PHK from phenol hydroxylase of Pseudomonas sp. OX1. Insight into the role of an accessory protein in bacterial multicomponent monooxygenases

Bacterial multicomponent monooxygenases (BMMs) are members of a wide family of diiron enzymes that use molecular oxygen to hydroxylate a variety of aromatic compounds. The presence of genes encoding for accessory proteins not involved in catalysis and whose role is still elusive, is a common feature of the gene clusters of several BMMs, including phenol hydroxylases and several soluble methane monooxygenases. In this study we have expressed, purified, and partially characterized the accessory component PHK of the phenol hydroxylase from Pseudomonas sp. OX1, a bacterium able to degrade several aromatic compounds. The phenol hydroxylase (ph) gene cluster was expressed in Escherichia coli/JM109 cells in the absence and in the presence of the phk gene. The presence of the phk gene lead to an increase in the hydroxylase activity of whole recombinant cells with phenol. PHK was assessed for its ability to interact with the active hydroxylase complex. Our results show that PHK is neither involved in the catalytic activity of the phenol hydroxylase complex nor required for the assembly of apo-hydroxylase. Our results suggest instead that this component may be responsible for enhancing iron incorporation into the active site of the apo-hydroxylase

Evidences on overweight of regular blood donors in a center of Southern Italy

Objective: Smoking, unhealthy diet and obesity, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders. Blood centers can play a public health role in obesity surveillance and interventions. The purpose study was to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. Methods: A descriptive cross-sectional face-to-face questionnaire was developed. It included a 41 item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. Donors were regular repeat blood donors, eligible to donate. Results: Of the 2468 blood donors enrolled between July 2017 and January 2018, 1390 were repeat donors. Only 205 agreed to respond to the questionnaire. Data showed that donors followed mainly a Mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. The prevalence of overweight was found 50.7% in men and 16.9% in women. Conclusions: Overweight and obesity are common among regular blood donors and it is more frequent in men than women. The female blood donors seem to be characterized by better knowledge on the relationship between lifestyle and health, and by a better “putting into practice” of the information possessed

Nanocarriers for topical delivery of resveratrol.

Purpose: The aim of the present work was to develop a nanocarrier-based formulation for topical delivery of resveratrol. Methods: Trans-resveratrol (t-res) was encapsulated in liposomes, ethosomes or transferosomes, by a modified hand-shaking method followed by extrusion. All the formulations were characterised in terms of mean diameter, size distribution (I.P.), t-res loading, t-res stability upon encapsulation during storage. The nanocarriers containing t-res were then introduced in cellulose-based gel to allow their final administration on the skin and the viscoelastic properties of the resulting formulation were investigated. Finally, we studied the inhibition of reactive oxygen species (ROS) in human keratinocyte (HaCaT) cell line stimulated with H2O2 for 24 h and then incubated with the t-res containing nanocarriers. Results: All the t-res containing carriers were characterised by a very high (close to 100%) encapsulation efficiency, a negligible t-res release at 4°C and stability of resveratrol in its trans form. The carriers only slightly influenced the viscoelastic characteristics of cellulose-based gels. Nanocarriers encapsulating t-res reduced, in a concentration-dependent manner, ROS production induced by H2O2 and this effect was higher when using t-res-encapsulating nanocarrier, with the higher effect observed in the case of ethosomes. Conclusions: In this work nanocarriers with high encapsulation efficiency, high physical stability and negligible t-res release during storage at 4°C were prepared. To allow their final administration on the skin, the nanocarrier can be easilisy loaded in cellulose-based gels without altering its rheological properties. Moreover, the use of t-res-encapsulating ethosomes led to an efficient antioxidant activity. Further ex vivo and in vivo studies will clarify the role of the different carrier when administered on the skin

Metabolic Alterations in Obstructive Jaundice: Effect of Duration of Jaundice and Bile-Duct Decompression

We examined the effect of prolonged bile duct obstruction, and subsequent biliary decompression, on biochemical and metabolic parameters, using a reversible jaundice model in male Fischer 344 rats. The animals were studied after biliary obstruction for varying periods (4 days, one week, and two weeks) and following decompression. They were sacrificed one or two weeks following decompression. All the rats were compared to sham operated, pair-fed, controls. Obstructive jaundice rapidly increased bilirubin, liver enzymes, serum free fatty acid, and triglyceride levels. Glucose levels were significantly decreased in the jaundice rats compared to their pair-fed controls. Only after two weeks of jaundice was significant hypoalbuminemia observed. Following decompression, all biochemical and metabolic values gradually returned to normal levels, except for albumin. Hypoalbuminemia was not reversed within the two-week post-decompression period. The rats jaundiced for two weeks had significantly higher mortality, compared to the other groups. We conclude that prolonged jaundice adversely affects the metabolic capacity of the rats, with albumin concentration being markedly decreased, and that biliary decompression could not reverse completely all the alterations seen with cholestasis, especially following two weeks of bile duct obstruction

Regional Body-Wave Attenuation Using a Coda Source Normalization Method: Application to MEDNET Records of Earthquakes in Italy

We develop a new methodology to determine apparent attenuation for the regional seismic phases Pn, Pg, Sn, and Lg using coda-derived source spectra. The local-to-regional coda methodology (Mayeda, 1993; Mayeda and Walter, 1996; Mayeda et al., 2003) is a very stable way to obtain source spectra from sparse networks using as few as one station, even if direct waves are clipped. We develop a two-step process to isolate the frequency-dependent Q. First, we correct the observed direct wave amplitudes for an assumed geometrical spreading. Next, an apparent Q, combining path and site attenuation, is determined from the difference between the spreading-corrected amplitude and the independently determined source spectra derived from the coda methodology. We apply the technique to 50 earthquakes with magnitudes greater than 4.0 in central Italy as recorded by MEDNET broadband stations around the Mediterranean at local-to-regional distances. This is an ideal test region due to its high attenuation, complex propagation, and availability of many moderate sized earthquakes. We find that a power law attenuation of the form Q(f) = Q{sub 0}f{sup Y} fit all the phases quite well over the 0.5 to 8 Hz band. At most stations, the measured apparent Q values are quite repeatable from event to event. Finding the attenuation function in this manner guarantees a close match between inferred source spectra from direct waves and coda techniques. This is important if coda and direct wave amplitudes are to produce consistent seismic results

Robust Classification via Support Vector Machines

Classification models are very sensitive to data uncertainty, and finding robust classifiers that are less sensitive to data uncertainty has raised great interest in the machine learning literature. This paper aims to construct robust Support Vector Machine (SVM) classifiers under feature data uncertainty via two probabilistic arguments. The first classifier, Single Perturbation (SP-SVM), reduces the local effect of data uncertainty with respect to one given feature and acts as a local test that could confirm or refute the presence of significant data uncertainty for that particular feature. The second classifier, Extreme Empirical Loss (EEL-SVM), aims to reduce the aggregate effect of data uncertainty with respect to all features, which is possible via a trade-off between the number of prediction model violations and the size of these violations. Both methodologies are computationally efficient and our extensive numerical investigation highlights the advantages and possible limitations of the two robust classifiers on synthetic and real-life data

Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxyge

Value within otolaryngology: Assessment of the cost-utility analysis literature

AbstractObjectiveTo assess the characteristics and quality of cost utility analyses (CUA) related to otolaryngology within the CEA registry and to summarize their collective results.MethodsAll cost-utility analyses published between 1976 and 2011 contained in the Cost-Effectiveness Analysis Registry (CEA Registry) were evaluated. Topics that fall within the care of an otolaryngologist were included in the review regardless of the presence of an otolaryngologist author. Potential associations between various study characteristics and CEA registry quality scores were evaluated using the Pearson product moment correlation coefficient.ResultsSixty-one of 2913 (2.1%) total CUA publications screened were related to otolaryngology. Eighteen of 61 (29.5%) publications included an otolaryngologist as an author. Fourteen studies agreed on the cost effectiveness of at least unilateral cochlear implantation and six of seven (85.7%) studies demonstrated the cost effectiveness of continuous positive airway pressure (CPAP) for obstructive sleep apnea (OSA). Forty-six percent (28 of 61) of all manuscripts were published between 2008 and 2011. A more recent publication year was associated with a higher CEA registry quality score while the presence of an otolaryngologist author and journal impact factor had no significant correlation with the quality of the CUA.ConclusionBased on current evidence in the CEA registry, unilateral cochlear implantation for hearing loss and CPAP for OSA are both cost-effective therapeutic interventions. Although CUAs in otolaryngology have increased in quantity and improved in quality in more recent years, there is a relative lack of CUAs in otolaryngology in comparison to other subspecialties