bluuebunny/crossref_metadata_2025 huggingface dataset:
The Minimum Information About A Biosynthetic Gene Cluster Standard as a Means of Organizing Bioinformatic Data
Authors: Epstein Samuel C. , Medema Marnix H. , Charkoudian Louise K.
Published: June 2021

Therapeutic agents play an increasingly large role in our daily lives, and it is important to study the source of these powerful chemical compounds. Biosynthetic gene clusters found in a diverse set of organisms are capable of manufacturing structurally complex organic compounds in a systematic way, by expressing suites of a enzymes to build the natural products. There is a growing need for a centralized database to connect data for related genes, enzymes, and compounds. This information is currently stored in a siloed fashion, across several repositories, creating an obstacle for modern researchers. The Minimum Information about a Biosynthetic Gene cluster (MIBiG) database aims to solve this problem by utilizing user‐inputted data to translate scientific discoveries into a format that can be analyzed computationally to connect genes to chemistry, understand biosynthetic gene clusters in the context of environmental diversity, and develop computer‐generated gene cluster engineering. As an effort to make MiBIG more accessible to an expanding scientific community, we developed a workflow, Excel templates, a tutorial video, a collection of review literature, and educational tools to facilitate the entry process have all been developed. To verify the effectiveness of the protocol and associated resources, MIBiG was deployed as a course‐based undergraduate research experience (CURE) in an upper‐level undergraduate course.

Support or Funding Information

We would like to acknowledge an NSF CAREER Award #R15GM120704 to Louise K. Charkoudian as a source of funding for this project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intravenous Self‐Administration of Synthetic Cathinones in Rhesus Monkeys
Authors: de Moura Fernando Barreto , Sherwood Alexander , Prisinzano Thomas Edward , Kohut Stephen John , Bergman Jack
Published: June 2021

The recreational use of synthetic cathinones (“bath salts”) is a persistent public health challenge. However, there currently is a paucity of information regarding the reinforcing effects of varying types of cathinones in nonhuman primates. In this study, self‐administration of the cathinones methcathinone (MCAT), methylenedioxypyrovalerone (MDPV), methylone, and the norepinephrine (NE)‐preferring substituted phenethylamine PAL‐329 was compared to behavior maintained by the nonselective monoamine transporter blocker cocaine and the monoamine “releasers” d‐methamphetamine (dMA) and 3,4‐methylenedioxymethamphetamine (MDMA) in rhesus monkeys (N=4) responding under a FR30/TO60‐s schedule of IV drug injections. All drugs produced inverted U‐shape dose‐response functions characteristic for drug‐maintained behavior, with a rank‐order potency of MDPV≥MCAT≥d‐MA>cocaine>MDMA>methylone≥PAL‐329. The peak number of infusions per session at optimal unit doses was comparable for cocaine, MCAT, MDPV, and d‐MA (40–50) and lower for PAL‐329 (30) and methylone or MDMA (16). Although the role of pharmacokinetic (especially time‐course) differences among drugs cannot be discounted, it is noteworthy that the cathinones MDPV (a transporter blocker) and MCAT (a monoamine releaser), which maintained the high levels of intake observed with cocaine or methamphetamine, have preferentially dopaminergic actions. In contrast, both methylone and PAL‐329 maintained lower levels of self‐administration, which may reflect the more prominent influence of their, respectively, serotonergic and noradrenergic actions. These studies provide a framework for the further evaluation of abuse‐related effects of novel cathinones based upon their neurochemical mechanisms of action.

Support or Funding Information

Supported by DA 002519

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autotaxin and Glutamate Dysregulation in Alzheimer's Mice Model
Authors: Jones Ellery , Govindarajulu Manoj , Knowlton Stella , Suppiramaniam Vishnu
Published: June 2021

Without the development of new therapeutics, the number of cases of Alzheimer's disease (AD) – currently estimated to be greater than 5.4 million – will rapidly increase in the coming decades. As well, the total estimated worldwide cost of dementia is US$818 billion, according to World Alzheimer's Report 2016. Current treatments provide, at best, only modest and temporary symptomatic relief, failing to alter the underlying pathophysiology, which leads to the onset and mediates the progression of the disease. Autotaxin, is an adipokine that is upregulated in type 2 diabetes mellitus and obesity, indicating a potential involvement of insulin resistance. Mounting evidence suggests that hyperactivation of the Autotaxin (ATX) pathway helps drive neurologic dysfunction in Alzheimer's disease (AD). The deleterious effects of Autotaxin activity on neuronal function appear to be strongly linked to the dysregulation of glutamate homeostasis. The goals of the proposed research were to test the involvement of the Autotaxin (ATX) pathway in glutamate dysregulation associated with AD. We utilized intracerebroventricular (ICV) Streptozotocin (STZ) treated mice to induce brain insulin resistance at 2 months of age and compared with control mice. Autotaxin levels in the hippocampus and cortex were quantified by performing western blot and rt‐PCR, measuring autotaxin mRNA at 4, 8 and 12 months of age. Behavioral studies and electrophysiological studies were performed to assess the cognitive deficits. Finally, biochemical assays to quantify levels of glutamate transporters, other glutamate regulatory proteins, expression of PSD‐95 and Arc proteins essential for synaptic maturity and plasticity and P38 MAPkinase were conducted with hippocampal and cortical lysates. Compared with controls, STZ mice showed significant memory impairment, reduction of synaptic long‐term potentiation and decreased synaptic proteins, which were mediated by the activation of the P38 MAPK Kinase pathway. We believe our studies demonstrate, for the first time, that an increase in autotaxin in the central nervous system directly triggers behavioral and cognitive deficits by promoting synaptic dysfunction, thereby uncovering the molecular mechanisms of autotaxin‐regulated glutamate dyshomeostasis in AD, and possibly other neurodegenerative disorders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammation Produced by Alcohol Synergizes with Methamphetamine to Cause Dopaminergic Deficits
Authors: Blaker Amanda L. , Yamamoto Bryan K.
Published: June 2021

Alcohol and methamphetamine (Meth) are often co‐abused, in that ~77% of Meth addicts also display an alcohol use disorder. However, the neurochemical consequences of this comorbid drug taking behavior are unknown. We tested the hypothesis that serial exposure to ethanol (EtOH) and Meth produces greater neurotoxicity compared to either drug alone. Sprague Dawley rats were allowed drinking access to a 2 bottle choice of 10% EtOH and water, every other day for 4 weeks, followed by one day of binge Meth (10 mg/kg × 4 injections). Over 4 weeks, both EtOH intake (g/kg) and preference (% total volume) increased (p<0.05). EtOH drinking alone caused inflammation, evidenced by significant increases in lipopolysaccharide (LPS) in serum and increased LPS and cyclooxygenase‐2 (COX‐2) in the striatum 24 hours after the last day of EtOH compared to water drinking (*p<0.05 vs. Water). 7 days after Meth or saline injections, striatal dopamine (DA) was depleted by 50% in Meth‐treated rats and by 95% in EtOH+Meth treated rats (*p<0.05 vs. Water+Saline). The DA transporter, DAT, followed a similar trend. Importantly, EtOH alone did not affect DA or DAT concentrations in the brain. Additionally, the amount of EtOH imbibed significantly correlated with the extent of Meth‐induced DA depletions (r2=0.89; p<0.001). The significantly enhanced DA depletions produced by the serial exposure to EtOH drinking and Meth (Meth alone vs. EtOH+Meth; p<0.05) were blocked by the COX inhibitor, ketoprofen (5 mg/kg) administered twice daily on the intermittent withdrawal days during the EtOH drinking period. To determine whether the loss of DA was paralleled by changes in the substantia nigra pars compacta (SNpc), cell bodies were stained for tyrosine hydroxylase (TH). Immunofluorescence showed a significant loss of TH‐positive cells in the SNpc. To ascertain if the loss of DA at the cell body and terminal levels resulted in Parkinsonian‐like motor deficits, motor function was tested 7 days after the last drug exposure. Rotarod test results showed that neither EtOH alone nor Meth alone affected motor behavior, but the serial exposure to the drugs significantly decreased the latency to fall off the rod. These results show the inflammation during EtOH drinking increases the sensitivity to Meth neurotoxicity in the striatum. Future studies will examine if inflammation mediates the loss of TH and motor function, and the extent to which glutamate‐mediated excitotoxicity plays a role in this DAergic loss.

Support or Funding Information

NIH DA007606

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adenosine G Protein‐Coupled Receptor Biased Agonism to Treat Ischemic Heart Disease
Authors: May Lauren Therese , Baltos Jo‐Anne , Chuo Chung H. , Vecchio Elizabeth A. , Wang Bing H. , Kompa Andrew R. , Christopoulos Arthur , Scammells Peter J. , White Paul J.
Published: June 2021

Ischemic heart disease and secondary heart failure places an immense burden on society. Stimulation of adenosine G protein‐coupled receptors (ARs) represents a powerful protective mechanism to decrease cardiac ischemia‐reperfusion injury (IRI). Despite this, therapeutic targeting of ARs has been largely unsuccessful due to on‐target adverse effects, including pronounced bradycardia, atrioventricular block and hypotension (1). Biased agonism has the potential to overcome these limitations by enabling the separation of therapeutic from adverse effects (2).

Aim

To profile prototypical and biased AR agonism in isolated cardiac cells. Subsequent studies investigated the in vivo profile of AR biased agonism in acute and longer‐term rat models of cardiac ischemia‐reperfusion injury.

Results

In isolated rat cardiomyocytes, prototypical and biased AR agonists stimulated reperfusion injury salvage kinase (RISK) signalling pathways, including Akt and ERK1/2, and promoted cardioprotection. Exposure to in vitro simulated ischemia‐reperfusion conditions differentially influenced the RISK signalling stimulated by prototypical and biased AR agonists. Within an in vivo model of acute myocardial ischemia‐reperfusion injury, both biased and prototypical agonists stimulated an A1AR‐mediated reduction in infarct size and an improvement in cardiac function post‐IRI. However, in contrast to prototypical agonists, our biased agonist had no significant adverse hemodynamic effects. Furthermore, in a longer‐term in vivo model of myocardial IRI, an AR biased agonist improved cardiac function and decreased cardiac hypertrophy and fibrosis.

Discussion

Insights into AR signalling in cardiomyocytes under normoxic and simulated ischemia‐reperfusion conditions suggests that context‐specific biased agonism stimulates a preferential signalling profile. Furthermore, AR biased agonism can promote cardioprotection and decrease pathological cardiac remodelling in the absence of bradycardia, a profile suggestive of ligand bias. Collectively, our findings demonstrate that AR biased agonism can stimulate potent cardioprotection in the absence of adverse hemodynamic effects and therefore represents an attractive therapeutic approach for the treatment of ischemic heart disease.

Support or Funding Information

This work was supported by Project Grant APP1084487 of the National Health and Medical Research Council of Australia (NHMRC). J‐A.B., CHC and E.A.V are recipients of the Australian Government Research Training Program Scholarship. A.C. is a Senior Principal Research Fellow of the NHMRC. L.T.M. is an Australian Heart Foundation Future Leaders Fellow.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Distal Helix 5 as a Determinant of GPCR‐G protein Coupling Selectivity
Authors: Okashah Najeah , Wan Qingwen , Inoue Asuka , Lambert Nevin
Published: June 2021

G protein‐coupled receptors (GPCRs) interact with heterotrimeric G proteins to mediate important physiological responses, and compounds that bind to GPCRs represent approximately one third of FDA‐approved drugs. In humans, hundreds of GPCRs converge onto 16 G alpha subunits that are grouped into four families, and individual receptors select between these subtypes. Prior work suggests that the distal half of helix 5 (H5), the extreme C‐terminus of the G alpha subunit, is a key determinant of GPCR‐G protein coupling selectivity. The objective of this study was to determine the relative contributions of the extreme C‐terminus (H5.17‐H5.26) and the rest of the G alpha subunit (HN.1‐H5.16) to GPCR‐G protein coupling selectivity.

We constructed a panel of G alpha subunits that included a wild‐type G protein from each of the four families (Gs‐long, Gi1, Gq, and G12), and all twelve possible chimeras where the last 10 amino acids (H5.17‐H5.26) of each G alpha subunit is replaced by those of another (e.g. Gsi‐10). These formed heterotrimers with venus‐labeled betagamma subunits, and coupling to Rluc8‐labeled GPCRs was measured using bioluminescence resonance energy transfer (BRET). BRET was measured in the presence and absence of both agonists and nucleotides in digitonin‐permeabilized HEK293 Gs/q/12 knockout cells which, together with pertussis toxin, were used to minimize interference from endogenous G proteins.

Our results suggest that distal H5 influences GPCR‐G protein coupling, but by itself is not sufficient to determine selectivity. Furthermore, for some receptors, exchanging distal H5 had little effect on G protein coupling. For example, M3 muscarinic acetylcholine receptors coupled to Gqs‐10 more strongly than to Gsq‐10, suggesting that for this receptor the key selectivity determinants reside proximal to distal H5. In addition, the distal H5 of G12 was generally permissive, e.g. A1 adenosine receptors coupled to Gi12‐10 nearly as well as wild‐type Gi1. Taken together our results suggest that GPCR‐G protein selectivity determinants are likely to be distributed throughout the G protein alpha subunit, and distal H5 is only one such determinant. Moreover, G protein specificity determinants are likely to vary for different GPCRs. Future experiments will specifically address proposed specificity‐determining sites outside of distal H5.

Support or Funding Information

Supported by NIH grants GM078319 and GM109879.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct regulatory domains on the N‐ and C‐termini of Kv7.4 and Kv7.5 contribute to both their voltage‐dependence of activation and their responses to PKA and PKC.
Authors: Byron Kenneth L. , Brueggemann Lyubov I. , Cribbs Leanne L.
Published: June 2021

We previously determined that homomeric Kv7.4 potassium channels expressed in smooth muscle cells are insensitive to regulation by protein kinases A and C (PKA or PKC), whereas homomeric Kv7.5 channels are robustly enhanced by PKA and robustly inhibited by PKC activation. To determine the molecular basis for these different responses of Kv7.4 and Kv7.5 channels, using A7r5 vascular smooth muscle cells as an expression system, we examined the regulation of chimeric channels, constructed by inserting subdomains of Kv7.5 into Kv7.4, to determine whether particular parts of Kv7.5 can confer sensitivity onto Kv7.4. A putative PKA phosphorylation site, S53, is located on the N‐terminus of Kv7.5, but is missing from Kv7.4. We found that replacing the Kv7.4 N‐terminus with the Kv7.5 N‐terminus (Q5N‐Q4) rendered Kv7.4 significantly more responsive to a PKA‐activating stimulus than was wild‐type Kv7.4, but this Q5N‐Q4 chimera was still insensitive to AVP (a PKC‐activating stimulus). PKC is thought to target sites on the C‐terminus of Kv7.5, so we switched the C‐terminus of Kv7.4 with that from Kv7.5 (Q5C‐Q4). Preliminary results suggest that this Q5C‐Q4 chimera is sensitive to PKC‐dependent current suppression. The Q5N‐Q4 chimera had voltage‐dependence of activation similar to WT Kv7.4 (V0.5=−31 ± 3 mV for Q5N‐Q4 and −34 ± 2 mV for WT Kv7.4), whereas the Q5C‐Q4 chimera activated at voltages similar to WT Kv7.5 (V0.5=−44 ± 1 mV for Q5C‐Q4 and −45 ± 2 mV for WT Kv7.5). Our findings suggest that distinct regulatory domains on the N‐ and C‐termini of Kv7.5 and Kv7.4 contribute to both the voltage‐dependence of activation and the differential regulation of the channels by protein kinases.

Support or Funding Information

The project described was supported by Award Number I01BX007080 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The G Protein Gamma Subunit, Ste18, is a Phosphorylation‐Dependent pH Sensor that Controls MAPK Activation in Yeast
Authors: Toosi Zahra Nassiri , Torres Matthew P.
Published: June 2021

As essential transducers of extracellular stimuli such as hormones and neurotransmitters, G protein signaling (GPS) systems – comprised of seven transmembrane receptors (GPCRs), heterotrimeric G‐proteins (Gαβγ), and regulators of G‐protein signaling (RGS) proteins – are important regulators of cell/environment homeostasis in all eukaryotes. Yeast harbor a single canonical GPS system that regulates a pheromone‐dependent mating response, which has long served as a tool for discovering regulators fundamental for all eukaryotic GPS systems. In wild type yeast, the activation rate and amplitude of pheromone signaling is severely dampened by negative feedback phosphorylation of the canonical Gγ subunit, Ste18, and the MAPK scaffold, Ste5. Here, we show that Ste18 is a pH sensor that undergoes combinatorial phosphorylation/dephosphorylation in response to fluctuations in intracellular pH (pHi). Using a site‐specific phosphorylation‐dependent electrophoretic mobility assay and a pHi probe (pHlourin), we show that Ste18S3 is rapidly phosphorylated in response to intracellular acidification, while Ste18S7 is rapidly phosphorylated in response to mating pheromone. When pHi is lowered, basal phosphorylation on Ste18S7 decreases concomitantly with increasing phosphorylation on Ste18S3. In contrast, pheromone stimulation activates phosphorylation on Ste18S7, but not Ste18S3. We show that phosphomimic mutations on either phosphosite function to prohibit phosphorylation of the other non‐mutated site, demonstrating that phosphorylation is mutually exclusive. Furthermore, phosphorylation at either site, mimicked by glutamate mutations, is sufficient to inhibit ultra‐fast and intense output from the pheromone pathway. Thus, our findings suggest that Ste18Nt acts as a phosphorylation‐dependent pH‐sensor that is capable of restricting pheromone pathway output under acid stress conditions when yeast mating is sub‐optimal. Considering that dysregulation of pHi is linked to chronic and life‐threatening diseases such as cancer, stroke, and Alzheimer's disease, our data reveal the possibility that Gγ subunits serve as pH sensors important for regulating G protein signaling in such diseases.

Support or Funding Information

National Institutes of Health (NIH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen‐dependent epigenetic regulation of soluble epoxide hydrolase via DNA methylation
Authors: Yang Yang‐Ming , Sun Dong , Kandhi Sharath , Froogh Ghezal , Hammock Bruce D. , Huang An
Published: June 2021

We have demonstrated that female‐specific downregulation of soluble epoxide hydrolase (sEH) protein expression in cardiac, pulmonary, and vascular tissues favors a greater contribution of epoxyeicosatrienoic acids (EETs) in females. Thus, to elucidate molecular mechanism(s) responsible for the sexually dimorphic phenotype of sEH expression, we tested the hypothesis that female‐specific downregulation of sEH expression is driven by estrogen‐dependent methylation of the Ephx2 gene.

Methods

Mesenteric arteries isolated from male, female, ovariectomized female (OV) and OV with estrogen replacement (OVE) mice, as well as human cell line (HEK293T) were used.

Results

Methylation‐specific PCR (MSP) and bisulfite genomic sequencing (BGS) analysis indicate significant increases in DNA/CG methylation in vessels of female and OVE compared to those of male and OV mice. The same increase in CG methylation was also observed in male vessels incubated with a physiological concentration of 17b‐estradiol (17b‐E2) for 48 hours. All vessels that displayed increases in CG methylation were concomitantly associated with decreases in their Ephx2 mRNA and protein, suggesting a methylation‐induced gene silencing. Transient transfection assays indicate that the activity of Ephx2 promoter‐coding luciferase was significantly attenuated in HEK293T cells treated with 17b‐E2, which was prevented by additional treatment with an estrogen receptor (ER) antagonist (ICI). Chromatin immunoprecipitation (ChIP) analysis indicates significantly reduced binding activities of transcription factors (including SP1, AP‐1 and NFκB with their binding elements located in the Ephx2 promoter) in vessels of female mice and human cells treated with 17b‐E2, responses that were prevented by ICI and Decitabine (DNA methyltransferase inhibitor), respectively. In conclusion, estrogen/ER‐dependent methylation of the promoter of Ephx2 gene silences sEH expression, which is involved in specific transcription factor‐directed regulatory pathways.

Support or Funding Information

(This work was supported by grants NIH HL070653 and HL129797, and also partially supported by NIEHS grant R01 ES02710 and NIEHS Superfund Research Program P42 ES04699).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitor of Soluble Epoxide Hydrolase Attenuates Decline in Learning and Memory of Diabetic Rats
Authors: Goswami Sumanta Kumar , Minaz Nathani , Razdan Rema , Hammock Bruce D.
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are epoxide derivatives formed from arachidonic acid and are neuroprotectant. These bioactive lipids are degraded by soluble epoxide hydrolase (sEH). Pharmacological inhibition of sEH enhances the synaptic function in the central nervous system and has a protective role in the age‐related cognitive decline. We have evaluated the hypothesis that the sEH inhibitor TPPU might prevent a diabetes‐induced decline in learning and memory which is associated with alteration in the level of neurotransmitter and oxidative stress. Type 1 diabetes (streptozotocin 52 mg/kg, i.p.) was induced in rats and the animals were treated with TPPU (0.1 and 0.3 mg/kg body weight; p.o.) for 8 weeks. The learning and memory functions were assessed by Barnes maze and step down test. Level of oxidative stress indicators (reduced glutathione and malondialdehyde) and neurotransmitters (γ‐Aminobutyric acid, norepinephrine, and dopamine), and activity of acetylcholinesterase and were measured in the discrete regions of the brain. Our results revealed that treatment with TPPU which penetrates the blood‐brain barrier significantly improved learning and memory performance in diabetic rats with a parallel decrease in the level of blood sugar. Moreover, treatment with TPPU significantly minimized the diabetes‐induced alteration in levels of neurotransmitters and activity of acetylcholinesterase. In addition, TPPU protected anti‐oxidant defense system. Based on our experimental findings, it can be concluded that the inhibition of sEH is a viable strategy for retrieving diabetes‐induced decline in learning and memory.

Support or Funding Information

This study was partially funded by the National Institute of Environmental Health Sciences (NIEHS) [Grant R01 ES002710], NIEHS Superfund Research Program [Grant P42 ES004699], West Coast Central Comprehensive Metabolomics Center [Grant U24 DK097154] to BDH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Soluble Epoxide Hydrolase Protects the Brain from Permanent Middle Cerebral Artery Occlusion
Authors: Koehler Raymond C. , Tu Ranran , Zhang Xiaofan
Published: June 2021

Inhibition of soluble epoxide hydrolase (sEH) has been shown to reduce infarct volume after transient middle cerebral artery occlusion (MCAO), but to the best of our knowledge, has not been tested in a model of permanent MCAO. Here, we produced permanent MCAO in male C57Bl6 mice by coagulation of the distal MCA and administered vehicle or the sEH inhibitor TPPU [1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl)urea] at 90 min after MCAO and daily over the subsequent 6 days. Protein expression of sEH was measured at 1, 3, 7, 14, and 28 days after MCAO and was found to nearly double at 3 and 7 days after MCAO before decreasing at 14 days and returning to the surgical sham control levels at 28 days. Tissue activity of sEH tripled 3 days after MCAO and was attenuated by 2.7, 64.5, and 62.6% with ip administration of 0.3, 1, and 3 mg/kg/day TPPU. Subsequent experiments used a dose of 1 mg/kg/day. Infarct volume was significantly decreased from 23.9±8.2% (±SD) in the vehicle group to 16.6±4.1% in the TPPU group (P<0.01, n=13). Likewise, sensorimotor function, as assessed by the time to recognize adhesive tape on the affected paw, was significantly improved at 1, 3, 7, and 14 days after MCAO in the TPPU‐treated group. Tissue cytokine mRNA was measured at 1, 3, and 7 days after MCAO. Relative to vehicle treatment, TPPU attenuated the increase in IL‐1β and TNFα mRNA at 3 days after MCAO and augmented the increase in IL‐10 mRNA at 7 days after MCAO. These results indicate that ischemic neuroprotection by administration of a sEH inhibitor does not require reperfusion and that TPPU limits expression of pro‐inflammatory cytokine expression and augments the delayed increase in reparative IL‐10 expression after permanent focal cerebral ischemia.

Support or Funding Information

Supported by NIH NS060703

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Potent Soluble Epoxide Hydrolase Inhibitors for Veterinarian Usage
Authors: Morisseau Christophe , Shihadih Diyala S. , Harris Todd R. , Kodani Sean D. , Hwang Sung‐Hee , Lee Kin Sing S. , Hamamoto Briana , Guedes Alonso , Hammock Bruce D.
Published: June 2021

The veterinary pharmacopeia to treat pain and inflammation in companion animals is very limited. Currently, cyclooxygenase inhibitors (both NSAIDs and COXIBs) are the main class of drugs available to reduce pain and inflammation in cats, dogs and horses. However, these drugs often have insufficient efficacy and a great number of known adverse effects, underlying the need for novel pain medication for companion animals. Soluble epoxide hydrolase (sEH) inhibitors are a new class of anti‐inflammatory and analgesic drugs being tested in humans. They block the hydrolysis, and thus increase endogenous concentrations, of signaling molecules called epoxy‐fatty acids (EpFAs) that modulate inflammation and nociception. In addition to their use in human, sEH inhibitors may be a suitable alternative to cyclooxygenase inhibitors in companion animals. However, the compounds optimized for usage in primates and rodents are typically not sufficiently potent inhibitors of other animals' sEH for in vivo usage. Here, we screened a library of 2,300 inhibitors developed for the human enzyme with partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six candidate sEH inhibitors that are very potent across species (IC50 < 1nM 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 drug‐able solvents. Based on this work, trans‐4‐{4‐[3‐(4‐trifluoromethoxy‐phenyl)‐ureido]‐cyclohexyloxy}‐benzoic acid was selected for further testing in veterinary clinical trials of pain and inflammation in companion animals.

Support or Funding Information

This work was supported by the National Institute of Environmental Health Sciences grant numbers ES002710 and ES004699.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Cytochrome P450 2J2 and Soluble Epoxide Hydrolase in Drug Cardiotoxicity
Authors: Solanki Meetal Satishkumar , Jones Barry , Dawkins‐Hall Laurence , Pointon Amy , Herbert Karl
Published: June 2021

Cytochrome P450 2J2 (CYP2J2), a multifunctional enzyme that is abundant in cardiac tissue, metabolises endogenous arachidonic acid to epoxyeicosatrienoic acids (EETs) which protect the heart. EETs dilate blood vessels increasing blood flow to heart muscle cells, and reduce inflammation which may protect against development of coronary artery disease. Moreover, it is reported that EETs mitigate the harmful effects of the cancer chemotherapeutic drug, doxorubicin, which causes cardiotoxicity in some patients. In vivo, EETs are rapidly metabolised by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which are less biologically active. Several studies have shown that sEH may play an influential role in the development of atherosclerosis and cardiovascular disease, however, its role in cardiotoxicity has not been well reported. The current study aimed to investigate in vitro the role of EETs in the cardiotoxicity of a drug known to interact with CYP2J2, astemizole.

Human cardiac myocytes (HCM) and human umbilical vein endothelial cell‐derived EA.hy926 cells, were incubated with astemizole in the absence or presence of inhibitors which were hypothesised to either enhance or diminish EET production. Changes to gene and protein expression were determined and toxicity was assessed.

mRNA and protein expression of CYP2J2 and sEH was established in both cell models with higher expression in HCM in line with literature reports. Investigation of sEH expression showed the apparent presence of multiple mRNA isoforms but the predominant isoform was assessed. Treatment of cells with astemizole showed a toxic effect in both cell types accompanied by increased mRNA expression of sEH and CYP2J2 in EA.hy926 cells and HCM. Addition of the sEH inhibitor trans‐4‐[4‐3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxyl]‐benzoic acid, (t‐AUCB), which increases levels of EETs in cells and exogenous 11,12‐EET protected cells against the damaging effects of six hours exposure to astemizole. In response to t‐AUCB in astemizole treated cells sEH expression was increased and CYP2J2 mRNA and protein was decreased; presumably a feedback mechanism exists following sEH inhibition that involves both CYP2J2 and sEH.

Taken together, these data show that enhancing EET levels protected against astemizole toxicity in cardiovascular cells in vitro, indicating that the balance between endogenous CYP2J2 and sEH activities may play key role in counteracting damage to the heart by certain drugs. Agents that promote EETs in the heart may be useful adjuncts to counteract the cardiotoxicity due to certain drugs.

Support or Funding Information

This research was supported by Biotechnology and Biosciences Research Council, UK (ref: BB/M503368/1) and AstraZeneca.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A NOVEL INTERACTION BETWEEN AT1 RECEPTOR AND SOLUBLE EPOXIDE HYDROLASE IN DIABETIC RETINOPATHY
Authors: Wang Mong Heng , Al‐Shabrawey Mohamed
Published: June 2021

There is a critical need to identify new therapeutic targets to treat the microvascular damage associated with diabetic retinopathy (DR) because nearly all patients with type 1 diabetes and 60% of those with type 2 diabetes develop DR. Activation of the renin‐angiotensin system (RAS) contributes to retinal hyperpermeability and neovascularization in DR. Clinical studies have suggested that RAS may be a promising therapeutic target in treating DR. Blockade of RAS has reduced the incidence of DR in clinical studies, yet, RAS blockade did not decrease the incidence of diabetic macular edema or reduce the progression of DR. Hence, the FDA still does not approve the use of RAS blockade to treat DR. In short, lack of understanding of the molecular mechanism of retinal microvascular damage induced by RAS is a critical barrier to the use of RAS blockade to prevent or treat DR. Here we show that 1) Angiotensin II (Ang II) increases retinal soluble epoxide hydrolase (sEH) levels by 2.8 fold (2.84 ± 1.1 vs. 1 ± 0.08, P < 0.05, n = 4), which is blunted by an AT1 blocker; 2) both sEH blockade and knockout (KO), which accumulate epoxyeicosatrienoic acids (EETs), promote Ang II‐induced retinal albumin leakage (an index of blood‐retinal barrier (BRB) injury) by 1.58 (1.58 ± 0.4 vs. 1 ± 0.13, P < 0.05, n = 4) and 2.8 fold (2.8 ± 1.7 vs. 1 ± 0.09, P < 0.05, n = 4), respectively; 3) treatment with synthetic 11,12‐EET potentiates intravitreal Ang II‐induced retinal albumin leakage by 2.2 fold (2.2 ± 0.5 vs. 1 ± 0.18, P < 0.05, n = 4); 4) while sEH KO and blockade reduce Ang II‐induced retinal vascular remodeling, sEH KO causes retinal vascular leakage (fluorescein angiogram analysis) in sEH‐null mice treated with Ang II; and 5) sEH KO potentiates diabetes (STZ)‐induced retinal damage via promoting retinal vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT‐1). Overall, our data demonstrate that in DR, activation of retinal AT1 triggers a compensatory mechanism by increasing retinal sEH, but decreasing EETs. Our studies hold the promise of providing a new strategy, the use of combined EETs blockade with AT1 blocker, to prevent or reduce DR.

Support or Funding Information

AHA Grant‐in‐Aid AHASE00144 (M.H.W.) and R01 EY023315 (M.A.S.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Podocyte Exosome Release in Glomerular Injury induced by NLRP3 Inflammasome Activation during Hyperhomocysteinemia
Authors: Zhang Qinghua , Li Guangbi , Hong Jinni , Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Li Ningjun , Li Pin‐Lan
Published: June 2021

The NOD‐like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis to elevated homocysteine (Hcys) levels in vitro and in vivo. However, it remains unknown how the products of NRLP3 inflammasome activated in cytoplasm are secreted out of podocytes and whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in Hcys‐induced kidney injury. In the present study, we tried to answer these questions using Smpd1trg/Podocre (a podocye‐specific sphingomyelin phosphodiesterase 1 (Smpd1) transgenic mouse strain) because Smpd1 encodes acid sphingomyelinase that produces ceramide in lysosomes and regulates lysosome function and potentially controls exosome release. It was found that Smpd1trg/Podocre mice had obvious overexpression of Smpd1 gene and remarkable increase in ceramide concentrations in podocytes. These Smpd1trg/Podocre mice were fed a folate‐free (FF) diet to induce hyperhomocysteinemia (hHcys), and ~ 3 folds increase in colocalization of inflammasome proteins, NLRP3 with ASC or with caspase‐1 and ~ 4 folds of IL‐1β production were found in the glomeruli of these FF‐treated Smpd1trg/Podocre mice, suggesting NLRP3 inflammasome formation and activation. Increased IL‐1β production in the glomeruli was accompanied by decreased podocin levels, but enhanced desmin expression in podocytes with glomerular injury as shown by morphological examinations. We also observed significantly increased colocalization of Annexin‐2 and alkaline phosphatase (exosome markers) in glomeruli of FF‐treated Smpd1trg/Podocre mice than their control littermates, indicating the increased exosome release in mice with Smpd1 gene overexpression in podocytes. By Nanoparticle Tracking Analysis, we found that urinary exosomes (60–140 nm in size) were significantly increased especially from 90nm to 140nm in FF‐treated Smpd1trg/Podocre mice, but not in their control littermates. These increased exosomes in urine from FF‐treated Smpd1trg/Podocre mice contained more detectable podocin protein than control littermates, meaning enrichment of podocyte‐derived exosomes in urine from the podocyte‐specific Smpd1 transgenic mice. Taken together, our results suggest that increased ceramide in podocytes due to Smpd1 gene overexpression may increase exosome release and thereby enhance excretion of inflammasome products to induce podocyte dysfunction and glomerular injury.

Support or Funding Information

supported by NIH grants DK54927 to P.L. and, DK107991 to N.L.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thioredoxin Interacting Protein Deficiency Protects Against Obesity‐Induced Podocyte Injury and Glomerular Sclerosis
Authors: Mohammad Razia Sultana , Singh Gurinder Bir , Kshirasagar Naresh , Li Xiang , Hussain Tahir , Li Ningjun , Li Pin‐Lan , Koka Saisudha , Boini Krishna M.
Published: June 2021

Recently we have shown that activation and formation of NLRP3 inflammasomes in podocytes is implicated in the development of obesity‐induced glomerular injury. Moreover, we demonstrated that NADPH oxidase dependent O2•− production (ROS) mediates the obesity‐induced NLRP3 inflammasomes activation and thereby results in podocyte injury and glomerular damage. However, it remains unknown how NADPH oxidase‐derived ROS is sensed by NLRP3 inflammasomes in podocytes upon visfatin stimulation. The current study tested whether thioredoxin‐interacting protein (TXNIP) mediates obesity‐induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, confocal microscopic analysis showed that visfatin treatment significantly increased the colocalization of NLRP3 with Asc or caspase‐1 in podocytes compared to control cells. Pretreatment with TXNIP siRNA abolished the visfatin‐induced inflammasome formation. Correspondingly, visfatin treatment significantly increased the caspase‐1 activity and IL‐1β production compared to control cells. The TXNIP siRNA transfection significantly attenuated the visfatin‐induced caspase‐1 activity and IL‐1β production. Further immunofluorescence analysis showed that visfatin treatment significantly decreased the podocin expression (podocyte damage) compared to control cells. However, pretreatment with TXNIP siRNA attenuated the visfatin‐induced podocin reduction. In in vivo studies, adult C57BL/6J male mice were fed a high fat diet for 14 weeks to induce obesity, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water). High fat diet significantly increased the plasma visfatin levels compared to control mice. In addition, RT‐PCR analysis showed that high fat diet significantly increased the Nlrp3 expression in glomeruli of wild type mice compared to control mice but not in verapamil treated mice. Furthermore, TXNIP inhibition significantly reduced IL‐1β production in glomeruli of mice with obesity. Correspondingly, TXNIP inhibitor, verapamil attenuated obesity‐induced proteinuria, glomerular damage and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for obesity‐induced NLRP3 inflammasome formation and activation and subsequent glomerular injury

Support or Funding Information

NIH grant, DK104031

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐liver fibrotic effect of triggering receptor expressed on myeloid cells 2 (TREM2) via macrophage phenotype switching
Authors: Kang Byeongseok , Kim Nayoun , Yang Jin Won , Kang Keon Wook
Published: June 2021

Macrophage phenotype switching is critical for the progress of inflammatory diseases. The aim of this study is to investigate the potential role of triggering receptor expressed on myeloid cells‐2 (TREM2) in macrophage phenotype switching and liver fibrosis. TREM2 protein expression was increased in fibrotic livers from human and mouse. Transforming growth factor‐β1 (TGFβ1), a representative stimulus for the differentiation of M2c macrophage, specifically increased TREM2 expression in Raw264.7 macrophage cells. Co‐culture of hepatic stellate cells (HSCs) with TREM2‐overexpressing Raw264.7 cells showed the decreased expression of IL‐6 compared to co‐culture of HSCs and mock‐Raw264.7 cells. TREM2‐transgenic mice showed the decreased liver fibrosis in CCl4‐induced liver fibrosis model, whereas TREM2‐null mice showed the aggravated liver fibrosis compared to wild‐type mice. The basal mRNA level of interleukin‐10 was enhanced and the basal level of monocyte chemoattractant protein‐1 was decreased in liver samples from TREM2‐transgenic mice. Therefore, the overexpressed TREM2 in macrophages from fibrotic liver suppresses the progress of hepatic fibrogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanistic Effects of JNK and p53 Knockdown on Hepatocyte Viability Following Dipyridyl Herbicide Exposure
Authors: Nguyen Ngoc U. , Stamper Brendan
Published: June 2021

Objective

The objective of this study is to characterize key pathways that contribute to the hepatotoxic effects of two dipyridyl herbicides, paraquat and diquat.

Methods

Dose‐dependent toxicity assays were performed in an in vitro liver model (TGF‐alpha transgenic mouse hepatocytes; TAMH) to determine LC50 values for paraquat and diquat. Transient knockdown of JNK and p53 was performed using siRNA. Target knockdowns were confirmed using protein immunoblotting. Cell viability assays were then conducted to determine how each transfection affected cell survival following exposure to either paraquat or diquat.

Results

LC50 values were determined to be 57μM and 18μM for paraquat and diquat, respectively. siRNA‐based knockdown decreased JNK protein expression by 31%. This correlated with significant increases in cell viability for transfected cells exposed to either paraquat (21%) or diquat (15%). siRNA‐based knockdown decreased p53 protein expression by 66%. This correlated with a significant but small decrease in the viability of transfected cells exposed to paraquat only (6%).

Conclusion

Experimental LC50 values revealed diquat to be more hepatotoxic than paraquat in the TAMH line. The siRNA‐based knockdown of JNK provides evidence that JNK plays a crucial role in propagating toxicity caused by these herbicides. In contrast, effects from the p53 knockdown experiments had either no effect (diquat exposure) or propagated toxicity to a small but significant extent (paraquat). These observations suggest affecting targets that precede p53 during apoptotic signaling are crucial to rescuing hepatocytes from the toxic effects of dipyridyl herbicides.

Support or Funding Information

This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellow (SURF) Individual Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Steroid and Drug Metabolism by a Cytochrome P450 Oxidoreductase Variant Found in Apparently Normal Population
Authors: Pandey Amit V. , Parween Shaheena , Roucher‐Boulez Florence , Morel Yves
Published: June 2021

Background and Aims

A broad spectrum of human diseases, including abnormalities in steroidogenesis, are caused by mutations in the cytochrome P450 oxidoreductase (POR) (1–3). POR transfers electrons from NADPH to small molecules, non‐P450 redox partners and microsomal cytochrome P450 proteins. Our aim was to check if POR variations from non‐clinical samples (1000 genomes database), can be disruptive. The Y607C variant of POR (rs72557954, NM_000941.2:c. 1820A>G) has been reported in population studies and is prevalent in South Asians (4), but was predicted to be likely pathogenic. We performed detailed enzymatic and biochemical characterizations of Y607C variant of POR to study its effect on different substrate and redox partners.

Methods

We analysed the ability of POR wild type (WT) and Y607C variant to reduce ferricyanide, MTT, cytochrome c and drug, and steroid metabolizing cytochrome 450 enzymes. The POR WT and Y607C were expressed and produced as recombinant proteins while CYP19A1 and CYP3A4 were produced as His‐tagged recombinant proteins and purified by affinity chromatography. The effect of mutation on cofactor (FAD/FMN) binding and activity under varying substrate and cofactor conditions was performed.

Results

We found varied effects of Y607C mutation on activities with different partners. As compared to WT, Y607C variant showed 66% cytochrome c and 91 % ferricyanide reduction activity but had only 13 % MTT reduction activity. The POR Y607C did not affect flavin content but NADPH binding was severely affected. With varying NADPH concentration, Y607C showed ~95% decrease in supporting CYP19A1 and CYP3A4 activity. This mutation was later identified in patients with POR deficiency.

Conclusions

Identification of severe effects of this mutation on both drug and steroid metabolizing cytochrome P450s indicates that likely pathogenic mutations may be found in apparently normal (non‐clinical) population. Their combination as compound heterozygotes or homozygous may lead to severe impact on both steroid and drug metabolism by modification of POR redox partner activities. Variations in POR need to be evaluated individually. Most importantly, advanced identification of disease causing variants in POR will help in understanding the POR deficiency in patients if the same mutations are later identified.

Support or Funding Information

This research was funded by grants to Amit V Pandey from the Swiss National Science Foundation (31003A_134926).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.