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Chronic respiratory diseases are complex multifactorial disorders whose pathogenesis depends on the interplay between host and environmental factors. To fully understand them and to identify novel treatments, a holistic approach that integrates multiple types and levels of clinical and biological data is necessary. Toward this end, the application of systems biology-based strategies, in particular, network analysis, offers great potential. These systems-based approaches rely heavily on computational methods that can be challenging for the nonspecialist. Accordingly, this Pulmonary Perspective: (1) outlines the basic concepts of networks in biology and the fundamentals of network analysis, and (2) discusses recent applications of network analysis to understand respiratory diseases. The intent of this Perspective is to provide readers with increased understanding of the strengths and weaknesses of network analysis methods as well as their usefulness in addressing research questions involving chronic respiratory diseases.

Background: Prostaglandins that constrict and relax airways are synthesized in reactions catalyzed by either COX-1 or COX-2. It is not known whether selective inhibition of COX-2 makes asthmatic responses better or worse., Objective: To determine the effects of the selective COX-2 inhibitor, etoricoxib, on allergen-induced bronchoconstriction in asthmatic subjects., Methods: Sixteen subjects with mild atopic asthma underwent rising dose inhalation challenges with allergen or methacholine to determine PD20 FEV1 during a control study period or after 10 to 13 days of treatment with etoricoxib (90 mg once daily). The order of study periods was randomized with at least 2-week washout periods. Induced sputum cells and fractional exhaled nitric oxide levels were used to assess airway inflammation and blood assays for COX-1 and COX-2 activity to assess enzyme inhibition. Urinary excretion of lipids was used to assess prostaglandin biosynthesis., Results: Etoricoxib did not change baseline lung function, nor airway responsiveness to allergen or to methacholine. Neither were the allergen-induced increase in sputum eosinophils and fractional exhaled nitric oxide levels affected by treatment. The biochemical effectiveness of the treatment was established both in the blood assays and by the distinct inhibitory effect of etoricoxib on urinary excretion of tetranor-prostaglandin E2 (P < .001)., Conclusions: This first study of COX-2 inhibition in provoked asthma found no negative effects of etoricoxib on allergen-induced airflow obstruction and sputum eosinophils, basal lung function, or methacholine responsiveness. The study suggests that short-term use of COX-2 inhibitors is safe in subjects with asthma., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.

Background: Nitrate, which is an inorganic anion abundant in vegetables, increases the efficiency of isolated human mitochondria. Such an effect might be reflected in changes in the resting metabolic rate (RMR) and formation of reactive oxygen species. The bioactivation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species., Objective: We studied effects of inorganic nitrate, in amounts that represented a diet rich in vegetables, on the RMR in healthy volunteers., Design: In a randomized, double-blind, crossover study, we measured the RMR by using indirect calorimetry in 13 healthy volunteers after a 3-d dietary intervention with sodium nitrate (NaNO₃) or a placebo (NaCl). The nitrate dose (0.1 mmol · kg⁻¹ · d⁻¹) corresponded to the amount in 200-300 g spinach, beetroot, lettuce, or other vegetable that was rich in nitrate. Effects of direct nitrite exposure on cell respiration were studied in cultured human primary myotubes., Results: The RMR was 4.2% lower after nitrate compared with placebo administration, and the change correlated strongly to the degree of nitrate accumulation in saliva (r² = 0.71). The thyroid hormone status, insulin sensitivity, glucose uptake, plasma concentration of isoprostanes, and total antioxidant capacity were unaffected by nitrate. The administration of nitrite to human primary myotubes acutely inhibited respiration., Conclusions: Dietary inorganic nitrate reduces the RMR. This effect may have implications for the regulation of metabolic function in health and disease.

Oxylipins are oxidised fatty acids that can exert lipid mediator functions in inflammation, and several oxylipins derived from arachidonic acid are linked to asthma. This study quantified oxylipin profiles in different regions of the lung to obtain a broad-scale characterisation of the allergic asthmatic inflammation in relation to healthy individuals. Bronchoalveolar lavage fluid (BALF), bronchial wash fluid and endobronchial mucosal biopsies were collected from 16 healthy and 16 mildly allergic asthmatic individuals. Inflammatory cell counts, immunohistochemical staining and oxylipin profiling were performed. Univariate and multivariate statistics were employed to evaluate compartment-dependent and diagnosis-dependent oxylipin profiles in relation to other measured parameters. Multivariate modelling showed significantly different bronchial wash fluid and BALF oxylipin profiles in both groups (R(2)Y[cum]=0.822 and Q(2)[cum]=0.759). Total oxylipin concentrations and five individual oxylipins, primarily from the lipoxygenase (LOX) pathway of arachidonic and linoleic acid, were elevated in bronchial wash fluid from asthmatics compared to that from healthy controls, supported by immunohistochemical staining of 15-LOX-1 in the bronchial epithelium. No difference between the groups was found among BALF oxylipins. In conclusion, bronchial wash fluid and BALF contain distinct oxylipin profiles, which may have ramifications for the study of respiratory diseases. Specific protocols for sampling proximal and distal airways separately should be employed for lipid mediator studies.

Respiratory diseases are multifactorial heterogeneous diseases that have proved recalcitrant to understanding using focused molecular techniques. This trend has led to the rise of 'omics approaches (e.g., transcriptomics, proteomics) and subsequent acquisition of large-scale datasets consisting of multiple variables. In 'omics technology-based investigations, discrepancies between the number of variables analyzed (e.g., mRNA, proteins, metabolites) and the number of study subjects constitutes a major statistical challenge. The application of traditional univariate statistical methods (e.g., t-test) to these "short-and-wide" datasets may result in high numbers of false positives, while the predominant approach of p-value correction to account for these high false positive rates (e.g., FDR, Bonferroni) are associated with significant losses in statistical power. In other words, the benefit in decreased false positives must be counterbalanced with a concomitant loss in true positives. As an alternative, multivariate statistical analysis (MVA) is increasingly being employed to cope with 'omics-based data structures. When properly applied, MVA approaches can be powerful tools for integration and interpretation of complex 'omics-based datasets towards the goal of identifying biomarkers and/or subphenotypes. However, MVA methods are also prone to over-interpretation and misuse. A common software used in biomedical research to perform MVA-based analyses is the SIMCA package, which includes multiple MVA methods. In this opinion piece, we propose guidelines for minimum reporting standards for a SIMCA-based workflow, in terms of data preprocessing (e.g., normalization, scaling) and model statistics (number of components, R2, Q2, and CV-ANOVA p-value). Examples of these applications in recent COPD and asthma studies are provided. It is expected that readers will gain an increased understanding of the power and utility of MVA methods for applications in biomedical research.

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration-dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration-dependent pathway was independent of peroxisome proliferator-activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid-mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an "unbiased" approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the 'omics field. The contributions of the individual 'omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease.

Eicosanoids (e.g., prostaglandins and leukotrienes) are inflammatory signaling molecules that are metabolized and excreted in urine. The quantification of eicosanoid metabolites in human urine has been demonstrated to provide insight into the inflammatory and oxidative stress status of the individual. However, urine is a complex matrix that can exhibit profound matrix effects for quantification via liquid chromatography coupled to mass spectrometry (LC-MS/MS). This phenomenon can lead to impairment and biasing of results, because the sample background is dependent on the fluid intake and water-salt balance. Herein we describe an analytical methodology to address these limitations via the normalization of extracted urine volume by the ratio of absorbance at 300 nm to an optimized reference material. The platform is composed of 4 LC-MS/MS methods that collectively quantify 26 lipid mediators and their metabolites, with on-column limits of detection between 0.55 and 15 fmol. Prior to optimization, internal standards exhibited strong matrix effects with up to 50% loss of signal. Notably, the accuracy of exact deuterated structural analogues was found to vary based upon the number of incorporated deteurium. The platform was used to analyze urine from 16 atopic asthmatics under allergen provocation, showing increases in metabolites of prostaglandin D2, cysteinyl leukotrienes, and isoprostanes following the challenge. This method presents a functional and reproducible approach to addressing urine-specific matrix effects that can be readily formatted for quantifying large numbers of samples.

Lipids are heterogeneous biological molecules that possess multiple physiological roles including cell structure, homeostasis, and restoration of tissue functionality during and after inflammation. Lipid metabolism constitutes a network of pathways that are related at multiple biosynthetic hubs. Disregulation of lipid metabolism can lead to pathophysiological effects and multiple lipid mediators have been described to be involved in physiological processes, (e.g. inflammation). Accordingly, a thorough description of these pathways may shed light on putative relations in multiple complex diseases, including chronic obstructive pulmonary disease, asthma, Alzheimer's disease, multiple sclerosis, obesity, and cancer. Due to the structural complexity of lipids and the low abundance of many lipid mediators, mass spectrometry is the most commonly employed method for analysis. However, multiple challenges remain in the efforts to analyze every lipid subfamily. In this review, the biological role of sphingolipids, glycerolipids, oxylipins (e.g. eicosanoids), endocannabinoids, and N-acylethanolamines in relation to health and disease and the state-of-the-art analyses are summarized. The characterization and understanding of these pathways will increase our ability to examine for interrelations among lipid pathways and improve the knowledge of biological mechanisms in health and disease., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: In contrast to well-characterized PUFA levels in serum, little is known regarding their downstream metabolic products. However, many of these compounds are lipid mediators with prominent roles during pro- and antiinflammatory processes., Methods and Results: In this double blind crossover study on asthmatics, shifts in serum levels of ω-3 and ω-6 PUFA-derived oxidized fatty acids (e.g. eicosanoids, oxylipins) were quantified following dietary fish oil supplementation. Serum was obtained from subjects following fasting at three occasions; (i) prior to supplementation, (ii) following a 3-week supplement intake of either placebo or fish oil, and (iii) following a 3-week washout period with a subsequent 3-week period of either fish oil or placebo supplement. A total of 87 oxylipins representing cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS. The primary alterations observed were in CYP- and 15-LOX-derived EPA- and CYP-derived DHA oxylipins., Conclusion: The results indicate that intake of an ω-3 rich diet alters not only the PUFA ratio, but also the ratio of downstream oxylipins. These data further support that dietary manipulation with ω-3 PUFAs affects not only PUFA levels, but importantly also the downstream metabolic profile., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mast cells are inflammatory cells that play key roles in health and disease. They are distributed in all tissues and appear in two main phenotypes, connective tissue and mucosal mast cells, with differing capacities to release inflammatory mediators. A metabolic profiling approach was used to obtain a more comprehensive understanding of the ability of mast cell phenotypes to produce eicosanoids and other lipid mediators. A total of 90 lipid mediators (oxylipins) were characterized using liquid chromatography-tandem mass spectrometry (LC-MS/MS), representing the cyclooxygenase (COX), lipoxygenase (LO), and cytochrome P450 (CYP) metabolic pathways. In vitro-derived murine mucosal-like mast cells (MLMC) and connective tissue-like mast cells (CTLMC) exhibited distinct mRNA expression patterns of enzymes involved in oxylipin biosynthesis. Oxylipins produced by 5-LO and COX pathways were the predominant species in both phenotypes, with 5-LO products constituting 90 ± 2% of the CTLMCs compared with 58 ± 8% in the MLMCs. Multivariate analyses demonstrated that CTLMCs and MLMCs secrete differing oxylipin profiles at baseline and following calcium ionophore stimulation, evidencing specificity in both a time- and biosynthetic pathway-dependent manner. In addition to the COX-regulated prostaglandin PGD(2) and 5-LO-regulated cysteinyl-leukotrienes (e.g., LTC(4)), several other mediators evidenced phenotype-specificity, which may have biological implications in mast cell-mediated regulation of inflammatory responses.

Metabolomics is the global unbiased analysis of all the small-molecule metabolites within a biological system, under a given set of conditions. These methods offer the potential for a holistic approach to clinical medicine, as well as improving disease diagnosis and understanding of pathological mechanisms. Respiratory diseases including asthma and chronic obstructive pulmonary disorder are increasing globally, with the latter predicted to become the third leading cause of global mortality by 2020. The root causes for disease onset remain poorly understood and no cures are available. This review presents an overview of metabolomics followed by in-depth discussion of its application to the study of respiratory diseases, including the design of metabolomics experiments, choice of clinical material collected and potentially confounding experimental factors. Particular challenges in the field are presented and placed within the context of the future of the applications of metabolomics approaches to the study of respiratory diseases.

Systems biology methods using large-scale "omics" data sets face unique challenges: integrating and analyzing near limitless data space, while recognizing and removing systematic variation or noise. Herein we propose a complementary multivariate analysis workflow to both integrate "omics" data from disparate sources and analyze the results for specific and unique sample correlations. This workflow combines principal component analysis (PCA), orthogonal projections to latent structures discriminate analysis (OPLS-DA), orthogonal 2 projections to latent structures (O2PLS), and shared and unique structures (SUS) plots. The workflow is demonstrated using data from a study in which ApoE3Leiden mice were fed an atherogenic diet consisting of increasing cholesterol levels followed by therapeutic intervention (fenofibrate, rosuvastatin, and LXR activator T-0901317). The levels of structural lipids (lipidomics) and free fatty acids in liver were quantified via liquid chromatography-mass spectrometry (LC-MS). The complementary workflow identified diglycerides as key hepatic metabolites affected by dietary cholesterol and drug intervention. Modeling of the three therapeutics for mice fed a high-cholesterol diet further highlighted diglycerides as metabolites of interest in atherogenesis, suggesting a role in eliciting chronic liver inflammation. In particular, O2PLS-based SUS2 plots showed that treatment with T-0901317 or rosuvastatin returned the diglyceride profile in high-cholesterol-fed mice to that of control animals.

Background: Asthma is a chronic inflammatory airway disease influenced by genetic and environmental factors that affects ~300 million people worldwide, leading to ~250,000 deaths annually. Glucocorticoids (GCs) are well-known therapeutics that are used extensively to suppress airway inflammation in asthmatics. The airway epithelium plays an important role in the initiation and modulation of the inflammatory response. While the role of GCs in disease management is well understood, few studies have examined the holistic effects on the airway epithelium., Methods: Gene expression data were used to generate a co-transcriptional network, which was interrogated to identify modules of functionally related genes. In parallel, expression data were mapped to the human protein-protein interaction (PPI) network in order to identify modules with differentially expressed genes. A common pathways approach was applied to highlight genes and pathways functionally relevant and significantly altered following GC treatment., Results: Co-transcriptional network analysis identified pathways involved in inflammatory processes in the epithelium of asthmatics, including the Toll-like receptor (TLR) and PPAR signaling pathways. Analysis of the PPI network identified RXRA, PPARGC1A, STAT1 and IRF9, among others genes, as differentially expressed. Common pathways analysis highlighted TLR and PPAR signaling pathways, providing a link between general inflammatory processes and the actions of GCs. Promoter analysis identified genes regulated by the glucocorticoid receptor (GCR) and PPAR pathways as well as highlighted the interferon pathway as a target of GCs., Conclusions: Network analyses identified known genes and pathways associated with inflammatory processes in the airway epithelium of asthmatics. This workflow illustrated a hypothesis generating experimental design that integrated multiple analysis methods to produce a weight-of-evidence based approach upon which future focused studies can be designed. In this case, results suggested a mechanism whereby GCs repress TLR-mediated interferon production via upregulation of the PPAR signaling pathway. These results highlight the role of interferons in asthma and their potential as targets of future therapeutic efforts.

Background: Asthma is a respiratory tract disorder characterized by airway hyper-reactivity and chronic inflammation. Allergic asthma is associated with the production of allergen-specific IgE and expansion of allergen-specific T-cell populations. Progression of allergic inflammation is driven by T-helper type 2 (Th2) mediators and is associated with alterations in the levels of lipid mediators., Objectives: Responses of the respiratory system to birch allergen provocation in allergic asthmatics were investigated. Eicosanoids and other oxylipins were quantified in the bronchoalveolar lumen to provide a measure of shifts in lipid mediators associated with allergen challenge in allergic asthmatics., Methods: Eighty-seven lipid mediators representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS following off-line extraction of bronchoalveolar lavage fluid (BALF). Multivariate statistics using OPLS were employed to interrogate acquired oxylipin data in combination with immunological markers., Results: Thirty-two oxylipins were quantified, with baseline asthmatics possessing a different oxylipin profile relative to healthy individuals that became more distinct following allergen provocation. The most prominent differences included 15-LOX-derived ω-3 and ω-6 oxylipins. Shared-and-Unique-Structures (SUS)-plot modeling showed a correlation (R(2) = 0.7) between OPLS models for baseline asthmatics (R(2)Y[cum] = 0.87, Q(2)[cum] = 0.51) and allergen-provoked asthmatics (R(2)Y[cum] = 0.95, Q(2)[cum] = 0.73), with the majority of quantified lipid mediators and cytokines contributing equally to both groups. Unique structures for allergen provocation included leukotrienes (LTB(4) and 6-trans-LTB(4)), CYP-derivatives of linoleic acid (epoxides/diols), and IL-10., Conclusions: Differences in asthmatic relative to healthy profiles suggest a role for 15-LOX products of both ω-6 and ω-3 origin in allergic inflammation. Prominent differences at baseline levels indicate that non-symptomatic asthmatics are subject to an underlying inflammatory condition not observed with other traditional mediators. Results suggest that oxylipin profiling may provide a sensitive means of characterizing low-level inflammation and that even individuals with mild disease display distinct phenotypic profiles, which may have clinical ramifications for disease.

Oxylipins (e.g. eicosanoids) are endogenous signaling molecules that are formed from fatty acids by mono- or dioxygenase-catalyzed oxygenation and have been shown to play an important role in pathophysiological processes in the lung. These lipid mediators have been extensively studied for their role in inflammation in a broad swathe of respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and extrinsic allergic alveolitis. Traditional efforts have employed analytical methods (e.g. radio- and enzyme-immunoassay techniques) capable of measuring a limited number of compounds simultaneously. The advent of the omics technologies is changing this approach and methods are being developed for the quantification of small molecules (i.e. metabolomics) as well as lipid-focused efforts (i.e. lipidomics). This review examines in detail the breadth of oxylipins and their biological activity in the respiratory system. In addition, the state-of-the-art methodology in profiling of oxylipins via mass spectrometry is summarized including sample work-up and data processing. These methods will greatly increase our ability to probe oxylipin biology and examine for cross-talk between biological pathways as well as specific compartments in the body. These new data will increase our insight into disease processes and have great potential to identify new biomarkers for disease diagnosis as well as novel therapeutic targets.

Lipoxygenase (ALOX) enzymes are implicated in both pro- and anti-atherogenic processes. The aim of this study was to investigate mRNA expression of 12- and 15-lipoxygenases (ALOX12, ALOX12B, ALOX15, ALOX15B) and the atypical ALOXE3 in human carotid atherosclerotic lesions, in relation to cerebrovascular symptoms and risk factors. The Biobank of Karolinska Endarterectomies (BiKE) collection of human carotid plaque tissue and associated clinical data was utilized (n=132). Lesion mRNA levels were analyzed by TaqMan qPCR (n=132) and microarray hybridization (n=77). Of the investigated mRNAs, only ALOX15B (15-LOX-2; epidermis-type 15-LOX) was readily detected in all plaque samples by qPCR, and thus suitable for quantitative statistical evaluation. ALOX12, ALOX12B, ALOX15 and ALOXE3 were detected with lower frequency and at lower levels, or virtually undetected. Microarray analysis confirmed ALOX15B as the most abundant 12- or 15-lipoxygenase mRNA in carotid lesions. Comparing plaques with or without attributable cerebrovascular symptoms (amaurosis fugax, transient ischemic attack, or stroke), ALOX15B mRNA levels were higher in symptomatic than asymptomatic plaques (1.31 [1.11-1.56], n=102; and 0.79 [0.55-1.15], n=30, respectively; p=0.008; mean [95% CI], arbitrary units). Multiple regression analysis confirmed symptomatic/asymptomatic status as a significant determinant of ALOX15B mRNA levels, independently of potentially confounding factors. Immunohistochemical analyses showed abundant ALOX15B expression in macrophage-rich areas of carotid lesions, and lipidomic analyses demonstrated the presence of typical ALOX15B products in plaque tissue. In summary, we observed associations between high ALOX15B expression in carotid lesions and a history of cerebrovascular symptoms. These findings suggest a link between ALOX15B and atherothrombotic events that merits further investigation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Asthma is a chronic inflammatory lung disease that causes significant morbidity and mortality worldwide. Air pollutants such as particulate matter (PM) and oxidants are important factors in causing exacerbations in asthmatics, and the source and composition of pollutants greatly affects pathological implications., Objectives: This randomized crossover study investigated responses of the respiratory system to Stockholm subway air in asthmatics and healthy individuals. Eicosanoids and other oxylipins were quantified in the distal lung to provide a measure of shifts in lipid mediators in association with exposure to subway air relative to ambient air., Methods: Sixty-four oxylipins representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of bronchoalveolar lavage (BAL)-fluid. Validations through immunocytochemistry staining of BAL-cells were performed for 15-LOX-1, COX-1, COX-2 and peroxisome proliferator-activated receptor gamma (PPARγ). Multivariate statistics were employed to interrogate acquired oxylipin and immunocytochemistry data in combination with patient clinical information., Results: Asthmatics and healthy individuals exhibited divergent oxylipin profiles following exposure to ambient and subway air. Significant changes were observed in 8 metabolites of linoleic- and α-linolenic acid synthesized via the 15-LOX pathway, and of the COX product prostaglandin E(2) (PGE(2)). Oxylipin levels were increased in healthy individuals following exposure to subway air, whereas asthmatics evidenced decreases or no change., Conclusions: Several of the altered oxylipins have known or suspected bronchoprotective or anti-inflammatory effects, suggesting a possible reduced anti-inflammatory response in asthmatics following exposure to subway air. These observations may have ramifications for sensitive subpopulations in urban areas.

Background: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity., Objective: We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis., Methods: The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system., Results: Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-β1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-β1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma., Conclusion: Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg(-1) chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (< or = 1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Antigenic variation is a common mechanism employed by many pathogenic organisms to avoid recognition of surface proteins by the host immune system. The malaria parasite, Plasmodium falciparum, among many others, exploits this mechanism and manages to survive in an otherwise hostile environment. Although similarities in the mechanisms used among different species to generate antigenic variation are broadly recognized, there is a lack of studies using cross-species data. The varDB project (http://www.vardb.org) was created to study antigenic variation at a range of different levels, both within and among species. The project aims to serve as a resource to increase our understanding of antigenic variation by providing a framework for comparative studies. In this review we describe the varDB project, its construction, and the overall organization of information with the intent of increasing the utility of varDB to the research community. The current version of varDB supports 27 species involved in 19 different diseases affecting humans as well as other species. These data include 42 gene families that are represented by over 67,000 sequences. The varDB project is still in its infancy but is expected to continue to grow with the addition of new organisms and gene families as well as input from the general research community., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Eicosanoids are a family of oxygenated metabolites of arachidonic acid, including the prostaglandins, thromboxanes, leukotrienes and lipoxins. These lipid mediators play essential roles in normal cellular homeostasis as well as in a number of disease states. This review will focus on recent advances in the field of eicosanoids and highlight specific discoveries and achievements. Emphasis will be placed on structure and receptor biology, which are of significant pharmacological and clinical relevance., (2010 Elsevier Inc. All rights reserved.)

Systems biology offers the potential to provide new insights into our understanding of the pathogenesis of complex diseases such as atherosclerosis. It seeks to comprehend the system properties of the non-linear interactions of the multiple biomolecular components that characterize a living organism. An important component of this research approach is identifying the biological networks that connect the differing elements of a system and in the process describe the characteristics that define a shift in equilibrium from a healthy to a diseased state. The utility of this method becomes clear when applied to multifactorial diseases with complex etiologies such as inflammatory-related diseases, herein exemplified by cardiovascular disease. In this study, the application of network theory to systems biology is described in detail and an example is provided using data from a clinical biobank database of carotid endarterectomies from the Karolinska University Hospital (Biobank of Karolinska Endarterectomies, BiKE). Data from 47 microarrays were examined using a combination of Bioconductor modules and the Cytoscape resource with several associated plugins to analyze the transcriptomics data and create a combined gene association and correlation network of atherosclerosis. The methodology and workflow are described in detail, with a total of 43 genes found to be differentially expressed on a gender-specific basis, of which 15 were not directly linked to the sex chromosomes. In particular, the APOC1 gene was 2.1-fold down-regulated in plaques in women relative to men and was selected for further analysis based upon a purported role in cardiovascular disease. The resulting network was identified as a scale-free network that contained specific sub-networks related to immune function and lipid biosynthesis. These sub-networks link atherosclerotic-related genes to other genes that may not have previously known roles in disease etiology and only evidence small alterations, which are challenging to find by statistical and comparison-based methods. A number of Gene Ontology (GO), BioCarta and KEGG pathways involved in the atherosclerotic process were identified in the constructed sub-network, with 19 GO pathways related to APOC1 of which 'phospholipid efflux' evidenced the strongest association. The utility and functionality of network analysis and the different Cytoscape plugins employed are discussed. Lastly, the applications of these methods to cardiovascular disease are discussed with focus on the current limitations and future visions of this emerging field.

Lipid mediator is the collective term for prostanoids, leukotrienes, lysophospholipids, platelet-activating factor, endocannabinoids and other bioactive lipids, that are involved in various physiological functions including inflammation, immune regulation and cellular development. They act by binding to their ligand-specific G-protein coupled receptors (GPCRs). Since 1990's a number of lipid GPCRs have been cloned in humans, with a few more identified in other vertebrates. However, the conservation of these receptors has been poorly investigated in other eukaryotes. Herein we performed a phylogenetic analysis by collecting their orthologs in 13 eukaryotes with complete genomes. The analysis shows that orthologs for prostanoid receptors are likely to be conserved in the 13 eukaryotes. In contrast, those for lysophospholipid and cannabinoid receptors appear to be conserved only in vertebrates and chordates. Receptors for leukotrienes and other bioactive lipids are limited to vertebrates. These results indicate that the lipid mediators and their receptors have coevolved with the development of highly modulated physiological functions such as immune regulation and the formation of the central nervous system. Accordingly, examining the presence and role of lipid mediator GPCR orthologs in invertebrate species can provide insight into the development of fundamental biological processes across diverse taxa.

The varDB project (http://www.vardb.org) aims to create and maintain a curated database of antigenic variation sequences as well as a platform for online sequence analysis. Along with the evolution of drug resistance, antigenic variation presents a moving target for public health endeavors and greatly complicates vaccination and eradication efforts. However, careful analysis of a large number of variant forms may reveal structural and functional constraints that can be exploited to identify stable and cross-reactive targets. VarDB attempts to facilitate this effort by providing streamlined interfaces to standard tools to help identify and prepare sequences for various forms of analysis. We have newly implemented such tools for codon usage, selection, recombination, secondary and tertiary structure, and sequence diversity analysis. Just as the adaptive immune system encodes a mechanism for dynamically generating diverse receptors instead of encoding a receptor for every possible epitope, many pathogens take advantage of heritable diversity generating mechanisms to produce progeny able to evade immune recognition. Instead of merely cataloging the observed variation, a major goal of varDB is to characterize and predict the potential range of antigenic variation within a pathogen by investigating the mechanisms by which it attempts to expand its implicit genome. We believe that the new sequence analysis tools will improve the usefulness and range of varDB.

Summary: Whole-genome microarrays allow us to interrogate the entire transcriptome of a cell. Affymetrix microarrays are constructed using several probes that match to different regions of a gene and a summarization step reduces this complexity into a single value, representing the expression level of the gene or the expression level of an exon in the case of exon arrays. However, this simplification eliminates information that might be useful when focusing on specific genes of interest. To address these limitations, we present a software package for the R platform that allows detailed analysis of expression at the probe level. The package matches the probe sequences against a target gene sequence (either mRNA or DNA) and shows the expression levels of each probe along the gene. It also features functions to fit a linear regression based on several genetic models that enables study of the relationship between gene expression and genotype., Availability and Implementation: The software is implemented as a platform-independent R package available through the Bioconductor repository at http://www.bioconductor.org/. It is licensed as GPL 2.0., Supplementary Information: Supplementary data are available at Bioinformatics online.

Systems biology aims to understand the nonlinear interactions of multiple biomolecular components that characterize a living organism. One important aspect of systems biology approaches is to identify the biological pathways or networks that connect the differing elements of a system, and examine how they evolve with temporal and environmental changes. The utility of this method becomes clear when applied to multifactorial diseases with complex etiologies, such as inflammatory-related diseases, herein exemplified by atherosclerosis. In this paper, the initial studies in this discipline are reviewed and examined within the context of the development of the field. In addition, several different software tools are briefly described and a novel application for the KEGG database suite called KegArray is presented. This tool is designed for mapping the results of high-throughput omics studies, including transcriptomics, proteomics and metabolomics data, onto interactive KEGG metabolic pathways. The utility of KegArray is demonstrated using a combined transcriptomics and lipidomics dataset from a published study designed to examine the potential of cholesterol in the diet to influence the inflammatory component in the development of atherosclerosis. These data were mapped onto the KEGG PATHWAY database, with a low cholesterol diet affecting 60 distinct biochemical pathways and a high cholesterol exposure affecting 76 biochemical pathways. A total of 77 pathways were differentially affected between low and high cholesterol diets. The KEGG pathways "Biosynthesis of unsaturated fatty acids" and "Sphingolipid metabolism" evidenced multiple changes in gene/lipid levels between low and high cholesterol treatment, and are discussed in detail. Taken together, this paper provides a brief introduction to systems biology and the applications of pathway mapping to the study of cardiovascular disease, as well as a summary of available tools. Current limitations and future visions of this emerging field are discussed, with the conclusion that combining knowledge from biological pathways and high-throughput omics data will move clinical medicine one step further to individualize medical diagnosis and treatment.

A laboratory-based study was conducted to determine the basal carboxylesterase (CbE) activity in different tissues of the earthworm Lumbricus terrestris, and its sensitivity to the organophosphate (OP) pesticide chlorpyrifos-oxon (CPx). Carboxylesterase activity was found in the pharynx, crop, gizzard, anterior intestine, wall muscle and reproductive tissues of L. terrestris, and multiple tissue-specific isozymes were observed by native gel electrophoresis. Esterase activity and sensitivity to CPx inhibition varied on a tissue- and substrate-specific basis, suggesting isoforms-specific selectivity to OP-mediated inhibition. Three practical issues are recommended for the use of earthworm CbE activity as a biomarker of pesticide exposure: (i) CbE should be measured using several routine substrates, (ii) it should be determined in selected tissues instead of whole organism homogenate, and (iii) earthworm CbE activity should be used in conjuncture with other common biomarkers (e.g., ChE) within a multibiomarker approach to assess field exposure of OPs, and potentially other agrochemicals.

Owing to their importance in cellular physiology and pathology as well as to recent technological advances, the study of lipids has reemerged as a major research target. However, the structural diversity of lipids presents a number of analytical and informatics challenges. The field of lipidomics is a new postgenome discipline that aims to develop comprehensive methods for lipid analysis, necessitating concomitant developments in bioinformatics. The evolving research paradigm requires that new bioinformatics approaches accommodate genomic as well as high-level perspectives, integrating genome, protein, chemical and network information. The incorporation of lipidomics information into these data structures will provide mechanistic understanding of lipid functions and interactions in the context of cellular and organismal physiology. Accordingly, it is vital that specific bioinformatics methods be developed to analyze the wealth of lipid data being acquired. Herein, we present an overview of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and application of its tools to the analysis of lipid data. We also describe a series of software tools and databases (KGML-ED, VANTED, MZmine, and LipidDB) that can be used for the processing of lipidomics data and biochemical pathway reconstruction, an important next step in the development of the lipidomics field.

Carboxylesterases are enzymes that hydrolyze a broad suite of endogenous and exogenous ester-containing compounds to the corresponding alcohol and carboxylic acid. These enzymes metabolize a number of therapeutics including the anti-tumor agent CPT-11, the anti-viral drug oseltamivir, and the anti-thrombogenic agent clopidogrel as well as many agrochemicals. In addition, carboxylesterases are involved in lipid homeostasis, including cholesterol metabolism and transport with a proposed role in the development of atherosclerosis. Several different scaffolds capable of inhibiting carboxylesterases have been reported, including organophosphates, carbamates, trifluoromethyl ketone-containing structures (TFKs), and aromatic ethane-1,2-diones. Of these varied groups, only the 1,2-diones evidence carboxylesterase isoform-selectivity, which is an important characteristic for therapeutic application and probing biological mechanisms. This study constructed a series of classical and 3D-QSAR models to examine the physiochemical parameters involved in the observed selectivity of three mammalian carboxylesterases: human intestinal carboxylesterase (hiCE), human carboxylesterase 1 (hCE1), and rabbit carboxylesterase (rCE). CoMFA-based models for the benzil-analogs described 88%, 95% and 76% of observed activity for hiCE, hCE1 and rCE, respectively. For TFK-containing compounds, two distinct models were constructed using either the ketone or gem-diol form of the inhibitor. For all three enzymes, the CoMFA ketone models comprised more biological activity than the corresponding gem-diol models; however the differences were small with described activity for all models ranging from 85-98%. A comprehensive model incorporating both benzil and TFK structures described 92%, 85% and 87% of observed activity for hiCE, hCE1 and rCE, respectively. Both classical and 3D-QSAR analysis showed that the observed isoform-selectivity with the benzil-analogs could be described by the volume parameter. This finding was successfully applied to examine substrate selectivity, demonstrating that the relative volumes of the alcohol and acid moieties of ester-containing substrates were predictive for whether hydrolysis was preferred by hiCE or hCE1. Based upon the integrated benzil and TFK model, the next generation inhibitors should combine the A-ring and the 1,2-dione of the benzil inhibitor with the long alkyl chain of the TFK-inhibitor in order to optimize selectivity and potency. These new inhibitors could be useful for elucidating the role of carboxylesterase activity in fatty acid homeostasis and the development of atherosclerosis as well as effecting the controlled activation of carboxylesterase-based prodrugs in situ.

Eicosanoids are oxygenated, endogenous, unsaturated fatty acids derived from arachidonic acid. Detection and quantification of these compounds are of great interest because they play important roles in a number of significant diseases, including asthma, chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Because the endogenous levels of eicosanoids are quite low, sensitive and specific analytical methods are required to reliably quantify these compounds. High-performance liquid chromatography mass spectrometry (HPLC/MS) has emerged as one of the main techniques used in eicosanoid profiling. Herein, we describe the main LC/MS techniques and principles as well as their application in eicosanoid analysis. In addition, a protocol is given for extracting eicosanoids from biological samples, using bronchoalveolar lavage fluid (BALF) as an example. The method and instrument optimization procedures are presented, followed by the analysis of eicosanoid standards using reverse phase HPLC interfaced with an ion trap mass spectrometer (LC/MS/MS). This protocol is intended to provide a broad description of the field for readers looking for an introduction to the methodologies involved in eicosanoid quantification.

Unlabelled: Infectious diseases are a major threat to global public health and prosperity. The causative agents consist of a suite of pathogens, ranging from bacteria to viruses, including fungi, helminthes and protozoa. Although these organisms are extremely varied in their biological structure and interactions with the host, they share similar methods of evading the host immune system. Antigenic variation and drift are mechanisms by which pathogens change their exposed epitopes while maintaining protein function. Accordingly, these traits enable pathogens to establish chronic infections in the host. The varDB database was developed to serve as a central repository of protein and nucleotide sequences as well as associated features (e.g. field isolate data, clinical parameters, etc.) involved in antigenic variation. The data currently contained in varDB were mined from GenBank as well as multiple specialized data repositories (e.g. PlasmoDB, GiardiaDB). Family members and ortholog groups were identified using a hierarchical search strategy, including literature/author-based searches and HMM profiles. Included in the current release are>29,00 sequences from 39 gene families from 25 different pathogens. This resource will enable researchers to compare antigenic variation within and across taxa with the goal of identifying common mechanisms of pathogenicity to assist in the fight against a range of devastating diseases., Availability: varDB is freely accessible at http://www.vardb.org/

Analysis of ethyl 3-(2-chlorophenyl)propenoate by electron ionization mass spectrometry showed the distinct loss of an ortho chlorine. To characterize the structural requisites for the observed mass fragmentation, a series of 30 halogen-substituted 3-phenylpropenoate-related structures were examined. All ester-containing alkene derivatives exhibited loss of the distinctive chlorine from the 2-position of the phenyl ring. Analogous derivatives with the halogen (chlorine or bromine) in the para position did not evidence selective halogen loss. Results demonstrated that substituted 3-phenylpropenoates and their analogs fragment via the formation of a previously reported benzopyrylium intermediate. To understand the correlation between the intramolecular radical substitution and the abundance and selectivity of the chlorine (or other halogen) displacement, density functional theory calculations were performed to determine the charge on the principal cation involved in the chlorine loss (in the ortho, meta, and para positions), the charge for the neutral radical (noncation), the excess alpha-electron density on the relevant atom and the energy to form the cation from the neutral atom (ionization energy). Results showed that the selectivity and extent of halogen displacement correlated highly to the electrophilicity of the radical cation as well as the neutral radical. These data further support the proposed fragmentation mechanism involving intramolecular radical elimination., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.

Pyridine is a prototypical inducer of cytochrome P450 (CYP) 2E1, an enzyme associated with cellular oxidative stress and membrane damage. To better understand the effect of this treatment on cellular lipids, the influence of pyridine exposure (100 mg/kg/day i.p. for 5 days) on fatty acids, fatty esters, and fatty alcohol ethers in brain, heart, liver, and adipose tissue from male Swiss Webster mice was investigated. Lipid levels in cholesterol esters, triglycerides, free fatty acids, cardiolipin, sphingomyelin, and glycerylphospholipids were quantified. Pyridine altered the level and composition of lipids involved in membrane structure (i.e., sphingomyelin, phosphatidylethanolamines, and plasmalogens), energy metabolism (i.e., free fatty acids), and long-chain fatty acid transport (i.e., cholesterol esters) in a tissue-specific manner. Subtle changes in cholesterol esters were observed in all tissues. Sphingomyelin in the brain and heart were depleted in monounsaturated fatty acids (1.4- and 1.5-fold, respectively), while the liver sphingomyelin concentrations increased (1.5-fold). Pyridine exposure also increased heart free fatty acids by 1.3-fold, enriched cardiac phosphatidylethanolamine in long-chain polyunsaturated fatty acids by 1.3-fold, and depleted cardiolipin-associated plasmalogens by 3.8-fold. Phosphatidylethanolamines in the brain were also enriched in both saturated fatty acids (1.2-fold) and polyunsaturated fatty acids (1.3-fold) but were depleted in plasmalogens (2.9-fold). In particular, the levels of phosphatidylethanolamine-associated arachidonic (AA) and docosahexaenoic acid (DHA) in both brain and cardiac tissues significantly decreased following pyridine exposure. Considering the hypothetical role of plasmalogens as membrane-bound reactive oxygen scavengers, the current findings suggest that the brain and heart should be the focus of future studies on the toxicity of pyridine, as well as other CYP 2E1 inducers.

Carboxylesterases metabolize numerous exogenous and endogenous ester-containing compounds including the chemotherapeutic agent CPT-11, anti-influenza viral agent oseltamivir, and many agrochemicals. Trifluoromethyl ketone (TFK)-containing compounds with a sulfur atom beta to the ketone moiety are some of the most potent carboxylesterase and amidase inhibitors identified to date. This study examined the effects of alkyl chain length (i.e., steric effects) and sulfur oxidation state upon TFK inhibitor potency (IC50) and binding kinetics (k(i)). The selective carboxylesterase inhibitor benzil was used as a non-TFK containing control. These effects were examined using two commercial esterases (porcine and rabbit liver esterase) and two human recombinant esterases (hCE-1 and hCE-2) as well as human recombinant fatty acid amide hydrolase (FAAH). In addition, the inhibition mechanism was examined using a combination of 1H NMR, X-ray crystallography, and ab initio calculations. Overall, the data show that while sulfur oxidation state profoundly affects both inhibitor potency and binding kinetics, the steric effects dominate and override the contributions of sulfur oxidation. In addition, the data suggest that inclusion of a sulfur atom beta to the ketone contributes an increase (approximately 5-fold) in inhibitor potency due to effects upon ketone hydration and/or intramolecular hydrogen bond formation. These results provide further information on the nature of the TFK binding interaction and will be useful in increasing our understanding of this basic biochemical process.

This review has examined a number of issues surrounding the use of carboxylesterase activity in environmental monitoring. It is clear that carboxylesterases are important enzymes that deserve increased study. This class of enzymes appears to have promise for employment in environmental monitoring with a number of organisms and testing scenarios, and it is appropriate for inclusion in standard monitoring assays. Given the ease of most activity assays, it is logical to report carboxylesterase activity levels as well as other esterases (e.g., acetylcholinesterase). Although it is still unclear as to whether acetylcholinesterase or carboxylesterase is the most "appropriate" biomarker, there are sufficient data to suggest that at the very least further studies should be performed with carboxylesterases. Most likely, data will show that it is optimal to measure activity for both enzymes whenever possible. Acetylcholinesterase has the distinct advantage of a clear biological function, whereas the endogenous role of carboxylesterases is still unclear. However, a combination of activity measurements for the two enzyme systems will provide a much more detailed picture of organism health and insecticide exposure. The main outstanding issues are the choice of substrate for activity assays and which tissues/organisms are most appropriate for monitoring studies. Substrate choice is very important, because carboxylesterase activity consists of multiple isozymes that most likely fluctuate on an organism- and tissue-specific basis. It is therefore difficult to compare work in one organism with a specific substrate with work performed in a different organism with a different substrate. An attempt should therefore be made to standardize the method. The most logical choice is PNPA (p-nitrophenyl acetate), as this substrate is commercially available, requires inexpensive optics for assay measurements, and has been used extensively in the literature. However, none of these beneficial properties indicates that the substrate is an appropriate surrogate for a specific compound, e.g., pyrethroid-hydrolyzing activity. It will most likely be necessary to have more specific surrogate substrates for use in assays that require information on the ability to detoxify/hydrolyze specific environmental contaminants. The use of carboxylesterase activity in TIE protocols appears to have excellent promise, but there are further technical issues that should be addressed to increase the utility of the method. The main concerns include the large amount of nonspecific protein added to the testing system, which can lead to undesirable side effects including nonspecific reductions in observed toxicity, decrease in dissolved oxygen content, and organism growth. It is probable that these issues can be resolved with further assay development. The ideal solution would be to have a commercial recombinant carboxylesterase that possessed elevated pyrethroid-hydrolysis activity and which was readily available, homogeneous, and inexpensive. The availability of such an enzyme would address nearly all the current method shortcomings. Such a preparation would be extremely useful for the aquatic toxicology community. Further work should focus on screening available esterases for stability, cost, and activity on pyrethroids, with specific focus on esterases capable of distinguishing type I from type II pyrethroids. It would also be beneficial to identify esterases that are not sensitive to OP insecticides. Many esterases and lipases are available as sets to test chemical reactions for green chemistry, enabling large-scale screening. Other potential approaches to increase the utility of the enzyme include derivatization with polyethylene glycol (PEG) or cyanuric acid chloride to increase stability and reduce microbial degradation. It is also possible that the enzyme could be formulated in a sol gel preparation to increase stability. It is likely that the use of carboxylesterase addition will increase for applications in sediment TIEs. Carboxylesterases are an interesting and useful enzyme family that deserves further study for applications in environmental monitoring as well as to increase our understanding of the fundamental biological role(s) of these enzymes. There are, of course, other enzymes that show high esterase activity on pyrethroids but are not technically carboxylesterases in the alpha/beta-hydrolase fold protein family. These enzymes should also be examined for use in TIE protocols and "esterase" arrays as well as for general applications in environmental monitoring. One can envision the creation of a standardized screen of enzymes with esterase activity to (1) identify environmental contaminants, (2) estimate the potential toxic effects of new compounds on a range of organisms, and (3) monitor organism exposure to agrochemicals (and potentially other contaminants). This approach would provide a multibiomarker integrative assessment of esterase-inhibiting potential of a compound or mixture. In conclusion, much is still unknown about this enzyme family, indicating that this area is still wide open to researchers interested in the applications of carboxylesterase activity as well as basic biological questions into the nature of enzyme activity and the endogenous role of the enzyme.

We present a SAR method that can predict estrogen-like endocrine disrupting chemical (EDC) activity as well as key biodegradation steps for detoxification. This method is based on a recent graph-mining algorithm developed by Kudo et al., which generates a set of descriptors from all potent chemical fragments (including rings). This method is novel in that it achieves chemical diversity in the training data set by sampling another data set of larger diversity. The model achieved an 83% accuracy prediction rate, and identified 1291 EDC candidates from the KEGG database. From this set of candidate compounds, bisphenol A was chosen for assay validation and biodegradation pathway analysis. Results showed that bisphenol A exhibited estrogen-like activity and was degraded in three distinct reactions. The prediction model provided information on the mechanism of the ligand-target binding, such as key functional groups involved. We focused on the enzyme commission number, which is useful for analyses of biodegradation pathways. Results identified oxygenases, ether hydrolases, and carbon-halide lyases as being important in the biodegradation pathway. This combined approach provided new information regarding the biodegradation of EDCs, and can potentially be extended to applications with transcriptomic, proteomic, and metabolomic data to provide a quick screen of biological activity and biodegradation pathway(s).

Background and Purpose: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction., Experimental Approach: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy., Key Results: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA., Conclusions and Implications: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.

An infection with Plasmodium falciparum may lead to severe malaria as a result of excessive binding of infected erythrocytes in the microvasculature. Vascular adhesion is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP1), which is encoded for by highly polymorphic members of the var-gene family. Here, we profile var gene transcription in fresh P. falciparum trophozoites from Ugandan children with malaria through var-specific DBL1alpha-PCR amplification and sequencing. A method for subsectioning region alignments into homology areas (MOTIFF) was developed to examine collected sequences. Specific PfEMP1-DBL1alpha amino acid motifs correlated with rosetting and severe malaria, with motif location corresponding to distinct regions of receptor interaction. The method is potentially applicable to other families of variant proteins and may be useful in identifying sequence-phenotype relationships. The results suggest that certain PfEMP1 sequences are predisposed to inducing severe malaria.

Endogenous 2-arachidonoylglycerol (2-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of 2-AG. In contrast, exogenous 2-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2-AG. In PC-3 cells, arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) concentrations increased along with exogenously added 2-AG, and 12-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 12(S)-HETE but not 12(R)-HETE. The exogenous 2-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP, an inhibitor of 2-AG hydrolysis) and baicalein (a 12-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 12-HETE producing lipoxygenases (LOs), platelet-type 12-LO (P-12-LO) and leukocyte-type 12-LO (L-12-LO), in PC-3 cells. These results suggest that exogenous 2-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12-LO of AA to 12(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, 2-AG, and 12-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2-AG hydrolysis and concentration of 2-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. (c) 2007 Wiley-Liss, Inc., ((c) 2007 Wiley-Liss, Inc.)

Peroxisome proliferator activated receptor alpha (PPARα) agonists are anti-hyperlipidemic drugs that influence fatty acid combustion, phospholipid biosynthesis and lipoprotein metabolism. To evaluate impacts on other aspects of lipid metabolism, we applied targeted metabolomics to liver, heart, brain and white adipose tissue samples from male Swiss-Webster mice exposed to a 5 day, 500 mg/kg/day regimen of i.p. clofibrate. Tissue concentrations of free fatty acids and the fatty acid content of sphingomyelin, cardiolipin, cholesterol esters, triglycerides and phospholipids were quantified. Responses were tissue-specific, with changes observed in the liver > heart ≫ brain > adipose. These results indicate that liver saturated fatty acid-rich triglycerides feeds clofibrate-induced monounsaturated fatty acid (MUFA) synthesis, which were incorporated into hepatic phospholipids and sphingomyelin. In addition, selective enrichment of docosahexeneoic acid in the phosphatidylserine of liver (1.7-fold), heart (1.6-fold) and brain (1.5-fold) suggests a clofibrate-dependent systemic activation of phosphatidylserine synthetase 2. Furthermore, the observed ~20% decline in cardiac sphingomyelin is consistent with activation of a sphingomeylinase with a substrate preference for polyunsaturate-containing sphingomyelin. Finally, perturbations in the liver, brain, and adipose cholesterol esters were observed, with clofibrate exposure elevating brain cholesterol arachidonyl-esters ~20-fold. Thus, while supporting previous findings, this study has identified novel impacts of PPARα agonist exposure on lipid metabolism that should be further explored.

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.

Juvenile hormone (JH) is an insect hormone containing an alpha,beta-unsaturated ester consisting of a small alcohol and long, hydrophobic acid. JH degradation is required for proper insect development. One pathway of this degradation is through juvenile hormone esterase (JHE), which cleaves the JH ester bond to produce methanol and JH acid. JHE is a member of the functionally divergent alpha/beta-hydrolase family of enzymes and is a highly efficient enzyme that cleaves JH at very low in vivo concentrations. We present here a 2.7 A crystal structure of JHE from the tobacco hornworm Manduca sexta (MsJHE) in complex with the transition state analogue inhibitor 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) covalently bound to the active site. This crystal structure, the first JHE structure reported, contains a long, hydrophobic binding pocket with the solvent-inaccessible catalytic triad located at the end. The structure explains many of the interactions observed between JHE and its substrates and inhibitors, such as the preference for small alcohol groups and long hydrophobic backbones. The most potent JHE inhibitors identified to date contain a trifluoromethyl ketone (TFK) moiety and have a sulfur atom beta to the ketone. In this study, sulfur-aromatic interactions were observed between the sulfur atom of OTFP and a conserved aromatic residue in the crystal structure. Mutational analysis supported the hypothesis that these interactions contribute to the potency of sulfur-containing TFK inhibitors. Together, these results clarify the binding mechanism of JHE inhibitors and provide useful observations for the development of additional enzyme inhibitors for a variety of enzymes.

Increases in the use and application of pyrethroid insecticides have resulted in concern regarding potential effects on aquatic ecosystems. Methods for the detection of pyrethroids in receiving waters are required to monitor environmental levels of these insecticides. One method employed for the identification of causes of toxicity in aquatic samples is the toxicity identification evaluation (TIE); however, current TIE protocols do not include specific methods for pyrethroid detection. Recent work identified carboxylesterase treatment as a useful method for removing/detecting pyrethroid-associated toxicity. The present study has extended this earlier work and examined the ability of carboxylesterase activity to remove permethrin- and bifenthrin-associated toxicity to Ceriodaphnia dubia and Hyalella azteca in a variety of matrices, including laboratory water, Sacramento River (CA, USA) water, and Salinas River (CA, USA) interstitial water. Esterase activity successfully removed 1,000 ng/L of permethrin-associated toxicity and 600 ng/L of bifenthrin-associated toxicity to C. dubia in Sacramento River water. In interstitial water, 200 ng/L of permethrin-associated toxicity and 60 ng/L of bifenthrin-associated toxicity to H. azteca were removed. The selectivity of the method was validated using heat-inactivated enzyme and bovine serum albumin, demonstrating that catalytically active esterase is required. Further studies showed that the enzyme is not significantly inhibited by metals. Matrix effects on esterase activity were examined with municipal effluent and seawater in addition to the matrices discussed above. Results confirmed that the esterase retains catalytic function in a diverse array of matrices, suggesting that this technique can be adapted to a variety of aquatic samples. These data demonstrate the utility of carboxylesterase treatment as a viable step to detect the presence of pyrethroids in receiving waters.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.

We have previously reported the synthesis of four alpha-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight alpha-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the alpha-cyano ether compounds suggest that they are possibly good general P450 substrates.

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 microg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 microg/l), but not a low dose (1.2 microg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were approximately 30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish.