Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Enhancing cancer immunotherapy via inhibition of soluble epoxide hydrolase.

Cancer therapy, including immunotherapy, is inherently limited by chronic inflammation-induced tumorigenesis and toxicity within the tumor microenvironment. Thus, stimulating the resolution of inflammation may enhance immunotherapy and improve the toxicity of immune checkpoint inhibition (ICI). As epoxy-fatty acids (EpFAs) are degraded by the enzyme soluble epoxide hydrolase (sEH), the inhibition of sEH increases endogenous EpFA levels to promote the resolution of cancer-associated inflammation. Here, we demonstrate that systemic treatment with ICI induces sEH expression in multiple murine cancer models. Dietary omega-3 polyunsaturated fatty acid supplementation and pharmacologic sEH inhibition, both alone and in combination, significantly enhance anti-tumor activity of ICI in these models. Notably, pharmacological abrogation of the sEH pathway alone or in combination with ICI counter-regulates an ICI-induced pro-inflammatory and pro-tumorigenic cytokine storm. Thus, modulating endogenous EpFA levels through dietary supplementation or sEH inhibition may represent a unique strategy to enhance the anti-tumor activity of paradigm cancer therapies.

Modulating the sEH/EETs Axis Restrains Specialized Proresolving Mediator Impairment and Regulates T Cell Imbalance in Experimental Periodontitis.

Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids are short-acting lipids involved in resolution of inflammation. Their short half-life, due to its metabolism by soluble epoxide hydrolase (sEH), limits their effects. Specialized proresolving mediators (SPMs) are endogenous regulatory lipids insufficiently synthesized in uncontrolled and chronic inflammation. Using an experimental periodontitis model, we pharmacologically inhibited sEH, examining its impact on T cell activation and systemic SPM production. In humans, we analyzed sEH in the gingival tissue of periodontitis patients. Mice were treated with sEH inhibitor (sEHi) and/or EETs before ligature placement and treated for 14 d. Bone parameters were assessed by microcomputed tomography and methylene blue staining. Blood plasma metabololipidomics were carried out to quantify SPM levels. We also determined T cell activation by reverse transcription-quantitative PCR and flow cytometry in cervical lymph nodes. Human gingival samples were collected to analyze sEH using ELISA and electrophoresis. Data reveal that pharmacological sEHi abrogated bone resorption and preserved bone architecture. Metabololipidomics revealed that sEHi enhances lipoxin A4, lipoxin B4, resolvin E2, and resolvin D6. An increased percentage of regulatory T cells over Th17 was noted in sEHi-treated mice. Lastly, inflamed human gingival tissues presented higher levels and expression of sEH than did healthy gingivae, being positively correlated with periodontitis severity. Our findings indicate that sEHi preserves bone architecture and stimulates SPM production, associated with regulatory actions on T cells favoring resolution of inflammation. Because sEH is enhanced in human gingivae from patients with periodontitis and connected with disease severity, inhibition may prove to be an attractive target for managing osteolytic inflammatory diseases.

Enhancement of the liver's neuroprotective role ameliorates traumatic brain injury pathology.

Traumatic brain injury (TBI) is a pervasive problem worldwide for which no effective treatment is currently available. Although most studies have focused on the pathology of the injured brain, we have noted that the liver plays an important role in TBI. Using two mouse models of TBI, we found that the enzymatic activity of hepatic soluble epoxide hydrolase (sEH) was rapidly decreased and then returned to normal levels following TBI, whereas such changes were not observed in the kidney, heart, spleen, or lung. Interestingly, genetic downregulation of hepatic Ephx2 (which encodes sEH) ameliorates TBI-induced neurological deficits and promotes neurological function recovery, whereas overexpression of hepatic sEH exacerbates TBI-associated neurological impairments. Furthermore, hepatic sEH ablation was found to promote the generation of A2 phenotype astrocytes and facilitate the production of various neuroprotective factors associated with astrocytes following TBI. We also observed an inverted V-shaped alteration in the plasma levels of four EET (epoxyeicosatrienoic acid) isoforms (5,6-, 8,9-,11,12-, and 14,15-EET) following TBI which were negatively correlated with hepatic sEH activity. However, hepatic sEH manipulation bidirectionally regulates the plasma levels of 14,15-EET, which rapidly crosses the blood-brain barrier. Additionally, we found that the application of 14,15-EET mimicked the neuroprotective effect of hepatic sEH ablation, while 14,15-epoxyeicosa-5(Z)-enoic acid blocked this effect, indicating that the increased plasma levels of 14,15-EET mediated the neuroprotective effect observed after hepatic sEH ablation. These results highlight the neuroprotective role of the liver in TBI and suggest that targeting hepatic EET signaling could represent a promising therapeutic strategy for treating TBI.

Hepatic Transcriptome and Its Regulation Following Soluble Epoxide Hydrolase Inhibition in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N6-methyladenosine and 2-methylthio-N6-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.

Effects of sEH inhibition on the eicosanoid and cytokine storms in SARS-CoV-2-infected mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves an initial viral infection phase followed by a host-response phase that includes an eicosanoid and cytokine storm, lung inflammation and respiratory failure. While vaccination and early anti-viral therapies are effective in preventing or limiting the pathogenic host response, this latter phase is poorly understood with no highly effective treatment options. Inhibitors of soluble epoxide hydrolase (sEH) increase levels of anti-inflammatory molecules called epoxyeicosatrienoic acids (EETs). This study aimed to investigate the impact of sEH inhibition on the host response to SARS-CoV-2 infection in a mouse model with human angiotensin-converting enzyme 2 (ACE2) expression. Mice were infected with SARS-CoV-2 and treated with either vehicle or the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU). At day 5 post-infection, SARS-CoV-2 induced weight loss, clinical signs, a cytokine storm, an eicosanoid storm, and severe lung inflammation with ~50% mortality on days 6-8 post-infection. SARS-CoV-2 infection induced lung expression of phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX) pathway genes, while suppressing expression of most cytochrome P450 genes. Treatment with the sEH inhibitor TPPU delayed weight loss but did not alter clinical signs, lung cytokine expression or overall survival of infected mice. Interestingly, TPPU treatment significantly reversed the eicosanoid storm and attenuated viral-induced elevation of 39 fatty acids and oxylipins from COX, LOX and P450 pathways, which suggests the effects at the level of PLA2 activation. The suppression of the eicosanoid storm by TPPU without corresponding changes in lung cytokines, lung inflammation or mortality reveals a surprising dissociation between systemic oxylipin and cytokine signaling pathways during SARS-CoV-2 infection and suggests that the cytokine storm is primarily responsible for morbidity and mortality in this animal model.

sEH-derived metabolites of linoleic acid drive pathologic inflammation while impairing key innate immune cell function in burn injury.

Fatty acid composition in the Western diet has shifted from saturated to polyunsaturated fatty acids (PUFAs), and specifically to linoleic acid (LA, 18:2), which has gradually increased in the diet over the past 50 y to become the most abundant dietary fatty acid in human adipose tissue. PUFA-derived oxylipins regulate a variety of biological functions. The cytochrome P450 (CYP450)­formed epoxy fatty acid metabolites of LA (EpOMEs) are hydrolyzed by the soluble epoxide hydrolase enzyme (sEH) to dihydroxyoctadecenoic acids (DiHOMEs). DiHOMEs are considered cardioprotective at low concentrations but at higher levels have been implicated as vascular permeability and cytotoxic agents and are associated with acute respiratory distress syndrome in severe COVID-19 patients. High EpOME levels have also correlated with sepsis-related fatalities; however, those studies failed to monitor DiHOME levels. Considering the overlap of burn pathophysiology with these pathologies, the role of DiHOMEs in the immune response to burn injury was investigated. 12,13-DiHOME was found to facilitate the maturation and activation of stimulated neutrophils, while impeding monocyte and macrophage functionality and cytokine generation. In addition, DiHOME serum concentrations were significantly elevated in burn-injured mice and these increases were ablated by administration of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), a sEH inhibitor. TPPU also reduced necrosis of innate and adaptive immune cells in burned mice, in a dose-dependent manner. The findings suggest DiHOMEs are a key driver of immune cell dysfunction in severe burn injury through hyperinflammatory neutrophilic and impaired monocytic actions, and inhibition of sEH might be a promising therapeutic strategy to mitigate deleterious outcomes in burn patients.

Kurarinone alleviated Parkinson's disease via stabilization of epoxyeicosatrienoic acids in animal model.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by loss of dopaminergic neurons in the substantia nigra (SN), causing bradykinesia and rest tremors. Although the molecular mechanism of PD is still not fully understood, neuroinflammation has a key role in the damage of dopaminergic neurons. Herein, we found that kurarinone, a unique natural product from Sophora flavescens, alleviated the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral deficits and dopaminergic neurotoxicity, including the losses of neurotransmitters and tyrosine hydroxylase (TH)-positive cells (SN and striatum [STR]). Furthermore, kurarinone attenuated the MPTP-mediated neuroinflammation via suppressing the activation of microglia involved in the nuclear factor kappa B signaling pathway. The proteomics result of the solvent-induced protein precipitation and thermal proteome profiling suggest that the soluble epoxide hydrolase (sEH) enzyme, which is associated with the neuroinflammation of PD, is a promising target of kurarinone. This is supported by the increase of plasma epoxyeicosatrienoic acids (sEH substrates) and the decrease of dihydroxyeicosatrienoic acids (sEH products), and the results of in vitro inhibition kinetics, surface plasmon resonance, and cocrystallization of kurarinone with sEH revealed that this natural compound is an uncompetitive inhibitor. In addition, sEH knockout (KO) attenuated the progression of PD, and sEH KO plus kurarinone did not further reduce the protection of PD in MPTP-induced PD mice. These findings suggest that kurarinone could be a potential natural candidate for the treatment of PD, possibly through sEH inhibition.

COX-2/sEH-Mediated Macrophage Activation Is a Target for Pulmonary Protection in Mouse Models of Chronic Obstructive Pulmonary Disease.

Effective inhibition of macrophage activation is critical for resolving inflammation and restoring pulmonary function in patients with chronic obstructive pulmonary disease (COPD). In this study, we identified the dual-enhanced cyclooxygenase-2 (COX-2)/soluble epoxide hydrolase (sEH) as a novel regulator of macrophage activation in COPD. Both COX-2 and sEH were found to be increased in patients and mice with COPD and in macrophages exposed to cigarette smoke extract. Pharmacological reduction of the COX-2 and sEH by 4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl)-benzenesulfonamide (PTUPB) effectively prevented macrophage activation, downregulated inflammation-related genes, and reduced lung injury, thereby improving respiratory function in a mouse model of COPD induced by cigarette smoke and lipopolysaccharide. Mechanistically, enhanced COX-2/sEH triggered the activation of the NACHT, LRR, and PYD domains-containing protein 3 inflammasome, leading to the cleavage of pro-IL-1ß into its active form in macrophages and amplifying inflammatory responses. These findings demonstrate that targeting COX-2/sEH-mediated macrophage activation may be a promising therapeutic strategy for COPD. Importantly, our data support the potential use of the dual COX-2 and sEH inhibitor PTUPB as a therapeutic drug for the treatment of COPD.

Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Smartphone-integrated visual inspection for enhancing agricultural product quality and safety: a review.

The increasing emphasis on the quality and safety of agricultural products, which are vital to global trade and consumer health, has driven the innovation of cost-effective, convenient, and rapid smart detection technologies. Smartphones, with their interdisciplinary functionalities, have become valuable tools in quantification and analysis research. Acting as portable, affordable, and user-friendly analytical devices, smartphones are equipped with high-resolution cameras, displays, memory, communication modules, sensors, and operating systems (Android or IOS), making them powerful, palm-sized remote computers. This review delves into how visual inspection technology and smartphones have enhanced the quality and safety of agricultural products over the past decade. It also evaluates the key features and limitations of existing smart rapid inspection methods for agricultural products and anticipates future advancements, offering insights into the application of smart rapid inspection technology in agriculture.

Biosynthesis of magnetosome-nanobody complex in Magnetospirillum gryphiswaldense MSR-1 and a magnetosome-nanobody-based enzyme-linked immunosorbent assay for the detection of tetrabromobisphenol A in water.

In this study, two mutant strains, TBC and TBC+, able to biosynthesize a novel functional magnetosome-nanobody (Nb), were derived from the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1. The magnetosome-Nbs biosynthesized by TBC+ containing multi-copies of the Nb gene had a higher binding ability to an environmental pollutant, tetrabromobisphenol A (TBBPA), than those biosynthesized by TBC containing only one copy of the Nb gene. The magnetosome-Nbs from TBC+ can effectively bind to TBBPA in solutions with high capacity without being affected by a broad range of NaCl and methanol concentrations as well as pH. Therefore, a magnetosome-Nb-based enzyme-linked immunosorbent assay (ELISA) was developed and optimized for the detection of TBBPA, yielding a half-maximum signal inhibition concentration of 0.23 ng/mL and a limit of detection of 0.025 ng/mL. The assay was used to detect TBBPA in spiked river water samples, giving average recoveries between 90 and 120% and coefficients of variation of 2.5-6.3%. The magnetosome-Nb complex could be reused 4 times in ELISA without affecting the performance of the assay. Our results demonstrate the potential of magnetosome-Nbs produced by TBC+ as cost-effective and environment-friendly reagents for immunoassays to detect small molecules in environmental waters.

Eicosanoid regulation of debris-stimulated metastasis.

Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

A novel sustainable immunoassay for sensitive detection of atrazine based on the anti-idiotypic nanobody and recombinant full-length antibody.

Immunoassays have been widely used to determine small-molecule compounds in food and the environment, meeting the challenge of obtaining false positive or negative results because of the variance in the batches of antibodies and antigens. To resolve this problem, atrazine (ATR) was used as a target, and anti-idiotypic nanobodies for ATR (AI-Nbs) and a recombinant full-length antibody against ATR (ATR-rAb) were prepared for the development of a sustainable enzyme-linked immunosorbent assay (ELISA). AI-Nb-7, AI-Nb-58, and AI-Nb-66 were selected from an immune phage display library. ATR-rAb was produced in mammalian HEK293 (F) cells. Among the four detection methods explored, the assay using AI-Nb-66 as a coating antigen and ATR-rAb as a detection reagent yielded a half maximal inhibitory concentration (IC50) of 1.66 ng mL-1 for ATR and a linear range of 0.35-8.73 ng mL-1. The cross-reactivity of the assay to ametryn was 64.24%, whereas that to terbutylazine was 38.20%. Surface plasmon resonance (SPR) analysis illustrated that these cross-reactive triazine compounds can bind to ATR-rAb to varying degrees at high concentrations; however, the binding/dissociation kinetic curves and the response values at the same concentration are different, which results in differences in cross-reactivity. Homology modeling and molecular docking revealed that the triazine ring is vital in recognizing triazine compounds. The proposed immunoassay exhibited acceptable recoveries of 84.40-105.36% for detecting fruit, vegetables, and black tea. In conclusion, this study highlights a new strategy for developing sustainable immunoassays for detecting trace pesticide contaminants.

Development of a sensitive direct competitive chemiluminescent enzyme immunoassay for gentamicin based on the construction of a specific single-chain variable fragment-alkaline phosphatase fusion protein.

A sensitive chemiluminescent enzyme immunoassay (CLEIA) was established for the determination of gentamicin (GEN) residue levels in animal tissue. This assay is based on a fusion protein of single-chain variable fragment (scFv) and alkaline phosphatase (AP). Initially, VL and VH derived from anti-gentamicin monoclonal antibody were linked by a short peptide to construct a scFv. Subsequently, the constructed scFv sequence was accessed into the pLIP6/GN vector, and a soluble scFv-AP fusion protein was generated. The scFv-AP fusion protein was used to develop a direct competitive CLEIA (dcCLEIA) for the determination of gentamicin. In the dcCLEIA, the half inhibitory concentration (IC50) and limit of detection (LOD) were 1.073 ng/mL and 0.380 ng/mL, respectively. The average recoveries of gentamicin spiked in animal tissue samples ranged from 78% to 96%. These results showed a strong correlation with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The above results suggest that the anti-GEN scFv-AP fusion protein is suitable for detecting gentamicin residues in edible animal tissues.

Preclinical Evaluation of Soluble Epoxide Hydrolase Inhibitor AMHDU against Neuropathic Pain.

Inhibition of soluble epoxide hydrolase (sEH) is a promising therapeutic strategy for treating neuropathic pain. These inhibitors effectively reduce diabetic neuropathic pain and inflammation induced by Freund's adjuvant which makes them a suitable alternative to traditional opioids. This study showcased the notable analgesic effects of compound AMHDU (1,1'-(hexane-1,6-diyl)bis(3-((adamantan-1-yl)methyl)urea)) in both inflammatory and diabetic neuropathy models. While lacking anti-inflammatory properties in a paw edema model, AMHDU is comparable to celecoxib as an analgesic in 30 mg/kg dose administrated by intraperitoneal injection. In a diabetic tactile allodynia model, AMHDU showed a prominent analgesic activity in 10 mg/kg intraperitoneal dose (p < 0.05). The effect is comparable to that of gabapentin, but without the risk of dependence due to a different mechanism of action. Low acute oral toxicity (>2000 mg/kg) and a high therapeutic index makes AMHDU a promising candidate for further structure optimization and preclinical evaluation.

Role of soluble epoxide hydrolase in the abnormal activation of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by enigmatic pathogenesis. Polyunsaturated fatty acids (PUFAs) are implicated in RA's development and progression, yet their exact mechanisms of influence are not fully understood. Soluble epoxide hydrolase (sEH) is an enzyme that metabolizes anti-inflammatory epoxy fatty acids (EpFAs), derivatives of PUFAs. In this study, we report elevated sEH expression in the joints of CIA (collagen-induced arthritis) rats, concomitant with diminished levels of two significant EpFAs. Additionally, increased sEH expression was detected in both the synovium of CIA rats and in the synovium and fibroblast-like synoviocytes (FLS) of RA patients. The sEH inhibitor TPPU attenuated the migration and invasion capabilities of FLS derived from RA patients and to reduce the secretion of inflammatory factors by these cells. Our findings indicate a pivotal role for sEH in RA pathogenesis and suggest that sEH inhibitors offer a promising new therapeutic strategy for managing RA.

Mix-and-Read Nanobody-Based Sandwich Homogeneous Split-Luciferase Assay for the Rapid Detection of Human Soluble Epoxide Hydrolase.

The soluble epoxide hydrolase (sEH) is possibly both a marker for and target of numerous diseases. Herein, we describe a homogeneous mix-and-read assay for the detection of human sEH based on using split-luciferase detection coupled with anti-sEH nanobodies. Selective anti-sEH nanobodies were individually fused with NanoLuc Binary Technology (NanoBiT), which consists of a large and small portion of NanoLuc (LgBiT and SmBiT, respectively). Different orientations of the LgBiT and SmBiT-nanobody fusions were expressed and investigated for their ability to reform the active NanoLuc in the presence of the sEH. After optimization, the linear range of the assay could reach 3 orders of magnitude with a limit of detection (LOD) of 1.4 ng/mL. The assay has a high sensitivity to human sEH and reached a similar detection limit to our previously reported conventional nanobody-based ELISA. The procedure of the assay was faster (30 min total) and easy to operate, providing a more flexible and simple way to monitor human sEH levels in biological samples. In general, the immunoassay proposed here offers a more efficient detection and quantification approach that can be easily adapted to numerous macromolecules.

Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

Neuroprotective effect of herbal extracts inhibiting soluble epoxide hydrolase (sEH) and cyclooxygenase (COX) against chemotherapy-induced cognitive impairment in mice.

Chemotherapy-induced cognitive impairment (CICI) is a novel clinical condition characterized by memory, learning, and motor function deficits. Oxidative stress and inflammation are potential factors contributing to chemotherapy's adverse effects on the brain. Inhibition of soluble epoxide hydrolase (sEH) has been proven effective in neuroinflammation and reversal of memory impairment. The research aims to evaluate the memory protective effect of sEH inhibitor and dual inhibitor of sEH and COX and compare its impact with herbal extracts with known nootropic activity in an animal model of CICI. In vitro sEH, the inhibitory activity of hydroalcoholic extracts of Sizygium aromaticum, Nigella sativa, and Mesua ferrea was tested on murine and human sEH enzyme as per the protocol, and IC50 was determined. Cyclophosphamide (50 mg/kg), methotrexate (5 mg/kg), and fluorouracil (5 mg/kg) combination (CMF) were administered intraperitoneally to induce CICI. The known herbal sEH inhibitor, Lepidium meyenii and the dual inhibitor of COX and sEH (PTUPB) were tested for their protective effect in the CICI model. The herbal formulation with known nootropic activity viz Bacopa monnieri and commercial formulation (Mentat) were also used to compare the efficacy in the CICI model. Behavioral parameter such as cognitive function was assessed by Morris Water Maze besides investigating oxidative stress (GSH and LPO) and inflammatory (TNFα, IL-6, BDNF and COX-2) markers in the brain. CMF-induced CICI, which was associated with increased oxidative stress and inflammation in the brain. However, treatment with PTUPB or herbal extracts inhibiting sEH preserved spatial memory via ameliorating oxidative stress and inflammation. S. aromaticum and N. sativa inhibited COX2, but M. Ferrea did not affect COX2 activity. Lepidium meyenii was the least effective, and mentat showed superior activity over Bacopa monnieri in preserving memory. Compared to untreated animals, the mice treated with PTUPB or hydroalcoholic extracts showed a discernible improvement in cognitive function in CICI.