Antibacterial effects of plant extracts with hurdle technology against Vibrio cholerae.

Vibrio cholerae is an etiological cause of cholera implicated in several pandemics. Antibacterial activity of plant extracts has been established. However, these extracts exhibit activity at a concentration that may alter organoleptic attributes of water and food, hence limiting their application. In this light, there is need to device ways of reducing plant extracts' effective levels in order to widen their application. Thus, this study was conducted to improve activities of plant ethanolic extracts through combination with other generally recognized as safe antimicrobials. Combination of plant extracts with sodium acetate (NaOAc) 0.4% at pH 7.0 reduced minimum inhibitory concentrations (MICs) of clove, lemon eucalyptus, rosemary and sage from 0.2 to 0.025%. At pH 6.4, combinations were more effective reducing MICs of clove, lemon eucalyptus, rosemary and sage from 0.2 to 0.0125% with NaOAc at 0.2%. At pH 7.0, the combination resulted in additive effect. Nevertheless, at pH 6.4, synergic effect was established. No interactive effect was observed with combinations involving glycine. Combination of plant extracts with NaOAc at mildly acidic pH creates a hurdle effect that may have potential application to control the growth of V. cholerae.

Speciation of inorganic selenium in environmental water samples by inductively coupled plasma optical emission spectrometry after preconcentration by using a mesoporous zirconia coating on coal cinder.

A simple, novel, and selective flow-injection solid-phase extraction with inductively coupled plasma optical emission spectrometry method was developed for the speciation of inorganic selenium in environmental water samples. A mesoporous zirconia film was simply introduced to coat coal cinder by means of the sol-gel technique, and the adsorptive performance of the coated material for Se(IV)/Se(VI) was investigated in different media. Both Se(IV) and Se(VI) can be retained quantitatively by the material in HCl/NaOH (pH 1.0-9.0) media, while only Se(IV) was adsorbed quantitatively in sodium acetate buffer (pH 3.5-6.0). Thus, the assay of Se(VI) is based on subtracting Se(IV) from total selenium by controlling different adsorptive media without employing any redox procedure. Under the optimum conditions, the detection limit of Se(IV) is 9.0 ng/L with an enrichment factor of 100, and the relative standard deviation is 3.6% (n = 9, C = 5.0 ng/mL). The developed method was successfully applied to the speciation of inorganic selenium in environmental water samples with satisfactory results. In order to further verify the accuracy of the developed method, it was applied to analysis of total selenium in GSBZ 50031-94 certified reference environmental water, and the determined values coincided with the certified values very well.

Quantitative evaluation of myoglobin unfolding in the presence of guanidinium hydrochloride and ionic liquids in solution.

The use of ionic liquids in biochemical and biophysical applications has increased dramatically in recent years due to their interesting properties. We report results of a thermodynamic characterization of the chaotrope-induced denaturation of equine myoglobin in two different ionic liquid aqueous environments using a combined absorption/fluorescence spectroscopic approach. Denaturation by guanidinium hydrochloride was monitored by loss of heme absorptivity and limited unfolding structural information was obtained from Förster resonance energy transfer experiments. Results show that myoglobin unfolding is generally unchanged in the presence of ethylmethylimidazolium acetate (EMIAc) in aqueous solution up to 150 mM concentration but is facilitated by butylmethylimidazolium boron tetrafluoride (BMIBF4) in solution. The presence of 150 mM BMIBF4 alone does not induce unfolding but destabilizes the structure as observed by a decrease in threshold denaturant concentration for unfolding and an 80% decrease in the magnitude of ΔGunfolding from 44 kJ/mol in the absence of BMIBF4 to 8 kJ/mol in the presence of 150 mM BMIBF4. Thus, the BMIBF4 significantly destabilizes the myoglobin structure while the EMIAc does not, likely due to differences in anion interaction capabilities. This is confirmed with control studies using NaAc and LiBF4 solutions. EMIAc may be chosen as cosolvent additive with minimal effects on protein structure while BMIBF4 may be used as a supplement in protein folding experiments, potentially allowing access to proteins which have been traditionally difficult to denature as well as designing ionic liquids to match protein characteristics.

Combination of solid phase extraction and dispersive liquid-liquid microextraction for separation/preconcentration of ultra trace amounts of uranium prior to its fiber optic-linear array spectrophotometry determination.

A simple and sensitive method for the separation and preconcentration of the ultra trace amounts of uranium and its determination by spectrophotometry was developed. The method is based on the combination of solid phase extraction and dispersive liquid-liquid microextraction. Thus, by passing the sample through the basic alumina column, the uranyl ion and some cations are separated from the sample matrix. The retained uranyl ion along with the cations are eluted with 5 mL of nitric acid (2 mol L(-1)) and after neutralization of the eluent, the extracted uranyl ion is converted to its anionic benzoate complex and is separated from other cations by extraction of its ion pair with malachite green into small volume of chloroform using dispersive liquid-liquid microextraction. The amount of uranium is then determined by the absorption measurement of the extracted ion pair at 621 nm using flow injection spectrophotometry. Under the optimum conditions, with 500 mL of the sample, a preconcentration factor of 1980, a detection limit of 40 ng L(-1), and a relative standard deviation of 4.1% (n=6) at 400 ng L(-1) were obtained. The method was successfully applied to the determination of uranium in mineral water, river water, well water, spring water and sea water samples.

Effects of different amine fluoride concentrations on enamel remineralization.

OBJECTIVES: The aim of this study was to investigate the effects of decreasing fluoride concentrations on repeated demineralizing challenges on human enamel. MATERIALS AND METHODS: In 24 teeth, 3mm×3mm windows were prepared on the buccal and lingual sides and treated in a cycling demineralization-remineralization model. Remineralization was achieved with 100, 10 and 0.1 ppm fluoride from anime fluoride. Coronal sections were cut through the artificial lesions, and three sections per tooth were investigated using polarized light microscopy and scanning electron microscopy with quantitative element analysis. RESULTS: The morphology of the lesions was studied, and the extensions of the superficial layer and the body of the lesion were measured. Using element analysis, the Ca, P and F content were determined. The body of the lesion appeared remineralized after application of 100 ppm fluoride, while remineralization of the lesion was less successful after application of 10 and 0.1 ppm fluoride. The thickness of the superficial layer increased with decreasing fluoride concentrations, and also the extension of the body of the lesion increased. Ca and P content increased with increasing fluoride concentrations. CONCLUSIONS: The effectiveness of fluoride in enamel remineralization increased with increasing fluoride concentration. CLINICAL RELEVANCE: A consistently higher level of fluoride in saliva should be a goal in caries prevention.

Determination of phenolic acids and flavonoids in Taraxacum formosanum Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization technique.

A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for the determination of phenolic acids and flavonoids in a medicinal Chinese herb Taraxacum formosanum Kitam. Initially, both phenolic acids and flavonoids were extracted with 50% ethanol in a water-bath at 60 °C for 3 h and eventually separated into acidic fraction and neutral fraction by using a C(18) cartridge. A total of 29 compounds were separated within 68 min by employing a Gemini C(18) column and a gradient solvent system of 0.1% formic acid and acetonitrile at a flow rate of 1.0 mL/min. Based on the retention behavior as well as absorption and mass spectra, 19 phenolic acids and 10 flavonoids were identified and quantified in T. formosanum, with the former ranging from 14.1 µg/g to 10,870.4 µg/g, and the latter from 9.9 µg/g to 325.8 µg/g. For further identification of flavonoids, a post-column derivatization method involving shift reagents such as sodium acetate or aluminum chloride was used and the absorption spectral characteristics without or with shift reagents were compared. An internal standard syringic acid was used for quantitation of phenolic acids, whereas (±) naringenin was found suitable for quantitation of flavonoids. The developed LC-MS/MS method showed high reproducibility, as evident from the relative standard deviation (RSD) values for intra-day and inter-day variability being 1.0-6.8% and 2.0-7.7% for phenolic acids and 3.7-7.4% and 1.5-8.1% for flavonoids, respectively, and thus may be applied for simultaneous determination of phenolic acids and flavonoids in Chinese herb and nutraceuticals.

Insights in starch acetylation in sub- and supercritical CO2.

An in-depth study on the acetylation of starch with acetic anhydride (Ac(2)O) and sodium acetate (NaOAc) as the catalyst in pressurized carbon dioxide (scCO(2)) in a broad pressure range (8-25MPa) and a temperature of 90°C is provided. Highest degrees of substitution (DS) of 0.29 (1h reaction time) and 0.62 (24h reaction time) were found near the critical point of the mixture (15MPa). The phase behavior of the system CO(2), starch and acetic anhydride (Ac(2)O) was studied in a high pressure view cell. The critical points were a clear function of the temperature and increased from the range of 9.4-10MPa to 14.5-14.8MPa when going from 50 to 90°C (Ac(2)O mole fraction at the critical point in the range of 0.08-0.09). Acetylation experiments with a range of starch particles sizes showed a clear relation between the DS and the particle size.

[Formation of the phosphorus removal granular sludge and phosphorus removal characteristics of the anaerobic/oxic and anaerobic/anoxic/oxic granular sludge process in SBR].

Phosphorous removal & denitrifying phosphate uptake of the granular sludge was investigated in this study. Inoculated with flocculation sludge, the granulation of the biological phosphorous removal sludge was realized in a sequencing batch reactor (SBR) fed with sodium acetate by means of hydraulic selection under an anaerobic/oxic alternating operation (referred to as an A/O). Then the biological phosphorous removal granular sludge was induced into the denitrifying phosphate uptake granular sludge under an anaerobic/anoxic/oxic alternating operation (referred to as an A/A/O). The properties of the two kinds of granular sludge were studied. The biological phosphorus removal granular sludge was completed on the 82nd day. The biological phosphorus removal granule sludge showed some characteristics, e.g. pallideflavens in color, 0.5-1.5 mm in diameter, 20-30 m/h in settling velocity, 94% in water content, 1.043 9 in specific gravity, and below 50 mL/g in SVI. The max. specific release phosphorus rate (SRPR), the max. specific uptake phosphorus rate (SUPR) and the phosphorus content of the MLSS (TP/SS) was 67.7 mg/(g x h), 43.2 mg/(g x h) and 6.5% respectively on the 437th day. On the 448th day the operation of the reactor was changed into A/A/O. The max. SRPR, the max. anoxic SUPR and the TP/SS of the denitrifying phosphate uptake granular sludge was 30 mg/(g x h), 27.9 mg/(g x h) and 6.3% respectively on the 653rd day. The two kinds of granular sludge had potential to carry out phosphorus removal.

Phase change material for thermotherapy of Buruli ulcer: modelling as an aid to implementation.

A mathematical model was developed and validated to predict the thermal behaviour of a heat application device based on a phase change material (pcm) for the heat treatment of Mycobacterium ulcerans infection (Buruli ulcer). The thermal model allows the prediction of skin surface temperatures and an optimization of the amount of pcm with respect to discharge time. A first prototype of such a pcm bandage was manufactured and used in a proof-of-principal trial in Cameroon. The experimental data were analysed and yielded no difference in thermoregulatory response between people living in hot or moderate climate. Short-term maximum skin surface temperatures of 42 degrees C are tolerable; the pcm bandage keeps the skin surface temperature above 40 degrees C for about four to five hours. This makes such pcm bandages an ideal device for the heat treatment of Buruli ulcer. The pcm bandage is easy to apply, cheap, and thus is well suited for use in low-resource countries.

Preparation and ion chromatographic properties of a new core-shell chromatographic support Al2O3/SiO2-10.

A new stationary phase Al(2)O(3)/SiO(2)-10 was prepared and characterized by XPS, XRD, SEM and surface analysis. The anion exchanger properties of this new stationary phase were investigated by the separation of inorganic anions in ion chromatography (IC). pH of the mobile phase, concentration and strength of the Lewis base of the elute, and the organic modifier of the mobile phase strongly affect the separation of inorganic anions, and anion exchange selectivities of the analyte on the new support are significantly different from quaternary ammonium styrene based anion exchangers. The result of separation of inorganic anions shows that the new stationary phase provides excellent column efficiency, well-defined chromatographic peaks and favorable retention times.

Fabrication of colloidosomes at low temperature for the encapsulation of thermally sensitive compounds.

A method using safe and mild conditions to prepare water-core colloidosomes of a few micrometers in size is examined. Using poly(styrene-co-butylacrylate) colloidal particles of 180 nm in size, with a low-temperature glass transition of 30 or 42 degrees C, the self-assembly into micrometer-sized water-core polymer shell particles is demonstrated. The effect of oil phase type, surfactant and cosolvent, sintering time, and the method of transfer into an aqueous phase are all examined. The work demonstrates the production of water-core rigid-shell colloidosomes at low temperatures, between 35 and 65 degrees C.

An enzymatic hydrolysis approach for characterizing labile phosphorus forms in dairy manure under mild assay conditions.

Characterizing labile P forms in animal manure is a challenge due to their susceptibility to hydrolysis. In this study, we enzymatically characterized P forms in dairy manure (no bedding), collected from a representative dairy farm in New York, by separating into soluble and residual components under mild assay conditions using water and sodium acetate buffer (pH 5.0). About 75% of total manure P in the fresh manure was characterized, with the remainder (25%) regarded as recalcitrant or biochemically unidentified P. The hydrolyzable organic P in soluble and residual fractions was then characterized by using phosphatase enzymes to simple monoester P, polynucleotide P, phytate-like P, and non-hydrolyzable P. Of the total P in water extracts, 77% was inorganic P, 11% hydrolyzable organic P and 12% non-hydrolyzable P. In the residual resuspension, the distribution of characterized P was 25% spontaneous labile P, 32% simple monoester P, 7% polynucleotide P, 9% phytate-like P, and 26% non-hydrolyzable P. Ultrasonication increased the P(i) release from the manure residues, but the deviation in Pi concentrations due to the sampling variance was greater that the increase in P(i) due to sonication. Autoclaving sped up the release of both spontaneously labile P and enzymatically hydrolyzable P trapped in the manure residual matrix. Quantifying labile P forms by this approach may advance our ability to predict amount of manure P that will be hydrolyzed and eventually become bioavailable. The information obtained by our modified method would be complementary to that obtained by other methods (such as P-31 NMR and sequential fractionation) for a full spectrum of P species in animal manure.

Quantitative determination of saccharides in dietary glyconutritional products by anion-exchange liquid chromatography with integrated pulsed amperometric detection.

A new technique for the assay of carbohydrates is described in which separation and quantification of neutral saccharides, aminosaccharides, glycuronic acids, and disaccharides may be accomplished in less than 50 min of total run time. This method involves optimized anion-exchange liquid chromatography coupled with integrated pulse amperometric detection. Complex carbohydrates from various sources, including dietary supplements, were hydrolyzed in a dilute solution of trifluoroacetic acid, freeze-dried, and reconstituted in water containing 2-deoxygalactose as the internal standard. The solution was filtered and separated on CarboPac PA20 column. The eluted saccharides were detected by oxidation on a gold electrode with quadruple-pulsed integrated amperometry. The calibration plots for the saccharides were linear with an average correlation coefficient of 0.999. Method precision regarding peak retention time and resolution used in the peak identifications was verified. With this method, previously difficult-to-separate saccharides, such as galactosamine, glucosamine, and N-acetylglucosamine, were successfully resolved from the neutral saccharides rhamnose, arabinose, and galactose. Mannose was also resolved from xylose, and de-acetylation of aminosaccharides prior to separation was not necessary. This technique provides an accurate and efficient means to assay carbohydrates in dietary supplements, which new federal regulations will soon mandate.

[Screening of microbial secondary metabolites inhibiting cholesterol biosynthesis with the use of hepatoblastoma G2]. / Otbor mikrobnykh vtorichnykh matabolitov--ingibitorov biosinteza kholesterina s pomozh'iu kul'tury kletok gepatoblastomy G2.

The culture of hepatoblastoma G2 (Hep G2) cells is proposed as an effective model for screening of microbial metabolites--inhibitors of sterol biosynthesis. This model can be applied at early stages of screening procedures and is quite effective for testing of crude extracts of producers' culture broth. The test is based on measurement inhibition of the radiolabelled precursors incorporation in cholesterol and separate fractions of lipids by microbial metabolites in Hep G2 cells. That allows not only to reveal inhibitors of cholesterol biosynthesis, but also to evaluate mechanism of action, including ability to inhibit the synthesis of cholesterol ethers. The cholesterol biosynthesis inhibition was tested at 150 microbial cultures (actinomycetes and imperfect fungi), isolated from soil. The ability to inhibit 14C-acetate incorporation into cholesterol was found in 15-20% of microbial cultures possessing antifungal activity of extracts (culture broth and mycelium).

Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase.

A method was developed that enabled the study of non-esterified galacturonic acid sequences (so-called blocks) in pectin. Endopolygalacturonase of Kluyveromyces fragilis was used to extensively degrade pectin, and the composition of the galacturonic acid molecules produced was determined with high-performance anion-exchange chromatography at pH 5. With this technique, the amount of non-esterified mono-, di-, and trigalacturonic acid released was determined. In addition, the relative amounts of methyl-esterified oligomers--up to 10 galacturonic acid residues could be observed. By comparing the percentages of non-esterified mono-, di-, and trigalacturonic acids released, pectins with large enzyme-degradable blocks could be distinguished from pectins with small enzyme-degradable blocks. High percentages of mono- and digalacturonic acid were found for pectins containing small non-esterified blocks. The total area of all peaks corresponding to methyl-esterified oligomers was found to be indicative for the distribution of these blocks. The higher the ratio of the methyl- to non-esterified peak areas, the more closely associated blocks are present. Randomly esterified pectins, with degrees of methyl esterification of 50 and higher, contained smaller, more clustered blocks than commercial extracted pectins of comparable degrees of esterification. The approach developed enables a very detailed study of the methyl-ester distribution of pectin to be carried out and is a very important addition in the study of the functional behavior of this complex polymer.

A re-examination in vivo of the phosphatidylcholine-galactolipid metabolic relationship during plant lipid biosynthesis.

It remains unclear how and in what form the lipids synthesized in plant endoplasmic reticulum are exported to chloroplasts and used as precursors for the biosynthesis of plastid galactolipids, which are the most abundant lipids on Earth. Neither the mechanism of transfer nor the nature of the lipids imported into plastids has been elucidated. To characterize events occurring in vivo, the labelling of lipids from 15-day-old leek seedlings (Allium porrum, var. furor) was studied using pulse-chase experiments. During the chase, a substantial decline in the radioactivity incorporated into phosphatidylcholine (and not in other phospholipids) was accompanied by an increase in the label found in galactolipids. The positional distribution of labelled fatty acids in phosphatidylcholine and galactolipids was further studied as a function of the chase time; whereas phosphatidylcholine was preferentially labelled at the sn-2 position, the increase in radioactivity in galactolipids mainly concerned the sn-1 position. These results strongly suggest that the diacylglycerol moiety of phosphatidylcholine might not be integrated as a whole in the galactolipid.