in the oral cavity. Microbial proteins with binding functions (such as adhesins and enzymes) very often contain critical tyrosine residues, supported by one or more asparagines in the binding cleft. This study investigates the reduction in adhesiveness and in proteolytic activity after treating P. gingivalis with the tyrosine- and asparagine-targeting enzymes polyphenol oxidase (PPO) and asparaginase (ASG). Cysteine protease activity was reduced by pretreatment with both enzymes, while the trypsin-like activity was affected only by PPO. Adhesion to buccal epithelial cells, laminin and fibronectin as well as hemagglutination was reduced by one or both of the enzymes. PPO, but not ASG, reduced the coaggregation of P. gingivalis with Actinomyces naeslundii. Treatment with these enzymes might provide an alternative to traditional antimicrobial strategies.

Study of Candida albicans strains isolated from women with various forms of vaginal candidiasis.

Adhesive activity of Candida albicans towards vaginal epitheliocytes is hormone-dependent. Two types of C. albicans adhesins sensitive to polyphenyloxidase and asparaginase were detected. Laser irradiation nonspecifically modulated both adhesin types. Population relationships between fungi and lactobacilli in patients with vaginal candidal infection and in C. albicans carriers were studied. Genodiagnostic method for identification of C. albicans and morphogenesis-associated genes of these yeast-like fungi was approved.

Rapid dot sputum and serum assay in pulmonary tuberculosis.

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.

Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin.

The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.

Combined effect of low-frequency laser and polyphenol oxidase on adhesive activity of Escherichia coli strains isolated from patients with urinary diseases.

The effects of polyphenol oxidase and low-frequency laser irradiation on adhesion of pathogenic Escherichia coli to human erythrocytes and buccal epithelial cells were studied. The maximum decrease in adhesive activity of these strains was observed after complex exposure to laser and enzyme.

Complex effects of pulsed infrared laser and asparaginase on Escherichia coli strains isolated from patients with urinary diseases.

Treatment with L-asparaginase and low-frequency laser decreased adhesion of uropathogenic Escherichia coli to human erythrocytes. The maximum effect was observed after combined treatment (laser exposure followed by enzyme treatment).

Effects of asparaginase and polyphenol oxidase on adhesive characteristics of microorganisms.

We studied the effects of polyphenol oxidase and asparaginase on microorganism adhesion to buccal epithelial cells. These enzymes reduced adhesion of pathogenic microorganisms (uropathogenic and Escherichia coli, Salmonella enteritidis, Entamoeba spp., Influenza virus, Candida albicans, Streptococcus spp.) and had virtually no effect on adhesive characteristics of probiotic variants of Escherichia coli and Lactobacillus fermentum.

Regulation of autolysins in teichuronic acid-containing Bacillus subtilis cells.

Bacillus subtilis cells grown under phosphate starvation induce teichuronic acid (TUA) synthesis while simultaneously repressing teichoic acid synthesis (TA). The turnover rates of TA-containing and TUA-containing walls are similar, indicating that autolysin function is similar and suggesting that modulation of autolytic function may be similar. In this study, it is demonstrated, utilizing fluorescein isothiocyanate (FITC)-dextran to probe the wall pH, that a low pH exists in the wall matrix. A second probe, cationized ferritin (CF), was used to observe cell surface protonation. Suspensions of B. subtilis cells containing either TA or TUA were aggregated with CF only after the addition of a proton-motive-force-dissipating agent. Respiring B. subtilis TUA-containing cells labelled with FITC-dextran exhibited little fluorescence. Conversely, fluorescence intensities exhibited by cells de-energized with nitrogen gas were significantly greater. The effects of protonmotive force on autolytic activity were studied by adding cell wall protein extract containing concentrated autolysin to exponentially growing TA-containing and TUA-containing B. subtilis cells. Both TUA-containing and TA-containing cells were lysed only after the addition of sodium azide. These data suggest that during normal growth the wall of TUA-containing B. subtilis cells is protonated, and proton-motive force influences autolytic regulation in both TUA-containing and TA-containing B. subtilis cells.

Fluoride modifies adhesion of Streptococcus pyogenes.

Streptococcus pyogenes grown in the presence of subinhibitory concentrations of sodium fluoride had a diminished ability, compared to control cells, to adhere to buccal cells, collagen, fibronectin, and laminin. In addition, sodium fluoride was a competitive inhibitor of streptococcal adhesion to collagen and fibronectin, but not laminin. It is suggested that sodium fluoride may be useful in therapy or prophylaxis in infections involving group A streptococci.

Fluoride exposure attenuates expression of Streptococcus pyogenes virulence factors.

Fluoridation causes an obvious reduction of dental caries by interference with cariogenic streptococci. However, the effect of fluoride on group A streptococci that causes rheumatic fever and acute poststreptococcal glomerulonephritis is not known. We have used proteomic analysis to create a reference proteome map for Streptococcus pyogenes and to determine fluoride-induced protein changes in the streptococci. Cellular and extracellular proteins were resolved by two-dimensional polyacrylamide gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. 183 protein spots were visualized, and 74 spots representing 60 unique proteins were identified. A 16-h exposure to sodium fluoride caused decreased expression of proteins required to respond to cellular stress, including anti-oxidants, glycolytic enzymes, transcriptional and translational regulators, and protein folding. Fluoride caused decreased cellular expression of two well-characterized S. pyogenes virulence factors. Fluoride decreased expression of glyceraldehyde-3-phosphate dehydrogenase, which acts to bind fibronectin and promote bacterial adherence. We also performed proteomic analysis of protein released by S. pyogenes into the culture supernatant and observed decreased expression of M proteins following fluoride exposure. These data provide evidence that fluoride causes decreased expression by S. pyogenes proteins used to respond to stress, virulence factors, and implicated in non-suppurative complications of S. pyogenes, including glomerulonephritis and rheumatic fever.

Evidence that the cell wall of Bacillus subtilis is protonated during respiration.

Several independent experiments suggest that cell walls of Bacillus subtilis are protonated during growth. When cells were grown in the presence of fluorescein-labeled dextran to saturate the cell walls, centrifuged, and suspended in PBS, fluorescence-activated cell sorter analyses revealed the bacteria were only poorly fluorescent. In contrast, when the bacteria were purged with N(2) to dissipate protonmotive force (pmf) fluorescence became intense. Upon reconstitution of the pmf with phenazine methosulfate, glucose, and oxygen, fluorescence declined. Another approach used pH-dependent chemical modification of cell walls. The walls of respiring B. subtilis cells were amenable to carboxylate modification by [(14)C]ethanolamine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The carbodiimide activation of carboxylate groups occurs only in acidic conditions. Upon dissipation of pmf the walls were refractory to chemical modification. Ammonium groups can be condensed with FITC in alkaline medium, but the condensation is very slow in acidic buffers. It was found that the derivatization of the walls with FITC could occur in the absence of pmf. The use of pH-dependent fluorophores and pH-dependent chemical modification reactions suggest that cell walls of respiring B. subtilis cells have a relatively low pH environment. This study shows a bacterium has a protonated compartment. Acidification of cell walls during growth may be one means of regulating autolytic enzymes.

Polycarboxylates inhibit the glucan-binding lectin of Streptococcus sobrinus.

Polycarboxylates, such as carboxymethylcellulose and hyaluronan, were found to be reversible inhibitors of the glucan-binding lectin of Streptococcus sobrinus. When the carboxylate groups were coupled to ethylenediamine, or reduced with carbodiimide-borohydride, inhibitory powers were lost. Similarly, N-deacetylated hyaluronan had poor inhibitory powers, probably due to the introduction of positive charges into the polymer. Other polymers, such as chondroitin sulfates, dextran sulfate, fetuin, heparin were not inhibitors. It appears that inhibition is based on repeating carboxylates, free of influence from ammonium groups. Such polymers have the property of complexing with metals. Earlier studies had concluded that the streptococcal lectin depended on manganese for activity. It is likely the carboxymethylcellulose and hyaluronan perturb essential metal coordination centers in the lectin. Polycarboxylates may have value in oral health care by acting on glucan-dependent microbial adhesion and biofilm formation.

Inhibitory effects of plant polyphenoloxidase on colonization factors of Streptococcus sobrinus 6715.

Exogenously added polyphenoloxidase (EC 1.14.18.1), an enzyme which oxidizes tyrosine residues and is commonly found in many dietary components, abolished the aggregation of Streptococcus sobrinus 6715 by high-molecular-weight dextran. The enzyme decreased glucan-binding lectin and/or glucosyltransferase I activities.

Sensitivity of bacterial coaggregation to chelating agents.

Coaggregation between pairs of microorganisms was found to be inhibited by chelating agents, such as acetylacetone, citrate, EDTA and carboxymethylcellulose. Assays were conducted on eight pairs of periodontopathogens and one pair consisting of Escherichia coli and Saccharomyces cerevisiae. The inhibitory effects of the chelating agents were reversible except for Actinomyces naeslundii 12104, the adhesin of which was irreversibly inactivated. Even though the bacteria possessed different kinds of adhesins, their sensitivity to chelating agents appears to be a common property. Non-toxic chelating agents, such as carboxymethylcellulose and citrate, may prove to be useful anti-adhesins.