Lipoxygenase Products Research Articles

Anti-Inflammatory Potential of Pygeum africanum Bark Extract: An In Vitro Study of Cytokine Release by Lipopolysaccharide-Stimulated Human Peripheral Blood Mononuclear Cells.

Pygeum africanum bark has been shown to inhibit the production of pro-inflammatory prostaglandins in the prostate and reduces the production of leukotrienes and other 5-lipoxygenase (5-LO) metabolites. It has been suggested that inflammation plays an important role in the pathophysiology of benign prostatic hyperplasia (BPH). Data from clinical trials have shown that P. africanum improves the symptoms and objective measures of BPH. This in vitro study aimed to assess the anti-inflammatory potential of a proprietary Pygeum bark standardized extract (Prunera®) on cytokine release from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (PBMCs). PBMCs were obtained from four donors, and a bead-based assay (ProcartaPlex™ panel) was used for the detection and quantitation of cytokines. Pygeum africanum bark standardized extract (PABE) induced a statistically significant decrease (p < 0.05) of IL-6 in three donors. Other effects were as follows: IL-2 was lowered in all donors in the absence of a clear dose-response relationship; IL-4, IL-5, IL-9, and IL-13 levels were decreased in most donors; IL-22 levels seemed to be suppressed only for donor 4 at lower and medium concentrations; and IL-27 and TNF-α levels decreased at all PABE concentrations in all donors. The anti-inflammatory effect of PABE, particularly the reduction in IL-6 as a marker of inflammation, supports the potential use of this natural compound in the management of BPH and other conditions in which pro-inflammatory cytokines are involved in their underlying pathophysiological mechanisms.

Polyunsaturated fatty acids stimulate immunity and eicosanoid production in Drosophila melanogaster

AbstractEicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450–derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

Apolipoprotein D3 and LOX product play a role in immune-priming of a lepidopteran insect, Spodoptera exigua

Immune-priming occurs in insects after a prior pathogen exposure. However, its underlying mechanism in insects remains elusive. In the present work, immune-priming was detected in a lepidopteran insect, Spodoptera exigua. Specifically, a prior infection with a heat-killed pathogenic bacterium, Escherichia coli, led to increased survival upon the second infection of different pathogens. Plasma collected from larvae with the prior infection possessed the immune-priming factor(s) that significantly up-regulated cellular and humoral immune responses of naïve larvae. Our study also finds that variations in the timing of plasma collection for priming larvae resulted in distinct impacts on both cellular and humoral responses. However, when the active plasma exhibiting the immune-priming was heat-treated, it lost this priming activity, therefore suggesting that protein factor(s) play a role in this immune-priming. An immunofluorescence assay showed that the hemocytes collected from the immune-primed larvae highly reacted to a polyclonal antibody specific to a vertebrate lipocalin, apolipoprotein D (ApoD). Among 27 ApoD genes (Se-ApoD1 ∼ Se-ApoD27) of S. exigua, Se-ApoD3 was found to be highly induced during the immune-priming, in which it was shown to be expressed in hemocytes and fat body from a fluorescence in situ hybridization analysis. RNA interference of Se-ApoD3 expression significantly impaired the immune-priming of S. exigua larvae. Moreover, the inhibition of eicosanoid biosynthesis suppressed the immune-priming, in which treatment with a lipoxygenase (LOX) inhibitor—and not treatment with a cyclooxygenase inhibitor—suppressed immune-priming. Further, an addition of LOX product such as lipoxin A4 or lipoxin B4 significantly rescued the lost immune-priming activity. Taken together, these results suggest that a complex of ApoD3 and LOX product mediates the immune-priming activity of S. exigua.

Single and multiple inhibitors of the biosynthesis of 5-, 12-, 15-lipoxygenase products derived from cinnamyl-3,4-dihydroxy-α-cyanocinnamate: Synthesis and structure-activity relationship.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site.

Unfractionated heparin reverses aspirin inhibition of platelets during coronary artery bypass graft surgery

Unfractionated heparin (UFH) is an effective antithrombotic during surgery but has known adverse effects, in particular on platelets. A marked increase in platelet responsiveness has previously been observed in patients within minutes of receiving UFH, despite adequate inhibition by aspirin prior to heparin. We studied this phenomenon in patients undergoing cardiac artery bypass grafting (n = 17) to determine whether the effects of heparin were systemic or platelet-specific. All patients’ platelets were fully inhibited by aspirin prior to surgery, but within 3 min of receiving heparin spontaneous aggregation and responses to arachidonic acid (AA) and ADP increased significantly (p ≥ 0.0002), and activated platelets were found in the circulation. While there was no rise in thromboxane in the plasma following heparin, levels of the major platelet 12-lipoxygenase product, 12-HETE, rose significantly. Mixing experiments demonstrated that the changes caused by heparin resided primarily in the platelets, while addition of AA pathway inhibitors, and analysis of oxylipins provided evidence that, following heparin, aggregating platelets regained their ability to synthesise thromboxane. These findings highlight potentially unrecognised pro-thrombotic and pro-inflammatory changes during CABG surgery, and provide further evidence of adverse effects associated with UFH.

Algerian Propolis from Distinct Geographical Locations: Chemical Profiles, Antioxidant Capacity, Cytotoxicity and Inhibition of 5-Lipoxygenase Product Biosynthesis.

Propolis was collected from honeybee hives in three geographically distinct Algerian climates and extracts were characterized for composition and bioactivity. Bees were identified as native subspecies using an in-silico DraI mtDNA COI-COII test. Over 20 compounds were identified in extracts by LC-MS. Extracts from the Medea region were more enriched in phenolic content (302±28 mg GAE/g of dry extract) than those from Annaba and Ghardaia regions. Annaba extracts had the highest flavonoid content (1870±385 mg QCE/g of dry extract). Medea extracts presented the highest free-radical scavenging activity (IC50=13.5 μg/mL) using the DPPH radical assay while Ghardaia extracts from the desert region were weak (IC50>100 μg/mL). Antioxidant activities measured using AAPH oxidation of linoleic acid were similar in all extracts with IC50 values ranging from 2.9 to 4.9 μg/mL. All extracts were cytotoxic (MTT assay) and proapoptotic (Annexin-V) against human leukemia cell lines in the low μg/mL range, although the Annaba extract was less active against the Reh cell line. Extracts inhibited cellular 5-lipoxygenase product biosynthesis with IC50 values ranging from 0.6 to 3.2 μg/mL. Overall, examined propolis extracts exhibited significant biological activity that warrant further characterization in cellular and in vivo models.

Brown Adipose Tissue Activation in Humans Increases Plasma Levels of Lipid Mediators.

Activation of brown adipose tissue (BAT) thermogenesis improves insulin sensitivity and is beneficial in obesity. Emerging evidence indicates that BAT activation increases lipid mediators that play autocrine and endocrine roles to regulate metabolism and inflammation. The goal of the study was to determine the relationship between 2 distinct approaches of BAT activation (cold exposure and mirabegron treatment) with lipid mediators in humans. Healthy female subjects (n = 14) were treated with the β3-adrenergic receptor agonist mirabegron (100 mg) daily for 28 days. A subset of female subjects (n = 8) was additionally exposed to cold temperatures (14-16 °C) for 2 hours using a cooling vest prior to initiating mirabegron treatment. A panel of lipid mediators was assessed in plasma using targeted liquid chromatography-tandem mass spectrometry, and their relationship to anthropometric and metabolic parameters was determined. Activation of BAT with cold exposure acutely increased levels of lipoxygenase and cyclooxygenase products, including 12-hydroxyeicosapentaenoic acid, 12-hydroxyeicosatetraenoic acid (HETE), 5-HETE, 14-hydroxydocosahexaenoic acid (HDHA), an isomer of maresin 2 (MaR2), 17-HDHA, protectin D1 (PD1), and prostaglandin E2. Mirabegron treatment similarly increased these products acutely, although levels of some mediators were blunted after chronic mirabegron treatment. Selected lipid mediators, including an MaR2 isomer, 17-HDHA, 5-HETE, and 15-HETE, positively correlated with nonesterified fatty acids and negatively correlated with the respiratory quotient, while PD1, 15-HETE, and 5-HETE positively correlated with adiponectin. These results indicate that selected lipid mediators may serve as biomarkers of BAT activation.

Exploring the Anti-Inflammatory Potential of Ajuga integrifolia Leaves Extract: In Vitro Dual Inhibition of Cyclooxygenase and Lipoxygenase Enzymes.

This study investigated the anti-inflammatory properties of Ajuga integrifolia, an herbal preparation. Qualitative and quantitative phytochemical analyses were conducted to identify active compounds in the preparation. The researchers also assessed its ability to inhibit the production of pro-inflammatory enzymes, cyclooxygenases (COX-1, COX-2), and lipoxygenase (5-LOX) in vitro. The extracts demonstrated dose-dependent inhibition of these enzymes, with some extracts showing IC50 values comparable to standard anti-inflammatory drugs. The ethanol extract exhibited significant inhibition of 5-LOX (52.99 μg/mL), compared to the standard drug zileuton (32.41 μg/mL), while the inhibition of COX-1 (66.00 μg/mL) and COX-2 (71.62 μg/mL) was comparable to the standard drug indomethacin (40.57 and 54.39 μg/mL, respectively). These findings suggest that A. integrifolia has the potential to be used as a herbal remedy for treating inflammatory conditions. By inhibiting pro-inflammatory enzymes, the extracts may effectively reduce inflammation and promote tissue healing or repair. The inhibition potential of extract of this plant can be taken as a good candidate of anti-inflammatory agent.

Deep phenotyping of the lipidomic response in COVID-19 and non-COVID-19 sepsis.

Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs-CoV-2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID-19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α-III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12-HETE, exhibited relative specificity for COVID-19 and correlated with sPLA2 and the interleukin-13 receptor to drive lymphopenia, a marker of disease severity. Pro-inflammatory eicosanoids remained correlated with severity in COVID-19 28 days after admission. Amongst non-COVID ICU patients, elevations in 5- and 15-HETE and 9- and 13-HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflected disease severity in COVID-19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non-B non-T cell activation. In COVID-19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids - ChoE 18:3, LPC-O-16:0 and PC-O-30:0 - were altered specifically in COVID. LPC-O-16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi-omic inflammatory pathways and disease severity. A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12-HETE and the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients and correlate with the inflammatory response to link to disease severity.

Comparative Analysis of Bio- and Chemo-Catalysts for the Synthesis of Flavour Compound Hexanal from Linoleic Acid

Hexanal, hexenal, nonenal and their corresponding alcohols are used as green notes in the fragrance and flavour industry. The production of bio-based hexanal starts from linoleic acid, which can be obtained from sunflower or safflower oil. The biocatalytic process utilizes C13-specific lipoxygenase (LOX) for hydroperoxidation and consecutive splitting with hydroperoxide lyase (HPL). In this study, we investigated the chemical splitting of the LOX product 13-HPODE in comparison to HPL catalysis. In addition, 13-HPODE was synthesized using enriched linoleic acid from safflower oil. Varying amounts of soybean flour suspension as a source of LOX yielded up to 60% HPODE with a regioselectivity of 92% towards 13-HPODE. Using low-toxicity Lewis acids like AlCl3 and ZrCl4, cleavage of the produced 13-HPODE was possible. A maximum hexanal yield of 22.9% was reached with AlCl3 under mild reaction conditions, though product degradation was an interfering process. Comparative trials with N-terminal truncated HPL from papaya revealed hexanal recovery within a comparable range. Additionally, we successfully demonstrated the viability of Hock rearrangement of 13-HPODE through heterogeneous catalysts. Notably, Beta zeolite and Montmorillonite K10 exhibited a turnover frequency (TOF) on par with common heterogeneous catalysts employed in industrial processes.

Impact of geographical habitat and soil characteristics on morphological features, phytochemical composition and enzymatic activities of Erodium glaucophyllum

The impact of geographical habitat and soil types on morphological aspect, phytochemical composition and enzymes activity of Erodium glaucophyllum was assessed. Different soil textures were identified including sandy loam, clay and sandy clay loam. The plants exhibiting the highest degree of morphological development were identified in the Gabes region. The best nutrient composition value was recorded for the Gafsa and the Gabes regions. Among the mineral elements detected, the highest amounts of Mg and Ca were found in the Sidi Bouzid region with 29.61 and 50.07 mg kg−1, respectively. They depended on geographical location, altitude, average rainfall and the physiological condition of the plant. Plants from the Gafsa region exhibited the highest levels of phenolic and flavonoid contents measuring 187.47 mg GAE/g and 29.76 mg QE/g, respectively. Antioxidant activity was in accordance with total phenols of the three localities and the lowest IC50 values for DPPH scavenging and FRAP assay was recorded for the Gafsa region. The findings demonstrated that the studied species has the potential to serve as a valuable ecological natural source for bioactive antioxidant compounds. Furthermore, it shows promise as an alternative source for the production of β-amylase, L-asparaginase and lipoxygenase.

Caffeic acid phenethyl ester analogues as selective inhibitors of 12-lipoxygenase product biosynthesis in human platelets.

The inflammatory response is an essential process for the host defence against pathogens. Lipid mediators are important in coordinating the pro-inflammatory and pro-resolution phases of the inflammatory process. However, unregulated production of these mediators has been associated with chronic inflammatory diseases such as arthritis, asthma, cardiovascular diseases, and several types of cancer. Therefore, it is not surprising that enzymes implicated in the production of these lipid mediators have been targeted for potential therapeutic approaches. Amongst these inflammatory molecules, the 12-hydroxyeicosatetraenoic acid (12(S)-HETE) is abundantly produced in several diseases and is primarily biosynthesized via the platelet's 12-lipoxygenase (12-LO) pathway. To this day, very few compounds selectively inhibit the 12-LO pathway, and most importantly, none are currently used in the clinical settings. In this study, we investigated a series of polyphenol analogues of natural polyphenols that inhibit the 12-LO pathway in human platelets without affecting other normal functions of the cell. Using an ex vivo approach, we found one compound that selectively inhibited the 12-LO pathway, with IC50 values as low as 0.11µM, with minimal inhibition of other lipoxygenase or cyclooxygenase pathways. More importantly, our data show that none of the compounds tested induced significant off-target effects on either the platelet's activation or its viability. In the continuous search for specific and better inhibitors targeting the regulation of inflammation, we characterized two novel inhibitors of the 12-LO pathway that could be promising for subsequent in vivo studies.

Metabolomic Profiling of Long-Chain Polyunsaturated Fatty Acid Oxidation in Adults with Retinal Vein Occlusion: A Case-Control Study

BackgroundLong-chain polyunsaturated fatty acids (LCPUFAs) and their metabolites are closely related to neovascular eye diseases. However, the clinical significance of their oxylipins in retinal vein occlusion (RVO) remains inconclusive. ObjectivesThis case-control study aimed to explore metabolomic profiles of LCPUFA oxidation in RVO and to identify potential indicators for diagnosis and pathologic progression. MethodsThe plasma concentrations of ω-3 (n–3) and ω-6 (n–6) LCPUFA and their oxylipins in 44 adults with RVO and 36 normal controls were analyzed using ultraperformance liquid chromatography tandem mass spectrometry. Univariate analysis combined with principal component and orthogonal projections to latent structure discriminant analysis was used to screen differential metabolites. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of 5-oxo-eicosatetraenoic acids (ETE) on angiogenesis ex vivo. Tubule formation and wound healing assays were performed to verify its effects on human retinal microvascular endothelial cell functions. ResultsHigher ω-6 and lower ω-3 LCPUFA plasma concentrations were measured in the adults with RVO compared with control (odds ratio [OR]: 2.34; 95% confidence interval [CI]: 1.42, 3.86; P < 0.001; OR: 0.28; 95% CI: 0.15, 0.51; P < 0.001). Metabolomic analysis revealed 20 LCPUFA and their oxylipins dysregulated in RVO, including increased arachidonic acid (ω-6, OR: 1.85; 95% CI: 1.18, 2.90; P < 0.001) and its lipoxygenase product 5-oxo-ETE (OR: 11.76; 95% CI: 3.73, 37.11; P < 0.001), as well as decreased docosahexaenoic acid (ω-3, OR: 0.13; 95% CI: 0.05, 0.33; P < 0.001). Interestingly, 5-oxo-ETE was downregulated in ischemic compared with nonischemic central RVO. Exogenous 5-oxo-ETE attenuated aortic ring and choroidal explant sprouting and inhibited tubule formation and migration of human retinal microvascular endothelial cells in a dose-dependent manner, possibly via suppressing the vascular endothelial growth factor signaling pathway. ConclusionsThe plasma concentrations of ω-6 and ω-3 LCPUFA and their oxylipins were associated with RVO. The ω-6 LCPUFA-derived metabolite 5-oxo-ETE was a potential marker of RVO development and progression.

The same boat, different storm: stress volatile emissions in response to biotrophic fungal infections in primary and alternate hosts

ABSTRACT Rust infection results in stress volatile emissions, but due to the complexity of host-pathogen interaction and variations in innate defense and capacity to induce defense, biochemical responses can vary among host species. Fungal-dependent modifications in volatile emissions have been well documented in numerous host species, but how emission responses vary among host species is poorly understood. Our recent experiments demonstrated that the obligate biotrophic crown rust fungus (P. coronata) differently activated primary and secondary metabolic pathways in its primary host Avena sativa and alternate host Rhamnus frangula. In A. sativa, emissions of methyl jasmonate, short-chained lipoxygenase products, long-chained saturated fatty acid derivatives, mono- and sesquiterpenes, carotenoid breakdown products, and benzenoids were initially elicited in an infection severity-dependent manner, but the emissions decreased under severe infection and photosynthesis was almost completely inhibited. In R. frangula, infection resulted in low-level induction of stress volatile emissions, but surprisingly, in enhanced constitutive isoprene emissions, and even severely-infected leaves maintained a certain photosynthesis rate. Thus, the same pathogen elicited a much stronger response in the primary than in the alternate host. We argue that future work should focus on resolving mechanisms of different fungal tolerance and resilience among primary and secondary hosts.

Ketols Emerge as Potent Oxylipin Signals Regulating Diverse Physiological Processes in Plants.

Plants produce an array of oxylipins implicated in defense responses against various stresses, with about 600 oxylipins identified in plants to date. Most known oxylipins are the products of lipoxygenase (LOX)-mediated oxygenation of polyunsaturated fatty acids. One of the most well-characterized oxylipins produced by plants is the hormone jasmonic acid (JA); however, the function of the vast majority of oxylipins remains a mystery. One of the lesser-studied groups of oxylipins is comprised of ketols produced by the sequential action of LOX, allene oxide synthase (AOS), followed by non-enzymatic hydrolysis. For decades, ketols were mostly considered mere by-products of JA biosynthesis. Recent accumulating evidence suggests that ketols exhibit hormone-like signaling activities in the regulation of diverse physiological processes, including flowering, germination, plant-symbiont interactions, and defense against biotic and abiotic stresses. To complement multiple reviews on jasmonate and overall oxylipin biology, this review focuses specifically on advancing our understanding of ketol biosynthesis, occurrence, and proposed functions in diverse physiological processes.

Abstract 240: Bioactive Lipids In Circulation: Biomarker Of Ageing?

Blood transports cells and nutrients. However, it is less known that the major plasma compound is the lipid family in the broad sense. Among these lipids, cholesterol, triglyceride but also the family of polyunsaturated fatty acids (PUFAs) and their metabolites. These lipid mediators are involved in several chronic, inflammatory diseases such as rheumatoid arthritis. But, ageing is associated with chronic inflammation and affects almost 20% of the world’s population. Thus, it is by far the most important population before cardiovascular disease and cancer. Using mass spectrometry analyses, we evaluated the levels of different classes of lipid mediators from healthy young and elderly donors (single donor apheresis). Our data showed a significant decrease of lysophosphatidic acid species‘s expression, modulation of the 5-lipoxygenase product’s, as well as lipid mediators derived from PUFA oxidation. Their effect on blood vessel and circulating cells influenced their function. Indeed, lipid mediator intake is involved in transfusion reactions, and we observed an activation of blood cells, especially platelet, with significant increase of p-selectin. These data highlighted the importance of lipid mediator level in a context of transfusion and ageing of blood donor independently of the storage time of single donor apheresis. They can serve as biomarker of ageing.

Nonsteroidal anti-inflammatory drugs as a targeted therapy for bone marrow failure in Ghosal hematodiaphyseal dysplasia

AbstractAdvances in genomic diagnostics hold promise for improved care of rare hematologic diseases. Here, we describe a novel targeted therapeutic approach for Ghosal hematodiaphyseal dysplasia, an autosomal recessive disease characterized by severe normocytic anemia and bone abnormalities due to loss-of-function mutations in thromboxane A synthase 1 (TBXAS1). TBXAS1 metabolizes prostaglandin H2 (PGH2), a cyclooxygenase (COX) product of arachidonic acid, into thromboxane A2. Loss-of-function mutations in TBXAS result in an increase in PGH2 availability for other PG synthases. The current treatment for Ghosal hematodiaphyseal dysplasia syndrome consists of corticosteroids. We hypothesize that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit COX-1 and COX-2, could ameliorate the effects of TBXAS1 loss and improve hematologic function by reducing prostaglandin formation. We treated 2 patients with Ghosal hematodiaphyseal dysplasia syndrome, an adult and a child, with standard doses of NSAIDs (aspirin or ibuprofen). Both patients had rapid improvements concerning hematologic parameters and inflammatory markers without adverse events. Mass spectrometry analysis demonstrated that urinary PG metabolites were increased along with proinflammatory lipoxygenase (LOX) products 5-hydroxyeicosatetraenoic acid and leukotriene E4. Our data show that NSAIDs at standard doses surprisingly reduced both COX and LOX products, leading to the resolution of cytopenia, and should be considered for first-line treatment for Ghosal hematodiaphyseal dysplasia syndrome.

Engineering sigma factors and chaperones for enhanced heterologous lipoxygenase production in Escherichia coli

BackgroundLipoxygenase (EC. 1.13.11.12, LOX) can catalyze the addition of oxygen into polyunsaturated fatty acids to produce hydroperoxides, which are widely used in the food, chemical, and pharmaceutical industries. In recent years, the heterologous production of LOX by Escherichia coli has attracted extensive attention. However, overexpressed recombinant LOX in E. coli aggregates and forms insoluble inclusion bodies owing to protein misfolding.ResultsIn this study, a split green fluorescent protein-based screening method was developed to screen sigma (σ) factors and molecular chaperones for soluble LOX expression. Three mutant libraries of Skp, GroES, and RpoH was analyzed using the high-throughput screening method developed herein, and a series of mutants with significantly higher yield of soluble heterologous LOX were obtained. The soluble expression level of LOX in the isolated mutants increased by 4.2- to 5.3-fold. Further, the highest LOX activity (up to 6240 ± 269 U·g-DCW−1) was observed in E. coli REopt, with the regulatory factor mutants, RpoH and GroES. Based on RNA-Seq analysis of the selected strains, E. coli Eopt, E. coli Sopt, E. coli Ropt, and wild type, amino acid substitutions in σ factors and molecular chaperones regulated the expression level of genes related to gene replication, recombination, and repair. Furthermore, the regulatory factor mutants were identified to be beneficial to the soluble expression of two other heterologous proteins, amylase and bone morphological protein 12.ConclusionIn this study, a high-throughput screening method was developed for improved soluble LOX expression. The obtained positive mutants of the regulatory factor were analyzed and employed for the expression of other heterologous proteins, thus providing a potential solution for the inclusion-body protein.

INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage.

Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.

Effects of Levofloxacin, Aztreonam, and Colistin on Enzyme Synthesis by P. aeruginosa Isolated from Cystic Fibrosis Patients

(1) Background: Cystic fibrosis (CF) is characterized by chronic pulmonary inflammation and persistent bacterial infections. P. aeruginosa is among the main opportunistic pathogens causing infections in CF. P. aeruginosa is able to form a biofilm, decreasing antibiotic permeability. LOX, a lipoxygenase enzyme, is a virulence factor produced by P. aeruginosa and promotes its persistence in lung tissues. The aim of this study is to evaluate if antibiotics currently used for aerosol therapy in CF are able to interfere with the production of lipoxygenase from open isolates of P. Aeruginosa from patients with CF. (2) Methods: Clinical isolates of P. aeruginosa from patients with CF were grown in Luria broth (LB). Minimum inhibitory concentration (MIC) was performed and interpreted for all isolated strains according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. We selected four antibiotics with different mechanisms of action: aztreonam, colistin, amikacin, and levofloxacin. We used human pulmonary epithelial NCI-H929 cells to evaluate LOX activity and its metabolites according to antibiotic action at increasing concentrations. (3) Results: there is a correlation between LOX secretion by clinical isolates of P. aeruginosa and biofilm production. Levofloxacin exhibits highly significant inhibitory activity compared to the control. Amikacin also exhibits significant inhibitory activity against LOX production. Aztreonam and colistin do not show inhibitory activity. These results are also confirmed for LOX metabolites. (4) Conclusions: among the evaluated antibiotics, levofloxacin and amikacin have an activity on LOX secretion.