Free Release Research Articles

Proton pumping in cytochrome c oxidase: Energetic requirements and the role of two proton channels

Cytochrome c oxidase is a superfamily of membrane bound enzymes catalyzing the exergonic reduction of molecular oxygen to water, producing an electrochemical gradient across the membrane. The gradient is formed both by the electrogenic chemistry, taking electrons and protons from opposite sides of the membrane, and by proton pumping across the entire membrane. In the most efficient subfamily, the A-family of oxidases, one proton is pumped in each reduction step, which is surprising considering the fact that two of the reduction steps most likely are only weakly exergonic. Based on a combination of quantum chemical calculations and experimental information, it is here shown that from both a thermodynamic and a kinetic point of view, it should be possible to pump one proton per electron also with such an uneven distribution of the free energy release over the reduction steps, at least up to half the maximum gradient. A previously suggested pumping mechanism is developed further to suggest a reason for the use of two proton transfer channels in the A-family. Since the rate of proton transfer to the binuclear center through the D-channel is redox dependent, it might become too slow for the steps with low exergonicity. Therefore, a second channel, the K-channel, where the rate is redox-independent is needed. A redox-dependent leakage possibility is also suggested, which might be important for efficient energy conservation at a high gradient. A mechanism for the variation in proton pumping stoichiometry over the different subfamilies of cytochrome oxidase is also suggested. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Identification of the C4′-Oxidized Abasic Site as the Most Abundant 2-Deoxyribose Lesion in Radiation-Damaged DNA Using a Novel HPLC-Based Approach

A novel analytical high-performance liquid chromatography (HPLC)-based method of quantification of the yields of C4'-oxidized abasic sites, 1, in oxidatively damaged DNA has been elaborated. This new approach is based on efficient conversion of 1 into N-substituted 5-methylene-Δ(3)-pyrrolin-2-ones, 2, upon treatment of damaged DNA with primary amines in neutral or slightly acidic solutions with subsequent quantification of 2 by HPLC. The absolute and relative radiation-chemical yields of 1 in irradiated DNA solutions were re-evaluated using this method. The yields were compared with those of other 2-deoxyribose degradation products including 5-methylene-2(5H)-furanone, malondialdehyde, and furfural resulting from the C1', C4' and C5'-oxidations, respectively. The yield of free base release (FBR) determined in the same systems was employed as an internal measure of the total oxidative damage to the 2-deoxyribose moiety. Application of this technique identifies 1 as the most abundant sugar lesion in double-stranded (ds) DNA irradiated under air in solution (36% FBR). In single-stranded (ss) DNA this product is second by abundance (33% FBR) after 2-deoxyribonolactones (C1'-oxidation; 43% FBR). The production of nucleoside-5'-aldehydes (C5'-oxidation; 14% and 5% FBR in dsDNA and ssDNA, respectively) is in the third place. Taken together with the parallel reaction channel that converts C4'-radicals into malondialdehyde and 3'-phosphoglycolates, our results identify the C4'-oxidation as a prevalent pathway of oxidative damage to the sugar-phosphate backbone (50% or more of all 2-deoxyribose damages) in indirectly damaged DNA.

An Application Method of Free Burn HRR Data to Room Fire Scenarios

An application method of free burn heat release rate (HRR) data for a single seater sofa to predict the burning behavior in a compartment is proposed. A single seater sofa was burnt in an open environment to measure the HRR using a furniture calorimeter. The time-HRR curve was fitted with a model of burning of a cubic polyurethane block developed earlier. The model included the flame spread over horizontal, downward and lateral directions. The flame spread rates were increased if the block received radiative heat from external heat sources other than from the flame. The thermal radiation feedback from the flame, smoke layer, and heated wall surfaces was coupled with the burning model. Using the coupled model, the burning and spread rate of several sofas and a table in a small compartment was calculated and compared with experimental results. The model could reproduce the trend for the increase in HRR qualitatively. However, the time to spread to an adjacent object was not in good agreement. If the time to spread was given input to the model, other parameters such as compartment temperature and so on could be calculated with reasonable agreement.

Why do firms give away their patents for free?

Within the trend of increasing patent commercialisation and open innovation, a recent phenomenon where firms give away their patents free of charge can be observed. This seems contradictory to the original intention of the patent system (enabling firms to create temporary monopolies to appropriate returns from their R&D investments). Consequently, this paper explores why firms make their patents available for free and which benefits they may gain from this behaviour. Adopting the open source software phenomenon as a background and using firm data from 26 patent release cases, we identify a typology consisting of four motives of ‘free patent release approaches’: profit making, cost cutting, innovation catalysing, technology providing. Further, we discuss the motives of these firms to offer their patents as ‘open source’. We find that firms may obtain valuable technological input for subsequent innovations as well as social benefits in return for their free patent release.

Optimization of a measurement facility for radioactive waste free release by Monte Carlo simulation

A novel free release measurement facility (FRMF) was developed within the joint research project “Metrology for Radioactive Waste Management” of the European Metrology Research Programme. Before and during FRMF design and construction, Monte Carlo calculations with MCNPX and PENELOPE codes were used to optimize the thickness of the shielding, the dimensions of the container, and the shape of detector collimators. Validation of the numerical models of the FRMF detectors and the results of the optimization are discussed in the paper.

Effect of controlled extracellular oxidation–reduction potential on microbial metabolism and proteolysis in buckwheat sourdough

The aim of this study was to keep constant the extracellular oxidation–reduction potential (ORP) and to observe the microbial activity changes in gluten-free (GF) sourdough fermentations with lactic acid bacteria (Weissella cibaria, Pediococcus pentosaceus and coculture of both microorganisms). ORP (Eh7) was held constant at ca. +350 and −300 mV by gas sparging with air and N2/H2, respectively, to achieve oxidizing or reducing conditions during buckwheat sourdough fermentations. Microbial metabolism, free thiols, proteolysis and volatile compounds were monitored. Oxidizing conditions increased the acidification rate of W. cibaria and mixed culture (mix), which contains P. pentosaceus and W. cibaria. Reducing conditions exhibited a slow acidification rate and low microbial cell density upon fermentation. Oxidizing conditions changed lactic to acetic acid ratio of W. cibaria and mix culture from 7.9 ± 1.3 and 16.0 ± 0.6 (control conditions) to 0.5 ± 0.1 and 1.2 ± 0.2 (oxidizing conditions). A release of glucose and fructose was observed by W. cibaria and P. pentosaceus under oxidizing conditions. Free thiols content was increased by reducing conditions in fermentations with P. pentosaceus. Free amino nitrogen and free amino acid content were highly increased by reducing conditions in fermentations with W. cibaria. Free amino acid release was mainly influenced by low ORP and low pH values. Extracellular redox potential changes exhibited profile modifications of volatile compounds. Oxidizing conditions promoted a higher variety in volatile compounds as compared to the profile obtained under reducing conditions. It has been demonstrated that the extracellular ORP control has an influence on the microbial activity in buckwheat sourdoughs. Oxidizing and reducing conditions can influence the microbial activity differently and thus the final quality of GF sourdough. This is a new alternative approach for GF sourdough production and it could provide an improvement of raw materials, which can be used for GF bread production.

DNA Damage by the Direct Effect of Ionizing Radiation: Products Produced by Two Sequential One-Electron Oxidations

It has long been assumed that the population of radicals trapped in irradiated DNA (that is, the radicals escaping recombination) would quantitatively account for the lesions observed in DNA. Recent results indicate that this is not the case. The yield of DNA lesions exceed the yield of trappable radicals. To account for a portion of this shortfall, it is thought that some of the initially formed 2'-deoxyribose radicals undergo a second oxidation by nearby base cation radicals to form 2'-carbocations. The carbocations react to give strand breaks and free base release. Schemes are presented to account for the major oxidation products observed including 8-oxoGua, 8-oxoAde, 5-OHMeUra, and free base release. Theoretical calculations were performed to ascertain the likelihood of the second oxidation step in these reaction pathways actually occurring, and to account for base sequence dependence and various levels of hydration.

Inhibitory Effects of Ethyl Acetate-Soluble Fraction from Morus alba on Lipid Accumulation in 3T3-L1 Cells

Fruits of mulberry (Morus alba) have been widely used for therapeutic purposes in Asian countries for centuries. Treatment of 3T3-L1 cells with ethanolic extracts of M. alba decreased adipocyte differentiation at 100 microg/mL by 18.6%. Treatment suppressed mRNA levels of PPARgamma and C/EBPalpha expression in 3T3-L1 cells. However, the extract did not change free glycerol release from mature adipocytes. Thus, M. alba inhibited lipid accumulation by regulating transcription factors in 3T3-L1 adipocytes without a lipolytic effect. Among the soluble- fractions, the ethyl acetate-soluble fraction had the highest antiadipogenic effects on 3T3-L1 cells. This fraction decreasing intracellular lipid accumulation by 38.5% in response to treatment with 100 microg/mL. In addition, HPLC analysis of the ethyl acetate-soluble fraction of M. alba contained 167.7 microM of protocatechulic acid in 1 mg/mL of fraction, which inhibited lipid accumulation by 44.8% in response to treatment with 100 microM. From these results, M. alba is a possible candidate for regulating lipid accumulation in obesity.

Stability assessment of hypromellose acetate succinate (HPMCAS) NF for application in hot melt extrusion (HME)

HPMCAS is a widely used polymer in the pharmaceutical industry as an excipient. In this work, the physicochemical stability of HPMCAS was investigated for hot melt extrusion (HME) application. The reduction in zero rate viscosity (η0) of the polymer with the increase in temperature was determined using rheological evaluation prior to HME processing. The energy of activation for AS-MF determined by fitting Arrhenius model to the temperature dependent reduction in η0 was found to be slightly lower than that for the other grades of HPMCAS. Glassy yellowish HMEs were obtained using Haake Mini-Lab MicroCompounder operated at 160, 180, and 200°C and 100, 200, and 300rpm for all the grades at each temperature. Various physicochemical properties of HPMCAS such as glass transition temperature, semi-crystalline nature, solid state functional group properties, moisture content, and solution viscosity were not significantly affected by the HME processing. The most significant change was the release of acetic and succinic acid with the increase in HME temperature and speed. The free acid content release due to HME was directly proportional to the speed at lower operating temperatures. AS-LF was found to be the most stable with the lowest increase in total free acid content even at higher HME temperature and speed. Although the dissolution time was not affected due to HME for AS-LF and AS-MF grades, it was notably increased for AS-HF, perhaps due to significant reduction of succinoyl content. In conclusion, the HME processing conditions for solid dispersions of HPMCAS should be based on the acceptance levels of free acid for the drug and the drug product.

Brain susceptibility to oxidative stress in the perinatal period

Oxidative stress (OS) occurs at birth in all newborns as a consequence of the hyperoxic challenge due to the transition from the hypoxic intrauterine environment to extrauterine life. Free radical (FRs) sources such as inflammation, hyperoxia, hypoxia, ischaemia-reperfusion, neutrophil and macrophage activation, glutamate and free iron release, all increases the OS during the perinatal period. Newborns, and particularly preterm infants, have reduced antioxidant defences and are not able to counteract the harmful effects of FRs. Energy metabolism is central to life because cells cannot exist without an adequate supply of ATP. Due to its growth, the mammalian brain can be considered as a steady-state system in which ATP production matches ATP utilisation. The developing brain is particularly sensitive to any disturbances in energy generation, and even a short-term interruption can lead to long-lasting and irreversible damage. Whenever energy failure develops, brain damage can occur. Accumulating evidence indicates that OS is implicated in the pathogenesis of many neurological diseases, such as intraventricular haemorrhage, hypoxic-ischaemic encephalopathy and epilepsy.

Pathophysiology of NASH: Perspectives for a Targeted Treatment

Non alcoholic steatohepatitis (NASH) is the more severe form of nonalcoholic fatty liver disease. In NASH, fatty liver, hepatic inflammation, hepatocyte injury and fibrogenesis are associated, and this condition may eventually lead to cirrhosis. Current treatment of NASH relies on the reduction of body weight and increase in physical activity, but there is no pharmacologic treatment approved as yet. Emerging data indicate that NASH progression results from parallel events originating from the liver as well as from the adipose tissue, the gut and the gastrointestinal tract. Thus, dysfunction of the adipose tissue through enhanced flow of free fatty acids and release of adipocytokines, and alterations in the gut microbiome generate proinflammatory signals that underlie NASH progression. Additional 'extrahepatic hits' include dietary factors and gastrointestinal hormones. Within the liver, hepatocyte apoptosis, ER stress and oxidative stress are key contributors to hepatocellular injury. In addition, lipotoxic mediators and danger signals activate Kupffer cells which initiate and perpetuate the inflammatory response by releasing inflammatory mediators that contribute to inflammatory cell recruitment and development of fibrosis. Inflammatory and fibrogenic mediators include chemokines, the cannabinoid system, the inflammasome and activation of pattern-recognition receptors. Here we review the major mechanisms leading to appearance and progression of NASH, focusing on both extrahepatic signals and local inflammatory mechanisms, in an effort to identify the most promising molecular targets for the treatment of this condition.

Investigation of the radiological impact on the coastal environment surrounding a fertilizer plant

This investigation was carried out in order to assess the marine environmental radioactive pollution and the radiological impact caused by a large production plant of phosphate fertilizer, located in the Lebanese coastal zone. Natural radionuclides (238U, 235U, 232Th, 226Ra, 210Po, 210Pb, 40K) and anthropogenic 137Cs were measured by alpha and gamma spectrometry in seawater, sediment, biota and coastal soil samples collected from the area impacted by this industry. The limited environmental monitoring program within 2 km of the plant indicates localized contamination with radionuclides of the uranium decay chain mainly due to the transport, the storage of raw materials and the free release of phosphogypsum waste.

Iron (III) isomaltoside 1000

Intravenous (iv.) iron is now the recommended treatment for iron deficiency anemia if oral preparations have failed or in those undergoing hemodialysis. Iron isomaltoside is a new iv. iron preparation, licensed since 2009 in the UK and Europe. The iron is tightly bound within a nonionic isomaltoside carbohydrate matrix, as opposed to most other iv. iron preparations that use branched polymers to form a carbohydrate shell. This conformation produces a low immunogenic potential, which allows high single-dose infusions to adequately replenish stores. Two Phase III, open-label, noncomparative, multicenter clinical trials have investigated the safety profile of iron isomaltoside in chronic kidney disease and chronic heart failure. Two serious adverse events were observed (Staphylococcus aureus sepsis and angina pectoris), although their relationship to the drug was questioned. Significant hemoglobin and serum ferritin rises were seen in the chronic kidney disease group. The chronic heart failure group showed a significant serum ferritin rise and improved ‘overall quality of life’ but a nonsignificant hemoglobin rise. Preparations of iv. iron can cause renal injury, possibly through oxidative stress. Modern preparations, such as iron isomaltoside and ferumoxytol, have demonstrated less free iron release and hence may theoretically cause less renal damage. The cost of iron isomaltoside is greater than some of the current standard preparations used in most hospitals in the UK and Europe. However, when overheads and patient throughput are calculated, it may be a more cost-effective therapy than current therapies in the UK, owing to its faster infusion rate. Currently, there remains limited data on efficacy, safety and cost–effectiveness. Although initial data are encouraging, they come from only three published small trials, thus restricting the conclusions that can be made. Future research needs to concentrate on comparative analyses with other iv. iron therapies.

In the eye of the storm: Iodinated contrast medium induced thyroid storm presenting as cardiopulmonary arrest

The administration of iodinated contrast medium may lead to excess free thyroid hormone release and cause thyroid storm. A woman presented to the emergency department with dyspnea, hemoptysis, and intermittent bilateral lower extremities edema. Physical examination revealed mildly enlarged thyroid. Patient underwent a computed tomography scan of the chest with intravenous iodinated contrast medium to rule out pulmonary embolism, the patient developed a thyroid storm second to iodinated contrast medium injection. Proper treatment was provided and the patient had a good outcome. We present this case of an unusual presentation of a thyroid storm with cardiac arrest. This case illustrates that evaluating thyroid function tests in patients with an enlarged thyroid prior to the administration of iodinated contrast medium could prevent the development of thyroid storm.

Comparison of measured and calculated concrete and rebar specific activity during decommissioning of the Dalhousie SLOWPOKE-2 reactor

Following the defuelling and dismantling of the Dalhousie University SLOWPOKE-2 Reactor (DUSR) in 2011, the reactor pool concrete and rebar were sampled to support the unconditional free release of the material such that the facility could be classified for unrestricted use. A detailed MCNP5 model of the critical core assembly was simulated to calculate the thermal, intermediate and fast neutron flux profile below the reactor pool floor. The neutron fluxes were used to calculate the specific activity of significant radionuclides in the concrete and rebar. The calculated specific activity and consequently the calculated neutron fluxes were validated at a number of sample locations. The calculated concrete and rebar specific activity were found to be in good agreement with the measured specific activity at the sample locations. The unrestricted use of the facility was granted through the approval of the licence to abandon the facility in August 2011.

Design, fabrication, testing and packaging of a silicon micromachined radio frequency microelectromechanical series (RF MEMS) switch

RF characterization and packaging of a single pole single throw (SPST) direct contact microelectromechanical (MEMS) series radio frequency (RF) switch is reported. Precise thickness of the silicon MEMS structure is achieved using a specially developed silicon Deep Reactive Ion Etching (DRIE) thinning process. A stress free release process is employed which ensures a high yield of released microstructures. The design of the device is based on stiffness equations derived from first principles. Displacement of the actuator under applied field is measured to confirm electrostatic pull in, which occurs in the 30–50 V range. The variation of contact resistance with time has been measured and is found to have a power law decay, in agreement with theoretical models. At the bare die level the insertion loss, return loss and the isolation of the switch were measured to be −0.43 dB, −25 dB and −21 dB, respectively at 10 GHz. The devices were packaged in commercially available RF packages and mounted in alumina boards for post package characterization. Due to the presence of bond wires in the signal path of the packaged devices, the RF performance was found to degrade at high frequencies. However, losses were measured to be at acceptable levels up to 2 GHz. Factors contributing to insertion loss at the die and package device levels are discussed in detail with possible solutions.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl−) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV–visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10µM), precluding the enzyme from functioning at maximum activity (80–90% inhibition). MPO heme destruction occurred only in the presence of Cl−. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO–Compound I–Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron.

Effect of Ichnocarpus frutescens (L.) R. Br. hexane extract on preadipocytes viability and lipid accumulation in 3T3-L1 cells

ObjectiveTo investigate the crude extracts of Ichnocarpus frutescens (I. frutescens) for antiobesity effect. MethodsLeaves of I. frutescens were sequentially extracted with hexane, ethyl acetate, and methanol and their effect on viability of 3T3-L1 preadipocytes were evaluated. Based on this the apoptosis on preadipocytes was confirmed by DNA fragmentation and LDH (Lactate dehydrogenase) leakage assays. Anti-adipogenesis was performed by oil red O (ORO) staining and free glycerol release in the medium of differentiated adipocytes. ResultsThe hexane extract of I. frutescens (IFHE) inhibited cell viability in a time- and dose-related manner. An increased release of LDH, as a marker of membrane integrity, was observed at a dose of 200 μ g/mL. The discontinuous DNA fragments on agarose gel electrophoresis showed the apoptotic effect of the IFHE. Morphological observations of cells stained with ORO showed a decrease in cellular lipid content at the concentrations tested compared to the induced control cells. In the experiment of lipolytic activity, treatment with IFHE enhanced glycerol secretion with the rates of approximately 28%, 55%, and 46% at the concentrations of 100, 200 and 300 μ g/mL, respectively. ConclusionsThe observed properties clearly revealed the medicinal property of I. frutescens in the treatment of obesity.

Abstract 5658: Development and optimization of CZ48-loaded microbubbles for controlled drug delivery .

Abstract CZ48, the 20-O-propionate ester of camptothecin, is a prodrug requiring activation by converting to parental CPT in vivo. To circumvent the problems of low water solubility, lack of absorption, and poor bioavailability, novel CZ48-loaded microbubbles were investigated in this study. CZ48-loaded microbubbles were prepared at particle sizes ranging from 0.5 μm to 6.7 μm using a double emulsion technique (W/O/W). The satisfactory encapsulation efficiency and drug-loading content were 85.73±2.41% and 26.07±0.76%, respectively. The successful encapsulation of the microbubbles was confirmed by confocal laser scanning microscope (CLSM) observations and X-ray diffraction (XRD) patterns. The comparison between the controlled drug release from CZ48-loaded microbubbles and the free drug release from plain CZ48 crystals was performed in vitro, showing that 55% of the CZ48 was steadily released by the CZ48-loaded microbubbles in the first 48 hrs while more than 90% of the CZ48 was rapidly released by CZ48 crystals within the first 5 hrs. In vivo pharmacokinetic studies indicated that the bioavailabilities of CZ48 and CPT from CZ48-loaded microbubbles raised 121% and 517%, respectively, after intravenous administration compared to the plain CZ48. Compared to the plain CZ48, CZ48-loaded microbubbles also showed a higher mean residence time (MRT) and a longer elimination half-life (T1/2), indicating that CZ48-loaded microbubbles were indeed able to release CZ48 drug in a finely controlled manner. These results demonstrated that such microbubbles could be a promising approach for the construction of an effectively controlled drug delivery system for CZ48 as well as for other drugs with lower water solubility. Citation Format: Yang Wang, Siyang Li, Jian Zheng, Xiufeng Yan, Zhisong Cao, Dana Vardeman, Beppino Giovanella. Development and optimization of CZ48-loaded microbubbles for controlled drug delivery . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5658. doi:10.1158/1538-7445.AM2013-5658

A composite equation of state for the modeling of sonic carbon dioxide jets in carbon capture and storage scenarios

The development of a novel composite two‐phase method to predict the thermodynamic physical properties of carbon dioxide (CO2) above and below the triple point, applied herein in the context of Reynolds‐Averaged Navier–Stokes computational modeling has been detailed here. A number of approaches have been combined to make accurate predictions in all three phases (solid, liquid, and gas) and at all phase changes for application in the modeling of releases of CO2 at high pressure into the atmosphere. Predictions of a free release of CO2 into the atmosphere from a reservoir at a pressure of 10 MPa and a temperature of 283 K, typical of transport conditions in carbon capture and storage scenarios, is examined. A comparison of the results shows that the sonic CO2 jet that forms requires a three‐phase equation of state including the latent heat of fusion to realistically simulate its characteristics. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3928–3942, 2013