RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics.

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.

A molecular beacon design for a colorimetric loop-mediated isothermal amplification assay.

In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.

You are what you eat-ecological niche and microhabitat influence venom activity and composition in aquatic bugs.

True water bugs (Nepomorpha) are mostly predacious insects that live in aquatic habitats. They use their piercing-sucking mouthparts to inject venomous saliva that facilitates the capture and extra-oral digestion of prey animals, but their venom can also be deployed for defence. In Central Europe, nepomorph species representing different families coexist in the same habitat. However, their feeding ecology, including venom composition and deployment, has not been investigated in detail. We used an integrated proteotranscriptomic and bioactivity-based approach to test whether venom composition and activity differ between four water bug species sharing the same habitat but occupying different ecological niches. We found considerable species-dependent differences in the composition of digestive enzymes and venom components that probably evolved as adaptations to particular food sources, foraging strategies and/or microhabitats. The venom of Corixa punctata differed substantially from that of the three strictly predatory species (Ilyocoris cimicoides, Notonecta glauca and Nepa cinerea), and the abundance of herbivory-associated proteins confirms a mostly plant-based diet. Our findings reveal independent adaptations of the digestive and defensive enzyme repertoires accompanied by the evolution of distinct feeding strategies in aquatic bugs.

Rapid detection of Shiga-toxin-producing Escherichia coli O157:H7 based on a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon paired with HRPzyme.

Contamination by Escherichia coli O157:H7 is considered a threat in the livestock and food industries. Therefore, it is necessary to develop methods for the convenient and rapid detection of Shiga-toxin-producing E. coli O157:H7. This study aimed to develop a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon to rapidly detect E. coli O157:H7. Primers and a molecular beacon were designed for targeting the Shiga-toxin-producing virulence genes (stx1 and stx2) as molecular markers. Additionally, Bst polymerase concentration and amplification conditions for bacterial detection were optimized. The sensitivity and specificity of the assay were also investigated and validated on artificially tainted (100-104 CFU/g) Korean beef samples. The cLAMP assay could detect 1 × 101 CFU/g at 65 °C for both genes, and the assay was confirmed to be specific for E. coli O157:H7. The cLAMP takes about an hour and does not require expensive devices (e.g., thermal cycler and detector). Hence, the cLAMP assay proposed herein can be used in the meat industry as a fast and simple way to detect E. coli O157:H7.

Investigating Activity Recognition for Hemiparetic Stroke Patients Using Wearable Sensors: A Deep Learning Approach with Data Augmentation.

Measuring the daily use of an affected limb after hospital discharge is crucial for hemiparetic stroke rehabilitation. Classifying movements using non-intrusive wearable sensors provides context for arm use and is essential for the development of a home rehabilitation system. However, the movement classification of stroke patients poses unique challenges, including variability and sparsity. To address these challenges, we collected movement data from 15 hemiparetic stroke patients (Stroke group) and 29 non-disabled individuals (ND group). The participants performed two different tasks, the range of motion (14 movements) task and the activities of daily living (56 movements) task, wearing five inertial measurement units in a home setting. We trained a 1D convolutional neural network and evaluated its performance for different training groups: ND-only, Stroke-only, and ND and Stroke jointly. We further compared the model performance with data augmentation from axis rotation and investigated how the performance varied based on the asymmetry of movements. The joint training of ND + Stroke yielded an increased F1-score by a margin of 31.6% and 10.6% compared to ND-only training and Stroke-only training, respectively. Data augmentation further enhanced F1-scores across all conditions by an average of 11.3%. Finally, asymmetric movements decreased the F1-score by 25.9% compared to symmetric movements in the Stroke group, indicating the importance of asymmetry in movement classification.

Surface-Tailored Medium Entropy Alloys as Radically Low Overpotential Oxygen Evolution Electrocatalysts.

Numerous studies have explored new materials for electrocatalysts, but it is difficult to discover materials that surpass the catalytic activity of current commercially available noble metal electrocatalysts. In contrast to conventional transition metal alloys, high-entropy alloys (HEAs) have immense potential to maximize their catalytic properties because of their high stability and compositional diversity as oxygen evolution reactions (OERs). This work presents medium-entropy alloys (MEAs) as OER electrocatalysts to simultaneously satisfy the requirement of high catalytic activity and long-term stability. The surface of MEA electrocatalyst is tailored to suit the OER via anodizing and cyclic voltammetry activation methods. Optimized electrical properties and hydrophilicity of the surface enable an extremely low overpotential of 187 mV for achieving the current density of 10 mA cm-2 alkaline media. Furthermore, a combined photovoltaic-electrochemical system with MEA electrocatalyst and a perovskite/Si tandem solar cell exhibits a solar-to-hydrogen conversion efficiency of 20.6% for an unassisted hydrogen generation system. These results present a new pathway for designing sustainable high efficiency water splitting cells.

Emerging Two-Dimensional-Based Nanostructured Catalysts: Applications in Sustainable Organic Transformations.

The extension of green and sustainable materials in the preparation of heterogeneous catalysts for organic transformations has increased over the past few decades. Because of their unique and intriguing physical and chemical properties, two-dimensional (2D) nanostructured materials have attracted widespread attention and have been used in a variety of applications, such as catalysis, electronics, and energy storage. A promising pathway to enhance the performance of 2D nanomaterials is their coupling with other functional materials to form heterogeneous or hybrid structures. Herein, we discuss the use of 2D-based nanostructured catalysts for enhancing organic transformations and highlight selected examples to demonstrate the synthesis, advantages, challenges, efficiency, and reusability of the introduced heterogeneous catalysts for cross-coupling and reduction reactions.

Colorimetric detection of norovirus by helicase-dependent amplification method based on specific primers integrated with HRPzyme.

Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2 h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.

Assessment of Factors Influencing Personal Exposure to Air Pollution on Main Roads in Bogota: A Mixed-Method Study.

Background and Objectives: Particulate Matter (PM), particles of variable but small diameter can penetrate the respiratory system via inhalation, causing respiratory and/or cardiovascular diseases. This study aims to evaluate the association of environmental particulate matter (PM2.5) and black carbon (BC) with respiratory health in users of different transport modes in four roads in Bogotá. Materials and Methods: this was a mixed-method study (including a cross sectional study and a qualitative description of the air quality perception), in 300 healthy participants, based on an exploratory sequential design. The respiratory effect was measured comparing the changes between pre- and post-spirometry. The PM2.5 and black carbon (BC) concentrations were measured using portable devices. Inhaled doses were also calculated for each participant according to the mode and route. Perception was approached through semi-structured interviews. The analysis included multivariate models and concurrent triangulation. Results: The concentration of matter and black carbon were greater in bus users (median 50.67 µg m-3; interquartile range (-IR): 306.7). We found greater inhaled dosages of air pollutants among bike users (16.41 µg m-3). We did not find changes in the spirometry parameter associated with air pollutants or transport modes. The participants reported a major sensory influence at the visual and olfactory level as perception of bad air quality. Conclusions: We observed greater inhaled doses among active transport users. Nevertheless, no pathological changes were identified in the spirometry parameters. People's perceptions are a preponderant element in the assessment of air quality.

The relationship between person-centred care and the intensive care unit experience of critically ill patients: A multicentre cross-sectional survey.

BACKGROUND: Person-centred care has the potential to improve the patient experience in the intensive care unit (ICU). However, the relationship between person-centred care perceived by critically ill patients and their ICU experience has yet to be determined. OBJECTIVES: The aim of this study was to investigate the relationship between person-centred care and the ICU experience of critically ill patients. METHODS: This study was a multicentre, cross-sectional survey involving 19 ICUs of four university hospitals in Busan, Korea. The survey was conducted from June 2019 to July 2020, and 787 patients who had been admitted to the ICU for more than 24 hours participated. We measured person-centred care using the Person-Centered Critical Care Nursing perceived by Patient Questionnaire. Participants' ICU experience was measured by the Korean version of the Intensive Care Experience Questionnaire that consists of four subscales. We analysed the relationship between person-centred care and each area of the ICU experience using multivariate linear regression. RESULTS: Person-centred care was associated with 'awareness of surroundings' (ß = 0.29, p < .001), 'frightening experiences' (ß = -0.31, p < .001), and 'satisfaction with care' (ß = 0.54, p < .001). However, there was no significant association between person-centred care and 'recall of experience'. CONCLUSIONS: We observed that person-centred care was positively related to most of the ICU experiences of critically ill patients except for recall of experience. Further studies on developing person-centred nursing interventions are needed.

Boosting Unassisted Alkaline Solar Water Splitting Using Silicon Photocathode with TiO2 Nanorods Decorated by Edge-Rich MoS2 Nanoplates.

To construct a highly efficient photoelectrochemical tandem device with silicon photocathode operating in alkaline conditions, it is desirable to develop stable and active catalysts which enable the photocathode to reliably perform under an alkaline environment. With nanostructured passivation layer and edge-exposed transition metal disulfides, silicon photocathode provides new opportunities for achieving unbiased alkaline solar water splitting. Here, the TiO2 nanorod arrays decorated by edge-rich MoS2 nanoplates are elaborately synthesized and deposited on p-Si. The vertically aligned TiO2 nanorods fully stabilize the Si surface and improve anti-reflectance. Moreover, MoS2 nanoplates with exposed edge sites provide catalytically active regions resulting in the kinetically favored hydrogen evolution under an alkaline environment. Interfacial energy band bending between p-Si and catalyst layers facilitates the transport of photogenerated electrons under steady-state illumination. Consequently, the MoS2 nanoplates/TiO2 nanorods/p-Si photocathode exhibits significantly improved photoelectrochemical-hydrogen evolution reaction (PEC-HER) performance in alkaline media with a high photocurrent density of 10 mA cm-2 at 0 V versus RHE and high stability. By integrating rationally designed photocathode with earth-abundant Fe60 (NiCo)30 Cr10 anode and perovskite/Si tandem photovoltaic cell, an unassisted alkaline solar water splitting is accomplished with a current density of 5.4 mA cm-2 corresponding to 6.6% solar-to-hydrogen efficiency, which is the highest among p-Si photocathodes.

Enhanced Oxygen Evolution Electrocatalysis in Strained A-Site Cation Deficient LaNiO3 Perovskite Thin Films.

As the BO6 octahedral structure in perovskite oxide is strongly linked with electronic behavior, it is actively studied for various fields such as metal-insulator transition, superconductivity, and so on. However, the research about the relationship between water-splitting activity and BO6 structure is largely lacking. Here, we report the oxygen evolution reaction (OER) of LaNiO3 (LNO) by changing the NiO6 structure using compositional change and strain. The 5 atom % La deficiency in LNO resulted in an increase of the Ni-O-Ni bond angle and an expansion of bandwidth, enhancing the charge transfer ability. In-plane compressive strain derives the higher dz2 orbital occupancy, leading to suitable metal-oxygen bond strength for OER. Because of the synergistic effect of A-site deficiency and compressive strain, the overpotential (η) of compressively strained L0.95NO film is reduced to 130 mV at j = 30 µA/cm2 compared with nonstrained LNO (η = 280 mV), indicating a significant enhancement in OER.

Lead-Free Dual-Phase Halide Perovskites for Preconditioned Conducting-Bridge Memory.

Organometallic and all-inorganic halide perovskites (HPs) have recently emerged as promising candidate materials for resistive switching (RS) nonvolatile memory due to their current-voltage hysteresis caused by fast ion migration. Lead-free and all-inorganic HPs have been researched for non-toxic and environmentally friendly RS memory devices. However, only HP-based devices with electrochemically active top electrode (TE) exhibit ultra-low operating voltages and high on/off ratio RS properties. The active TE easily reacts to halide ions in HP films, and the devices have a low device durability. Herein, RS memory devices based on an air-stable lead-free all-inorganic dual-phase HP (AgBi2 I7 -Cs3 Bi2 I9 ) are successfully fabricated with inert metal electrodes. The devices with Au TE show filamentary RS behavior by conducting-bridge involving Ag cations in HPs with ultra-low operating voltages (<0.15 V), high on/off ratio (>107 ), multilevel data storage, and long retention times (>5 × 104 s). The use of a closed-loop pulse switching method improves reversible RS properties up to 103 cycles with high on/off ratio above 106 . With an extremely small bending radius of 1 mm, the devices are operable with reasonable RS characteristics. This work provides a promising material strategy for lead-free all-inorganic HP-based nonvolatile memory devices for practical applications.

Thalamic nuclei volumes and network in juvenile myoclonic epilepsy.

OBJECTIVE: The aim of this study was to investigate the alterations of thalamic nuclei volumes and intrinsic thalamic networks in patients with juvenile myoclonic epilepsy (JME) compared to healthy controls. METHODS: We enrolled 50 patients with JME and 42 healthy controls. We obtained structural volumes of the individual thalamic nuclei based on T1-weighted imaging and performed intrinsic thalamic network analysis using graph theoretical analysis. We analyzed the differences of thalamic nuclei volumes and intrinsic thalamic networks between the patients with JME and healthy controls. RESULTS: In the patients with JME, there were significant alterations of thalamic nuclei volumes compared to healthy controls. Right laterodorsal and left suprageniculate nuclei volumes were significantly increased (0.0019% vs 0.0014%, P < .0001; 0.0011% vs 0.0008%, P = .0006, respectively), whereas left ventral posterolateral, left ventromedial, and left pulvinar inferior nuclei volumes (0.0572% vs 0.0664%, P = .0001; 0.0013% vs 0.0015%, P = .0002; 0.0120% vs 0.0140%, P < .0001, respectively) were decreased in the patients with JME. Furthermore, the intrinsic thalamic network of the patients with JME was significantly different from that of the healthy controls. The modularity in the patients with JME was significantly increased over that in healthy controls (0.0785 vs 0.0212, P = .039). CONCLUSION: We found that there were significant alterations of thalamic nuclei volumes and intrinsic thalamic networks in patients with JME compared to healthy controls. These findings might contribute to the underlying pathogenesis of in JME.

Quantification of thalamic nuclei in patients diagnosed with temporal lobe epilepsy and hippocampal sclerosis.

PURPOSE: The aim of this study is to investigate the changes in the volume of individual thalamic nuclei in patients with temporal lobe epilepsy (TLE) and hippocampal sclerosis (HS). METHODS: We enrolled 80 TLE patients with HS and 40 healthy controls. All of the subjects underwent 3D T1-weighted imaging. The hippocampus subfields and thalamic nuclei were segmented using the FreeSurfer program. We investigated volume changes in thalamic nuclei according to the HS side involved, and types or antiepileptic drug (AED) response compared with healthy controls. RESULTS: Compared with healthy controls, patients with HS showed atrophy of thalamic nuclei involving right and left parafascicular nuclei. In the right HS, the atrophy of the right thalamic nucleus was more prominent than that of the left thalamic nucleus, whereas the reduction in the volume of left thalamic nuclei was more prominent in patients with left HS. The reduction in thalamic nuclear volumes was more prominent in HS type 1 (atrophy of both CA1 and CA4 regions) than in other HS types. The suprageniculate nuclear volumes were significantly increased in patients with drug-controlled epilepsy. CONCLUSIONS: Our study demonstrates thalamic nuclear atrophy in TLE patients with HS. In addition, the atrophy of individual thalamic nuclei varied according to the HS side, the HS types, and the AED response. These findings suggest that the role of thalamic nuclei varied in TLE with HS subtypes and displayed varying degrees of vulnerability in the pathology networks associated with the hippocampus.

Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

Fap2 of Fusobacterium nucleatum is a galactose-inhibitable adhesin involved in coaggregation, cell adhesion, and preterm birth.

Fusobacterium nucleatum is a common oral anaerobe involved in periodontitis that is known to translocate and cause intrauterine infections. In the oral environment, F. nucleatum adheres to a large diversity of species, facilitating their colonization and creating biological bridges that stabilize the multispecies dental biofilm. Many of these interactions (called coadherences or coaggregations) are galactose sensitive. Galactose-sensitive interactions are also involved in the binding of F. nucleatum to host cells. Hemagglutination of some F. nucleatum strains is also galactose sensitive, suggesting that a single galactose-sensitive adhesin might mediate the interaction of fusobacteria with many partners and targets. In order to identify the fusobacterial galactose-sensitive adhesin, a system for transposon mutagenesis in fusobacteria was created. The mutant library was screened for hemagglutination deficiency, and three clones were isolated. All three clones were found to harbor the transposon in the gene coding for the Fap2 outer membrane autotransporter. The three fap2 mutants failed to show galactose-inhibitable coaggregation with Porphyromonas gingivalis and were defective in cell binding. A fap2 mutant also showed a 2-log reduction in murine placental colonization compared to that of the wild type. Our results suggest that Fap2 is a galactose-sensitive hemagglutinin and adhesin that is likely to play a role in the virulence of fusobacteria.

B-cell deficiency and severe autoimmunity caused by deficiency of protein kinase C δ.

Primary B-cell disorders comprise a heterogeneous group of inherited immunodeficiencies, often associated with autoimmunity causing significant morbidity. The underlying genetic etiology remains elusive in the majority of patients. In this study, we investigated a patient from a consanguineous family suffering from recurrent infections and severe lupuslike autoimmunity. Immunophenotyping revealed progressive decrease of CD19(+) B cells, a defective class switch indicated by low numbers of IgM- and IgG-memory B cells, as well as increased numbers of CD21(low) B cells. Combined homozygosity mapping and exome sequencing identified a biallelic splice-site mutation in protein C kinase δ (PRKCD), causing the absence of the corresponding protein product. Consequently, phosphorylation of myristoylated alanine-rich C kinase substrate was decreased, and mRNA levels of nuclear factor interleukin (IL)-6 and IL-6 were increased. Our study uncovers human PRKCD deficiency as a novel cause of common variable immunodeficiency-like B-cell deficiency with severe autoimmunity.

Early-onset inflammatory bowel disease and common variable immunodeficiency-like disease caused by IL-21 deficiency.

BACKGROUND: Alterations of immune homeostasis in the gut can result in development of inflammatory bowel disease (IBD). Recently, Mendelian forms of IBD have been discovered, as exemplified by deficiency of IL-10 or its receptor subunits. In addition, other types of primary immunodeficiency disorders might be associated with intestinal inflammation as one of their leading clinical presentations. OBJECTIVE: We investigated a large consanguineous family with 3 children who presented with early-onset IBD within the first year of life, leading to death in infancy in 2 of them. METHODS: Homozygosity mapping combined with exome sequencing was performed to identify the molecular cause of the disorder. Functional experiments were performed to assess the effect of IL-21 on the immune system. RESULTS: A homozygous mutation in IL21 was discovered that showed perfect segregation with the disease. Deficiency of IL-21 resulted in reduced numbers of circulating CD19(+) B cells, including IgM(+) naive and class-switched IgG memory B cells, with a concomitant increase in transitional B-cell numbers. In vitro assays demonstrated that mutant IL-21(Leu49Pro) did not induce signal transducer and activator of transcription 3 phosphorylation and immunoglobulin class-switch recombination. CONCLUSION: Our study uncovers IL-21 deficiency as a novel cause of early-onset IBD in human subjects accompanied by defects in B-cell development similar to those found in patients with common variable immunodeficiency. IBD might mask an underlying primary immunodeficiency, as illustrated here with IL-21 deficiency.

Soluble epoxide hydrolase dimerization is required for hydrolase activity.

Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity.