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Highlights • Lipophilic extracts of ground roast Arabica coffees were authenticated by benchtop NMR. • Small amounts of esterified 16-O-methylcafestol were found in Arabica coffees. • The compound identity was confirmed by NMR and MS experiments. • 16-OMC remains a useful marker for non-Arabicas as these contain much higher amounts. • 6 out of 60 retail Arabicas contained significant amounts of non-Arabica species., High-field and low-field proton NMR spectroscopy were used to analyse lipophilic extracts from ground roast coffees. Using a sample preparation method that produced concentrated extracts, a small marker peak at 3.16 ppm was observed in 30 Arabica coffees of assured origin. This signal has previously been believed absent from Arabicas, and has been used as a marker for detecting adulteration with robusta. Via 2D 600 MHz NMR and LC-MS, 16-O-methylcafestol and 16-O-methylkahweol were detected for the first time in Arabica roast coffee and shown to be responsible for the marker peak. Using low-field NMR, robusta in Arabica could be detected at levels of the order of 1–2% w/w. A surveillance study of retail purchased “100% Arabica” coffees found that 6 out of 60 samples displayed the 3.16 ppm marker signal to a degree commensurate with adulteration at levels of 3–30% w/w.

Graphical abstract, Highlights • A new screening method for identifying the species in ground roast coffees using low-field NMR spectroscopy. • A calibration-free statistical approach to detecting adulteration of arabica with robusta coffee. • A study of time-varying spectral changes in the 16-OMC component of lipophilic extracts following sample preparation., This work reports a new screening protocol for addressing issues of coffee authenticity using low-field (60 MHz) bench-top 1H NMR spectroscopy. Using a simple chloroform-based extraction, useful spectra were obtained from the lipophilic fraction of ground roast coffees. It was found that 16-O-methylcafestol (16-OMC, a recognized marker compound for robusta beans) gives rise to an isolated peak in the 60 MHz spectrum, which can be used as an indicator of the presence of robusta beans in the sample. A total of 81 extracts from authenticated coffees and mixtures were analysed, from which the detection limit of robusta in arabica was estimated to be between 10% and 20% w/w. Using the established protocol, a surveillance exercise was conducted of 27 retail samples of ground roast coffees which were labelled as “100% arabica”. None were found to contain undeclared robusta content above the estimated detection limit.

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. Central to the method is the identification of 'corresponding proteins' in the species of interest, in other words proteins that are nominally the same but possess species-specific sequence differences. When subject to proteolysis, corresponding proteins will give rise to some peptides which are likewise similar but with species-specific variants. These are 'corresponding peptides'. Species-specific peptides can be used as markers for species determination, while pairs of corresponding peptides permit relative quantitation of two species in a mixture. The peptides are detected using multiple reaction monitoring (MRM) mass spectrometry, a highly specific technique that enables peptide-based species determination even in complex systems. In addition, the ratio of MRM peak areas deriving from corresponding peptides supports relative quantitation. Since corresponding proteins and peptides will, in the main, behave similarly in both processing and in experimental extraction and sample preparation, the relative quantitation should remain comparatively robust. In addition, this approach does not need the standards and calibrations required by absolute quantitation methods. The protocol is described in the context of red meats, which have convenient corresponding proteins in the form of their respective myoglobins. This application is relevant to food fraud detection: the method can detect 1% weight for weight of horse meat in beef. The corresponding protein, corresponding peptide (CPCP) relative quantitation using MRM peak area ratios gives good estimates of the weight for weight composition of a horse plus beef mixture.

Epidemiological evidence suggests that cardiovascular disease is associated with osteoporosis, independent of age. Bone resorptive surface is increased in mice on a high-fat diet, and osteoclastic differentiation of bone marrow preosteoclasts is promoted by oxidized phospholipids. Because osteoclastic differentiation requires cytokines produced by osteoblasts, we hypothesized that the stimulatory mechanism of oxidized phospholipids is via induction of osteoclast-regulating cytokines in osteoblasts. To investigate the effects of oxidized phospholipids on expression of such cytokines, murine calvarial preosteoblasts, MC3T3-E1, were treated with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), an active component of oxidized lipoproteins. Results showed that ox-PAPC increased expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha. IL-6 expression was also elevated in calvarial tissues from hyperlipidemic but not in wild-type mice. Ox-PAPC also induced IL-6 protein levels in both MC3T3-E1 and primary calvarial cells. Promoter-reporter assay analysis showed that ox-PAPC, but not PAPC, induced murine IL-6 promoter activity. Effects of ox-PAPC on IL-6 expression and the promoter activity were attenuated by H89, a PKA inhibitor. Analysis of deletion and mutant IL-6 promoter constructs suggested that CAAT/enhancer binding protein (C/EBP) partly mediates the ox-PAPC effects. Taken together, the data suggest that oxidized phospholipids induce IL-6 expression in osteoblasts in part via C/EBP.

The EGFR/PI3K/Akt/mTOR signaling pathway is activated in many cancers including glioblastoma, yet mTOR inhibitors have largely failed to show efficacy in the clinic. Rapamycin promotes feedback activation of Akt in some patients, potentially underlying clinical resistance and raising the need for alternative approaches to block mTOR signaling. AMPK is a metabolic checkpoint that integrates growth factor signaling with cellular metabolism, in part by negatively regulating mTOR. We used pharmacological and genetic approaches to determine whether AMPK activation could block glioblastoma growth and cellular metabolism, and we examined the contribution of EGFR signaling in determining response in vitro and in vivo. The AMPK-agonist AICAR, and activated AMPK adenovirus, inhibited mTOR signaling and blocked the growth of glioblastoma cells expressing the activated EGFR mutant, EGFRvIII. Across a spectrum of EGFR-activated cancer cell lines, AICAR was more effective than rapamycin at blocking tumor cell proliferation, despite less efficient inhibition of mTORC1 signaling. Unexpectedly, addition of the metabolic products of cholesterol and fatty acid synthesis rescued the growth inhibitory effect of AICAR, whereas inhibition of these lipogenic enzymes mimicked AMPK activation, thus demonstrating that AMPK blocked tumor cell proliferation primarily through inhibition of cholesterol and fatty acid synthesis. Most importantly, AICAR treatment in mice significantly inhibited the growth and glycolysis (as measured by 18 fluoro-2-deoxyglucose microPET) of glioblastoma xenografts engineered to express EGFRvIII, but not their parental counterparts. These results suggest a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.

The Checkerboard Creek Rock Slope is located 1.5 km upstream of the Revelstoke Dam, which impounds the Columbia River in British Columbia, Canada. A detailed investigation completed in 2004 indicated that 2 to 3 million m3 of a rock slope was moving toward the reservoir at rate of approximately 10 mm/year. There was concern that, if a portion of the slow-moving rockslide accelerated rapidly into the reservoir, the resultant wave could overtop the dam. Distinct element analyses were carried out using UDEC to assess potential rockslide run-out characteristics (time histories of rock volume, thickness and velocity). A Voronoi tessellation scheme was used to create a rock fabric that allowed the moving rock slope to disaggregate. The range of slide behaviours was investigated by varying the following four factors: (1) type and amount of damping used to simulate the coefficient of restitution and, thus, energy loss during run-out; (2) friction angle of contacts during movement; (3) reservoir effects; and (4) r...

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

Poor nutrition, overweight and obesity have increasingly become a public health concern as they affect many metabolic disorders, including heart disease, diabetes, digestive system disorders, and renal failure. Study of the effects of life style including healthy nutrition will help further elucidate the mechanisms involved in the adverse effects of poor nutrition.Unhealthy life style including poor nutrition can result in imbalance in our oxidation/redox systems. Lipids can undergo oxidative modification by lipoxygenases, cyclooxygenases, myeloperoxidase, and other enzymes. Oxidized phospholipids can induce inflammatory molecules in the liver and other organs. This can contribute to inflammation, leading to coronary heart disease, stroke, renal failure, inflammatory bowl disease, metabolic syndrome, bone and joint disorders, and even certain types of cancer.Our antioxidant and antiinflammatory defense mechanisms contribute to a balance between the stimulators and the inhibitors of inflammation. Beyond a point, however, these systems might be overwhelmed and eventually fail. High-density lipoprotein is a potent inhibitor of the formation of toxic oxidized lipids. High-density lipoprotein is also an effective system for stimulating the genes whose products are active in the removal, inactivation, and elimination of toxic lipids. Supporting the high-density lipoprotein function should help maintain the balance in these systems. It is hoped that the present report would elucidate some of the ongoing work toward this goal.

The post-genomic technologies are generating vast quantities of data but many nutritional scientists are not trained or equipped to analyse it. In high-resolution NMR spectra of urine, for example, the number and complexity of spectral features mean that computational techniques are required to interrogate and display the data in a manner intelligible to the researcher. In addition, there are often multiple underlying biological factors influencing the data and it is difficult to pinpoint which are having the most significant effect. This is especially true in nutritional studies, where small variations in diet can trigger multiple changes in gene expression and metabolite concentration. One class of computational tools that are useful for analysing this highly multivariate data include the well-known ‘whole spectrum’ methods of principal component analysis and partial least squares. In this work, we present a nutritional case study in which NMR data generated from a human dietary Cu intervention study is analysed using multivariate methods and the advantages and disadvantages of each technique are discussed. It is concluded that an alternative approach, called feature subset selection, will be important in this type of work; here we have used a genetic algorithm to identify the small peaks (arising from metabolites of low concentration) that have been altered significantly following a dietary intervention.

We report a novel oil-in-water emulsion that is both buoyancy and refractive index matched. The dispersion is created using low-shear cross-flow membrane emulsification technology with a discrete phase comprising a mixture of n -hexane and the perfluorinated oil tetradecafluorohexane, in the form of FC-72. Three-dimensional confocal laser light imaging of a bulk-aggregated emulsion is demonstrated, showing explicit droplet-level detail of the emulsion interior. Magnetic resonance microscopy with a spatial resolution of tens of microns is also described. The proximity of a liquid–liquid binodal in the oil mixture can give rise to a ternary phase separation reminiscent of an O/O/W emulsion.

Objective— Oxidized 1-palmitoyl-2-arachidonyl- sn -3-glycero-phosphorylcholine (Ox-PAPC) and its component phospholipid, 1-palmitoyl-2-(5,6 epoxyisoprostanoyl)- sn -glycero-3-phosphocholine (PEIPC), which are present in atherosclerotic lesions, activate endothelial cells to induce a complex inflammatory and pro-oxidant response. Previously, we demonstrated induction of genes regulating chemotaxis, sterol biosynthesis, the unfolded protein response, and redox homeostasis by Ox-PAPC in human aortic endothelial cells (HAECs). Activation of the c-Src kinase/signal transducer and activator of transcription 3 and the endothelial nitric oxide synthase/sterol regulatory element binding protein (SREBP) pathways were shown to regulate several of these inflammatory effects of Ox-PAPC in HAECs. The goal of the current studies was to determine the role of high-density lipoprotein (HDL) in regulating Ox-PAPC signaling in HAECs. Methods and Results— Using quantitative real-time polymerase chain reaction, Western analysis, and functional studies, we demonstrated that pretreatment of HAECs with HDL reduced the induction of inflammatory, sterol biosynthetic, and unfolded protein response genes by Ox-PAPC and PEIPC; Ox-PAPC-induced chemotactic activity and monocyte binding were also decreased. These effects were associated with HDL inhibition of Ox-PAPC-induced c-Src , signal transducer and activator of transcription 3, and SREBP activation, alterations in endothelial nitric oxide synthase phosphorylation (previously associated with the inflammatory action of Ox-PAPC), and a decrease in superoxide formation. Finally, we demonstrated that treatment with HDL did not inhibit Ox-PAPC and PEIPC-induced activation of redox pathways, which protect the cell from the effects of oxidative stress. Conclusions— Taken together, these studies demonstrated that HDL inhibits the pro-inflammatory effects of Ox-PAPC and PEIPC, while maintaining the antioxidant activities of these lipids.

A rapid multiple reaction monitoring (MRM) mass spectrometric method for the detection and relative quantitation of the adulteration of meat with that of an undeclared species is presented. Our approach uses corresponding proteins from the different species under investigation and corresponding peptides from those proteins, or CPCP. Selected peptide markers can be used for species detection. The use of ratios of MRM transition peak areas for corresponding peptides is proposed for relative quantitation. The approach is introduced by use of myoglobin from four meats: beef, pork, horse and lamb. Focusing in the present work on species identification, by use of predictive tools, we determine peptide markers that allow the identification of all four meats and detection of one meat added to another at levels of 1% (w/w). Candidate corresponding peptide pairs to be used for the relative quantification of one meat added to another have been observed. Preliminary quantitation data presented here are encouraging.

Noise reduction, restoration, and segmentation methods are developed for the quantitative structural analysis in three dimensions of aggregated oil-in-water emulsion systems imaged by fluorescence confocal laser scanning microscopy. Mindful of typical industrial formulations, the methods are demonstrated for concentrated (30% volume fraction) and polydisperse emulsions. Following a regularized deconvolution step using an analytic optical transfer function and appropriate binary thresholding, novel application of the Euclidean distance map provides effective discrimination of closely clustered emulsion droplets with size variation over at least 1 order of magnitude. The a priori assumption of spherical nonintersecting objects provides crucial information to combat the ill-posed inverse problem presented by locating individual particles. Position coordinates and size estimates are recovered with sufficient precision to permit quantitative study of static geometrical features. In particular, aggregate morphology is characterized by a novel void distribution measure based on the generalized Apollonius problem. This is also compared with conventional Voronoi/Delauney analysis.

We introduce a simple force and flux balance model for sedimentation and creaming in high volume fraction, polydisperse colloidal suspensions. The model is set alongside monodisperse and bidisperse sedimentation data for latex spheres, and we suggest that the broadening of the larger species sedimentation profile observed in the bidisperse case is linked to the particle pressure gradient arising from the smaller species. The model gives a satisfactory qualitative description of real emulsion creaming data, but implies either that the effective droplet radius is larger than the measured droplet radius, or that the effective background viscosity is reduced. Increasing the particle pressure gradient results in interface broadening at short times. We propose that the smallest emulsion droplets contribute to this broadening.

Now, the real work starts. For over half a decade we have talked about moving from science, technology, engineering, and math (STEM) to science, technology, engineering, arts, and math (STEAM). While we must continue to advocate for STEAM education, the passing of the Every Student Succeeds Act (ESSA) in December 2015 has moved us into the implementation stage. ESSA is intended to empower state and local decision makers to build differentiated community-based systems for school improvement in ways that “cookie-cutter federal solutions like No Child Left Behind” never supported (White House Report, 2015, December 10, n.p.). In the reauthorization and amendment of the Elementary and Secondary Education Act of 1965 (ESEA) achieved under ESSA, “Arts and music education are specified as eligible uses for new, state-administered ‘Student Support and Academic Enrichment Grants’ including support for the arts in STEM education” (ESEA Reauthorization is Finalized as Every Student Succeeds Act [ESSA], n.p.). Now th...

Edible oils and fats consist almost entirely of triglycerides, thus triglycerides are of great economic and nutritional importance. Triglycerides are esters of glycerol comprising a glyceride backbone with three fatty acids. These three acyl residues need not be the same, thus edible oils and fats naturally exhibit diverse triglyceride compositions. There are numerous ways of analysing the triglyceride content of oils and fats however the presence of hydrogens in the different environments allows for proton NMR. Here, the authors outline recent results on triglyceride mixtures obtained using a new, low-field 1H NMR spectrometer called Pulsar. The Pulsar is based on permanent magnets rather than the superconducting magnets standard in modern high-field instruments. Any technique capable of analysing triglyceride mixtures is therefore able to establish key compositional properties of pure oils and fats and may be able to detect the addition of one oil to another. Using these approaches, low-field 1H NMR offers a viable method for testing the authenticity of meat and the detecting the adulteration of edible oils with one another.

Addition of the non-adsorbing polymer hydroxyethylcellulose (HEC) to oil-in-water emulsions induces flocculation via the depletion mechanism. In the present work, we report results on the delay time prior to creaming of hexadecane-in-water emulsions, flocculated by HEC. In addition, we report on oscillation rheology measurements of a comparable, density-matched system. We compare the two in the search for rheological signatures of delay, and for insight into the delay mechanism. Our results suggest that the commonly used low-frequency relaxation time does not map simply to delay time. We do, however, find that rheology can tell which of our compositions will exhibit delay, and which will not. In addition, those compositions having the greatest shear modulus have the longest delay. A simple model, in which the delay is attributed to a slow creaming stage over a very small distance, produces an acceptable correlation with the product of complex viscosity and the strain at the end of linearity. Thus the area under the linear portion of the complex viscosity–strain plot may provide a useful marker of delay time. The unexpected observation of a liquid-like regime at high oscillation frequency merits further investigation.

There is growing evidence for the accumulation of phospholipid oxidation products (some of which can also be formed enzymatically) in several chronic disease processes including atherosclerosis. There also is considerable evidence that enzymes involved in hydrolysis of these phospholipids (present in both lipoproteins and cells) may be important in regulation of atherogenesis. In vitro studies suggest that these lipids can activate vascular wall cells to states that contribute to the atherosclerotic process. This review focuses on two types of bioactive phospholipids: phosphatidyl cholines in which the sn-2 fatty acid has been modified by oxidation and lysophosphatidic acid in which both the sn-2 and sn-3 positions have been modified. The mechanism by which these phospholipid oxidation products activate cells has revealed the presence of several different receptors and signal transduction pathways.

There is considerable evidence to suggest that oxidation of LDL plays an important role in atherogenesis. Polyunsaturated fatty acids, a major oxidative target, are present as phospholipids in the outer core of the lipoprotein particle. Studies from several laboratories have shown an increase in the levels of phospholipid oxidation products in atherosclerotic lesions and of antibodies to oxidized phospholipids in mice and humans with lesions. Significantly, phospholipid oxidation products have been demonstrated (in vitro) to selectively activate processes in vascular wall cells that may contribute to atherogenesis. This review discusses activities, methods for isolation, identification and measurement of bioactive phospholipids. Past studies suggest that defined and relatively simple current technologies allow identification of bioactive phospholipid oxidation products and measurement of their levels in tissue.

Abstract Leukocyte binding to the endothelium is one of the earliest events in the occurrence of atherosclerosis. Leukocyte adhesion molecules involved in this process have not been definitely identified. We have found that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) for 24 hours caused a 2- to 3-fold increase of P-selectin protein, with little change in P-selectin surface expression. A 15-minute histamine treatment of cells exposed to MM-LDL caused a 50% to 100% increase in P-selectin surface expression compared with cells not treated with the lipoprotein. This increase resulted in a 2-fold increase in binding of leukocytes to the endothelium. Immunostaining of permeabilized HAECs after MM-LDL treatment also revealed a highly reproducible increase in intracellular P-selectin associated with rod-shaped structures, typical of Weibel-Palade bodies. Oxidized phospholipids were shown to be mainly responsible for the action of MM-LDL. This increased P-selectin expression was associated with MM-LDL–induced cAMP elevation. Like histamine, highly oxidized low-density lipoprotein, especially the oxidized fatty acids, caused immediate redistribution of P-selectin to the cell surface followed by reinternalization. Immunohistochemical staining showed that endothelial cells on human fatty streak lesions expressed increased levels of P-selectin compared with nonlesion areas. These studies suggest that P-selectin may play an important role in early recruitment of mononuclear cells to the subendothelium in human atherosclerosis and that oxidized lipoproteins may contribute to the increased expression of this molecule by increasing intracellular stores and causing redistribution to the cell surface.

Abstract Atherosclerotic calcification and osteoporosis often coexist in patients, yielding formation of bone mineral in vascular walls and its simultaneous loss from bone. To assess the potential role of lipoproteins in both processes, we examined the effects of minimally oxidized low-density lipoprotein (MM-LDL) and several other lipid oxidation products on calcifying vascular cells (CVCs) and bone-derived preosteoblasts MC3T3-E1. In CVCs, MM-LDL but not native LDL inhibited proliferation, caused a dose-dependent increase in alkaline phosphatase activity, which is a marker of osteoblastic differentiation, and induced the formation of extensive areas of calcification. Similar to MM-LDL, oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine (ox-PAPC) and the isoprostane 8-iso prostaglandin E 2 but not PAPC or isoprostane 8-iso prostaglandin F 2α induced alkaline phosphatase activity and differentiation of CVCs. In contrast, MM-LDL and the above oxidized lipids inhibited differentiation of the MC3T3-E1 bone cells, as evidenced by their stimulatory effect on proliferation and their inhibitory effect on the induction of alkaline phosphatase and calcium uptake. These results suggest that specific oxidized lipids may be the common factors underlying the pathogenesis of both atherosclerotic calcification and osteoporosis.

A total synthesis of the naturally occurring hydroxy ketone PEIPC 1, a compound that plays a role in endothelial activation in atherosclerosis, has been completed via a triply convergent preparation of a protected EI derivative 13 from 3,5-diacetoxycyclopentene 7, pentane-1,5-diol, and vinyllithium, using Sharpless epoxidation and enzymatic resolution as key steps. Final coupling with lyso-PC 16 and silyl group deprotection gave PECPC 2 and PEIPC 1, which showed the same activity as natural PECPC and PEIPC. [reaction: see text]

The earliest lesion in the development of an atherosclerotic plaque is the fatty streak. This chronic inflammatory reaction results from a sequence of events that begins with the trapping of low density lipoprotein (LDL) in the subendothelial space of the artery wall. The trapped LDL is seeded with oxidative species released by the overlying endothelium, and lipid oxidation is initiated within the LDL particle. Some of the lipids that result lead to the activation of NFkB-like transcription factors that cause the expression of genes whose protein products mediate monocyte binding, monocyte chemotaxis into the subendothelial space, and conversion into macrophages. At least 1 major gene modulates the oxidation of LDL lipids and/or the biologic response to these lipids. The inverse relation between high density lipoprotein (HDL) and atherosclerotic events may in part be due to enzymes associated with HDL that destroy the biologically active lipids generated in LDL.

Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), referred to as OxPAPC, and an active component, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphatidylcholine (PEIPC), accumulate in atherosclerotic lesions and regulate over 1,000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog of OxPAPC, covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine whether binding regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited the binding of OxPNB to HAEC proteins. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPNB and PEIPE-NB, the analog of PEIPC, bound to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This binding was observed with recombinant protein and in cells overexpressing H-Ras. OxPAPC and PEIPC compete with OxPNB for binding to H-Ras. 15dPGJ2 and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC, PEIPC, and 15dPGJ2 in HAEC, suggesting that some effects attributed to 15dPGJ2 may also be regulated by PEIPC because both molecules accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function.

Glioblastoma, the most common malignant brain tumor, is among the most lethal and difficult cancers to treat. Although epidermal growth factor receptor (EGFR) mutations are frequent in glioblastoma, their clinical relevance is poorly understood. Studies of tumors from patients treated with the EGFR-inhibitor lapatinib revealed that EGFR induces the cleavage and nuclear translocation of the master transcriptional regulator of fatty acid synthesis, sterol regulatory element-binding protein 1 (SREBP-1). This response was mediated by Akt; however, clinical data from rapamycin-treated patients showed that SREBP-1 activation was independent of the mammalian target of rapamycin complex 1 (mTORC1), possibly explaining rapamycin’s poor efficacy in the treatment of such tumors. Glioblastomas without constitutively active EGFR signaling were resistant to inhibition of fatty acid synthesis, whereas introduction of a constitutively active mutant form of EGFR, EGFRvIII, sensitized tumor xenografts in mice to cell death, which was augmented by the hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor atorvastatin. These results identify a previously undescribed EGFR-mediated pro-survival metabolic pathway, and suggest new therapeutic approaches to treating EGFR-activated glioblastomas.

Rationale: Matrix Gla protein (MGP) is a calcification inhibitor, which binds and inhibits bone morphogenetic protein (BMP)-2 and -4. Objective: The objective was to determine whether MGP also binds other proteins, which could interfere with its function. Methods and Results: We transfected bovine aortic endothelial cells with N-terminally FLAG-tagged MGP and used immunoprecipitation and liquid chromatographic–tandem mass spectrometric analysis to identify MGP-binding proteins. Heat shock protein (HSP)70, a stress-induced protein expressed in atherosclerotic lesions and soluble in serum, was identified as a novel MGP-binding protein. The interaction between MGP and HSP70 was confirmed by coimmunoprecipitation and chemical crosslinking, and blocked the interaction between MGP and BMP-4. In endothelial cells, HSP70 enhanced BMP-4–induced proliferation and tube formation, and in calcifying vascular cells, HSP70 enhanced BMP-induced calcium deposition. In addition, HSP70 mediated the procalcific effect of interleukin-6 on calcifying vascular cells. In apolipoprotein E–null mice, a model for atherosclerosis, levels of BMP-4, HSP70, MGP, and interleukin-6 were elevated in the aortic wall. Levels of BMP-4, HSP70, and interleukin-6 were also elevated in serum, and anti-HSP70 antibodies diminished its procalcific effect on calcifying vascular cells. Conclusion: HSP70 binds MGP and enhances BMP activity, thereby functioning as a potential link between cellular stress, inflammation, and BMP signaling.

An improved synthesis of the naturally occurring hydroxy ketone 1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC) 1, a compound that plays a role in endothelial activation...

An improved synthesis of the naturally occurring hydroxy ketone 1-palmitoyl-2-(5,6)-epoxyisoprostane E 2- sn-glycero-3-phosphocholine (PEIPC) 1, a compound that plays a role in endothelial activation in atherosclerosis, has been carried out using a PMB ether as the key protecting group. Opening of an intermediate with pentylamine shows that the allylic epoxide is the position of attack by nucleophiles.

Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (Ox-PAPC) are found in atherosclerotic lesions, apoptotic cells, and oxidized LDL and stimulate human aortic endothelial cells (HAECs) to produce inflammatory cytokines, leukocyte chemoattractants, and coagulation factors. This regulation is thought to be a receptor-mediated process in which oxidized phospholipids activate specific receptors on HAECs to evoke an inflammatory response. To characterize the HAEC proteins with which oxidized phospholipids interact, a biotinylated PAPC analog, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidyl-(N-biotinylethanolamine) (PAPE-N-biotin), was synthesized. Oxidation of PAPE-N-biotin in air generated a mixture of biotin-labeled oxidized lipids analogous to Ox-PAPC. Ox-PAPE-N-biotin, like Ox-PAPC, induced interleukin-8 (IL-8) protein synthesis and stimulated IL-8, low density lipoprotein receptor, heme oxygenase-1, and activating transcription factor-3 mRNA expression in HAECs. After treatment of HAECs with Ox-PAPE-N-biotin, the cellular proteins were isolated and separated by SDS-PAGE. Western analysis with streptavidin-HRP demonstrated at least 20 different biotinylated HAEC proteins to which the Ox-PAPE-N-biotin was associated, which were not detected with unoxidized PAPE-N-biotin treatment. This work suggests that oxidized phospholipids, such as those found in oxidized LDL, apoptotic cells, and atherosclerotic lesions, form tight interactions with specific endothelial cell proteins, which may be responsible for the inflammatory response. Identification of these putative oxidized phospholipid targets may reveal therapeutic targets to modulate inflammation and atherosclerosis.

High-density lipoprotein (HDL) is conventionally believed to possess many features that protect against atherosclerosis. However, these lipoproteins may be modified in certain individuals and/or circumstances to become pro-inflammatory. The ability of HDL to inhibit or paradoxically to enhance vascular inflammation, lipid oxidation, plaque growth, and thrombosis reflects changes in specific enzyme and protein components. The anti-inflammatory and pro-inflammatory functional properties of HDL can now be assessed using cell-based and cell-free assays. Acute or chronic systemic inflammation and the metabolic syndrome appear to render HDL pro-inflammatory. In contrast, statins and experimental agents such as apolipoprotein A-1 mimetics render HDL more anti-inflammatory. The 2 main classes of existing drugs for HDL modification are fibric acid derivatives, also known as "fibrates," and niacin- containing compounds. In several controlled and prospective intervention studies, patients with low HDL-C and additional risk factors benefited from treatment with fibrates or niacin. However, an increase in HDL-C did not lead to a decrease in cardiovascular events in all trials.

Objective— Previous studies have shown that oxidized products of PAPC (Ox-PAPC) regulate cell transcription of interleukin-8, LDL receptor, and tissue factor. This upregulation takes place in part through the activation of sterol regulatory element-binding protein (SREBP) and Erk 1/2. The present studies identify vascular endothelial growth factor receptor 2 (VEGFR2) as a major regulator in the activation of SREBP and Erk 1/2 in endothelial cells activated by Ox-PAPC. Methods and Results— Ox-PAPC induced the phosphorylation of VEGFR2 at Tyr 1175 in human aortic endothelial cells. Inhibitors and siRNA for VEGFR2 decreased the transcription of interleukin-8, LDL receptor, and tissue factor in response to Ox-PAPC and the activation of SREBP and Erk 1/2, which mediate this transcription. We provide evidence that the activation of VEGFR2 is rapid, sustained, and c-Src–dependent. Conclusions— These data point to a major role of VEGFR2 in endothelial regulation by oxidized phospholipids which accumulate in atherosclerotic lesions and apoptotic cells.

Food Colloids: Interactions, Microstructure and Processing describes the principles and practice underlying the formulation of food emulsions, dispersions, gels, and foams. Emphasis is on understanding how the functional properties of biopolymers and surfactants determine the texture and shelf-life of multiphase food materials. This book provides essential new findings by experts in the field on specific topics including: the interfacial rheological properties of proteins; the use of microscopy and image analysis to probe structure and phase transitions; the control of colloidal stability during thermal and mechanical processing; the interactions of proteins with polysaccharides and emulsifiers; the incorporation of neutraceuticals into food colloids; and the consumer perception of taste and texture. Food Colloids: Interactions, Microstructure and Processing provides a link between current research on the fundamental physical chemistry of colloidal systems and the requirements of the food technologist to use modern colloid science in new product formulation. It is suitable for postgraduates and researchers, both in industry and academia.

The earliest lesion in the development of an atherosclerotic plaque is the fatty streak. This chronic inflammatory reaction results from a sequence of events that begins with the retention of low density lipoprotein (LDL) in the subendothelial space of the artery wall. The trapped LDL is“seeded” with oxidative species generated by endothelial 12-lipoxygenase that can be released and initiate lipid oxidation within the LDL particle. Specific lipids that result, lead to the activation of molecules including NF-kB-like transcription factors that cause the expression of genes whose protein products mediate monocyte binding, monocyte chemotaxis into the subendothelial space, and their conversion into macrophages. High density lipoprotein (HDL) prevents these events and the inverse relation between HDL and atherosclerotic events may in part be due to enzymes associated with HDL that destroy the biologically active lipids generated in LDL. The potential role of one of these enzymes, paraoxonase 1 (PON1) is discussed below.

Abstract —Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn -2 position occurs when a critical concentration of “seeding molecules” derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.

The addition of nonadsorbing polymer to an alkane-in-water emulsion causes the droplets to flocculate into a space-spanning, stress-bearing network. We report rheological measurements of an emulsion of 1-bromohexadecane-in-water flocculated by hydroxy-ethylcellulose. Small-deformation oscillatory measurements allowed characterization of the structure during formation and an indication of the strength of the resulting network. Emulsions without polymer, and polymer solutions alone, showed essentially viscous behavior, with dominant viscous modulus over the whole frequency range (0.01-10 Hz). However, the emulsion containing polymer demonstrated a significant elastic modulus, dependent on the oil and polymer concentrations, attributable to interdroplet depletion interactions. Power-law relationships were observed between the elastic modulus, elastic strain limit, and oil volume fraction, but the indices were lower than those predicted by fractal models, giving unrealistic fractal dimensionalities. The modulus increased exponentially with polymer concentration, but the elastic strain limit was independent of added polymer. The rate of formation of the network was not consistent with diffusion-controlled aggregation. Copyright 2000 Academic Press.