Epoxide Hydrolase Research Articles

Metabolomics reveals soluble epoxide hydrolase as a therapeutic target for high-sucrose diet-mediated gut barrier dysfunction

Highsucrose diet (HSD) was reported as a causative factor for multiorgan injuries. The underlying mechanisms and therapeutic strategies remain largely uncharted. In the present study, by using a metabolomics approach, we identified the soluble epoxide hydrolase (sEH) as a therapeutic target for HSD-mediated gut barrier dysfunction. Specifically, 16-week feeding on an HSD caused gut barrier dysfunction, such as colon inflammation and tight junction impairment in a murine model. A metabolomics analysis of mouse colon tissue showed a decrease in the 5(6)-epoxyeicosatrienoic acid [5(6)-EET] level and an increase in soluble epoxide hydrolase, which is related to HSD-mediated injuries to the gut barrier. The mice treated with a chemical inhibitor of sEH and the mice with genetic intervention by intestinal-specific knockout of the sEH gene significantly attenuated HSD-caused intestinal injuries by reducing HSD-mediated colon inflammation and improving the impaired tight junction caused by an HSD. Further, in vitro studies showed that treatment with 5(6)-EET, but not its hydrolytic product 5,6-dihydroxyeicosatrienoic acid (5,6-DiHET), significantly ablated high sucrose-caused intestinal epithelial inflammation and impaired tight junction. Additionally, 5(6)-EET is anti-inflammatory and improves gut epithelial tight junction while 5,6-DiHET cannot do so. This study presents an underlying mechanism of and a therapeutic strategy for the gut barrier dysfunction caused by an HSD.

The role of soluble epoxide hydrolase in the intestine.

The soluble epoxide hydrolase (sEH; encoded by the EPHX2 gene) is an α/β hydrolase fold protein that is, widely distributed throughout the body. Recent studies have highlighted that sEH, in the metabolism of polyunsaturated fatty acids, plays a part in the pathogenesis of various diseases, including cardiovascular disease, Alzheimer's disease and intestine-associated disease. This review discusses the current findings on the role of sEH in the development of intestine- and intestine-associated diseases, including colitis, colorectal cancer, and other intestinal diseases, as well as the potential underlying mechanisms involved.

Soluble epoxide hydrolase deletion rescues behavioral and synaptic deficits. By AMPK-mTOR pathway in autism animals

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social defects often accompanied with emotional comorbidities. Aberrations in synaptic function and plasticity are the core feature in the pathophysiology of ASD. Targeting soluble epoxide hydrolase (sEH) has been found to exert protection in a wide-range of pathological conditions. However, the regulation of sEH deficiency on the synaptic deficits of ASD and the underlying mechanisms remain unclear. The valproate (VPA)-treated ASD animal model with genetic sEH knockout was applied in the present study. The results showed that the sEH expression was significantly increased in the prefrontal cortex of VPA-treated animals. Although no effect was found on tail malformation and body weight loss, genetic sEH deletion alleviated social deficits, and fear learning and memory extinction in the VPA-treated mice. After a series of electrophysiological assessments, we found that the beneficial effects of sEH deletion focused on the long-term synaptic plasticity, rather than presynaptic efficiency, in the VPA-treated mice. Furthermore, we observed that the dysregulated AMPK-mTOR pathway was restored under genetic sEH deletion in VPA-treated mice. Taken together, these findings uncovered an important role of sEH deficiency in the synaptic dysfunctions of ASD mediated by AMPK-mTOR pathway, providing a novel therapeutic target for ASD.

Aerobic exercise alleviates statin-induced PCSK9 upregulation by increasing epoxyeicosatrienoic acid levels through the FoxO3a-Sirt6 axis

BackgroundStatins are the cornerstone of low-density lipoprotein cholesterol (LDL-C)-lowering therapy; however, the therapeutic efficacy of statins in countering atherosclerotic cardiovascular disease (ASCVD) is compromised by the concurrent elevation of proprotein convertase subtilisin/kexin type 9 (PCSK9), a pivotal molecule that increases LDL-C levels. Aerobic exercise lowers PCSK9 levels, but the underlying mechanism remains unclear. Therefore, we investigated how aerobic exercise can ameliorate statin-induced increases in PCSK9 levels. MethodsThree-week-old male American Institute of Cancer Research (ICR) mice were fed a high-fat-cholesterol diet (HFD) for 12 weeks and then administered atorvastatin alone or atorvastatin combined with aerobic exercise (Statin+Ex). Moreover, a total of 165 participants with stable coronary heart disease (CHD) enrolled at the inpatient and outpatient departments of the Second Xiangya Hospital of Central South University from January 2018 to July 2020 were randomized into the Statin group (male/female = 51/33) and Statin+Ex group (male/female = 52/29). Patients in the Statin+Ex group underwent treadmill exercise of 45–60 min/day for 7 days. ResultsAerobic exercise effectively alleviated statin-induced PCSK9 upregulation in human patients with CHD and hypercholesterolemic ICR mice (all p< 0.05). Mechanistically, our findings revealed that aerobic exercise induced elevated epoxyeicosatrienoic acids (EETs) plasma levels while concurrently reducing the activity of soluble epoxide hydrolase (sEH) (all p < 0.05), an enzyme responsible for EETs degradation. Further, EETs significantly suppressed PCSK9 expression, subsequently reducing the LDL-C levels (all p < 0.05); this effect was mediated via the activation of the Forkhead box O3a-Silent mating type information regulation 2 homolog 6 (FoxO3a-Sirt6) axis, with no impact on the Sterol Regulatory Element Binding Protein 2 and 3-hydroxy-3-methylglutaryl-CoA reductase(SREBP2-HMGCR) pathway. ConclusionOur study sheds light on the paradigm of "Exercise is Medicine", providing evidence to support the use of statins combined with exercise in reducing LDL-C levels, and unveils potential avenues for clinical applications of sEH inhibitors, presenting novel prospects for therapeutic interventions in ASCVD.

Eicosanoids in coronary sinus negatively correlate with natriuretic peptides in heart failure with reduced ejection fraction: insights from Eicosanoids in Human Heart Failure Study

Abstract Background The diagnostic and therapeutic potential of eicosanoids, metabolites of arachidonic acid with positive cardio-renal activity in preclinical heart failure models, remains unclear in human chronic heart failure with reduced ejection fraction (HFrEF). Purpose The aim of this part of a traslational Eicosanoids in Human Heart Failure Study was to investigate levels of eicosanoids in different body compartments and their relation to natriuretic peptides. Methods Eleven consecutive patients with HFrEF indicated to cardiac resynchronisation therapy (CRT) were enrolled to measure plasmatic 14,15-epoxyeicosatrienoic acid (14,15-EET) and 14,15-dihydroxyicosatrienoic acid (14,15-DHET) levels from venous, arterial, and coronary sinus (CS) blood samples and correlate them with N-terminal pro B-type natriuretic peptide (NT-proBNP) from the same compartments. Results In CS, NT-proBNP levels negatively correlated with plasmatic 14,15-EET levels (r = -0.63, p = 0.03) and positively with the DHET/EET ratio (r = 0.73, p = 0.02). This correlation was not found in the other compartments. Plasmatic 14,15-EET nor 14,15-DHET levels in measured compartments did not differ statistically (p = 0.21, p = 0,64, respectively). In individual patients, the levels of both eicosanoids correlated across all compartments. Peripheral plasma 14,15-EET levels in controls were lower compared to HF patients. Conclusion Peripheral venous eicosanoid (14,15-EET, 14,15-DHET) levels correlate and do not differ from arterial and CS levels in patients with HFrEF indicated to CRT. In CS, NT-proBNP levels negatively correlated with plasmatic 14,15-EET levels and positively with the DHET/EET ratio, an indirect soluble epoxide hydrolase activity parameter.Graphical AbstractFigures

Soluble Epoxide Hydrolase Inhibitor Ameliorates Olfactory Dysfunction, Modulates Microglia Polarization, and Attenuates Neuroinflammation after Ischemic Brain Injury.

Olfactory bulb (OB) microglia activation and inflammation can lead to olfactory dysfunction, which often occurs after an ischemic stroke. Inhibition of soluble epoxide hydrolase (sEH) attenuates neuroinflammation in brain injuries by reducing the degradation of anti-inflammatory epoxyeicosatrienoic acids. However, whether sEH inhibitors can ameliorate olfactory dysfunction after an ischemic stroke remains unknown. Ischemic brain injury and olfactory dysfunction were induced by middle cerebral artery occlusion (MCAO) in Wistar Kyoto rats. The rats were administered 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a selective sEH inhibitor. Olfactory function, cerebral infarct volume, and the degree of degeneration, microglial polarization and neuroinflammation in OB were evaluated. Following treatment with AUDA, rats subjected to MCAO displayed mild cerebral infarction and OB degeneration, as well as better olfactory performance. In OB, AUDA triggered a modulation of microglial polarization toward the M2 anti-inflammatory type, reduction in proinflammatory mediators, and enhancement of the antioxidant process. The effectiveness of AUDA in terms of anti-inflammatory, neuroprotection and anti-oxidative properties suggests that it may have clinical therapeutic implication for ischemic stroke related olfactory dysfunction.

Impact of soluble epoxide hydrolase inhibition on silica-induced pulmonary fibrosis, ectopic lymphoid neogenesis, and autoantibody production in lupus-prone mice

Objective Acute intranasal (IN) instillation of lupus-prone NZBWF1 mice with crystalline silica (cSiO2) triggers robust lung inflammation that drives autoimmunity. Prior studies in other preclinical models show that soluble epoxide hydrolase (sEH) inhibition upregulates pro-resolving lipid metabolites that are protective against pulmonary inflammation. Herein, we assessed in NZBWF1 mice how acute IN cSiO2 exposure with or without the selective sEH inhibitor TPPU influences lipidomic, transcriptomic, proteomic, and histopathological biomarkers of inflammation, fibrosis, and autoimmunity. Methods Female 6-week-old NZBWF1 mice were fed control or TPPU-supplemented diets for 2 weeks then IN instilled with 2.5 mg cSiO2 or saline vehicle. Cohorts were terminated at 7 or 28 days post-cSiO2 instillation (PI) and lungs analyzed for prostaglandins, cytokines/chemokines, gene expression, differential cell counts, histopathology, and autoantibodies. Results cSiO2-treatment induced prostaglandins, cytokines/chemokine, proinflammatory gene expression, CD206+ monocytes, Ly6B.2+ neutrophils, CD3+ T cells, CD45R+ B cells, centriacinar inflammation, collagen deposition, ectopic lymphoid structure neogenesis, and autoantibodies. While TPPU effectively inhibited sEH as reflected by skewed lipidomic profile in lung and decreased cSiO2-induced monocytes, neutrophils, and lymphocytes in lung lavage fluid, it did not significantly impact other biomarkers. Discussion cSiO2 evoked robust pulmonary inflammation and fibrosis in NZBWF1 mice that was evident at 7 days PI and progressed to ELS development and autoimmunity by 28 days PI. sEH inhibition by TPPU modestly suppressed cSiO2-induced cellularity changes and pulmonary fibrosis. However, TPPU did not affect ELS formation or autoantibody responses, suggesting sEH minimally impacts cSiO2-triggered lung inflammation, fibrosis, and early autoimmunity in our model.

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa infects the airways of people with cystic fibrosis (CF) and produces a virulence factor Cif that is associated with worse outcomes. Cif is an epoxide hydrolase that reduces cell-surface abundance of the cystic fibrosis transmembrane conductance regulator (CFTR) and sabotages pro-resolving signals. Its expression is regulated by a divergently transcribed TetR family transcriptional repressor. CifR represents the first reported epoxide-sensing bacterial transcriptional regulator, but neither its interaction with cognate operator sequences nor the mechanism of activation has been investigated. Using biochemical and structural approaches, we uncovered the molecular mechanisms controlling this complex virulence operon. We present here the first molecular structures of CifR alone and in complex with operator DNA, resolved in a single crystal lattice. Significant conformational changes between these two structures suggest how CifR regulates the expression of the virulence gene cif. Interactions between the N-terminal extension of CifR with the DNA minor groove of the operator play a significant role in the operator recognition of CifR. We also determined that cysteine residue Cys107 is critical for epoxide sensing and DNA release. These results offer new insights into the stereochemical regulation of an epoxide-based virulence circuit in a critically important clinical pathogen.

Exploring the impact of sEH inhibition on intestinal cell differentiation and Colon Cancer: Insights from TPPU treatment

Inhibition of soluble epoxide hydrolase (sEH) appears to be promising for the treatment of many diseases. Studies have focused on the beneficial effects of epoxyeicosatrienoic acids (EETs), which are sEH substrates. However, our recent studies have shown that the sEH activity is crucial for the proper intestinal cell differentiation. In this recent study, we investigated the impact of TPPU, an inhibitor of sEH, on the colon cancer cell lines Caco2 and HT-29. We analysed the changes in the expression of the cytoskeletal protein ezrin and the phosphorylated protein kinase p38 (p-p38). Our results showed a decrease in ezrin expression in differentiated cells and an increase in p-p38 expression after TPPU treatment. Immunocytochemical staining revealed a higher staining intensity of p-p38 in the nuclei of HT-29 cells following TPPU treatment. Immunohistochemical staining was performed on human samples of normal colon tissue, grade 2 tumours, and embryonal/foetal tissues. The staining intensity of ezrin in tumours was reduced in the surface area compared to the crypts. Additionally, we observed the translocation of p-p38 expression from the cytoplasm to the nucleus during differentiation. The tumour samples exhibited higher levels of p-p38 in the cytoplasm, similar to normal undifferentiated tissue. To observe the disruption of the cytoskeleton after TPPU treatment, confocal microscopy was used. It was found that β-actin associated with ezrin forms clusters under the plasma membranes. All of these results are significant because sEH inhibitors are being tested in clinical trials, but they could cause an unexpected adverse effects.

Improving Hydrolytic Activity and Enantioselectivity of Epoxide Hydrolase from Phanerochaete chrysosporium by Directed Evolution.

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts.

Inhibiting the soluble epoxide hydrolase increases the EpFAs and ERK1/2 expression in the hippocampus of LiCl-pilocarpine post-status epilepticus rat model

PurposeThis study aimed to investigate the enzyme activity of soluble epoxide hydrolase (sEH) and quantify its metabolic substrates, namely epoxygenated fatty acids (EpFAs), and products of sEH in the hippocampus after administering TPPU [1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea], an inhibitor of sEH. Furthermore, it explored whether the extracellular signal-activated protein kinase 1/2 (ERK1/2) is involved in the anti-seizure effects of TPPU in the lithium chloride (LiCl)-pilocarpine induced post-status epilepticus (SE) rat model. MethodsThe rats were intraperitoneally (I.P.) injected with LiCl and pilocarpine to induce SE and then spontaneous recurrent seizures (SRS) were observed. Rats were randomly assigned into SRS + TPPU group (intragastrically administering 0.1 mg/kg/d TPPU), SRS + Vehicle group (administering the vehicle instead), and Control group. Enzyme-linked immunosorbent assay, Western-blot analysis, and ultra-high-performance liquid chromatography/mass spectrometry (LC/MS) were performed to measure the enzyme activity of sEH, the protein level of sEH and ERK1/2, and the concentration of TPPU and polyunsaturated fatty acids (PUFAs) metabolisms in the hippocampus. ResultsThe frequency of SRS events of Racine stage 3 or higher ranged from 0 to 19 per week in the SRS + Vehicle group, compared to 0–5 per week in the SRS + TPPU group. sEH enzyme activity and protein levels were significantly elevated in the SRS + Vehicle group compared to the Control group. After TPPU administration, the hippocampal TPPU concentration reached 10.94 ± 4.37 nmol/kg. sEH enzyme activity was significantly reduced in the LiCl-pilocarpine-induced post-SE rat model, although sEH protein levels did not decrease significantly. The regioisomers 8,9-, 11,12-, and 14,15-EETs, total EETs, the EETs/DHETs ratio, other EpFAs including 16(17)-EpDPA, and the 19(20)-EpDPA/19,20-DiHDPA ratio in the hippocampus were significantly increased. Additionally, the p-ERK1/2 to ERK1/2 ratio in the hippocampus was significantly elevated following TPPU administration. ConclusionThis study demonstrates that inhibiting sEH with TPPU increases the levels of EETs, other EpFAs, and ERK1/2 expression in the hippocampus of a LiCl-pilocarpine-induced post-SE rat model. These findings suggest that the anti-seizure effect of TPPU may be mediated through the EETs-ERK1/2 pathway.

Discovery of glycosidated glycyrrhetinic acid derivatives: Natural product-based soluble epoxide hydrolase inhibitors

There are few reports on soluble epoxide hydrolase (sEH) structure-activity relationship studies using natural product-based scaffolds. In this study, we discovered that C-30 urea derivatives of glycyrrhetinic acid such as 33, rather than C-20/C-3 urea derivatives, possess in vitro sEH inhibitory capabilities. Furthermore, we explored the impact of stereoconfigurations at C-3 and C-18 positions, and glycosidic bonds at the 3-OH on the compound's activity. Consequently, a glycoside of 33, specifically 49Cα containing alpha-oriented mannose, exhibited promising in vivo efficacy in alleviating carrageenan-induced paw edema and acetic acid-induced writhing. Meanwhile, 49Cα demonstrated potential in mitigating acute pancreatitis by modulating the ratios of anti-inflammatory epoxyeicosatrienoic acids (EETs) to pro-inflammatory dihydroxyeicosatrienoic acids (DHETs). The co-crystal structure of sEH in complex with 49Cα revealed that the N-tetrahydropyranylmethylene urea hydrogen bonded with the residues within the sEH tunnel, contrasting with the mannose component that extended beyond the tunnel's confines. Our findings highlight 49Cα (coded LQ-38) as a promising candidate for anti-inflammatory and analgesic effects, and pave the way for the future rational design of triterpenoid-based sEH inhibitors.

Elevated 12,13-diHOME level in maternal and umbilical cord blood complicated with preeclampsia.

Preeclampsia (PE) is a condition in pregnancy characterized by hypertension and proteinuria, thus leading to severe complications for both mother and fetus, including fetal growth restriction (FGR). However, there are still unclear aspects regarding the pathogenesis, prevention, and treatments. This study aimed to elucidate the characteristics of lipid metabolism in maternal and umbilical cord plasma complicated with PE using liquid chromatography-mass spectrometry (LC-MS). The study included singleton pregnant women at Osaka Metropolitan University Hospital from March 2023 to February 2024. PE was diagnosed based on new-onset hypertension after 20 weeks of gestation and other symptoms such as proteinuria and organ dysfunction. FGR was defined by ultrasound measurements below -1.5 standard deviation (SD). Plasma samples were collected from maternal and umbilical cord blood within 24 hours before delivery. Lipid metabolites were comprehensively analyzed using LC-MS, and the lipokine 12,13-diHOME, identified as elevated in the comprehensive analysis, was quantified. Immunohistochemistry was conducted on placental samples to assess soluble epoxide hydrolase (sEH) expression. The study involved 31 participants, with 20 in the control group and 11 in the PE group. A comprehensive analysis of maternal plasma samples identified a significant increase in 12,13-diHOME levels in the PE group compared to the control group. Quantification of 12,13-diHOME showed a significant increase in maternal plasma, umbilical venous plasma, and umbilical arterial plasma in the PE group compared to the control group (p = 0.007, p = 0.008, p = 0.005). PE with FGR showed significantly higher 12,13-diHOME concentrations in the umbilical arterial/venous ratio compared to the PE without FGR group (p = 0.03). Negative correlations were observed between 12,13-diHOME levels and birth weight in the PE group. Immunohistochemistry did not show significant differences in the sEH expression between the groups. This study demonstrated that 12,13-diHOME levels were significantly elevated in maternal and umbilical cord blood in PE patients, particularly in PE with FGR. Elevated 12,13-diHOME may reflect the progression of placental ischemia due to PE pathogenesis. This lipid metabolite could serve as a marker for the severity of preeclampsia, thus providing new insights into perinatal lipidomics and the potential role of 12,13-diHOME in PE.

Discovery of Orally Active Phenylquinoline-Based Soluble Epoxide Hydrolase Inhibitors with Anti-Inflammatory and Analgesic Activity.

Currently, there are no specific drugs for treating acute pancreatitis. Soluble epoxide hydrolase (sEH) inhibitors show promise, but face challenges like low blood drug concentrations and potential adverse effects on CYP enzymes and the human ether-a-go-go-related gene (hERG). In this study, an approach involving scaffold hopping and structure-activity guided optimization was employed to design a series of phenylquinoline-based sEH inhibitors. Among these compounds, DJ-53 exhibited potent in vitro and in vivo effects in alleviating pain and reducing inflammation. The in vivo mechanism of action involved inhibiting sEH enzyme activity, thereby increasing levels of anti-inflammatory epoxyeicosatrienoic acids (EETs) and decreasing levels of proinflammatory dihydroxyeicosatrienoic acids (DHETs). Importantly, DJ-53 showed exceptional oral bioavailability and pharmacokinetics, while avoiding inhibition of CYP enzymes or the hERG channel. These results highlight DJ-53's potential as a new lead compound for anti-inflammatory and analgesic applications and provide a safe and effective scaffold for developing sEH inhibitors.

Molecular Properties of Virulence and Antibiotic Resistance of Pseudomonas aeruginosa Causing Clinically Critical Infections.

The increase in the number of hospital strains of hypervirulent and multidrug resistant (MDR) Pseudomonas aeruginosa is a major health problem that reduces medical treatment options and increases mortality. The molecular profiles of virulence and multidrug resistance of P. aeruginosa-associated hospital and community infections in Mexico have been poorly studied. In this study, we analyzed the different molecular profiles associated with the virulence genotypes related to multidrug resistance and the genotypes of multidrug efflux pumps (mex) in P. aeruginosa causing clinically critical infections isolated from Mexican patients with community- and hospital-acquired infections. Susceptibility to 12 antibiotics was determined using the Kirby-Bauer method. The identification of P. aeruginosa and the detection of virulence and efflux pump system genes were performed using conventional PCR. All strains isolated from patients with hospital-acquired (n = 67) and community-acquired infections (n = 57) were multidrug resistant, mainly to beta-lactams (ampicillin [96.7%], carbenicillin [98.3%], cefalotin [97.5%], and cefotaxime [87%]), quinolones (norfloxacin [78.2%]), phenicols (chloramphenicol [91.9%]), nitrofurans (nitrofurantoin [70.9%]), aminoglycosides (gentamicin [75%]), and sulfonamide/trimethoprim (96.7%). Most strains (95.5%) isolated from patients with hospital- and community-acquired infections carried the adhesion (pilA) and biofilm formation (ndvB) genes. Outer membrane proteins (oprI and oprL) were present in 100% of cases, elastases (lasA and lasB) in 100% and 98.3%, respectively, alkaline protease (apr) and alginate (algD) in 99.1% and 97.5%, respectively, and chaperone (groEL) and epoxide hydrolase (cif) in 100% and 97.5%, respectively. Overall, 99.1% of the strains isolated from patients with hospital- and community-acquired infections carried the efflux pump system genes mexB and mexY, while 98.3% of the strains carried mexF and mexZ. These findings show a wide distribution of the virulome related to the genotypic and phenotypic profiles of antibiotic resistance and the origin of the strains isolated from patients with hospital- and community-acquired infections, demonstrating that these molecular mechanisms may play an important role in high-pathogenicity infections caused by P. aeruginosa.

A highly efficient chemoenzymatic process to produce (R)-6,7-dihydroxygeraniol

The efficient asymmetric dihydroxylation of 6,7-epoxygeraniol was performed using a chemoenzymatic process employing a yeast epoxide hydrolase (EH) resolution stereoinversion step, followed by stereoretentive chemical hydrolysis of the remaining epoxide to produce (6 R)-6,7-dihydroxygeraniol at unprecedented high enantiometic excess (ee) (>97.5%) and high isolated yield (72 mol % overall yield over 5 steps from commercially available geraniol). The enzymatic process was completed within 2 h at 250 g/L substrate loading reaching > 49.5 mass % conversion of the racemic epoxide. The reaction was self-limiting and furnished both the homochiral (6 R)-triol and the residual (6 R)-epoxide at > 99% ee, due to enantio-inversion of the (6S)-epoxide. The (6 R)-epoxide was subsequently chemically hydrolysed, without first needing to separate the epoxide and triol products, to afford the desired (6 R)-triol product in >97.5% ee, at multigram scale. This chemoenzymatic procedure offers an excellent alternative to chemical asymmetric epoxidation or dihydroxylation for the production of enantiopure 6,7-dihydroxygeranyl and 6,7-epoxygeranyl type compounds in general. It exemplifies the benefits of using greener EH bioprocesses to produce such compounds in high ee’s and high isolated yields.

Soluble Epoxide Hydrolase Inhibition Attenuates Proteinuria by Alleviating Renal Inflammation and Podocyte Injuries in Adriamycin-Induced Nephropathy.

Soluble epoxide hydrolase (sEH) has previously been demonstrated to play an important part in kidney diseases by hydrolyzing renoprotective epoxyeicosatrienoic acids to their less active diols. However, little is known about the role of sEH in primary glomerular diseases. Here, we investigated the effects of sEH inhibition on proteinuria in primary glomerular diseases and the underlying mechanism. The expression of sEH in the renal tubules of patients with minimal change disease, IgA nephropathy, and membranous nephropathy was significantly increased. Renal sEH expression level was positively correlated with the 24 h urine protein excretion and negatively correlated with serum albumin. In the animal model of Adriamycin (ADR)-induced nephropathy, renal sEH mRNA and protein expression increased significantly. Pharmacological inhibition of sEH with AUDA effectively reduced urine protein excretion and attenuated renal pathological damage. Furthermore, sEH inhibition markedly abrogated the abnormal expressions of nephrin and desmin in glomerular podocytes induced by ADR. More importantly, AUDA treatment inhibited renal NF-κB activation and reduced TNF-α levels in rats with ADR-induced nephropathy. Overall, our findings suggest that sEH inhibition ameliorates renal inflammation and podocyte injury, thus reducing proteinuria and exerting renoprotective effects. Targeting sEH might be a potential strategy for the treatment of proteinuria in primary glomerular diseases.

Soluble epoxide hydrolase inhibition impairs triggering receptor expressed on myeloid cells-1 in periodontal tissue.

Periodontitis is a prevalent inflammatory disorder affecting the oral cavity, driven by dysbiotic oral biofilm and host immune response interactions. While the major clinical focus of periodontitis treatment is currently controlling oral biofilm, understanding the immune response is crucial to prevent disease progression. Soluble epoxide hydrolase (sEH) inhibition has shown promise in preventing alveolar bone resorption. Triggering receptors expressed on myeloid cells (TREMs) play pivotal roles in regulating inflammation and bone homeostasis, and dysregulation of TREM signaling is implicated in periodontitis. Here, we investigated the impact of sEH inhibition on TREM 1 and 2 expression, associated with inflammatory cytokines, and histologically assessed the inflammatory infiltrate in periodontal tissue. The experimental periodontitis model was induced by placing a ligature around the upper second molar. For 14 days, animals were treated daily with a sEH inhibitor (TPPU) or vehicle. The alveolar bone loss was examined using a methylene blue stain. Gingival tissues were used to measure the mRNA expression of TREM-1, TREM-2, IKKβ, NF-κB, IL-1β, IL-6, IL-8, and TNF-α by RT-qPCR. Another set of experiments was performed to determine the histological inflammatory scores. In a ligature-induced periodontitis model, sEH inhibition prevented alveolar bone loss and reduced TREM1 expression, albeit with a slight elevation compared to the disease-free group. In contrast, TREM2 expression remained elevated, suggesting sustained immunomodulation favoring resolution. The inhibition of sEH reduced the expression of NF-κB, IL-1β, and TNF-α, while no differences were found in the expression of IL-6, IL-8, and IKKβ. In histological analysis, sEH inhibition reduced the inflammatory leukocyte infiltrate in periodontal tissues close to the ligature. These findings underscore the potential of sEH inhibition to modulate periodontal inflammation by regulating TREM-1 alongside decreased IL-1β and TNF-α expression, highlighting a promising therapeutic approach for periodontitis management.

Identification of Gene Signatures Associated with COVID-19 across Children, Adolescents, and Adults in the Nasopharynx and Peripheral Blood by Using a Machine Learning Approach.

Significant variations in immune profiles across different age groups manifest distinct clinical symptoms and prognoses in Coronavirus Disease 2019 (COVID-19) patients. Predominantly, severe COVID-19 cases that require hospitalization occur in the elderly, with the risk of severe illness escalating with age among young adults, children, and adolescents. This study aimed to delineate the unique immune characteristics of COVID-19 across various age groups and evaluate the feasibility of detecting COVID-19-induced immune alterations through peripheral blood analysis. By employing a machine learning approach, we analyzed gene expression data from nasopharyngeal and peripheral blood samples of COVID-19 patients across different age brackets. Nasopharyngeal data reflected the immune response to COVID-19 in the upper respiratory tract, while peripheral blood samples provided insights into the overall immune system status. Both datasets encompassed COVID-19 patients and healthy controls, with patients divided into children, adolescents, and adult age groups. The analysis included the expression levels of 62,703 genes per patient. Then, 9 feature-sequencing methods (least absolute shrinkage and selection operator, light gradient boosting machine, Monte Carlo feature selection, random forest, ridge regression, adaptive boosting, categorical boosting, extremely randomized trees, and extreme gradient boosting) were employed to evaluate the association of the genes with COVID-19. Key genes were then utilized to develop efficient classification models. The findings identified specific markers: insulin-like growth factor binding protein 3 (downregulated in the peripheral blood of COVID-19 patients), interferon alpha-inducible protein 27 (upregulated), and SERPING1 (upregulated in nasopharyngeal tissues). In addition, fibulin-2 was downregulated in adolescent patients, but upregulated in the other groups, while epoxide hydrolase 3 was upregulated in healthy controls, but downregulated in children and adolescents. This study offers valuable insights into the local and systemic immune responses of COVID-19 patients across age groups, aiding in identifying potential therapeutic targets and formulating personalized treatment strategies.

Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (S)- and (R)-Flutriafol.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase (RpEH), obtaining mutant Escherichia coli/RpehH336W/L360F with a 6.4-fold enhanced enantiomeric ratio (E) from 5.5 to 35.4. This enabled a gram-scale resolution of rac-1a by E. coli/RpehH336W/L360F, producing (S)-1a (98.2% ees) and (R)-1b (75.0% eep) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (S)-flutriafol (98.2% ee) and (R)-flutriafol (75.0% ee) were easily synthesized by a one-step chemocatalytic process from (S)-1a and a two-step chemocatalytic process from (R)-1b, respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of RpEH toward bulky 2,2-bisaryl-substituted oxirane 1a.