Higher harmonic large-amplitude fourier transformed alternating current voltammetry : analytical attributes derived from studies of the oxidation of ferrocenemethanol and uric acid at a glassy carbon electrode

An analytical evaluation of the higher ac harmonic components derived from large amplitude Fourier transformed voltammetry is provided for the reversible oxidation of ferrocenemethanol (FcMeOH) and oxidation of uric acid by an EEC mechanism in a pH 7.4 phosphate buffer at a glassy carbon (GC) electrode. The small background current in the analytically optimal fifth harmonic is predominantly attributed to faradaic current associated with the presence of electroactive functional groups on the GC electrode surface, rather than to capacitive current which dominates the background in the dc, and the initial three ac harmonics. The detection limits for the dc and the first to fifth harmonic ac components are 1.9, 5.89, 2.1, 2.5, 0.8, and 0.5 µM for FcMeOH, respectively, using a sine wave modulation of 100 mV at 21.46 Hz and a dc sweep rate of 111.76 mV s−1. Analytical performance then progressively deteriorates in the sixth and higher harmonics. For the determination of uric acid, the capacitive background current was enhanced and the reproducibility lowered by the presence of surface active uric acid, but the rapid overall 2e− rather than 1e– electron transfer process gives rise to a significantly enhanced fifth harmonic faradaic current which enabled a detection limit of 0.3 µM to be achieved which is similar to that reported using chemically modified electrodes. Resolution of overlapping voltammetric signals for a mixture of uric acid and dopamine is also achieved using higher fourth or fifth harmonic components, under very low background current conditions. The use of higher fourth and fifth harmonics exhibiting highly favorable faradaic to background (noise) current ratios should therefore be considered in analytical applications under circumstances where the electron transfer rate is fast.

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Methanesulfonic acid-catalyzed conversion of glucose and xylose mixtures to levulinic acid and furfural

Methanesulfonic acid (MSA) was compared with sulfuric acid for the conversion of glucose and xylose mixtures to produce levulinic acid and furfural. The interactions of glucose and xylose, the predominant sugars found in biomass, were found to influence product yields with furfural degradation reactions enhanced under higher reactant loadings. Fast heating rates allowed maximal yields (>60 mol%) of levulinic acid and furfural to be achieved under short reaction times. Under the range of conditions examined, sulfuric acid produced a slight increase in levulinic acid yield by 6% (P = 0.02), although there was no significant difference (P = 0.11) between MSA and sulfuric acid in levulinic acid formed from glucose alone. The amount and type of the solid residue is similar between MSA and sulfuric acid. As such, MSA is a suitable alternative because its use minimizes corrosion and disposal issues associated with mineral acid catalysts. The heating value of the residue was 22 MJ/kg implying that it is a suitable source of fuel. On the basis of these results, a two-stage processing strategy is proposed to target high levulinic acid and furfural yields, and other chemical products (e.g., lactic acid, xylitol, acetic acid and formic acid). This will result in full utilization of bagasse components.

An objective study of acid reflux and cough in children using an ambulatory pHmetry-cough logger

Objective There are no objective ambulatory studies on the temporal relationship between reflux and cough in children. Commercial pHmetry loggers have slow capture rates (0.25 Hz) that limit objective quantification of reflux and cough. The authors aimed to evaluate if there is a temporal association between cough and acid pH in ambulatory children with chronic cough. setting and patients The authors studied children (aged <14 years) with chronic cough, suspected of acid reflux and considered for pHmetry using a specifically built ambulatory pHmetry–cough logger that enabled the simultaneous ambulatory recording of cough and pH with a fast (10 Hz) capture rate. Main outcome measures Coughs within (before and after) 10, 30, 60 and 120 s of a reflux episode (pH<4 for >0.5 s). Results Analysis of 5628 coughs in 20 children. Most coughs (83.9%) were independent of a reflux event. Cough–reflux (median 19, IQR 3–45) and reflux–cough (24.5, 13–51) sequences were equally likely to occur within 120 s. Within the 10 and 30 s time frame, reflux–cough (10 s=median 2.5, IQR 0–7.25; 30 s=6.5, 1.25–22.25) sequences were significantly less frequent than reflux–no cough (10 s=27, IQR 15–65; 30 s=24.5, 14.5–55.5) sequences, (p=0.0001 and p=0.001, respectively). No differences were found for 60 and 120 s time frame. Cough–reflux sequence (median 1.0, IQR 0–8) within 10 s was significantly less (p=0.0001) than no cough–reflux sequences (median 29.5, 15–67), within 30 s (p=0.006) and 60 s (p=0.048) but not within 120 s (p=0.47). Conclusions In children with chronic cough and suspected of having gastro-oesophageal reflux disease, the temporal relationship between acid reflux and cough is unlikely causal.

The use of phospholipid fatty acid analysis to measure impact of acid rock drainage on microbial communities in sediments

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0-0.1 km), compared with sites further downstream (2.5-18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments' metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:2ω6; the sulfate-reducing bacteria marker 10Me16:1ω7; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.

Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.

Weight-related differences in glucose metabolism and free fatty acid production in two South African population groups

OBJECTIVE The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) alpha and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS Anthropometric and various metabolic indices were measured at fasting and during a 7h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of (13)CO(2) to (12)CO(2) in breath following ingestion of 1-(13)C-labelled glucose. SUBJECTS Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS Visceral fat levels were significantly higher (P < 0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects (P < 0.05 for both) and was found to correlate negatively with weight (r = -0.69, P < 0.01) after correcting for age. Basal TNF alpha concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OCTT was 24 +/- 12% in OWW and - 38 +/- 23% (P < 0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120 - 420 min) was significantly higher in OWW than OBW (P < 0.01) and LWW (P < 0.01) and correlated positively with visceral fat mass independent of age (r = 0.78, P < 0.05) in the OWW only. Leptin levels were higher (P < 0.01) both at fasting and during the course of the OCTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

No evidence for association of ataxia-telangiectasia mutated gene T2119C and C3161G amino acid substitution variants with risk of breast cancer

Background There is evidence that certain mutations in the double-strand break repair pathway ataxia-telangiectasia mutated gene act in a dominant-negative manner to increase the risk of breast cancer. There are also some reports to suggest that the amino acid substitution variants T2119C Ser707Pro and C3161G Pro1054Arg may be associated with breast cancer risk. We investigate the breast cancer risk associated with these two nonconservative amino acid substitution variants using a large Australian population-based case–control study. Methods The polymorphisms were genotyped in more than 1300 cases and 600 controls using 5' exonuclease assays. Case–control analyses and genotype distributions were compared by logistic regression. Results The 2119C variant was rare, occurring at frequencies of 1.4 and 1.3% in cases and controls, respectively (P = 0.8). There was no difference in genotype distribution between cases and controls (P = 0.8), and the TC genotype was not associated with increased risk of breast cancer (adjusted odds ratio = 1.08, 95% confidence interval = 0.59–1.97, P = 0.8). Similarly, the 3161G variant was no more common in cases than in controls (2.9% versus 2.2%, P = 0.2), there was no difference in genotype distribution between cases and controls (P = 0.1), and the CG genotype was not associated with an increased risk of breast cancer (adjusted odds ratio = 1.30, 95% confidence interval = 0.85–1.98, P = 0.2). This lack of evidence for an association persisted within groups defined by the family history of breast cancer or by age. Conclusion The 2119C and 3161G amino acid substitution variants are not associated with moderate or high risks of breast cancer in Australian women.