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Abstract Oxylipins are a group of bioactive fatty acid metabolites generated via enzymatic oxygenation. They are notably involved in inflammation, pain, vascular tone, hemostasis, thrombosis, immunity, and coagulation. Oxylipins have become the focus of therapeutic intervention since they are implicated in many conditions, such as nonalcoholic fatty liver disease, cardiovascular disease, and aging. The liver plays a crucial role in lipid metabolism and distribution throughout the organism. Long-term exposure to pesticides is suspected to contribute to hepatic carcinogenesis via notable disruption of lipid metabolism. Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. The amounts of prometryn documented in the environment, mainly waters, soil and plants used for human and domestic consumption are significantly high. Previous research revealed that prometryn decreased liver development during zebrafish embryogenesis. To understand the mechanisms by which prometryn could induce hepatotoxicity, the effect of prometryn (185 mg/kg every 48 h for seven days) was investigated on hepatic and plasma oxylipin levels in mice. Using an unbiased LC–MS/MS-based lipidomics approach, prometryn was found to alter oxylipins metabolites that are mainly derived from cytochrome P450 (CYP) and lipoxygenase (LOX) in both mice liver and plasma. Lipidomic analysis revealed that the hepatotoxic effects of prometryn are associated with increased epoxide hydrolase (EH) products, increased sEH and mEH enzymatic activities, and induction of oxidative stress. Furthermore, 9-HODE and 13-HODE levels were significantly increased in prometryn treated mice liver, suggesting increased levels of oxidation products. Together, these results support that sEH may be an important component of pesticide-induced liver toxicity.

Abstract Chronic pain represents a significant unmet medical need, affecting one‐fifth of the U.S. population. EC5026 is a small molecule inhibitor of the enzyme soluble epoxide hydrolase (sEH) which is being developed as a novel non‐opioid, non‐NSAID analgesic. EC5026 prolongs the action of epoxy fatty acids, endogenous analgesic lipid mediators that are rapidly metabolized by sEH. We evaluated the safety and pharmacokinetic profile of EC5026 in two phase I trials, a single‐ascending dose (SAD) study and a fed‐fasted study. The SAD study evaluated EC5026 doses ranging from 0.5 to 24 mg in healthy volunteers. EC5026 was well tolerated. No treatment‐emergent adverse events were considered related to EC5026. No apparent treatment‐ or dose‐related trends in laboratory results, vital signs, physical examinations, or electrocardiograms were observed. A linear, near‐dose‐proportional increase in exposure was observed with progressive doses in the SAD study; plasma exposure was below or near the lower limit of quantification after 0.5–2 mg doses. Mean half‐lives ranged from 41.8 to 59.1 h. for doses of 8–24 mg, supporting a once‐daily dosing regimen. In the fed‐fasted study using 8 mg EC5026 tablets, higher peak concentrations (66%) and total exposures (53%) were observed under the fed condition. Plasma concentrations declined in a monoexponential manner with mean half‐lives of 59.5 h. in the fed state and 66.9 h. in the fasted state. Future clinical trials using EC5026 for the treatment of pain are justified based on the favorable outcomes from both clinical trials along with preclinical evidence of analgesic activity.

IntroductionWe reported that Ca2+-independent phospholipase A2β (iPLA2β)–derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting β-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development. MethodsCD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2β+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid. ResultsIn mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%–50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive β-cells. Reduced iPLA2β led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B. DiscussionThese findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.

Simultaneous inhibition of soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) with a single small molecule represents a novel therapeutic approach in treating inflammatory pain, since both targets are involved in pain and inflammation processes. In this study using multi-target directed ligands methodology we designed and synthesized 7 quinolinyl-based dual sEH/FAAH inhibitors, using an optimized microwave-assisted Suzuki-Miyaura coupling reaction and tested their potency in human FAAH and human, rat, and mouse sEH inhibition assays. The structure-activity relationship study showed that quinolinyl moiety is well tolerated in the active sites of both enzymes, yielding several very potent dual sEH/FAAH inhibitors with the IC50 values in the low nanomolar range. The most potent dual inhibitor 4d was further evaluated in stability assay in human and rat plasma where it performed better than the standard Warfarin while in vivo study revealed that 1 mg/kg 4d can inhibit acute inflammatory pain in male rats to a similar degree as the traditional nonsteroidal anti-inflammatory drug ketoprofen (30 mg/kg) after intraperitoneal injection. ADMET prediction studies for this dual inhibitor show favorable pharmacokinetic properties which will guide the future in vivo evaluations.

Pain arising from trigeminal systems such as headache is common, debilitating, and current treatments (e.g., sumatriptan) are limited. New treatments that target novel mechanisms of action may be required to innovate both short- and long-term pain therapy. Fatty acid amide hydrolase and soluble epoxide hydrolase are two pain-related enzymes that regulate pain and inflammation via independent pathways. We have previously demonstrated that simultaneous inhibition of these enzymes using a novel dual inhibitor alleviates acute inflammatory pain in the hindpaw and does not depress wheel running in rats. Here, we expanded on these findings and performed structure-activity relationships of our lead compound, the 4-phenyl-thiazole-based dual inhibitor SW-17, to generate 18 analogs and tested them for their inhibition at both enzymes. Conversion of the sulfonamide group to a tertiary amine led to a general decrease in the potency for the sEH enzyme, while this change was well-tolerated at the FAAH enzyme yielding several strong inhibitors. Six selected inhibitors were evaluated in mouse and rat sEH inhibition assays and results showed a species difference, i.e. 4-phenyl-thiazole-based analogs are significantly less or not active in mouse sEH compared to human and rat enzymes. The most potent inhibitor, SW-17, was evaluated in a plasma stability assay in human and rat plasma and showed moderate stability. However, SW-17 did not alleviate orofacial inflammatory pain in female rats compared to the traditional anti-migraine agent sumatriptan. Although modification of 4-phenyl-thiazole-based dual inhibitor SW-17 changes potencies at both FAAH and sEH, these approaches may not produce antinociception against trigeminal pain. Key Words: polypharmacology, formalin, inflammation, enzyme inhibition, structure-activity relationship studies

Patulin, an emerging mycotoxin with high toxicity, poses great risks to public health. Considering the poor antibody production in patulin immunization, this study focuses on the four-dimensional data-independent acquisition (4D-DIA) quantitative proteomics to reveal the immune response of patulin in rabbits. The rabbit immunization was performed with the complete developed antigens of patulin, followed by the identification of the immune serum. A total of 554 differential proteins, including 292 up-regulated proteins and 262 down-regulated proteins, were screened; the differential proteins were annotated; and functional enrichment analysis was performed. The differential proteins were associated with the pathways of metabolism, gene information processing, environmental information processing, cellular processes, and organismal systems. The functional enrichment analysis indicated that the immunization procedures mostly resulted in the regulation of biochemical metabolic and signal transduction pathways, including the biosynthesis of amino acid (glycine, serine, and threonine), ascorbate, and aldarate metabolism; fatty acid degradation; and antigen processing and presentation. The 14 key proteins with high connectivity included G1U9T1, B6V9S9, G1SCN8, G1TMS5, G1U9U0, A0A0G2JH20, G1SR03, A0A5F9DAT4, G1SSA2, G1SZ14, G1T670, P30947, P29694, and A0A5F9C804, which were obtained by the analysis of protein–protein interaction networks. This study could provide potential directions for protein interaction and antibody production for food hazards in animal immunization.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

Series of 1,3-disubstituted ureas and diadamantyl disubstituted diureas with fluorinated and chlorinated adamantane residues were shown to inhibit human soluble epoxide hydrolase (sEH) with inhibition potency ranging from 40 pM to 9.2 nM. The measured IC50 values for some molecules were below the accuracy limit of the existing in vitro assays. Such an increase in activity was achieved by minimal structural modifications to the molecules of known inhibitors, including 4-[trans-4-(1-adamantylcarbamoylamino)cyclohexyl]oxybenzoic acid. For the chlorinated homologue of the latter the sharp jump in inhibitory activity can be (according to molecular dynamics data) the result of interactions – Cl-π interaction. Considering the extremely high inhibitory activity, acceptable solubility and partial blockage of metabolically sensitive centres in their structures, some compounds are of interest for further in vivo biotesting.

Abstract Background Patients suffering from chronic pain often also exhibit depression symptoms. Soluble epoxide hydrolase (sEH) inhibitors can decrease blood levels of inflammatory cytokines. However, whether inhibiting sEH signaling is beneficial for the comorbidity of pain and depression is unknown. Methods According to a sucrose preference test (SPT), spared nerve injury (SNI) mice were classified into pain with or without an anhedonia phenotype. Then, sEH protein expression and inflammatory cytokines were assessed in selected tissues. Furthermore, we used sEH inhibitor TPPU to determine the role of sEH in chronic pain and depression. Importantly, agonists and antagonists of aryl hydrocarbon receptor (AHR) and translocator protein (TSPO) were used to explore the pathogenesis of sEH signaling. Results In anhedonia-susceptible mice, the tissue levels of sEH were significantly increased in the medial prefrontal cortex (mPFC), hippocampus, spinal cord, liver, kidney, and gut. Importantly, serum CYP1A1 and inflammatory cytokines, such as interleukin 1β (IL-1β) and the tumor necrosis factor α (TNF-α), were increased simultaneously. TPPU improved the scores of mechanical withdrawal threshold (MWT) and SPT, and decreased the levels of serum CYP1A1 and inflammatory cytokines. AHR antagonist relieved the anhedonia behaviors but not the algesia behaviors in anhedonia-susceptible mice, whereas an AHR agonist abolished the antidepressant-like effect of TPPU. In addition, a TSPO agonist exerted a similar therapeutic effect to that of TPPU, whereas pretreatment with a TSPO antagonist abolished the antidepressant-like and analgesic effects of TPPU. Conclusions sEH underlies the mechanisms of the comorbidity of chronic pain and depression and that TPPU exerts a beneficial effect on anhedonia behaviors in a pain model via AHR and TSPO signaling.

Autophagy is a form of programmed cell degradation that enables the maintenance of homeostasis in response to extracellular stress stimuli. Autophagy is primarily activated by starvation and mediates the degradation, removal, or recycling of cell cytoplasm, organelles, and intracellular components in eukaryotic cells. Autophagy is also involved in the pathogenesis of human diseases, including several cancers. Human uveal melanoma (UM) is the primary intraocular malignancy in adults and has an extremely poor prognosis; at present there are no effective therapies. Several studies have suggested that autophagy is important in UM. By understanding the mechanisms of activation of autophagy in UM it may be possible to develop biomarkers to provide more definitive disease prognoses and to identify potential drug targets for the development of new therapeutic strategies. This article reviews the current information regarding autophagy in UM that could facilitate biomarker and drug development.

BackgroundAspergillus fumigatus is a well-known opportunistic pathogen that causes a range of diseases including the often-fatal disease, invasive pulmonary aspergillosis (IPA), in immunocompromised populations. The severity of IPA is dependent on both host- and pathogen-derived signaling molecules that mediate host immunity and fungal growth. Oxylipins are bioactive oxygenated fatty acids known to influence host immune response and Aspergillus developmental programs. Aspergillus synthesizes 8-HODE and 5,8-diHODE that have structural similarities to 9-HODE and 13-HODE, which are known ligands of the host G-protein-coupled receptor G2A (GPR132).Materials and methodsOxylipins were extracted from infected lung tissue to assess fungal oxylipin production and the Pathhunter β-arrestin assay was used to assess agonist and antagonist activity by fungal oxylipins on G2A. An immunocompetent model of A. fumigatus infection was used to assess changes in survival and immune responses for G2A-/- mice.ResultsHere we report that Aspergillus oxylipins are produced in lung tissue of infected mice and in vitro ligand assays suggest 8-HODE is a G2A agonist and 5,8-diHODE is a partial antagonist. To address the hypothesis that G2A could be involved in the progression of IPA, we assessed the response of G2A-/- mice to A. fumigatus infection. G2A-/- mice showed a survival advantage over wild-type mice; this was accompanied by increased recruitment of G2A-/- neutrophils and increased levels of inflammatory markers in A. fumigatus-infected lungs.ConclusionsWe conclude that G2A suppresses host inflammatory responses to Aspergillus fumigatus although it remains unclear if fungal oxylipins are involved in G2A activities.

Background: Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality with an estimated 80% of SHS-related deaths attributed to cardiovascular causes. Public health measures and smoking bans have been successful both in reducing SHS exposure and improving cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a promising target for mitigating SHS exposure-induced cardiovascular outcomes.Objectives: The objectives of this study were to determine 1) effects of environmentally relevant SHS exposure on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of an sEH inhibitor (TPPU) can reduce the adverse cardiovascular effects of SHS exposure.Methods: Male C57BL/6J mice (11 weeks old) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to filtered air or 3 mg/m3 of SHS (6 hr/d, 5 d/wk) for 12 weeks, followed by 4 weeks of recovery in filtered air. Some mice received TPPU in drinking water (15 mg/L) throughout SHS exposure. BP, heart rate (HR), HR variability (HRV), baroreflex sensitivity (BRS), and BP variability were determined monthly.Results: SHS exposure significantly decreased 1) short-term HRV by ∼20% (p < 0.05) within 4 weeks; 2) overall HRV with maximum effect at 12 weeks (−15%, p < 0.05); 3) pulse pressure (−8%, p < 0.05) as early as week 4; and 4) BRS with maximum effect at 12 weeks (−11%, p < 0.05). Four weeks of recovery following 12 weeks of SHS ameliorated all SHS-induced cardiovascular detriments. Importantly, mice exposed to TPPU in drinking water during SHS-related exposure were protected from SHS cardiovascular consequences.Discussion: The data suggest that 1) environmental relevant SHS exposure significantly alters cardiac autonomic function and BP regulation; 2) cardiovascular consequences from SHS can be reversed by discontinuing SHS exposure; and 3) inhibiting sEH can prevent SHS-induced cardiovascular consequences.

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

Chemotherapy induced peripheral neuropathy (CIPN) is a particularly pernicious form of neuropathy and the associated pain is the primary dose-limiting factor of life-prolonging chemotherapy treatment. The prevalence of CIPN is high and can last long after treatment has been stopped. Currently, late in the COVID-19 pandemic, there are still increased psychological pressures on cancer patients as well as additional challenges in providing analgesia for them. These include the risks of nonsteroidal anti-inflammatory drug (NSAID) analgesics potentially masking early infection symptoms and the immunosuppression of steroidal and opiate based approaches. Even without these concerns, CIPN is often inadequately treated with few therapies that offer significant pain relief. The experiments we report use soluble epoxide hydrolase inhibitors (sEHI) which relieved this intractable pain in preclinical models. Doses of EC5026, an IND candidate intended to treat neuropathic pain, elicited dose dependent analgesic responses in multiple models including platinum-based, taxane, and vinca alkaloid-based CIPN pain in Sprague Dawley rats. At the same time as a class, the sEHI are known to result in fewer debilitating side effects of other analgesics, likely due to their novel mechanism of action. Overall, the observed dose-dependent analgesia in both male and female rats across multiple models of chemotherapy induced neuropathic pain holds promise as a useful tool when translated to the clinic.

Aflatoxin B1 (AFB1) contamination in food and feed leads to severe global health problems. Acting as the frontier immunological barrier, the intestinal mucosa is constantly challenged by exposure to foodborne toxins such as AFB1 via contaminated diets, but the detailed toxic mechanism and endogenous regulators of AFB1 toxicity are still unclear. Here, we showed that AFB1 disrupted intestinal immune function by suppressing macrophages, especially M2 macrophages, and antimicrobial peptide-secreting Paneth cells. Using an oxylipinomics approach, we identified that AFB1 immunotoxicity is associated with decreased epoxy fatty acids, notably epoxyeicosatrienoic acids, and increased soluble epoxide hydrolase (sEH) levels in the intestine. Furthermore, sEH deficiency or inhibition rescued the AFB1-compromised intestinal immunity by restoring M2 macrophages as well as Paneth cells and their-derived lysozyme and α-defensin-3 in mice. Altogether, our study demonstrates that AFB1 exposure impairs intestinal immunity, at least in part, in a sEH-mediated way. Moreover, the present study supports the potential application of pharmacological intervention by inhibiting the sEH enzyme in alleviating intestinal immunotoxicity and associated complications caused by AFB1 global contamination.

Abstract Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

A previous report showed that 12-week lowering of dietary omega-6 linoleic acid (LA) coupled with increased omega-3 polyunsaturated fatty acid (PUFA) intake (H3-L6 diet) reduced headache frequency and improved quality of life in patients with chronic daily headaches (CDHs) compared to dietary LA reduction alone (L6 diet). The trial also showed that targeted dietary manipulation alters PUFA-derived lipid mediators and endocannabinoids. However, several additional classes of lipid mediators associated with pain in preclinical models were not measured. The current secondary analysis investigated whether the clinical benefits of the H3-L6 diet were related to changes in plasma unesterified PUFA-derived lipid mediators known to be involved in nociception, including prostanoids. Lipid mediators were measured by ultra-high-pressure liquid chromatography coupled with tandem mass-spectrometry. Compared to baseline, dietary LA lowering with or without added omega-3 fatty acids did not alter unesterified n-6 PUFA-derived lipid mediators, although several species derived from LA, di-homo-gamma-linolenic acid, and arachidonic acid were positively associated with headache frequency and intensity, as well as mental health burden. Alpha-linolenic acid (ALA)-derived metabolites were also associated with increased headache frequency and intensity, although they did not change from the baseline in either dietary group. Compared to baseline, docosahexaenoic acid (DHA)-derived epoxides were more elevated in the H3-L6 group compared to the L6 group. Diet-induced elevations in plasma DHA-epoxides were associated with reduced headache frequency, better physical and mental health, and improved quality of life (p < 0.05). Prostanoids were not detected, except for PGF2-alpha, which was not associated with any outcomes. This study demonstrates that diet-induced changes in DHA-epoxides were associated with pain reduction in patients with chronic headaches, whereas n-6 PUFA and ALA metabolites were associated with nociception. Lipid mediator associations with mental health and quality of life paralleled pain management outcomes in this population. The findings point to a network of multiple diet-modifiable lipid mediator targets for pain management in individuals with CDHs.

Pesticides are used on fruit and vegetable crops to obtain greater yield and quality. Residues can be detected in these crops or their products if applied pesticides do not degrade naturally. Therefore, this study aimed to estimate pesticide residues in some strawberry and tomato-based products available in the market for human consumption and associated dietary risks. Contamination with 3–15 pesticides in the tested samples was found. The total number of pesticides detected in the tested samples was 20, belonging to the group of insecticides (84%) and fungicides (16%). Pesticides of cypermethrin, thiamethoxam, chlorpyrifos, and lambda-cyhalothrin appeared at 100% in a number of samples, where the most detected was cypermethrin followed by thiamethoxam. The average values of pesticide residues detected in the tested samples ranged from 0.006 to 0.568 mg kg−1, where it was found that cypermethrin had the highest residue value and appeared in strawberry jam obtained from the market. The recovery rate of pesticides from fortified samples with pyrethroids ranged from 47.5% (fenvalerate) to 127% (lambda-cyhalothrin). Home processing of fortified tomato and strawberry samples had a significant effect on reducing residues in tomato sauce and strawberry jam, where the reduction reached 100%. The results of acute and chronic risk assessment showed that their values were much lower than 100%, indicating minimal risk of dietary intake.

Abstract Over the last decade oxylipins have become more recognized for their involvement in several diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are known to inhibit cyclooxygenase (COX) enzymes, but how NSAIDs affect oxylipins, in addition to COX products, in animal tissues is not well understood. Oxylipins in livers from male and female mice treated with 100 mg/kg/day of ibuprofen for 7 days were investigated. The results showed that ibuprofen treated male livers contained 7 times more altered oxylipins than ibuprofen treated female livers. In male and female livers some prostaglandins were altered, while diols, hydroxy fatty acids and epoxides were significantly altered in male livers. Some soluble epoxide hydrolase (sEH) products, such as 9,10-DiHODE were found to be decreased, while sEH substrates (such as 9(10)-EpODE and 5(6)-EpETrE) were found to be increased in male livers treated with ibuprofen, but not in ibuprofen treated female livers. The enzymatic activities of sEH and microsomal epoxide hydrolase (mEH) were elevated by ibuprofen in both males and females. Analyzing the influence of sex on the effect of ibuprofen on oxylipins and COX products showed that approximately 27% of oxylipins detected were influenced by sex. The results reveal that ibuprofen disturbs not only the COX pathway, but also the CYP450 and lipoxygenase pathways in male mice, suggesting that ibuprofen is likely to generate sex related differences in biologically active oxylipins. Increased sEH activity after ibuprofen treatment is likely to be one of the mechanisms by which the liver reduces the higher levels of EpODEs and EpETrEs.

Abstract Chimeric antigen receptor-based T-cell immunotherapy is a promising strategy for treatment of hematological malignant tumors; however, its efficacy towards solid cancer remains challenging. We therefore focused on developing nanobody-based CAR-T cells that treat the solid tumor. CD105 expression is upregulated on neoangiogenic endothelial and cancer cells. CD105 has been developed as a drug target. Here we show the generation of a CD105-specific nanobody, an anti-human CD105 CAR-T cells, by inserting the sequences for anti-CD105 nanobody-linked standard cassette genes into AAVS1 site using CRISPR/Cas9 technology. Co-culture with CD105+ target cells led to the activation of anti-CD105 CAR-T cells that displayed the typically activated cytotoxic T-cell characters, ability to proliferate, the production of pro-inflammatory cytokines, and the specific killing efficacy against CD105+ target cells in vitro. The in vivo treatment with anti-CD105 CAR-T cells significantly inhibited the growth of implanted CD105+ tumors, reduced tumor weight, and prolonged the survival time of tumor-bearing NOD/SCID mice. Nanobody-based CAR-T cells can therefore function as an antitumor agent in human tumor xenograft models. Our findings determined that the strategy of nanobody-based CAR-T cells engineered by CRISPR/Cas9 system has a certain potential to treat solid tumor through targeting CD105 antigen.

Background & Aims: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. Methods: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. Results: We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. Conclusions: These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

Abstract Background Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. Results Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78–101%. Conclusions The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

Abstract Aim Although opioids have been used as treatment of neuropathic pain, opioids have abuse potential in humans. Since soluble epoxide hydrolase (sEH) in the metabolism of polyunsaturated fatty acids plays a key role in the pain, sEH inhibitors would be promising new therapeutic drugs for neuropathic pain. In this study, we examined the effect of the sEH inhibitor TPPU on rewarding effects in mice using the conditioned place preference (CPP) paradigm. Methods The rewarding effects of morphine (10 mg/kg) and TPPU (3, 10, or 30 mg/kg) in mice were examined using CPP paradigm. Furthermore, the effect of TPPU (30 mg/kg) on morphine‐induced rewarding effects was examined. Results TPPU (3, 10, or 30 mg/kg) did not increase CPP scores in the mice whereas morphine significantly increased CPP scores in the mice. Furthermore, pretreatment with TPPU did not block the rewarding effects of morphine in the mice, suggesting that sEH does not play a role in the rewarding effect of morphine. Conclusion This study suggests that TPPU did not have rewarding effects in rodents. This would make sEH inhibitors potential therapeutic drugs without abuse potential for neuropathic pain.

The soluble epoxide hydrolase (sEH) is a potential target to treat cardiovascular, renal and neuronal diseases. A series of sEH inhibitors containing naturally occurring lipophilic groups (originating from camphor and fenchone) were developed. Inhibitory potency ranging from 0.7 nM to 6.47 μM was obtained. It was discovered that ureas derived from l-camphor were more active against sEH (2.3-fold average) than the corresponding analogues derived from d-camphor indicating enantiomeric preference of sEH. Ureas derived from fenchone possess lower activity against sEH (ca. 80-fold on average) than their camphor-derived analogs due to the specific structure of the lipophilic fragment and show less enantiomeric preference (1.75-fold on average). Moreover, fenchone-derived ureas show no consistency in enantiomeric preference. Endo/exo-form of compound L-3a derived from l-camphor is 4-fold more potent than the corresponding analogue prepared from d-camphor (IC50 = 0.7 nM vs 2.8 nM) making it the most promising sEH inhibitor among the tested series.

Asthma currently affects more than 339 million people worldwide. In the present preliminary study, we examined the efficacy of a new, inhalable soluble epoxide hydrolase inhibitor (sEHI), 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), to attenuate airway inflammation, mucin secretion, and hyper-responsiveness (AHR) in an ovalbumin (OVA)-sensitized murine model. Male BALB/c mice were divided into phosphate-buffered saline (PBS), OVA, and OVA+TPPU (2- or 6-h) exposure groups. On days 0 and 14, the mice were administered PBS or sensitized to OVA in PBS. From days 26–38, seven challenge exposures were performed with 30 min inhalation of filtered air or OVA alone. In the OVA+TPPU groups, a 2- or 6-h TPPU inhalation preceded each 30-min OVA exposure. On day 39, pulmonary function tests (PFTs) were performed, and biological samples were collected. Lung tissues were used to semi-quantitatively evaluate the severity of inflammation and airway constriction and the volume of stored intracellular mucosubstances. Bronchoalveolar lavage (BAL) and blood samples were used to analyze regulatory lipid mediator profiles. Significantly (p < 0.05) attenuated alveolar, bronchiolar, and pleural inflammation; airway resistance and constriction; mucosubstance volume; and inflammatory lipid mediator levels were observed with OVA+TPPU relative to OVA alone. Cumulative findings indicated TPPU inhalation effectively inhibited inflammation, suppressed AHR, and prevented mucosubstance accumulation in the murine asthmatic model. Future studies should determine the pharmacokinetics (i.e., absorption, distribution, metabolism, and excretion) and pharmacodynamics (i.e., concentration/dose responses) of inhaled TPPU to explore its potential as an asthma-preventative or -rescue treatment.

In agriculture industries, workers are at increased risk for developing pulmonary diseases due to inhalation of agricultural dusts, particularly when working in enclosed confinement facilities. Agricultural dusts inhalation leads to unresolved airway inflammation that precedes the development and progression of lung disease. We have previously shown beneficial effects of the omega-3 polyunsaturated fatty acid (ω-3 PUFA) DHA in protecting against the negative inflammatory effects of repetitive dust exposure in the lung. Dietary manipulation of pulmonary disease risk is an attractive and timely approach given the contribution of an increased ω-6 to ω-3 PUFA ratio to low grade inflammation and chronic disease in the Western diet. To prevent any confounding factors that comes with dietary supplementation of ω-3 PUFA (different sources, purity, dose, and duration), we employed a Fat-1 transgenic mouse model that convert ω-6 PUFA to ω-3 PUFA, leading to a tissue ω-6 to ω-3 PUFA ratio of approximately 1:1. Building on our initial findings, we hypothesized that attaining elevated tissue levels of ω-3 PUFA would attenuate agricultural dust-induced lung inflammation and its resolution. To test this hypothesis, we compared wild-type (WT) and Fat-1 transgenic mice in their response to aqueous extracts of agricultural dust (DE). We also used a soluble epoxide hydrolase inhibitor (sEH) to potentiate the effects of ω-3 PUFA, since sEH inhibitors have been shown to stabilize the anti-inflammatory P450 metabolites derived from both ω-3 and ω-6 PUFA and promote generation of specialized pro-resolving lipid mediators from ω-3 PUFA. Over a three-week period, mice were exposed to a total of 15 intranasal instillations of DE obtained from swine confinement buildings in the Midwest. We observed genotype and sex-specific differences between the WT vs. Fat-1 transgenic mice in response to repetitive dust exposure, where three-way ANOVA revealed significant main effects of treatment, genotype, and sex. Also, Fat-1 transgenic mice displayed reduced lymphoid aggregates in the lung following DE exposure as compared to WT animals exposed to DE, suggesting improved resilience to the DE-induced inflammatory effects. Overall, our data implicate a protective role of ω-3 FA in the lung following repetitive dust exposure.

In this study, a monoclonal antibody (mAb) specific to forchlorfenuron (CPPU) with high sensitivity and specificity was produced and designated (9G9). To detect CPPU in cucumber samples, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold nanobead immunochromatographic test strip (CGN-ICTS) were established using 9G9. The half-maximal inhibitory concentration (IC50) and the LOD for the developed ic-ELISA were determined to be 0.19 ng/mL and 0.04 ng/mL in the sample dilution buffer, respectively. The results indicate that the sensitivity of the antibodies prepared in this study (9G9 mAb) was higher than those reported in the previous literature. On the other hand, in order to achieve rapid and accurate detection of CPPU, CGN-ICTS is indispensable. The IC50 and the LOD for the CGN-ICTS were determined to be 27 ng/mL and 6.1 ng/mL. The average recoveries of the CGN-ICTS ranged from 68 to 82%. The CGN-ICTS and ic-ELISA quantitative results were all confirmed by liquid chromatography—tandem mass spectrometry (LC-MS/MS) with 84–92% recoveries, which indicated the methods developed herein are appropriate for detecting CPPU in cucumber. The CGN-ICTS method is capable of both qualitative and semiquantitative analysis of CPPU, which makes it a suitable alternative complex instrument method for on-site detection of CPPU in cucumber samples since it does not require specialized equipment.

Kidney injury from antiangiogenic chemotherapy is a significant clinical challenge, and we currently lack the ability to effectively treat it with pharmacological agents. Thus, we set out to investigate whether simultaneous soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) inhibition using a dual sEH/COX-2 inhibitor PTUPB could be an effective strategy for treating antiangiogenic therapy-induced kidney damage. We used a multikinase inhibitor, sorafenib, which is known to cause serious renal side effects. The drug was administered to male Sprague–Dawley rats that were on a high-salt diet. Sorafenib was administered over the course of 56 days. The study included three experimental groups; 1) control group (naïve rats), 2) sorafenib group [rats treated with sorafenib only (20 mg/kg/day p.o.)], and 3) sorafenib + PTUPB group (rats treated with sorafenib only for the initial 28 days and subsequently coadministered PTUPB (10 mg/kg/day i.p.) from days 28 through 56). Blood pressure was measured every 2 weeks. After 28 days, sorafenib-treated rats developed hypertension (161 ± 4 mmHg). Over the remainder of the study, sorafenib treatment resulted in a further elevation in blood pressure through day 56 (200 ± 7 mmHg). PTUPB treatment attenuated the sorafenib-induced blood pressure elevation and by day 56, blood pressure was 159 ± 4 mmHg. Urine was collected every 2 weeks for biochemical analysis. After 28 days, sorafenib rats developed pronounced proteinuria (9.7 ± 0.2 P/C), which intensified significantly (35.8 ± 3.5 P/C) by the end of day 56 compared with control (2.6 ± 0.4 P/C). PTUPB mitigated sorafenib-induced proteinuria, and by day 56, it reduced proteinuria by 73%. Plasma and kidney tissues were collected on day 56. Kidney histopathology revealed intratubular cast formation, interstitial fibrosis, glomerular injury, and glomerular nephrin loss at day 56 in sorafenib-treated rats. PTUPB treatment reduced histological features by 30%–70% compared with the sorafenib-treated group and restored glomerular nephrin levels. Furthermore, PTUPB also acted on the glomerular permeability barrier by decreasing angiotensin-II-induced glomerular permeability to albumin. Finally, PTUPB improved in vitro the viability of human mesangial cells. Collectively, our data demonstrate the potential of using PTUPB or dual sEH/COX-2 inhibition as a therapeutic strategy against sorafenib-induced glomerular nephrotoxicity.

The soluble epoxide hydrolase (sEH) enzyme is a major regulator of bioactive lipids. The enzyme is highly expressed in liver and kidney and modulates levels of endogenous epoxy-fatty acids, which have pleiotropic biological effects including limiting inflammation, neuroinflammation, and hypertension. It has been hypothesized that inhibiting sEH has beneficial effects on limiting obesity and metabolic disease as well. There is a body of literature published on these effects, but typically only male subjects have been included. Here, we investigate the role of sEH in both male and female mice and use a global sEH knockout mouse model to compare the effects of diet and diet-induced obesity. The results demonstrate that sEH activity in the liver is modulated by high-fat diets more in male than in female mice. In addition, we characterized the sEH activity in high fat content tissues and demonstrated the influence of diet on levels of bioactive epoxy-fatty acids. The sEH KO animals had generally increased epoxy-fatty acids compared to wild-type mice but gained less body weight on higher-fat diets. Generally, proinflammatory prostaglandins and triglycerides were also lower in livers of sEH KO mice fed HFD. Thus, sEH activity, prostaglandins, and triglycerides increase in male mice on high-fat diet but are all limited by sEH ablation. Additionally, these changes also occur in female mice though at a different magnitude and are also improved by knockout of the sEH enzyme.

Background: 4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl) -benzenesulfonamide (PTUPB), a dual cyclooxygenase-2 (COX-2)/soluble epoxide hydrolase (sEH) inhibitor, was found to alleviate renal, pulmonary fibrosis and liver injury. However, few is known about the effect of PTUPB on liver cirrhosis. In this study, we aimed to explore the role of PTUPB in liver cirrhosis and portal hypertension (PHT).Method: Rat liver cirrhosis model was established via subcutaneous injection of carbon tetrachloride (CCl4) for 16 weeks. The experimental group received oral administration of PTUPB (10 mg/kg) for 4 weeks. We subsequently analyzed portal pressure (PP), liver fibrosis, inflammation, angiogenesis, and intra- or extrahepatic vascular remodeling. Additionally, network pharmacology was used to investigate the possible mechanisms of PTUPB in live fibrosis.Results: CCl4 exposure induced liver fibrosis, inflammation, angiogenesis, vascular remodeling and PHT, and PTUPB alleviated these changes. PTUPB decreased PP from 17.50 ± 4.65 to 6.37 ± 1.40 mmHg, reduced collagen deposition and profibrotic factor. PTUPB alleviated the inflammation and bile duct proliferation, as indicated by decrease in serum interleukin-6 (IL-6), liver cytokeratin 19 (CK-19), transaminase, and macrophage infiltration. PTUPB also restored vessel wall thickness of superior mesenteric arteries (SMA) and inhibited intra- or extrahepatic angiogenesis and vascular remodeling via vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), etc. Moreover, PTUPB induced sinusoidal vasodilation by upregulating endothelial nitric oxide synthase (eNOS) and GTP-cyclohydrolase 1 (GCH1). In enrichment analysis, PTUPB engaged in multiple biological functions related to cirrhosis, including blood pressure, tissue remodeling, immunological inflammation, macrophage activation, and fibroblast proliferation. Additionally, PTUPB suppressed hepatic expression of sEH, COX-2, and transforming growth factor-β (TGF-β).Conclusion: 4-(5-phenyl-3-{3-[3-(4-trifluoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl)- benzenesulfonamide ameliorated liver fibrosis and PHT by inhibiting fibrotic deposition, inflammation, angiogenesis, sinusoidal, and SMA remodeling. The molecular mechanism may be mediated via the downregulation of the sEH/COX-2/TGF-β.

In this study, a previously unreported 3-((4-(isopropylamino)-6-(methylthio)-1,3,5-triazin-2-yl) amino) butyric acid hapten was designed and synthesized. This maximized the exposure of the antigen-determinant isopropyl of prometryn to the immune system in animals to induce the production of anticipated highly specific anti-prometryn antibodies. The hapten has a molecular weight of 285.37 Da. The compound was confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H NMR), nuclear magnetic resonance carbon spectroscopy (13C NMR), and high-resolution mass spectrometry (HRMS). By using the active ester approach, immunogens and coated antigens were created. Bovine serum albumin (BSA) was used as an immunogen, along with the successfully produced hapten, to immunize mice. The IC50 value of mouse monoclonal anti-prometryn antibody (mAb) 7D4 (the quantity of analyte that generated 50% prometryn inhibition) was 3.9 ng/mL. The anti-prometryn mAb was of the IgG1 subclass. The IC20 (80% binding level (B/B0) of prometryn)-IC80 (20% binding level (B/B0) of prometryn) range of the anti-prometryn monoclonal antibody standard curve working range was 0.9–18.1 ng/mL. The prepared mAb has good characteristics because it can specifically recognize prometryn, and the cross-reaction rates for ametryn, desmetryn, and terbumeton were 34.77%, 18.09%, and 7.64%, respectively. The cross-reaction rate with the other seven triazines was less than 1%. The hapten structure proposed can serve as an additional tool for modulating selectivity in detecting triazines.

The use of pesticides leads to an increase in agricultural production but also causes harmful effects on human health when excessively used. For safe consumption, pesticide residues should be below the maximum residual limits (MRLs). In this study, the residual levels of pesticides in vegetables and fruits collected from farmers’ markets in Sharkia Governorate, Egypt were investigated using LC-MS/MS and GC-MS/MS. A total number of 40 pesticides were detected in the tested vegetable and fruit samples. Insecticides were the highest group in detection frequency with 85% and 69% appearance in vegetables and fruits, respectively. Cucumber and apple samples were found to have the highest number of pesticide residues. The mean residue levels ranged from 7 to 951 µg kg−1 (in vegetable samples) and from 8 to 775 µg kg−1 (in fruit samples). It was found that 35 (40.7%) out of 86 pesticide residues detected in vegetables and 35 (38.9%) out of 90 pesticide residues detected in fruits exceeded MRLs. Results for lambda-cyhalothrin, fipronil, dimothoate, and omethoate in spinach, zucchini, kaki, and strawberry, respectively, can cause acute or chronic risks when consumed at 0.1 and 0.2 kg day−1. Therefore, it is necessary for food safety and security to continuously monitor pesticide residues in fruits and vegetables in markets.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an infectious disease that has become a serious burden on global public health. This study screened and yielded specific nanobodies (Nbs) against SARS-CoV-2 spike protein receptor binding domain (RBD), following testing its basic characteristics. A nanobody phage library was established by immunizing a camel with RBD protein. After three rounds of panning, the positive colonies were screened by enzyme-linked immunosorbent assay (ELISA). By sequencing, four different sequences of nanobody gene fragments were selected. The four nanobody fusion proteins were expressed and purified, respectively. The specificity and affinity of the four nanobodies were identified by ELISA. Our results showed that an immune phage display library against SARS-CoV-2 has been successfully constructed with a library capacity of which was 4.7 × 108 CFU. The four purified nanobodies showed specific high-affinity binding SARS-CoV-2 S-RBD. Among these, the antigen binding affinity of Nb61 was more comparable to that of commercial rabbit anti-SARS-CoV-2 S-RBD antibodies. In sum, our study has obtained four nanobody strains against SARS-CoV-2 S-RBD with significant affinity and specificity, therefore laying an essential foundation for further research as well as the applications of diagnostic and therapeutic tools of SARS-CoV-2.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Here we demonstrate a noncompetitive idiometric nanobodies phage immunoassay for the determination of FB1, utilizing two types of anti-idiotypic nanobody, that is specific for different epitopes within the hypervariable region of the primary mAb. Three alphatype anti-idiotypic nanobodies were obtained from a naive nanobodies phage-display library. A noncompetitive idiometric immunoassay was established with a combination of betatype anti-idiotypic nanobody and phage-displayed alphatype anti-idiotypic nanobody. The half-maximal saturation of the signal value (SC50) was 0.68 ng/mL, with the limit of detection (LOD) was 0.19 ng/mL. When the noncompetitive assay was compared to the competitive ELISA (LOD = 3.41 ng/mL), an over 17-fold improvement in sensitivity was observed. A correlation (R2) was 0.988 between the data of the noncompetitive assay and LC–MS/MS for the determination of FB1 in cereal samples. The noncompetitive idiometric immunoassay would have a broad utility for monitoring small molecules in food and environment.

Using newly designed epoxy group activated carbon shell magnetic beads as a goat against mouse antibody (G-Mab) carrier, a competitive immunoassay was conducted on the surface of G-Mab-coated magnetic beads (G-Mab-MBs). Mouse monoclonal antibody against Okadaic acid (MAb), Okadaic acid-horseradish peroxidase conjugate (OA-HRP) and OA were simultaneously added into a reaction system. The enzyme-linked immunosorbent assay (ELISA) was performed in a 96-well microplate incorporated with magnetic bars. Detection of the OA level by using the current approach provided an excellent linear relationship in the range of 0.35–25.0 µg L−1 with a half-maximal inhibitory concentration IC50 of 3.27 µg L−1 and LOD values of 0.35 µg L−1. The shellfish matrix and other algal toxins presented in the sample extraction did not produce obvious interference. As a comparison, tests were conducted via commercial ELISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding analytical results demonstrated the reliability of the current protocol.

In this study, we developed a double antibody sandwich chemiluminescent immunoassay (DAS-CLIA) based on phage-displayed nanobodies for the determination of Cry2A toxin. Nine phage-displayed nanobodies specifically to Cry2A were obtained from a naive nanobody library after four rounds of biopanning. The phage-displayed nanobody P2 with high affinity binding was selected as the detection antibody for the sensitive DAS-CLIA development. The proposed method exhibited high sensitivity with a limit of detection of 0.09 ng/mL, has a wide linear range for Cry2A toxin detection, in the range of 0.1–1000 ng/mL and negligible cross-reactivities to the other Bt toxins. The recoveries of Cry2A toxin spiked in cereal samples (rice, wheat, and corn) ranged from 82.7% to 118%, with a coefficient of variation of less than 10%. This study demonstrated that the DAS-CLIA procedure based on phage-displayed nanobodies may provide an alternative sensitive method for the detection of Bt toxins in agri-products.

Polyunsaturated fatty acids are metabolized into regulatory lipids important for initiating inflammatory responses in the event of disease or injury and for signaling the resolution of inflammation and return to homeostasis. The epoxides of linoleic acid (leukotoxins) regulate skin barrier function, perivascular and alveolar permeability and have been associated with poor outcomes in burn patients and in sepsis. It was later reported that blocking metabolism of leukotoxins into the vicinal diols ameliorated the deleterious effects of leukotoxins, suggesting that the leukotoxin diols are contributing to the toxicity. During quantitative profiling of fatty acid chemical mediators (eicosanoids) in COVID-19 patients, we found increases in the regioisomeric leukotoxin diols in plasma samples of hospitalized patients suffering from severe pulmonary involvement. In rodents these leukotoxin diols cause dramatic vascular permeability and are associated with acute adult respiratory like symptoms. Thus, pathways involved in the biosynthesis and degradation of these regulatory lipids should be investigated in larger biomarker studies to determine their significance in COVID-19 disease. In addition, incorporating diols in plasma multi-omics of patients could illuminate the COVID-19 pathological signature along with other lipid mediators and blood chemistry.

This study provides the first design and synthetic protocol for preparing highly sensitive and specific atrazine (ATR) monoclonal antibodies (mAbs). In this work, a previously unreported hapten, 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine, was designed and synthesized, which maximally exposed the characteristic amino group ATR to an animal immune system to induce the expected antibody. The molecular weight of the ATR hapten was 259.69 Da, and its purity was 97.8%. The properties of the anti-ATR mAb were systematically characterized. One 9F5 mAb, which can detect ATR, was obtained with an IC50 value (the concentration of analyte that produced 50% inhibition of ATR) of 1.678 µg/L for ATR. The molecular weight for the purified 9F5 mAb was approximately 52 kDa for the heavy chain and 15 kDa for the light chain. The anti-ATR mAb prepared in this study was the IgG1 type. The working range of the standard curve (IC20 (the concentration of analyte that produced 20% inhibition of ATR)—IC80 (the concentration of analyte that produced 80% inhibition of ATR)) was 0.384 to 11.565 µg/L. The prepared anti-ATR mAb had high specificity, sensitivity, and affinity with low cross-reactivity. The prepared anti-ATR mAb could provide the core raw material for establishing an ATR immunoassay.

The veterinary pharmacopeia available to treat pain and inflammation is limited in number, target of action and efficacy. Inhibitors of soluble epoxide hydrolase (sEH) are a new class of anti-inflammatory, pro-resolving and analgesic drugs being tested in humans that have demonstrated efficacy in laboratory animals. They block the hydrolysis, and thus, increase endogenous concentrations of analgesic and anti-inflammatory signaling molecules called epoxy-fatty acids. Here, we screened a library of 2,300 inhibitors of the sEH human against partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six very potent sEH inhibitors (IC50 < 1 nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in solvents suitable for the formulation of drugs. The trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB, 1,728) appears to be the best compromise between stability and potency across species. Thus, it was selected for further testing in veterinary clinical trials of pain and inflammation in animals.

Cyproheptadine hydrochloride (CYP), used as human and veterinary drug, has been used illegally as feed additive for food-producing animals, which could remain in food and jeopardize human health. There is a need for on-site detection of CYP residue in animal-derived food. In this study, a hapten was designed, and a specific monoclonal antibody (mAb) was developed to detect CYP with an IC50 of 1.38 ng/mL and negligible cross-reactivity (CR) for other analogs. Forthermore, a high sensitive immunochromatographic assay (QBs-ICA) was developed using quantum dot nanobeads as reporters. The assay showed the linear detection range (IC20-IC80) of 0.03–0.52 ng/mL, the limit of detection (LOD) and visual detection limit (VDL) reached to 0.01 and 0.625 ng/mL, respectively. Spiked recovery study in pig urine and pork confirmed that the QBs-ICA was applicable for on-site testing. This assay showed better sensitivity and speedy than the reported instrumental analysis and immunoassays.

Vicinal diols are important signaling metabolites of various inflammatory diseases, and some of them are potential biomarkers for some diseases. Utilizing the rapid reaction between diol and 6-bromo-3-pyridinylboronic acid (BPBA), a selective and sensitive approach was established to profile these vicinal diols using liquid chromatography-post column derivatization coupled with double precursor ion scan-mass spectrometry (LC-PCD-DPIS-MS). After derivatization, all BPBA-vicinal-diol esters gave a pair of characteristic isotope ions resulting from 79Br and 81Br. The unique isotope pattern generated two characteristic fragment ions of m/z 200 and 202. Compared to a traditional offline derivatization technique, the new LC-PCD-DPIS-MS method retained the capacity of LC separation. In addition, it is more sensitive and selective than a full scan MS method. As an application, an in vitro study of the metabolism of epoxy fatty acids by human soluble epoxide hydrolase was tested. These vicinal-diol metabolites of individual regioisomers from different types of polyunsaturated fatty acids were easily identified. The limit of detection (LOD) reached as low as 25 nM. The newly developed LC-PCD-DPIS-MS method shows significant advantages in improving the selectivity and therefore can be employed as a powerful tool for profiling vicinal-diol compounds from biological matrices.

In this study, a fluorometric and colorimetric dual-readout lateral flow immunoassay (LFIA) using antibody functionalized polydopamine-coated gold nanoparticles (Au@PDAs) as a probe was developed for the detection of carbendazim (CBD). Colloidal gold nanoparticles (AuNPs) were coated with polydopamines (PDA) by the oxidation of dopamine to synthesize Au@PDA nanoparticles. The Au@PDA nanoparticles mediated ZnCdSe/ZnS quantum dots (QDs) fluorescence quenching and recovery, resulting in a reverse fluorescence enhancement detection format of CBD. The CBD detection was obtained by the competition between the CBD and the immobilized antigen for Au@PDAs labelled antibody binding, resulting in a significant fluorescence increase and colorimetry decrease corresponded to the concentration of CBD. Dual readout modes were incorporated into the LFIA using the colorimetry signal under natural light and the fluorescence signal under UV light. The cut-off value in the mode of the colorimetric signal and fluorometric signal for CBD detection was 0.5 μg/mL and 0.0156 μg/mL, respectively. The sensitivity of LFIA of the fluorescence mode was 32 times higher than that of the colorimetry mode. There was negligible cross reactivity obtained by using LFIA for the determination of thiabendazole, benomyl, thiophanate-methyl, and thiophanate-ethyl. Consistent and satisfactory results have been achieved by comparing the results of Au@PDAs-QDs-LFIA and liquid chromatography-tandem mass spectrometry (LC—MS/MS) testing spiked cucumber and strawberry samples, indicating good reliability of the Au@PDAs-QDs-LFIA.