Sustainable and Eco-Friendly DLC Fabrication for Replacing Chrome on a Gravure Cylinder Surface.

This paper evaluates the potential of diamond-like carbon (DLC) as a durable surface protection to replace the chromium (Cr) layer, which is traditionally applied to gravure print cylinders and other components through a galvanic electroplating process. The fabrication of DLC is more eco-friendly and could reduce the environmental hazard posed by hexavalent chromium in liquid form that is used in Cr application and better adhere to environmental regulations. This could encourage businesses to bring the DLC fabrication process in-house, sharing resources such as materials, labor, and equipment, to help reduce costs. Four DLC variants (standard DLC, A-DLC, S-DLC, and organic silica) and chrome were analyzed and tested for their surface properties and durability. Data suggest that both standard DLC and S-DLC had higher surface free energy, allowing for good ink wetting on the surface when compared to chrome. In addition, the standard DLC and S-DLC surfaces are generally smoother than the chrome, resulting in lower relative hydrophilicity and allowing for easier removal of ink in the nonimage regions with the doctor blade. The elcometer adhesion test demonstrated that the bond strength of the DLC variants to their base layer was comparable to the bond strength of chrome, indicating that the adhesion strength of the two materials was similar. Furthermore, in the abrasion test, the wear on the standard DLC surface and the corresponding wear on the lamella tip of the metal doctor blades were notably lower than that observed on chrome. This distinction is particularly evident in each of the test trials, specifically run 1, which involved 2,000,000 wiping actions of a metal doctor blade in the presence of abrasive TiO2 ink pigments. Statistical analysis on standard DLC versus chrome suggests that DLC fabrication is effective and durable on plain and patterned surfaces. Therefore, from a sustainable and eco-friendly perspective, standard DLC and S-DLC would be good alternative durable surfaces for print cylinders and other components used in various industries due to superior wear resistance properties.

Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter.

Macrophages detect invading microorganisms via pattern recognition receptors that recognize pathogen-associated molecular patterns, or via sensing the activity of virulence factors that initiates effector-triggered immunity (ETI). Tissue damage that follows pathogen encounter leads to the release of host-derived factors that participate to inflammation. How these self-derived molecules are sensed by macrophages and their impact on immunity remain poorly understood. Here we demonstrate that, in mice and humans, host-derived oxidized phospholipids (oxPLs) are formed upon microbial encounter. oxPL blockade restricts inflammation and prevents the death of the host, without affecting pathogen burden. Mechanistically, oxPLs bind and inhibit AKT, a master regulator of immunity and metabolism. AKT inhibition potentiates the methionine cycle, and epigenetically dampens Il10, a pluripotent anti-inflammatory cytokine. Overall, we found that host-derived inflammatory cues act as "self" virulence factors that initiate ETI and that their activity can be targeted to protect the host against excessive inflammation upon microbial encounter.

Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation.

Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here, we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide. While cells activated by lipopolysaccharide rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine, and favor the accumulation of oxaloacetate in the cytoplasm. This metabolite potentiates interleukin-1ß production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.

Prostate cancer recognition based on mass spectrometry sensing data and data fingerprint recovery.

The high dimensionality and noisy spectra of Mass Spectrometry (MS) data are two of the main challenges to achieving high accuracy recognition. The objective of this work is to produce an accurate prediction of class content by employing compressive sensing (CS). Not only can CS significantly reduce MS data dimensionality, but it will also allow for full reconstruction of original data. We are proposing a weighted mixing of L1- and L2-norms via a regularization term as a classifier within compressive sensing framework. Using performance measures such as OSR, PPV, NPV, Sen and Spec, we show that the L2-algorithm with regularization terms outperforms the L1-algorithm and Q5 under all applicable assumptions. We also aimed to use Block Sparse Bayesian Learning (BSBL) to reconstruct the MS data fingerprint which has also shown better performance results that those of L1-norm. These techniques were successfully applied to MS data to determine patient risk of prostate cancer by tracking Prostate-specific antigen (PSA) protein, and this analysis resulted in better performance when compared to currently used algorithms such as L1 minimization. This proposed work will be particularly useful in MS data reduction for assessing disease risk in patients and in future personalized medicine applications.

By Capturing Inflammatory Lipids Released from Dying Cells, the Receptor CD14 Induces Inflammasome-Dependent Phagocyte Hyperactivation.

A heterogeneous mixture of lipids called oxPAPC, derived from dying cells, can hyperactivate dendritic cells (DCs) but not macrophages. Hyperactive DCs are defined by their ability to release interleukin-1 (IL-1) while maintaining cell viability, endowing these cells with potent aptitude to stimulate adaptive immunity. Herein, we found that the bacterial lipopolysaccharide receptor CD14 captured extracellular oxPAPC and delivered these lipids into the cell to promote inflammasome-dependent DC hyperactivation. Notably, we identified two specific components within the oxPAPC mixture that hyperactivated macrophages, allowing these cells to release IL-1 for several days, by a CD14-dependent process. In murine models of sepsis, conditions that promoted cell hyperactivation resulted in inflammation but not lethality. Thus, multiple phagocytes are capable of hyperactivation in response to oxPAPC, with CD14 acting as the earliest regulator in this process, serving to capture and transport these lipids to promote inflammatory cell fate decisions.

Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells.

Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.

An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells.

Dendritic cells (DCs) use pattern recognition receptors to detect microorganisms and activate protective immunity. These cells and receptors are thought to operate in an all-or-nothing manner, existing in an immunologically active or inactive state. Here, we report that encounters with microbial products and self-encoded oxidized phospholipids (oxPAPC) induce an enhanced DC activation state, which we call "hyperactive." Hyperactive DCs induce potent adaptive immune responses and are elicited by caspase-11, an enzyme that binds oxPAPC and bacterial lipopolysaccharide (LPS). oxPAPC and LPS bind caspase-11 via distinct domains and elicit different inflammasome-dependent activities. Both lipids induce caspase-11-dependent interleukin-1 release, but only LPS induces pyroptosis. The cells and receptors of the innate immune system can therefore achieve different activation states, which may permit context-dependent responses to infection.

HDL inhibits the effects of oxidized phospholipids on endothelial cell gene expression via multiple mechanisms.

Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phospholcholine (OxPAPC) and its component phospholipids accumulate in atherosclerotic lesions and regulate the expression of >1,000 genes, many proatherogenic, in human aortic endothelial cells (HAECs). In contrast, there is evidence in the literature that HDL protects the vasculature from inflammatory insult. We have previously shown that in HAECs, HDL attenuates the expression of several proatherogenic genes regulated by OxPAPC and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. We now demonstrate that HDL reverses >50% of the OxPAPC transcriptional response. Genes reversed by HDL are enriched for inflammatory and vascular development pathways, while genes not affected by HDL are enriched for oxidative stress response pathways. The protective effect of HDL is partially mimicked by cholesterol repletion and treatment with apoA1 but does not require signaling through scavenger receptor class B type I. Furthermore, our data demonstrate that HDL protection requires direct interaction with OxPAPC. HDL-associated platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes short-chain bioactive phospholipids in OxPAPC; however, inhibiting PAF-AH activity does not prevent HDL protection. Our results are consistent with HDL sequestering specific bioactive lipids in OxPAPC, thereby preventing their regulation of select target genes. Overall, this work implicates HDL as a major regulator of OxPAPC action in endothelial cells via multiple mechanisms.

An epoxyisoprostane is a major regulator of endothelial cell function.

The goal of these studies was to determine the effect of 5,6-epoxyisoprostane, EI, on human aortic endothelial cells (HAEC). EI can form as a phospholipase product of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine, PEIPC, a proinflammatory molecule that accumulates in sites of inflammation where phospholipases are also increased. To determine the effect of EI on HAEC, we synthesized several stereoisomers of EI using a convergent approach from the individual optically pure building blocks, the epoxyaldehydes 5 and 6 and the bromoenones 14 and 16. The desired stereoisomer of EI can be prepared from these materials in only six operations, and thus, large amounts of the product can be obtained. The trans/trans isomers had the most potent activity, suggesting specificity in the interaction of EI with the cell surface. EI has potent anti-inflammatory effects in HAEC. EI strongly inhibits the production of MCP-1, a major monocyte chemotactic factor, and either decreases or minimally increases the levels of 10 proinflammatory molecules increased by PEIPC. EI also strongly down-regulates the inflammatory effects of IL-1ß, a major inflammatory cytokine. Thus EI, a hydrolytic product of PEIPC, has potent anti-inflammatory function.

A novel approach to oral apoA-I mimetic therapy.

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR(-/-)) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.

Role of phospholipid oxidation products in atherosclerosis.

There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells, and HDL to promote atherogenesis. In the past few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long-term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects have been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways, some of which are proatherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have of the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.

Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function.

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.

Metalloproteinase processing of HBEGF is a proximal event in the response of human aortic endothelial cells to oxidized phospholipids.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease initiated by monocyte recruitment and retention in the vessel wall. An important mediator of monocyte endothelial interaction is the chemokine interleukin (IL)-8. The oxidation products of phospholipids, including oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC), accumulate in atherosclerotic lesions and strongly induce IL-8 in human aortic endothelial cells (HAECs). The goal of this study was to identify the proximal events leading to induction of IL-8 by Ox-PAPC in vascular endothelial cells. METHODS AND RESULTS: In a systems genetics analysis of HAECs isolated from 96 different human donors, we showed that heparin-binding EGF-like growth factor (HBEGF) transcript levels are strongly correlated to IL-8 induction by Ox-PAPC. The silencing and overexpression of HBEGF in HAECs confirmed the role of HBEGF in regulating IL-8 expression. HBEGF has been shown to be stored in an inactive form and activation is dependent on processing by a dysintegrin and metalloproteinases (ADAM) to a form that can activate the epidermal growth factor (EGF) receptor. Ox-PAPC was shown to rapidly induce HBEGF processing and EGF receptor activation in HAECs. Using siRNA we identified 3 ADAMs that regulate IL-8 induction and directly demonstrated that Ox-PAPC increases ADAM activity in the cells using a substrate cleavage assay. We provide evidence for one mechanism of Ox-PAPC activation of ADAM involving covalent binding of Ox-PAPC to cysteine on ADAM. Free thiol cysteine analogs showed inhibition of IL-8 induction by Ox-PAPC, and both a cysteine analog and a cell surface thiol blocker strongly inhibited ADAM activity induction by Ox-PAPC. Using microarray analyses, we determined that this ADAM pathway may regulate at least 30% of genes induced by Ox-PAPC in HAECs. CONCLUSIONS: This study is the first report demonstrating a role for the ADAM-HBEGF-EGF receptor axis in Ox-PAPC induction of IL-8 in HAECs. These studies highlight a role for specific ADAMs as initiators of Ox-PAPC action and provide evidence for a role of covalent interaction of Ox-PAPC in activation of ADAMs.

Characterization of a key trifunctional enzyme for aromatic amino acid biosynthesis in Archaeoglobus fulgidus.

In the aromatic amino acid biosynthesis pathway, chorismate presents a branch point intermediate that is converted to tryptophan, phenylalanine (Phe), and tyrosine (Tyr). In bacteria, three enzymes catalyze the conversion of chorismate to hydroxyphenylpyruvate or pyruvate. The enzymes, chorismate mutase (CM), prephenate dehydratase (PDT), and prephenate dehydrogenase (PDHG) are either present as distinct proteins or fusions combining two activities. Gene locus AF0227 of Archaeoglobus fulgidus is predicted to encode a fusion protein, AroQ, containing all three enzymatic domains. This work describes the first characterization of a trifunctional AroQ. The A. fulgidus aroQ gene was cloned and overexpressed in Escherichia coli. The recombinant protein purified as a homohexamer with specific activities of 10, 0.51, and 50 U/mg for CM, PDT, and PDHG, respectively. Tyr at 0.5 mM concentration inhibited PDHG activity by 50%, while at 1 mM PDT was activated by 70%. Phe at 5 muM inhibited PDT activity by 66% without affecting the activity of PDHG. A fusion of CM, PDT, and PDHG domains is evident in the genome of only one other organism sequenced to date, that of the hyperthermophilic archaeon, Nanoarchaeum equitans. Such fusions of contiguous activities in a biosynthetic pathway may constitute a primitive strategy for the efficient processing of labile metabolites.

Effect of growth temperature on ether lipid biochemistry in Archaeoglobus fulgidus.

The archaea are distinguished by their unique isoprenoid ether lipids, which typically consist of the sn-2,3-diphytanylglycerol diether or sn-2,3-dibiphytanyldiglycerol tetraether core modified with a variety of polar headgroups. However, many hyperthermophilic archaea also synthesize tetraether lipids with up to four pentacyclic rings per 40-carbon chain, presumably to improve membrane thermal stability at temperatures up to approximately 110 degrees C. This study aimed to correlate the ratio of tetraether to diether core lipid, as well as the presence of pentacyclic groups in tetraether lipids, with growth temperature for the hyperthermophilic archaeon, Archaeoglobus fulgidus. Analysis of the membrane core lipids of A. fulgidus using APCI-MS analysis revealed that the tetraether-to-diether lipid ratio increases from 0.3 +/- 0.1 for cultures grown at 70 degrees C to 0.9 +/- 0.1 for cultures grown at 89 degrees C. Thin-layer chromatography (TLC) followed by APCI-MS analysis provided evidence for no more than one pentacycle in the hydrocarbon chains of tetraether lipid from cultures grown at 70 degrees C and up to 2 pentacycles in the tetraether lipid from cultures grown at higher temperatures. Analysis of the polar lipid extract using TLC and negative-ion ESI-MS suggested the presence of diether and tetraether phospholipids with inositol, glycosyl, and ethanolamine headgroup chemistry.