Urinary glycosaminoglycans as biomarker for urothelial injury: Is it possible to discriminate damage from recovery?

OBJECTIVES The glycosaminoglycan (GAG) layer is referred to as a bladder protective factor. We reproduced an experimental model of urothelial damage to assess GAG metabolism in the process of injury and recovery of the urothelium. METHODS Wistar female rats were bladder catheterized and instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24-hour urine samples were obtained. The urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycan were determined in all groups and in nonmanipulated rats (day 0). Additionally, sulfated-GAG synthesis was assessed by the incorporation of [S-35]-inorganic sulfate. The bladders were analyzed by histochemical staining for HA and immunofluorescence for heparin sulfate and syndecan-4. RESULTS Urinary HA and sulfated-GAG were elevated after PS injection (P <0.05). A greater concentration of [S-35] -labeled GAG in the PS group animals on the fifth day and, especially, on the seventh day represented increased GAG synthesis at these periods (P <0.05). Bladder sections from the PS group animals on day 1 showed a greater amount of HA in the urothelium. PS instillation damaged the urothelium layer of heparin sulfate and syndecan-4 seen in the control animals. On day 5, patchy areas of a restored layer were seen, and, on day 7, this layer had completely regenerated. CONCLUSIONS Urinary GAG cannot differentiate urothelial damage from recovery. Elevated levels of urinary GAG can result from either desquamation of the surface cell GAG layer or increased GAG synthesis to regenerate the damaged urothelium.

Aerosol inorganic composition at a tropical site: Discrepancies between filter-based sampling and a semi-continuous method

The concentrations of the water-soluble inorganic aerosol species, ammonium (NH4+), nitrate (NO3-), chloride (Cl-), and sulfate (SO42-), were measured from September to November 2002 at a pasture site in the Amazon Basin (Rondnia, Brazil) (LBA-SMOCC). Measurements were conducted using a semi-continuous technique (Wet-annular denuder/Steam-Jet Aerosol Collector: WAD/SJAC) and three integrating filter-based methods, namely (1) a denuder-filter pack (DFP: Teflon and impregnated Whatman filters), (2) a stacked-filter unit (SFU: polycarbonate filters), and (3) a High Volume dichotomous sampler (HiVol: quartz fiber filters). Measurements covered the late dry season (biomass burning), a transition period, and the onset of the wet season (clean conditions). Analyses of the particles collected on filters were performed using ion chromatography (IC) and Particle-Induced X-ray Emission spectrometry (PIXE). Season-dependent discrepancies were observed between the WAD/SJAC system and the filter-based samplers. During the dry season, when PM2.5 (D-p <= 2.5 mu m) concentrations were similar to 100 mu g m(-3), aerosol NH4+ and SO42- measured by the filter-based samplers were on average two times higher than those determined by the WAD/SJAC. Concentrations of aerosol NO3- and Cl- measured with the HiVol during daytime, and with the DFP during day- and nighttime also exceeded those of the WAD/SJAC by a factor of two. In contrast, aerosol NO3- and Cl- measured with the SFU during the dry season were nearly two times lower than those measured by the WAD/SJAC. These differences declined markedly during the transition period and towards the cleaner conditions during the onset of the wet season (PM2.5 similar to 5 mu g m(-3)); when filter-based samplers measured on average 40-90% less than the WAD/SJAC. The differences were not due to consistent systematic biases of the analytical techniques, but were apparently a result of prevailing environmental conditions and different sampling procedures. For the transition period and wet season, the significance of our results is reduced by a low number of data points. We argue that the observed differences are mainly attributable to (a) positive and negative filter sampling artifacts, (b) presence of organic compounds and organosulfates on filter substrates, and (c) a SJAC sampling efficiency of less than 100%.

Fontes orgânicas e inorgânicas de zinco e cobre como melhoradores de desempenho em leitões desmamados

Foram conduzidos dois experimentos para avaliar fontes orgânicas e inorgânicas de zinco e cobre nas dietas e seus efeitos no desempenho de leitões desmamados aos 21 dias de idade. Em cada experimento, foram utilizados 90 leitões em delineamento experimental de blocos ao acaso, com cinco dietas, seis repetições e três animais por parcela. As dietas utilizadas nos experimentos 1 e 2 continham 120 ppm de zinco e 10 ppm de cobre na forma de sulfato. No experimento 1, foram suplementadas com 0, 300, 600 e 900 ppm de zinco na forma orgânica ou 2.400 ppm na forma de óxido (ZnO) e, no experimento 2, com 0, 50, 100 e 150 ppm de cobre na forma orgânica ou 240 ppm de cobre na forma de sulfato (CuSO4 H2O). No experimento 1, os níveis de zinco de fonte orgânica tiveram efeito linear no consumo de ração e no ganho de peso nos períodos de 0 a 15 dias e de 0 a 21 dias pós-desmame. O ganho de peso nas fases de 0 a 35 dias e de 0 a 42 dias pós-desmame e o consumo de ração dos leitões que receberam a dieta com 900 ppm de zinco de fonte orgânica não diferiram dos valores observados naqueles que receberam a dieta com 2.400 ppm de zinco na forma inorgânica. A suplementação da dieta com zinco na forma orgânica (900 ppm) ou inorgânica (2.400 ppm) aumentou o consumo de ração e o ganho de peso de leitões nas primeiras três semanas após o desmame. A suplementação da dieta com 2.400 ppm de zinco na forma inorgânica reduziu a incidência de diarreia nas primeiras três semanas pós-desmame. No experimento 2, os níveis de cobre de fonte orgânica tiveram efeito quadrático no consumo de ração dos leitões nos períodos de 0 a 31 e de 0 a 40 dias pós-desmame. A suplementação da dieta com cobre, tanto de fonte orgânica (90 ppm) como inorgânica (240 ppm), aumenta o consumo de ração e o ganho de peso de leitões nos primeiros 40 dias pós-desmame.

Photolysis of ferric ions in the presence of sulfate or chloride ions: implications for the photo-Fenton process

The photo-Fenton process (Fe(2+)/Fe(3+), H(2)O(2), UV light) is one of the most efficient and advanced oxidation processes for the mineralization of the organic pollutants of industrial effluents and wastewater. The overall rate of the photo-Fenton process is controlled by the rate of the photolytic step that converts Fe(3+) back to Fe(2+). In this paper, the effect of sulfate or chloride ions on the net yield of Fe(2+) during the photolysis of Fe(3+) has been investigated in aqueous solution at pH 3.0 and 1.0 in the absence of hydrogen peroxide. A kinetic model based on the principal reactions that occur in the system fits the data for formation of Fe(2+) satisfactorily. Both experimental data and model prediction show that the availability of Fe(2+) produced by photolysis of Fe(3+) is inhibited much more in the presence of sulfate ion than in the presence of chloride ion as a function of the irradiation time at pH 3.0.

Changes in cardiac heparan sulfate proteoglycan expression and streptozotocin-induced diastolic dysfunction in rats

Background: Changes in the proteoglycans glypican and syndecan-4 have been reported in several pathological conditions, but little is known about their expression in the heart during diabetes. The aim of this study was to investigate in vivo heart function changes and alterations in mRNA expression and protein levels of glypican-1 and syndecan-4 in cardiac and skeletal muscles during streptozotocin (STZ)-induced diabetes. Methods: Diabetes was induced in male Wistar rats by STZ administration. The rats were assigned to one of the following groups: control (sham injection), after 24 hours, 10 days, or 30 days of STZ administration. Echocardiography was performed in the control and STZ 10-day groups. Western and Northern blots were used to quantify protein and mRNA levels in all groups. Immunohistochemistry was performed in the control and 30-day groups to correlate the observed mRNA changes to the protein expression. Results: In vivo cardiac functional analysis performed using echocardiography in the 10-day group showed diastolic dysfunction with alterations in the peak velocity of early (E) diastolic filling and isovolumic relaxation time (IVRT) indices. These functional alterations observed in the STZ 10-day group correlated with the concomitant increase in syndecan-4 and glypican-1 protein expression. Cardiac glypican-1 mRNA and skeletal syndecan-4 mRNA and protein levels increased in the STZ 30-day group. On the other hand, the amount of glypican in skeletal muscle was lower than that in the control group. The same results were obtained from immunohistochemistry analysis. Conclusion: Our data suggest that membrane proteoglycans participate in the sequence of events triggered by diabetes and inflicted on cardiac and skeletal muscles.

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Cloud condensation nuclei in pristine tropical rainforest air of Amazonia: size-resolved measurements and modeling of atmospheric aerosol composition and CCN activity

Atmospheric aerosol particles serving as cloud condensation nuclei (CCN) are key elements of the hydrological cycle and climate. We have measured and characterized CCN at water vapor supersaturations in the range of S=0.10-0.82% in pristine tropical rainforest air during the AMAZE-08 campaign in central Amazonia. The effective hygroscopicity parameters describing the influence of chemical composition on the CCN activity of aerosol particles varied in the range of kappa approximate to 0.1-0.4 (0.16+/-0.06 arithmetic mean and standard deviation). The overall median value of kappa approximate to 0.15 was by a factor of two lower than the values typically observed for continental aerosols in other regions of the world. Aitken mode particles were less hygroscopic than accumulation mode particles (kappa approximate to 0.1 at D approximate to 50 nm; kappa approximate to 0.2 at D approximate to 200 nm), which is in agreement with earlier hygroscopicity tandem differential mobility analyzer (H-TDMA) studies. The CCN measurement results are consistent with aerosol mass spectrometry (AMS) data, showing that the organic mass fraction (f(org)) was on average as high as similar to 90% in the Aitken mode (D <= 100 nm) and decreased with increasing particle diameter in the accumulation mode (similar to 80% at D approximate to 200 nm). The kappa values exhibited a negative linear correlation with f(org) (R(2)=0.81), and extrapolation yielded the following effective hygroscopicity parameters for organic and inorganic particle components: kappa(org)approximate to 0.1 which can be regarded as the effective hygroscopicity of biogenic secondary organic aerosol (SOA) and kappa(inorg)approximate to 0.6 which is characteristic for ammonium sulfate and related salts. Both the size dependence and the temporal variability of effective particle hygroscopicity could be parameterized as a function of AMS-based organic and inorganic mass fractions (kappa(p)=kappa(org) x f(org)+kappa(inorg) x f(inorg)). The CCN number concentrations predicted with kappa(p) were in fair agreement with the measurement results (similar to 20% average deviation). The median CCN number concentrations at S=0.1-0.82% ranged from N(CCN,0.10)approximate to 35 cm(-3) to N(CCN,0.82)approximate to 160 cm(-3), the median concentration of aerosol particles larger than 30 nm was N(CN,30)approximate to 200 cm(-3), and the corresponding integral CCN efficiencies were in the range of N(CCN,0.10/NCN,30)approximate to 0.1 to N(CCN,0.82/NCN,30)approximate to 0.8. Although the number concentrations and hygroscopicity parameters were much lower in pristine rainforest air, the integral CCN efficiencies observed were similar to those in highly polluted megacity air. Moreover, model calculations of N(CCN,S) assuming an approximate global average value of kappa approximate to 0.3 for continental aerosols led to systematic overpredictions, but the average deviations exceeded similar to 50% only at low water vapor supersaturation (0.1%) and low particle number concentrations (<= 100 cm(-3)). Model calculations assuming aconstant aerosol size distribution led to higher average deviations at all investigated levels of supersaturation: similar to 60% for the campaign average distribution and similar to 1600% for a generic remote continental size distribution. These findings confirm earlier studies suggesting that aerosol particle number and size are the major predictors for the variability of the CCN concentration in continental boundary layer air, followed by particle composition and hygroscopicity as relatively minor modulators. Depending on the required and applicable level of detail, the information and parameterizations presented in this paper should enable efficient description of the CCN properties of pristine tropical rainforest aerosols of Amazonia in detailed process models as well as in large-scale atmospheric and climate models.

Hydrogen bonding in 2-(2-oxothiazolidin-3-yl)-4,5-dihydrothiazolium hydrogen sulfate monohydrate

The asymmetric unit of the title compound, C(6)H(9)N(2)OS(2)(+)center dot-HSO(4)(-)center dot H(2)O, contains a heterocyclic cation, a hydrogen sulfate anion and a water molecule. There are strong hydrogen bonds between the hydrogen sulfate anions and water molecules, forming an infinite chain along the [010] direction, from which the cations are pendent. The steric, electronic and geometric features are compared with those of similar compounds. In this way, structural relationships are stated in terms of the influence of the sulfate group on the protonation of the heterocycle and on the tautomeric equilibrium in the solid state.

On the template synthesis of nanostructured inorganic/organic hybrid films

Prussian Blue has been introduced as a mediator to achieve stable, sensitive, reproducible, and interference-free biosensors. However, Na(+), Li(+), H(+), and all group II cations are capable to block the activity of Prussian Blue and, because Na(+) can be found in most human fluids, Prussian Blue analogs have already been developed to overcome this problem. These analogs, such as copper hexacyanoferrate, have also been introduced in a conducting polypyrrole matrix to create hybrid materials (copper hexacyanoferrate/polypyrrole, CuHCNFe/Ppy) with improved mechanical and electrochemical characteristics. Nowadays, the challenges in amperometric enzymatic biosensors consist of improving the enzyme immobilization and in making the chemical signal transduction more efficient. The incorporation of nanostructured materials in biosensors can optimize both steps and a nanostructured hybrid CuHCNFe/Ppy mediator has been developed using a template of colloidal polystyrene particles. The nanostructured material has achieved sensitivities 7.6 times higher than the bulk film during H(2)O(2) detection and it has also presented better results in other analytical parameters such as time response and detection limit. Besides, the nanostructured mediator was successfully applied at glucose biosensing in electrolytes containing Prussian Blue blocking cations. (C) 2008 The Electrochemical Society.

Total and inorganic mercury determination in fish tissue by flow injection cold vapour atomic fluorescence spectrometry

A simple and reliable method for Hg determination in fish samples has been developed. Lyophilised fish tissue samples were extracted in a 25% (w/v) tetramethylammonium hydroxide (TMAH) solution; the extracts were then analysed by FI-CVAFS. This method can be used to determine total and inorganic Hg, using the same FI manifold. For total Hg determination, a 0.1% (w/v) KMnO(4) solution was added to the FI manifold at the sample zone, followed by the addition of a 0.5% (w/v) SnCl(2) solution, whereas inorganic Hg was determined by adding a 0.1% (w/v) L-cysteine solution followed by a 1.0% (w/v) SnCl(2) solution to the FI system. The organic fraction was determined as the difference between total and inorganic Hg. Sample preparation, reagent consumption and parameters that can influence the FI-CVAFS performance were also evaluated. The limit of detection for this method is 3.7 ng g(-1) for total Hg and 4.3 ng g(-1) for inorganic Hg. The relative standard deviation for a 1.0 mu gL(-1) CH(3)Hg standard solution (n = 20) was 1.1%, and 1.3% for a 1.0 mu gL(-1) Hg(2+) standard solution (n = 20). Accuracy was assessed by the analysis of Certified Reference Material (dogfish: DORM-2, NRCC). Recoveries of 99.1% for total Hg and 93.9% inorganic Hg were obtained. Mercury losses were not observed when sample solutions were re-analysed after a seven day period of storage at 4 degrees C.

Assessing sample representativeness for inorganic chemical investigation of invertebrates by INAA

The quality of environmental studies depends on the utilization of adequate sampling protocol and analytical method for obtaining reliable results and minimizing analytical uncertainties. In order to demonstrate the applicability of INAA for determining chemical element composition of invertebrates, this work evaluated sample representativeness in terms of subsampling and sample size. Br, Co, Fe, K, Na, Sc and Zn could be determined in very small samples despite increasing of analytical uncertainties. Special attention should be directed to invertebrate species with small structures because of the high chemical variation observed among different sample sizes tested.

Full-scale anaerobic sequencing batch biofilm reactor for sulfate-rich wastewater treatment

This paper describes the performance and biofilm characteristics of a full-scale anaerobic sequencing batch biofilm reactor (ASBBR; 20 m(3)) containing biomass immobilized on an inert support (mineral coal) for the treatment of industrial wastewater containing a high sulfate concentration. The ASBBR reactor was operated during 110 cycles (48 h each) at sulfate loading rates ranging from 6.9 to 62.4 kgSO(4)(2-)/cycle corresponding to sulfate concentrations of 0.58-5.2 gSO(4)(2-)/L. Domestic sewage and ethanol were utilized as electron donors for sulfate reduction. After 71 cycles the mean sulfate removal efficiency was 99%, demonstrating a high potential for biological sulfate reduction. The biofilm formed in the reactor occurred in two different patterns, one at the beginning of the colonization and the other of a mature biofilm. These different colonization patterns are due to the low adhesion of the microorganisms on the inert support in the start-up period. The biofilm population is mainly made up of syntrophic consortia among sulfate-reducing bacteria and methanogenic archaea such as Methanosaeta spp.

Influence of feed time and sulfate load on the organic and sulfate removal in an ASBR

The removal of sulfate and organic matter was assessed in an ASBR, which treated wastewater containing 500 mg COD L(-1) (3 g COD L(-1) d(-1)) in 8 h-cycles at 30 degrees C. The wastewater was enriched with sulfate at [COD/SO(4)(2-]) ratios of 1.34, 0.67 and 0.34 (8.8,4.5 and 2.2 gSO(4)(2-) L(-1) d(-1)). For each COD/[SO(4)(2-)] ratio fill times used were: 10 min (batch), 3 and 6 h (fed-batch), achieving sulfate reduction of 30%, 72% and 72% (COD/[SO(4)(2-)] of 1.34); 25%, 58% and 55% (COD/[SO(4)(2-)] of 0.67) and 23%, 37% and 27% (COD/[SO(4)(2-)] of 0.34), respectively, and organic matter removal of 87%, 68% and 80% (COD/[SO(4)(2-)] of 1.34); 78%, 75% and 69% (COD/[SO(4)(2-)] of 0.67) and 85%, 84% and 83% (COD/[SO(4)(2-)] of 0.34), respectively. The results showed that fed-batch operation improved sulfate reduction, whereas organic matter removals were similar for batch and fed-batch operation. In addition, increase in sulfate loading in the fed-batch operation improved organic matter removal. (C) 2010 Elsevier Ltd. All rights reserved.

Microbial processes and bacterial populations associated to anaerobic treatment of sulfate-rich wastewater

A pilot-scale (1.2 m(3)) anaerobic sequencing batch biofilm reactor (ASBBR) containing mineral coal for biomass attachment was fed with sulfate-rich wastewater at increasing sulfate concentrations. Ethanol was used as the main organic source. Tested COD/sulfate ratios were of 1.8 and 1.5 for sulfate loading rates of 0.65-1.90 kgSO(4)(2-)/cycle (48 h-cycle) or of 1.0 in the trial with 3.0 gSO(4)(2-) l(-1). Sulfate removal efficiencies observed in all trials were as high as 99%. Molecular inventories indicated a shift on the microbial composition and a decrease on species diversity with the increase of sulfate concentration. Beta-proteobacteria species affiliated with Aminomonas spp. and Thermanaerovibrio spp. predominated at 1.0 gSO(4)(2-) l(-1). At higher sulfate concentrations the predominant bacterial group was Delta-proteobacteria mainly Desulfovibrio spp. and Desulfomicrobium spp. at 2.0 gSO(4)(2-) l(-1), whereas Desulfurella spp. and Coprothermobacter spp. predominated at 3.0 gSO(4)(2-) l(-1). These organisms have been commonly associated with sulfate reduction producing acetate, sulfide and sulfur. Methanogenic archaea(Methanosaeta spp.)was found at 1.0 and 2.0 gSO(4)(2-) l(-1). Additionally, a simplified mathematical model was used to infer on metabolic pathways of the biomass involved in sulfate reduction. (C) 2009 Elsevier Ltd. All rights reserved.