Long-Lasting Priming of Endothelial Cells by Plasma Melatonin Levels

Background: Endothelial cells are of great interest for cell therapy and tissue engineering. Understanding the heterogeneity among cell lines originating from different sources and culture protocols may allow more standardized material to be obtained. In a recent paper, we showed that adrenalectomy interferes with the expression of membrane adhesion molecules on endothelial cells maintained in culture for 16 to 18 days. In addition, the pineal hormone, melatonin, reduces the adhesion of neutrophils to post-capillary veins in rats. Here, we evaluated whether the reactivity of cultured endothelial cells maintained for more than two weeks in culture is inversely correlated to plasma melatonin concentration. Methodology/Principal Findings: The nocturnal levels of melatonin were manipulated by treating rats with LPS. Nocturnal plasma melatonin, significantly reduced two hours after LPS treatment, returned to control levels after six hours. Endothelial cells obtained from animals that had lower nocturnal melatonin levels significantly express enhanced adhesion molecules and iNOS, and have more leukocytes adhered than cells from animals that had normal nocturnal levels of melatonin (naive or injected with vehicle). Endothelial cells from animals sacrificed two hours after a simultaneous injection of LPS and melatonin present similar phenotype and function than those obtained fromcontrol animals. Analyzing together all the data, taking into account the plasma melatonin concentration versus the expression of adhesion molecules or iNOS we detected a significant inverse correlation. Conclusions/Significance: Our data strongly suggest that the plasma melatonin level primes endothelial cells ""in vivo,"" indicating that the state of the donor animal is translated to cells in culture and therefore, should be considered for establishing cell banks in ideal conditions.

Retention of progenitor cell phenotype in otospheres from guinea pig and mouse cochlea

Background: Culturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy. Methods: Organs of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGF alpha). Immunofluorescence assays were conducted for phenotype characterization. Results: The TGF alpha group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells. Conclusions: Dissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.

EPR study of local symmetry sites of Ce(3+) in Pb(1-x)Ce(x)A (A=S, Se, and Te)

The local site symmetry of Ce(3+) ions in the diluted magnetic semiconductors Pb(1-x)Ce(x)A (A=S, Se, and Te) has been investigated by electron-paramagnetic resonance (EPR). The experiments were carried out on single crystals with cerium concentration x ranging from 0.001 to 0.035. The isotropic line due to Ce(3+) ions located at the substitutional Pb cation site with octahedral symmetry was observed for all the studied samples. We determined the effective Lande factors to be g=1.333, 1.364, and 1.402 for A=S, Se, and Te, respectively. The small difference with the predicted Lande factor g of 10/7 for the Gamma(7) (J=5/2) ground state was attributed to crystal-field admixture. In addition, EPR lines from Ce(3+) ions located at sites with small distortion from the original octahedral symmetry were also observed. Two distinct sites with axial distortion along the < 001 > crystallographic direction were identified and a third signal in the spectrum was attributed to sites with the cubic symmetry distorted along the < 110 > direction. The distortion at these distinct Ce sites is attributed to Pb lattice vacancies near the cerium ions that compensate for its donor activity.

Enzymatic Resolution of Racemic Sulcatol by Lipase from Candida Antarctica in a Large Scale

Large scale enzymatic resolution of racemic sulcatol 2 has been useful for stereoselective biocatalysis. This reaction was fast and selective, using vinyl acetate as donor of acyl group and lipase from Candida antarctica (CALB) as catalyst. The large scale reaction (5.0 g, 39 mmol) afforded high optical purities for S-(+)-sulcatol 2 and R-(+)-sulcatyl acetate 3, i.e., ee > 99 per cent and good yields (45 per cent) within a short time (40 min). Thermodynamic parameters for the chemoesterification of sulcatol 2 by vinyl acetate were evaluated. The enthalpy and Gibbs free energy values of this reaction were negative, indicating that this process is exothermic and spontaneous which is in agreement with the reaction obtained enzymatically.

Screening of lipases for the synthesis of xylitol monoesters by chemoenzymatic esterification and the potential of microwave and ultrasound irradiations to enhance the reaction rate

Lipases from different sources, Pseudomonas fluorescens (AK lipase), Burkholderia cepacia (PS lipase), Penicillium camembertii (lipase G) and Porcine pancreas lipase (PPL), previously immobilized on epoxy SiO(2)-PVA, were screened for the synthesis of xylitol monoesters by esterification of the protected xylitol using oleic acid as acyl donor group. Among all immobilized derivatives, the highest esterification yield was achieved by P. camembertii lipase, showing to be attractive alternative to bulk chemical routes to satisfy increasing commercial demands. Further experiments were performed to determine the influence of fatty acids chain size on the reaction yield and the feasibility of using non-conventional heating systems (microwave and ultrasound irradiations) to enhance the reaction rate. (C) 2010 Elsevier B.V. All rights reserved.

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica-PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 degrees C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1: 15 at 55 degrees C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production. (C) 2009 Elsevier Ltd. All rights reserved.

Organ donation: Acceptance and refusal among users of the public health system from Brazil

The present study sought to identify the perception of a given group of users of the Brazilian Public Health System (Sistema Unico de Saude) regarding organ donation and to implement an educational policy. Structured interviews were conducted with the aim of describing the profile of donor and nondonor subjects, the importance of organ donation, and the knowledge regarding donation and brain death. One hundred subjects were interviewed: 33% of them considered themselves potential donors; 40% were donors; 13% were nondonors; and 14% were ill-informed potential donors. However, only 40% of users have already officially expressed to their families a willingness to donate. Regarding their knowledge about the propitious moment for organ donation, only 64% of them associated the donation act with brain death. Although the present results revealed that users of the Brazilian Public Health System are prone to organ donation, there actually was a high amount of refusals, which may be due to lack of information and knowledge regarding the donation-transplantation process.

A novel aerobic-anoxic biological filter for nitrogen removal from UASB effluent using biogas compounds as electron donors for denitrification

The performance of a new trickling filter (TF) configuration composed of an upper compartment for nitrification and a lower compartment for denitrification of effluent from a UASB reactor treating domestic sewage was evaluated. The TF was packed with new plastic material characterized by its durability and high percentage of void spaces. The feasibility of using the reduced compounds present in the biogas produced by a UASB reactor as electron donor for denitrification was also evaluated. Efficient nitrification and denitrification was achieved for the mean hydraulic (5.6 m(3) m(-2) d(-1)) organic (0.26 kg COD m(-3) d(-1)) and ammonia-N (0.08 kg m(-3) d(-1)) loading rates applied, resulting in ammonia-N removal ranging from 60 to 74%. The final effluent presented ammonia-N lower than 13 mg L(-1). Despite the presence of dissolved oxygen (DO) in the denitrification compartment, its performance was considered quite satisfactory and final nitrate concentrations were lower than 10 mg L(-1). The results indicate that methane was the main electron donor used for denitrification. Additionally, denitrification can probably be improved by avoiding high DO concentration in the denitrification compartment and by enhancing biogas transfer in the anoxic zone.

Removal of ammonium via simultaneous nitrification-denitrification nitrite-shortcut in a single packed-bed batch reactor

A polyurethane packed-bed-biofilm sequential batch reactor was fed with synthetic substrate simulating the composition of UASB reactor effluents. Two distinct ammonia nitrogen concentrations (125 and 250 mg l(-1)) were supplied during two sequential long-term experiments of 160 days each (320 total). Cycles of 24 h under intermittent aeration for periods of 1 h were applied, and ethanol was added as a carbon source at the beginning of each anoxic period. Nitrite was the main oxidized nitrogen compound which accumulated only during the aerated phases of the batch cycle. A consistent decrease of nitrite concentration started always immediately after the interruption of oxygen supply and addition of the electron donor. Removal to below detection limits of all nitrogen soluble forms was always observed at the end of the 24 h cycles for both initial concentrations. Polyurethane packed-bed matrices and ethanol amendments conferred high process stability. Microbial investigation by cloning suggested that nitrification was carried out by Nitrosomonas-like species whereas denitrification was mediated by unclassified species commonly observed in denitrifying environments. The packed-bed batch bioreactor favored the simultaneous colonization of distinct microbial groups within the immobilized microbial biomass. The biofilm was capable of actively oxidizing ammonium and denitrification at high ratios in intermittent intervals within 24 h cycles. (c) 2008 Elsevier Ltd. All rights reserved.

Temperature and feed strategy effects on sulfate and organic matter removal in an AnSBB

The objective of this work was to analyze the interaction effects between temperature, feed strategy and COD/[SO(4)(2-)] levels, maintaining the same ratio, on sulfate and organic matter removal efficiency from a synthetic wastewater. This work is thus a continuation of Archilha et al. (2010) who studied the effect of feed strategy at 30 degrees C using different COD/[SO] ratios and levels. A 3.7-L anaerobic sequencing batch reactor with recirculation of the liquid phase and which contained immobilized biomass on polyurethane foam (AnSBBR) was used to treat 2.0 L synthetic wastewater in 8 h cycles. The temperatures of 15, 22.5 and 30 degrees C with two feed strategies were assessed: (a) batch and (b) batch followed by fed-batch. In strategy (a) the reactor was fed in 10 min with 2 L wastewater containing sulfate and carbon sources. In strategy (b) 1.2 L wastewater (containing only the sulfate source) was fed during the first 10 min of the cycle and the remaining 0.8 L (containing only the carbon source) in 240 min. Based on COD/[SO(4)(2-)] = 1 and on the organic matter (0.5 and 1.5 gCOD/L) and sulfate (0.5 and 1.5 gSO(4)(2-)/L) concentrations, the sulfate and organic matter loading rates applied were 1.5 and 4.5 g/L.d, i.e., same COD/[SO(4)(2-)] ratio (=1) but different levels (1.5/1.5 and 4.5/4.5 gCOD/gSO(4)(2-)). When reactor feed was 1.5 gCOD/L.d and 1.5 gSO(4)(2-)/L.d, gradual feeding (strategy b) showed to favor sulfate and organic matter removal in the investigated temperature range, indicating improved utilization of the electron donor for sulfate reduction. Sulfate removal efficiencies were 87.9; 86.3 and 84.4%, and organic matter removal efficiencies 95.2; 86.5 and 80.8% at operation temperatures of 30; 22.5 and 15 degrees C, respectively. On the other hand, when feeding was 4.5 gCOD/L.d and 4.5 gSO(4)(2-)/L.d, gradual feeding did not favor sulfate removal, indicating that gradual feeding of the electron donor did not improve sulfate reduction. (C) 2011 Elsevier Ltd. All rights reserved.

Feasibility of a Sequencing Reactor Operated in Batch and Fed-batch Mode Applied to Nitrification and Denitrification Processes

The objective of this work was to study the operational feasibility of nitrification and denitrification processes in a mechanically stirred sequencing batch reactor (SBR) operated in batch and fed-batch mode. The reactor was equipped with a draft-tube to improve mass transfer and contained dispersed (aerobic) and granulated (anaerobic) biomass. The following reactor variables were adjusted: aeration time during the nitrification step; dissolved oxygen concentration, feed time defining batch and fed-batch phases, concentration of external carbon source used as electron donor during the denitrification stage and volumetric ammonium nitrogen load in the influent. The reactor (5 L volume) was maintained at 30 +/- 1 degrees C and treated either 1.0 or 1.5 L wastewater in 8-h cycles. Ammonium nitrogen concentrations assessed were: 50 (condition 1) and 100 mgN-NH(4)(+).L(-1) (condition 2), resulting in 29 and 67 mgN-NH(4)(+).L-1-d(-1), respectively. A synthetic medium and ethanol were used as external carbon sources (ECS). Total nitrogen removal efficiencies were 94.4 and 95.9% when the reactor was operated under conditions 1 and 2, respectively. Low nitrite (0.2 and 0.3 mgN-NO(2)(-).L(-1), respectively) and nitrate (0.01 and 0.3 mgN-NO(3)(-).L(-1), respectively) concentrations were detected in the effluent and ammonium nitrogen removal efficiencies were 97.6% and 99.6% under conditions 1 and 2, respectively.

Influence of carbon source and inoculum type on anaerobic biomass adhesion on polyurethane foam in reactors fed with acid mine drainage

This paper analyzes the influence of carbon source and inoculum origin on the dynamics of biomass adhesion to an inert support in anaerobic reactors fed with acid mine drainage. Formic acid, lactic acid and ethanol were used as carbon sources. Two different inocula were evaluated: one taken from an UASB reactor and other from the sediment of a uranium mine. The values of average colonization rates and the maximum biomass concentration (C(max)) were inversely proportional to the number of carbon atoms in each substrate. The highest C(max) value (0.35 g TVS g(-1) foam) was observed with formic acid and anaerobic sludge as inoculum. Maximum colonization rates (v(max)) were strongly influenced by the type of inoculum when ethanol and lactic acid were used. For both carbon sources, the use of mine sediment as inoculum resulted in a v(max) of 0.013 g TVS g(-1) foam day(-1), whereas 0.024 g TVS g(-1) foam day(-1) was achieved with anaerobic sludge. (C) 2011 Elsevier Ltd. All rights reserved.

Violet ZnSe/ZnS as an Alternative to Green CdSe/ZnS in Nanocrystal-Fluorescent Protein FRET Systems

Fluorescent proteins from the green fluorescent protein family strongly interact with CdSe/ZnS and ZnSe/ZnS nanocrystals at neutral pH. Green emitting CdSe/ZnS nanocrystals and red emitting fluorescent protein dTomato constitute a 72% efficiency FRET system with the largest alteration of the overall photoluminescence profile, following complex formation, observed so far. The substitution of ZnSe/ZnS for CdSe/ZnS nanocrystals as energy donors enabled the use of a green fluorescent protein, GFP5, as energy acceptor. Violet emitting ZnSe/ZnS nanocrystals and green GFP5 constitute a system with 43% FRET efficiency and an unusually strong sensitized emission. ZnSe/ZnS-GFP5 provides a cadmium-free, high-contrast FRET system that covers only the high-energy part of the visible spectrum, leaving room for simultaneous use of the yellow and red color channels. Anisotropic fluorescence measurements confirmed the depolarization of GFP5 sensitized emission.

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40-CD40L system

The vascular effects of nitrolinoleate (LNO(2)), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO(2) was capable to deliver free radical nitric oxide ((center dot)NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO(2) for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO(2) were observed in vivo by intravital microscopy assays. LNO(2) decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO(2) reduced mRNA and protein expression of 2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO(2) on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO(2) involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO(2) inhibited adhesion molecules expression and promoted (center dot)NO inactivation of the CD40-CD40L system, both important processes of the inflammatory response. (C) 2010 Elsevier Inc. All rights reserved.

In Vitro Simultaneous Transfer of Lipids to HDL in Coronary Artery Disease and in Statin Treatment

The exchange of lipids with cells and other lipoproteins is a crucial process in HDL metabolism and for HDL antiatherogenic function. Here, we tested a practical method to quantify the simultaneous transfer to HDL of phospholipids, free-cholesterol, esterified cholesterol and triacylglycerols and to verify the lipid transfer in patients with coronary artery disease (CAD) or undergoing statin treatment. Twenty-eight control subjects without CAD, 27 with CAD and 25 CAD patients under simvastatin treatment were studied. Plasma samples were incubated with a donor nanoemulsion prepared by ultrasonication of the constituent lipids and labeled with radioactive lipids; % lipids transferred to HDL were quantified in the HDL-containing supernatant after chemical precipitation of non-HDL fractions and the nanoemulsion. The assay was precise and reproducible. Increase of temperature (4-37 A degrees C), of incubation period (5 min to 2 h), of HDL-cholesterol concentration (33-244 mg/dL) and of mass of nanoemulsion lipids (0.075-0.3 mg/mu L) resulted in increased lipid transfer from the nanoemulsion to HDL. In contrast, increasing pH (6.5-8.5) and albumin concentration (3.5-7.0 g/dL) did not affect lipid transfer. There was no difference between CAD and control non-CAD with regard to the lipid transfer, but statin treatment reduced the transfer to HDL of all four lipids. The test herein described is a valid and practical tool for exploring an important aspect of HDL metabolism.