Enhanced laccase-mediated transformation of diclofenac and flufenamic acid in the presence of bisphenol A and testing of an enzymatic membrane reactor

The inadequate removal of pharmaceuticals and other micropollutants in municipal wastewater treatment plants, as evidenced by their detection of these substances in the aquatic environment has led to the need for sustainable remediation strategies. Laccases possess a number of advantages including a broad substrate spectrum. To identify promoting or inhibitory effects of reaction partners in the remediation processes we tested not only single compounds-as has been described in most studies-but also mixtures of pollutants. The reaction of diclofenac (DCF) and flufenamic acid (FA), mediated by Trametes versicolor laccase resulted in the formation of products, which were more hydrophilic than the respective reactant (reactant concentration of 0.1 mM; laccase activity 0.5 U/ml). Analyses (HPLC, LC/MS) showed that the product 1a and 1b for DCF and FA, respectively, to be a para-benzoquinone imine derivative. The formation of 1a was enhanced by the addition of bisphenol A (BPA). After 6 days 97% more product was formed in the mixture of DCF and BPA compared with DCF tested alone. Product 1a was also detected in experiments with micropollutant-supplemented secondary effluent. Within 24 h 67% and 100% of DCF and BPA were transformed, respectively (25 U/ml). Experiments with a membrane reactor (volume 10 l; phosphate buffer, pH 7) were in good agreement with the results of the laboratory scale experiments (50 ml). EC50-values were also determined. The data support the use of laccases for the removal or detoxification of recalcitrant pollutants. Thus, the enzyme laccase may be a component of an additional environmentally friendly process for the treatment stage of wastewater remediation

Balance and gait performance after maximal and submaximal endurance exercise in seniors: is there a higher fall-risk?

Impaired balance and gait performance increase fall-risk in seniors. Acute effects of different exercise bouts on gait and balance were not yet addressed. Therefore, 19 healthy seniors (10 women, 9 men, age: 64.6±3.2years) were examined on 3days. After exhaustive treadmill testing, participants randomly completed a 2-km treadmill walking test (76±8% VO2max) and a resting control condition. Standing balance performance (SBALP) was assessed by single limb-eyes opened (SLEO) and double limb-eyes closed (DLEC) stance. Gait parameters were collected at comfortable walking velocity. A condition×time interaction of center of pressure path length (COPpath) was observed for both balance tasks (p<0.001). Small (Cohen's d=0.42, p=0.05) and large (d=1.04, p<0.001) COPpath increases were found after 2-km and maximal exercise during DLEC. Regarding SLEO, slightly increased COPpath occurred after 2-km walking (d=0.29, p=0.65) and large increases after exhaustive exercise (d=1.24, p<0.001). No significant differences were found for gait parameters. Alterations of SBALP after exhaustive exercise might lead to higher fall-risk in seniors. Balance changes upon 2-km testing might be of minor relevance. Gait is not affected during single task walking at given velocitie

Arxula adeninivorans Recombinant Urate Oxidase and Its Application in the Production of Food with Low Uric Acid Content

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food

Arxula adeninivorans Recombinant Guanine Deaminase and Its Application in the Production of Food with Low Purine Content

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods

Comparison of different microbial laccases as tools for industrial uses

6Laccases from different sources are employed in a number of biotechnological processes, each characterized by specific reaction constraints and thus requiring an enzyme with suitable properties. In order to avoid the bias generated by different assay methodologies, in this work we investigated the main properties of ten laccases from fungi and bacteria under identical conditions. As a general rule, the optimal activity was apparent at pH 3-4 and was lost at pH≥7.0 (all laccases were stable at pH≥7.0); enzymes active at neutral pH values were also identified. For all tested laccases, activity increased with temperature up to 80°C and stability was good at 25°C. Interestingly, laccases insensitive to high salt concentration were identified, this favoring their use in treating waste waters. Indeed, bacterial laccases retained a significant activity in the presence of DMSO (up to 40% final concentration) and of surfactants, suggesting that they can be applied in lignin degradation processes requiring solvents. The available laccases are versatile and satisfy requirements related to different processes. Notably, the recombinant laccase from Bacillus licheniformis favorably compares with the tested enzymes, indicating that it is well suited for different biotechnological applications.noneTonin, Fabio; Melis, Roberta; Cordes, Arno; Sanchez-Amat, Antonio; Pollegioni, Loredano; Rosini, ElenaTonin, Fabio; Melis, Roberta; Cordes, Arno; Sanchez Amat, Antonio; Pollegioni, Loredano; Rosini, Elen

Aerobic, resistance and combined exercise training on arterial stiffness in normotensive and hypertensive adults: A review

Exercise training has different effects on arterial stiffness according to training modalities. The optimal exercise modality for improvement of arterial function in normotensive and hypertensive individuals has not been well established. In this review, we aim to evaluate the effects of aerobic, resistance and combined aerobic and resistance training on arterial stiffness in individuals with and without hypertension. We systematically searched the Pubmed and Web of Science database from 1985 until December 2013 for relevant randomised controlled trials (RCTs). The data were extracted by one investigator and checked by a second investigator. The training effects on arterial stiffness were estimated using weighted mean differences of the relative changes (%) with 95% confidence intervals (CIs). We finally reviewed the results from 17 RCTs. The available evidence indicates that aerobic exercise tends to have a beneficial effect on arterial stiffness in normotensive and hypertensive patients, but does not affect arterial stiffness in patients with isolated systolic hypertension. Resistance exercise has differing effects on arterial stiffness depending on type and intensity. Vigorous resistance training is associated with an increase in arterial stiffness. There seem to be no unfavourable effects on arterial stiffness if the training is of low intensity, in a slow eccentric manner or with lower limb in healthy individuals. Combined training has neutral or even a beneficial effect on arterial stiffness. In conclusion, our review shows that exercise training has varying effects on arterial stiffness depending on the exercise modalities

Enhanced laccase-mediated transformation of diclofenac and flufenamic acid in the presence of bisphenol A and testing of an enzymatic membrane reactor

Abstract The inadequate removal of pharmaceuticals and other micropollutants in municipal wastewater treatment plants, as evidenced by their detection of these substances in the aquatic environment has led to the need for sustainable remediation strategies. Laccases possess a number of advantages including a broad substrate spectrum. To identify promoting or inhibitory effects of reaction partners in the remediation processes we tested not only single compounds—as has been described in most studies—but also mixtures of pollutants. The reaction of diclofenac (DCF) and flufenamic acid (FA), mediated by Trametes versicolor laccase resulted in the formation of products, which were more hydrophilic than the respective reactant (reactant concentration of 0.1 mM; laccase activity 0.5 U/ml). Analyses (HPLC, LC/MS) showed that the product 1a and 1b for DCF and FA, respectively, to be a para-benzoquinone imine derivative. The formation of 1a was enhanced by the addition of bisphenol A (BPA). After 6 days 97% more product was formed in the mixture of DCF and BPA compared with DCF tested alone. Product 1a was also detected in experiments with micropollutant-supplemented secondary effluent. Within 24 h 67% and 100% of DCF and BPA were transformed, respectively (25 U/ml). Experiments with a membrane reactor (volume 10 l; phosphate buffer, pH 7) were in good agreement with the results of the laboratory scale experiments (50 ml). EC50-values were also determined. The data support the use of laccases for the removal or detoxification of recalcitrant pollutants. Thus, the enzyme laccase may be a component of an additional environmentally friendly process for the treatment stage of wastewater remediation

Diurnal variation of arterial stiffness in healthy individuals of different ages and patients with heart disease

Arterial stiffness can be measured using various non-invasive methods. It is not well established whether it is necessary to standardize the time of the day when performing these measurements. The aim of the present study is to examine the effect of daytime on arterial stiffness in individuals with and without heart disease.; We investigated the diurnal variation of cardio-ankle vascular index (CAVI) and carotid femoral pulse wave velocity (cfPWV). CAVI and cfPWV were measured in 70 participants (23 healthy young individuals [HY], 22 healthy elderly individuals [HE], 25 patients with heart disease [HD]) at 09:00, 13:00 and 17:00 h.; There was a significant diurnal variation in CAVI with the highest values at 09:00 h in both univariate and multivariate analysis. After adjusting for age, sex and MAP (mean arterial pressure), CAVI maintained a significant highest values at 09:00 h, which was 4% higher than at 13:00 h (p = 0.022) and 5% higher than at 17:00 h (p = 0.002). However, a lack of diurnal variation was found in cfPWV in multivariate analysis in our study population.; Our findings suggest that it does not appear mandatory to measure cfPWV at the same time of day. However, standardizing the time of day for CAVI is important in routine clinical practice and longitudinal studies

Balance and gait performance after maximal and submaximal endurance exercise in seniors: is there a higher fall-risk?

Impaired balance and gait performance increase fall-risk in seniors. Acute effects of different exercise bouts on gait and balance were not yet addressed. Therefore, 19 healthy seniors (10 women, 9 men, age: 64.6 ± 3.2 years) were examined on 3 days. After exhaustive treadmill testing, participants randomly completed a 2-km treadmill walking test (76 ± 8 % VO(2max)) and a resting control condition. Standing balance performance (SBALP) was assessed by single limb-eyes opened (SLEO) and double limb-eyes closed (DLEC) stance. Gait parameters were collected at comfortable walking velocity. A condition × time interaction of center of pressure path length (COP(path)) was observed for both balance tasks (p &lt; 0.001). Small (Cohen's d = 0.42, p = 0.05) and large (d = 1.04, p &lt; 0.001) COP(path) increases were found after 2-km and maximal exercise during DLEC. Regarding SLEO, slightly increased COP(path) occurred after 2-km walking (d = 0.29, p = 0.65) and large increases after exhaustive exercise (d = 1.24, p &lt; 0.001). No significant differences were found for gait parameters. Alterations of SBALP after exhaustive exercise might lead to higher fall-risk in seniors. Balance changes upon 2-km testing might be of minor relevance. Gait is not affected during single task walking at given velocities