Search

Plants and algae play a crucial role in the earth's ecosystems. Through photosynthesis they convert light energy into chemical energy, capture CO2 and produce oxygen and energy-rich organic compounds. Photosynthetic organisms are primary producers and synthesize the essential omega 3 and omega 6 fatty acids. They have also unique and highly diverse complex lipids, such as glycolipids, phospholipids, triglycerides, sphingolipids and phytosterols, with nutritional and health benefits. Plant and algal lipids are useful in food, feed, nutraceutical, cosmeceutical and pharmaceutical industries but also for green chemistry and bioenergy. The analysis of plant and algal lipidomes represents a significant challenge due to the intricate and diverse nature of their composition, as well as their plasticity under changing environmental conditions. Optimization of analytical tools is crucial for an in-depth exploration of the lipidome of plants and algae. This review highlights how lipidomics analytical tools can be used to establish a complete mapping of plant and algal lipidomes. Acquiring this knowledge will pave the way for the use of plants and algae as sources of tailored lipids for both industrial and environmental applications. This aligns with the main challenges for society, upholding the natural resources of our planet and respecting their limits., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Limonene and linalool are used in cosmetic products for their floral scents, but their oxidation products are strong contact allergens whose mechanisms of action are still not fully understood., Objectives: The effects of limonene hydroperoxide (Lim-2-OOH) and linalool hydroperoxides (Lin-6/7-OOH) on the lipid profile of a human keratinocyte cell line (HaCaT) were evaluated. 2,4-Dinitrofluorobenzene (DNFB) was also included., Methods: Lim-2-OOH and Lin-6/7-OOH were synthesised according to previous methods. HaCaT cells were treated with allergens (10 μM) for 24 h and the cellular lipid extracts were analysed by C18 liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data analysis was performed using Lipostar software. Statistical analysis was carried out using Metaboanalyst and R software., Results: All three sensitisers used caused significant changes in the lipidome of HaCaT cells in a similar trend. There was an upregulation in several plasmanyl/plasmenyl phospholipids (O-/P-phosphatidylcholines [PC] and O-/P-phosphatidylethanolamines [PE]), sphingolipids (HexCer) and triacylglycerol lipid species, and a decrease in some polyunsaturated fatty acids-containing phospholipid (PE and PC) species suggesting oxidative stress and inflammation., Conclusions: This study is the first to evaluate the plasticity of the HaCaT cell lipidome in response to allylic hydroperoxide allergens Lim-2-OOH and Lin-6/7-OOH, together with the experimental contact allergen DNFB. These allergens are able to upregulate and downregulate certain lipid classes to a varying degree., (© 2024 The Author(s). Contact Dermatitis published by John Wiley & Sons Ltd.)

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most prevalent mitochondrial fatty acid β-oxidation disorder. In this study, we assessed the variability of the lipid profile in MCADD by analysing plasma samples obtained from 25 children with metabolically controlled MCADD (following a normal diet with frequent feeding and under l-carnitine supplementation) and 21 paediatric control subjects (CT). Gas chromatography-mass spectrometry was employed for the analysis of esterified fatty acids, while high-resolution C18-liquid chromatography-mass spectrometry was used to analyse lipid species. We identified a total of 251 lipid species belonging to 15 distinct lipid classes. Principal component analysis revealed a clear distinction between the MCADD and CT groups. Univariate analysis demonstrated that 126 lipid species exhibited significant differences between the two groups. The lipid species that displayed the most pronounced variations included triacylglycerols and phosphatidylcholines containing saturated and monounsaturated fatty acids, specifically C14:0 and C16:0, which were found to be more abundant in MCADD. The observed changes in the plasma lipidome of children with non-decompensated MCADD suggest an underlying alteration in lipid metabolism. Therefore, longitudinal monitoring and further in-depth investigations are warranted to better understand whether such alterations are specific to MCADD children and their potential long-term impacts., (© 2024 SSIEM.)

Autoimmune diseases (AID), such as systemic lupus erythematosus (SLE) and systemic sclerosis (SS), are complex conditions involving immune system dysregulation. Diagnosis is challenging, requiring biomarkers for improved detection and prediction of relapses. Lipids have emerged as potential biomarkers due to their role in inflammation and immune response. This study uses an untargeted C18 RP-LC-MS lipidomics approach to comprehensively assess changes in lipid profiles in patients with SLE and SS. By analyzing whole blood and plasma, the study aims to simplify the lipidomic analysis, explore cellular-level lipids, and compare lipid signatures of SLE and SS with healthy controls. Our findings showed variations in the lipid profile of SLE and SS. Sphingomyelin and ceramide molecular species showed significant increases in plasma samples from SS patients, suggesting an atherosclerotic profile and potentially serving as lipid biomarkers. Phosphatidylserine species in whole blood from SLE patients exhibited elevated levels supporting previously reported dysregulated processes of cell death and defective clearance of dying cells in this AID. Moreover, decreased phospholipids bearing PUFA were observed, potentially attributed to the degradation of these species through lipid peroxidation processes. Further studies are needed to better understand the role of lipids in the pathological mechanisms underlying SLE and SS.

The microalga Chlorella vulgaris is a popular food ingredient widely used in the industry, with an increasing market size and value. Currently, several edible strains of C. vulgaris with different organoleptic characteristics are commercialized to meet consumer needs. This study aimed to compare the fatty acid (FA) and lipid profile of four commercialized strains of C. vulgaris (C-Auto, C-Hetero, C-Honey, and C-White) using gas- and liquid-chromatography coupled to mass-spectrometry approaches, and to evaluate their antioxidant and anti-inflammatory properties. Results showed that C-Auto had a higher lipid content compared to the other strains and higher levels of omega-3 polyunsaturated FAs (PUFAs). However, the C-Hetero, C-Honey, and C-White strains had higher levels of omega-6 PUFAs. The lipidome signature was also different between strains, as C-Auto had a higher content of polar lipids esterified to omega-3 PUFAs, while C-White had a higher content of phospholipids with omega-6 PUFAs. C-Hetero and C-Honey showed a higher content of triacylglycerols. All extracts showed antioxidant and anti-inflammatory activity, highlighting C-Auto with greater potential. Overall, the four strains of C. vulgaris can be selectively chosen as a source of added-value lipids to be used as ingredients in food and nutraceutical applications for different market needs and nutritional requirements.

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

Coccolithophore microalgae, such as Emiliania huxleyi (EHUX) and Chrysotila pseudoroscoffensis (CP), are composed of calcium carbonate (CaCO 3 ) and contain bioactive compounds that can be explored to produce sustainable food packaging. In this study, for the first time, these microalgae were incorporated as fillers in starch-based films, envisioning the development of biodegradable and bioactive materials for food packaging applications. The films were obtained by solvent casting using different proportions of the filler (2.5, 5, 10, and 20%, w / w ). For comparison, commercial CaCO 3 , used as filler in the plastic industry, was also tested. The incorporation of CaCO 3 and microalgae (EHUX or CP) made the films significantly less rigid, decreasing Young's modulus up to 4.7-fold. Moreover, the incorporation of microalgae hydrophobic compounds as lipids turned the surface hydrophobic (water contact angles > 90°). Contrary to what was observed with commercial CaCO 3 , the films prepared with microalgae exhibited antioxidant activity, increasing from 0.9% (control) up to 60.4% (EHUX 20%) of ABTS radical inhibition. Overall, the introduction of microalgae biomass improved hydrophobicity and antioxidant capacity of starch-based films. These findings should be considered for further research using coccolithophores to produce active and sustainable food packaging material.

Fatty acid oxidation disorders (FAODs) are inborn errors of metabolism (IEMs) caused by defects in the fatty acid (FA) mitochondrial β-oxidation. The most common FAODs are characterized by the accumulation of medium-chain FAs and long-chain (3-hydroxy) FAs (and their carnitine derivatives), respectively. These deregulations are associated with lipotoxicity which affects several organs and potentially leads to life-threatening complications and comorbidities. Changes in the lipidome have been associated with several diseases, including some IEMs. In FAODs, the alteration of acylcarnitines (CARs) and FA profiles have been reported in patients and animal models, but changes in polar and neutral lipid profile are still scarcely studied. In this review, we present the main findings on FA and CAR profile changes associated with FAOD pathogenesis, their correlation with oxidative damage, and the consequent disturbance of mitochondrial homeostasis. Moreover, alterations in polar and neutral lipid classes and lipid species identified so far and their possible role in FAODs are discussed. We highlight the need of mass-spectrometry-based lipidomic studies to understand (epi)lipidome remodelling in FAODs, thus allowing to elucidate the pathophysiology and the identification of possible biomarkers for disease prognosis and an evaluation of therapeutic efficacy.

Dried blood spots (DBS) are being considered as an alternative sampling method of blood collection that can be used in combination with lipidomic and other omic analysis. DBS are successfully used in the clinical context to collect samples for newborn screening for the measurement of specific fatty acid derivatives, such as acylcarnitines, and lipids from whole blood for diagnostic purposes. However, DBS are scarcely used for lipidomic analysis and investigations. Lipidomic studies using DBS are starting to emerge as a powerful method for sampling and storage in clinical lipidomic analysis, but the major research work is being done in the pre- and analytical steps and procedures, and few in clinical applications. This review presents a description of the impact factors and variables that can affect DBS lipidomic analysis, such as the type of DBS card, haematocrit, homogeneity of the blood drop, matrix/chromatographic effects, and the chemical and physical properties of the analyte. Additionally, a brief overview of lipidomic studies using DBS to unveil their application in clinical scenarios is also presented, considering the studies of method development and validation and, to a less extent, for clinical diagnosis using clinical lipidomics. DBS combined with lipidomic approaches proved to be as effective as whole blood samples, achieving high levels of sensitivity and specificity during MS and MS/MS analysis, which could be a useful tool for biomarker identification. Lipidomic profiling using MS/MS platforms enables significant insights into physiological changes, which could be useful in precision medicine., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Complex lipids, phospholipids (PLs) and triacylglycerides (TAGs), are prone to modifications induced by reactive nitrated species and reactive oxygen species, generating a range of nitrated, nitrosated or nitroxidized derivatives, as nitro PLs and nitro TAGs. These modified lipids (epilipids) have been reported in vitro and in vivo using lipidomics approaches. However, their detection in living systems remains a challenge hampered by its complexity, high structural diversity, and low abundance. The advances in high-resolution mass spectrometry combined with the higher sensitivity of the instruments like Orbitrap-based mass spectrometers opened new opportunities for the detection of these modified complex lipids. This review summarizes the challenges and findings behind the identification of nitrated, nitrosated and nitroxidized PLs and TAGs fragmentation fingerprints based on collision-induced dissociation (CID) and higher energy CID (HCD) MS/MS approaches. Following what has already been reported for nitrated fatty acids, these complex lipids are found to act as endogenous mediators with potential electrophilic properties and can express bioactivities such as anti-inflammatory and antioxidant actions. This information can be used to design untargeted and targeted lipidomics strategies for these modified complex lipids in biological samples as well as in pathological, food and industrial settings, further unveiling their biological and signalling roles., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Glycolipids and phospholipids are the main reservoirs of omega polyunsaturated fatty acids in microalgae. Their extraction for the food industry requires food grade solvents, however, the use of these solvents is generally associated with low extraction yields. In this study, we evaluated the lipid extraction efficiency of food-grade ethanol, ultrasound-assisted ethanol (UAE) and dichloromethane/methanol (DCM) from Chlorella vulgaris cultivated under autotrophic and heterotrophic conditions. Yields of lipids, fatty acids (FA), and complex lipid profiles were determined by gravimetry, GC-MS, and LC-MS/MS, respectively. UAE and DCM showed the highest lipid yields with similar purity. The FA profiles were identical for all extracts. The polar lipidome of the DCM and UAE extracts was comparable, while the EtOH extracts were significantly different. These results demonstrated the effectiveness of UAE extraction to obtain high yields of polar lipids and omega-3 and -6-rich extracts from C. vulgaris that can be used for food applications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Phenylketonuria (PKU) is a disease of the catabolism of phenylalanine (Phe), caused by an impaired function of the enzyme phenylalanine hydroxylase. Therapeutics is based on the restriction of Phe intake, which mostly requires a modification of the diet. Dietary restrictions can lead to imbalances in specific nutrients, including lipids. In the present study, the plasma phospholipidome of PKU and healthy children (CT) was analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Using this approach, 187 lipid species belonging to nine different phospholipid classes and three ceramides were identified. Principal component analysis of the lipid species data set showed a distinction between PKU and CT groups. Univariate analysis revealed that 146 species of phospholipids were significantly different between both groups. Lipid species showing significant variation included phosphatidylcholines, containing polyunsaturated fatty acids (PUFA), which were more abundant in PKU. The high level of PUFA-containing lipid species in children with PKU may be related to a diet supplemented with PUFA. This study was the first report comparing the plasma polar lipidome of PKU and healthy children, highlighting that the phospholipidome of PKU children is significantly altered compared to CT. However, further studies with larger cohorts are needed to clarify whether these changes are specific to phenylketonuric children.

Macroalgae of the genus Ulva have long been used as human food. Local environmental conditions, among other factors, can have an impact on their nutrient and phytochemical composition, as well as on the value of the seaweed for food and non-food applications. This study is the first to initiate a comparison between commercial Ulva spp. from different European origins, France (FR, wild-harvested Ulva spp.), and Portugal (PT, farm-raised Ulva rigida ), in terms of proximate composition, esterified fatty acids (FA), and polar lipids. The ash content was higher in PT samples, while FR samples had higher levels of proteins, lipids, and carbohydrates and other compounds. The profile of esterified FA, as well as FA-containing polar lipids at the class and species levels were also significantly different. The FR samples showed about three-fold higher amount of n -3 polyunsaturated FA, while PT samples showed two-fold higher content of monounsaturated FA. Quantification of glycolipids and phospholipids revealed, respectively, two-fold and three-fold higher levels in PT samples. Despite the differences found, the polar lipids identified in both batches included some lipid species with recognized bioactivity, valuing Ulva biomass with functional properties, increasing their added value, and promoting new applications, namely in nutraceutical and food markets.

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MS n ) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewis x/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Polar lipids from microalgae have aroused greater interest as a natural source of omega-3 ( n -3) polyunsaturated fatty acids (PUFA), an alternative to fish, but also as bioactive compounds with multiple applications. The present study aims to characterize the polar lipid profile of cultured microalga Emiliania huxleyi using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-MS) and fatty acids (FA) analysis by gas chromatography (GC-MS). The lipidome of E. huxleyi revealed the presence of distinct n -3 PUFA (40% of total FA), namely docosahexaenoic acid (22:6 n -3) and stearidonic acid (18:4 n -3), which give this microalga an increased commercial value as a source of n -3 PUFA present in the form of polar lipids. A total of 134 species of polar lipids were identified and some of these species, particularly glycolipids, have already been reported for their bioactive properties. Among betaine lipids, the diacylglyceryl carboxyhydroxymethylcholine (DGCC) class is the least reported in microalgae. For the first time, monomethylphosphatidylethanolamine (MMPE) has been found in the lipidome of E. huxleyi . Overall, this study highlights the potential of E. huxleyi as a sustainable source of high-value polar lipids that can be exploited for different applications, namely human and animal nutrition, cosmetics, and pharmaceuticals.

Phenylketonuria (PKU) is the most prevalent inborn error of amino acid metabolism. The disease is due to the deficiency of phenylalanine (Phe) hydroxylase activity, which causes the accumulation of Phe. Early diagnosis through neonatal screening is essential for early treatment implementation, avoiding cognitive impairment and other irreversible sequelae. Treatment is based on Phe restriction in the diet that should be maintained throughout life. High dietary restrictions can lead to imbalances in specific nutrients, notably lipids. Previous studies in PKU patients revealed changes in levels of plasma/serum lipoprotein lipids, as well as in fatty acid profile of plasma and red blood cells. Most studies showed a decrease in important polyunsaturated fatty acids, namely DHA (22:6n-3), AA (20:4n-6) and EPA (20:5n-6). Increased oxidative stress and subsequent lipid peroxidation have also been observed in PKU. Despite the evidences that the lipid profile is changed in PKU patients, more studies are needed to understand in detail how lipidome is affected. As highlighted in this review, mass spectrometry-based lipidomics is a promising approach to evaluate the effect of the diet restrictions on lipid metabolism in PKU patients, monitor their outcome, namely concerning the risk for other chronic diseases, and find possible prognosis biomarkers., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The aquaculture of macroalgae for human consumption and other high-end applications is experiencing unprecedented development in European countries, with the brown algae Saccharina latissima being the flag species. However, environmental conditions in open sea culture sites are often unique, which may impact the biochemical composition of cultured macroalgae. The present study compared the elemental compositions (CHNS), fatty acid profiles, and lipidomes of S. latissima originating from three distinct locations (France, Norway, and the United Kingdom). Significant differences were found in the elemental composition, with Norwegian samples displaying twice the lipid content of the others, and significantly less protein (2.6%, while French and UK samples contained 6.3% and 9.1%, respectively). The fatty acid profiles also differed considerably, with UK samples displaying a lower content of n -3 fatty acids (21.6%), resulting in a higher n -6/ n -3 ratio. Regarding the lipidomic profile, samples from France were enriched in lyso lipids, while those from Norway displayed a particular signature of phosphatidylglycerol, phosphatidylinositol, and phosphatidylcholine. Samples from the UK featured higher levels of phosphatidylethanolamine and, in general, a lower content of galactolipids. These differences highlight the influence of site-specific environmental conditions in the shaping of macroalgae biochemical phenotypes and nutritional value. It is also important to highlight that differences recorded in the lipidome of S. latissima make it possible to pinpoint specific lipid species that are likely to represent origin biomarkers. This finding is relevant for future applications in the field of geographic origin traceability and food control., Competing Interests: The authors declare no conflict of interest.

It is now well established that exogenous β-1,4-xylanases improve the nutritive value of wheat-based diets for poultry. Among other factors, the mechanism of action of exogenous enzymes may involve a microbial route resulting from the generation of prebiotic xylo-oligosaccharides (XOS) in the birds' gastro-intestinal (GI) tract. In a series of three experiments, the effect of XOS on the performance of broilers fed wheat or corn-based diets was investigated. In experiment 1, birds receiving diets supplemented with XOS displayed an increased weight gain (P = 0.08). The capacity of XOS to improve the performance of animals during a longer trial (42 d) was investigated (Experiment 2). The data revealed that diet supplementation with XOS, tested at two incorporation rates (0.1 and 1 g/kg), or with an exogenous β-1,4-xylanase resulted in an increased nutritive value of the wheat-based diet. An improvement in animal performance was accompanied by a shift in the microbial populations colonizing the upper portions of the GI tract. XOS were also able to improve the performance of broilers fed a corn-based diet, although the effects were not apparent at incorporation rates of 10 g/kg. Together these studies suggest that in some cases the capacity of β-1,4-xylanases to improve the nutritive value of wheat-based diets is more related to their ability to produce prebiotic XOS than to their ability to degrade arabinoxylans. The extremely low quantities of XOS used in this study also challenge the depiction of a prebiotic being a quantitatively fermented substrate. These data also bring into question the validity of the "cell wall" mechanism, as XOS elicited an effect with clearly no action on endosperm cell wall integrity and yet the performance effects noted were equivalent or superior to the added enzymes.

The data presented here are related to the research paper entitled "Transglycosylation reactions, a main mechanism of phenolics incorporation in coffee melanoidins: inhibition by Maillard reaction" (Moreira et al., 2017) [1]. Methanolysis was applied in coffee fractions to quantify glycosidically-linked phenolics in melanoidins. Moreover, model mixtures mimicking coffee beans composition were roasted and analyzed using mass spectrometry-based approaches to disclose the regulatory role of proteins in transglycosylation reactions extension. This article reports the detailed chemical composition of coffee beans and derived fractions. In addition, it provides gas chromatography-mass spectrometry (GC-MS) chromatograms and respective GC-MS spectra of silylated methanolysis products obtained from phenolic compounds standards, as well as the detailed identification of all compounds observed by electrospray mass spectrometry (ESI-MS) analysis of roasted model mixtures, paving the way for the identification of the same type of compounds in other samples.

Extensive exposure to UVA is thought to increase the risk of malignancy and the progression of melanoma, the most serious type of skin cancer. It is well known that alterations in lipid metabolism represent an early event in carcinogenesis, but the impact of UVA exposure on the lipid composition of cancer cells is still largely unknown. In this study we aimed at investigating lipid remodeling in human melanoma cells in response to UVA exposure. After UVA irradiation, lipid extracts were either immediately collected from SK-MEL-28 cells or collected after a recovery period of 2 h or 24 h. The lipid profiles for each event were determined by liquid chromatography or gas chromatography coupled to mass spectrometry. UVA exposure led to major alterations in both fatty acids (FA) and phospholipid profiles. An increase of monounsaturated FA (MUFA) and FA18:0, as well as a decrease of FA16:0, were observed 24 h after irradiation. Moreover, phosphatidylcholine (PC) decreased and phosphatidylinositol (PI) increased after UVA exposure. Molecular alterations in the PC, lysoPC, PI, phosphatidylethanolamine (PE), ether-linked PE and phosphatidylglycerol (PG) profiles were also observed. The absence of cleaved caspase-3 after 2 h and 24 h of re-incubation is correlated with impairment of apoptosis. Overall, these data showed changes in membrane lipids, which may be associated with lipogenesis after UVA exposure which, in turn, is usually a determinant for cell survival.

The characterization of several commercial dextrins and the analysis of the potential of dextrin derived hydrogels for biomedical applications were performed in this work. The structural characterization of dextrins allowed the determination of the polymerization and branching degrees, which ranged from 6 to 17 glucose residues and 2 to 13%, respectively. Tackidex, a medical grade dextrin was choosen for further characterization. The combination of hydrogel with a dextrin nanogel and urinary bladder matrix was achieved without compromising the mechanical properties or microstructure. The encapsulation of cells, preserving its viability, confirms the biocompatibility of the injectable hydrogels, which have therefore great potential for biomedical applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)