bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Cannabidiol on Epileptic Processes through Microglial Modulation
Authors: Victor Tanya R. , Nissen Jillian C. , Elmes Matthew W. , Deutsch Dale G. , Tsirka Stella E. 
Published: June 2021

Epilepsy is a chronic disorder characterized by abnormal brain cell activity leading to recurrent, unprovoked seizures. Pharmacological therapies are commonly used for the treatment of epilepsy, although nearly 30% of patients do not respond to current medications. This highlights the need for new drug targets and treatments. Many recent studies have centered on the use of cannabidiol (CBD) to treat epilepsy. Early use of CBD, a non‐psychotropic component of Cannabis sativa, is reported to lessen the severity of seizure in pediatric cases of epilepsy, experimentally induced seizures in animals, and to suppress neuroinflammation in culture. Although CBD has been shown to increase intracellular levels of anandamide, an endogenous cannabinoid, its complete mechanism of action is still poorly understood. Previous studies have reported that in culture CBD can suppress neuroinflammation. We and others have shown that microglia, the immune cells of the central nervous system (CNS), are important mediators of seizure severity. Microglia also act as modulators of neurogenesis and synaptogenesis, contributing to the refinement of functional neuronal circuits. We postulate that CBD modulates the activation of microglial cells to exert beneficial effects in the brain. In this study, we evaluate the effects of CBD on cell death and inflammation in the hippocampus during recovery after seizures. Ongoing studies will evaluate microglial engulfment of newborn cells and the effect of CBD on signaling effectors, including Toll‐Like receptors.Support or Funding InformationThis work was supported by NIH‐IRACDA NY CAPS K12‐GM‐102778 and NIH T32GM007518.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‐ and Delay‐Dependent Effects of Δ9‐THC on Memory in Female Rats
Authors: DeLarge Alyssa Fournett , Winsauer Peter J. 
Published: June 2021

The cannabinoid receptor agonist, Δ9‐tetrahydrocannabinol (Δ9‐THC), is a psychoactive constituent of marijuana, and has been shown to produce sex‐dependent changes in learning and memory. This study examined the effects of estrogen on memory, and how these effects alter the acute disruptive effects of Δ9‐THC (0.32–3.2 mg/kg) on memory in female rats. To do this, female rats were trained to respond under a repeated acquisition and delayed‐performance procedure and then ovariectomized. During the acquisition phase of this procedure, rats acquired a 4‐response sequence on three response keys (center, left and right), and sequences changed daily (CRLC, LRCL, RLCR, etc). Sequence acquisition was followed by a delay phase (1 minute to 24 hours), and a delayed‐performance phase in which rats were required to emit the previously acquired sequence. Responding in the acquisition and delayed‐performance phases was maintained under a second‐order fixed‐ratio 3 schedule of food presentation. Δ9‐THC or vehicle (control) was administered 30 minutes prior to the delayed‐performance phase, regardless of delay duration, in order to specifically assess memory, which was quantified by a percent savings measure. Response rate and the percentage of errors were also recorded. To further examine the effects of estrogen, OVX female rats were chronically administered estradiol using a subcutaneous capsule containing a 25/75% ratio of estradiol to cholesterol. The disruptive effects of Δ9‐THC on memory were assessed prior to and during estradiol administration. Prior to estradiol administration, increasing delays ranging from 1 minute to 24 hours produced delay‐dependent decreases in percent savings. When the delay was 1 hour, acute administration of Δ9‐THC produced dose‐dependent decreases in response rate and percent savings, and increases in percent errors, that were significantly attenuated by estradiol administration. Increasing the delay from 1 to 3 hours enhanced the effects of low doses of Δ9‐THC (e.g., 0.56 mg/kg) and produced a significant decrease in percent savings. These results demonstrate the delay‐dependent effects of Δ9‐THC on memory and the involvement of estrogen in modulating Δ9‐THC's acute memory‐disrupting effects in female rats.Support or Funding Information–NIH‐NIDA: R01‐DA037255‐02S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting of the Histone 3 Lysine 9 Methyltransferase Pathway in Kras‐Induced Cell Growth and Pancreatic Cancer
Authors: Lomberk Gwen , Urrutia Guillermo , Caraballo Mariano Colon , Salmonson Ann , Missfeldt Mckenna , Mathison Angela , Tsai Susan , Adsay Nazmi Volkan , Evans Douglas , Iovanna Juan , Urrutia Raul 
Published: June 2021

Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl‐K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a‐GLP‐Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1‐CRE/LSL‐KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA‐Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs and have an upregulation of p21, a cyclin‐dependent kinase inhibitor that is a major effector of p53 activity and facilitates cell cycle arrest in response to DNA damage. Increased levels of p21 protein are also observed in lysates from Pdx1‐CRE/LSL‐KRASG12D/G9a−/− animals compared to Pdx1‐CRE/LSL‐KRASG12D control mice. Pharmacological inhibition and siRNA‐mediated knockdown of G9a in cells recapitulates this effect with p21 upregulation. Furthermore, we find G9a and its H3K9Me2 mark occupy the p21 promoter, suggesting G9a is involved in the direct regulation of this gene. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D‐induced cell proliferation, as well as pancreatic tumor growth in a subcutaneous xenograft model. Combined, these data provide evidence for a key role of the meK9H3‐G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC.Support or Funding InformationNIH R01CA178627; NIH R01DK58185; Advancing a Healthier Wisconsin EndowmentThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cyclooxygenase inhibitors for treating preterm labour? What is the evidence?
Authors: Liwa Anthony Cuthbert , Urrego Daniela , Cole William Crawford , Slater Donna Michelle 
Published: June 2021

BackgroundPreterm labour, a condition characterized by regular uterine contractions and cervical dilation prior to 37 weeks of pregnancy can result in premature birth of babies that may not be fully developed and/or possess serious health problems such as cerebral palsy and learning disabilities. Is associated with an increase in uterine prostaglandins (PGs) synthesis by cyclooxygenase (COX) enzymes which promotes inflammatory and contractile processes that drive labour. For this reason, COX inhibitors (e.g. indomethacin) have been used to treat/prevent preterm labour. Despite limited success, tocolysis by COX‐inhibition carries serious adverse fetal effects due to inhibition of prostaglandin‐dependent developmental homeostatic functions. The more recent discovery of a second, inducible COX isoform, COX‐2, presented an opportunity to spare homeostatic prostaglandin by selective inhibition of COX‐2. However, subsequent clinical studies have called into question the appropriateness of COX‐2 selective inhibition. This study reviewed the molecular evidence concerning the role of COX‐2 in human parturition that underpins the rationale of use of COX‐2 inhibitors in the treatment of preterm labour and presents evidence of COX‐2 gene expression in term and preterm human myometrium.MethodsFor our systematic literature review, we searched MEDLINE and EMBASE databases to identify papers reporting molecular evidence of COX enzyme expression and activity in gestational tissues at term and preterm. Two independent reviewers evaluated abstracts, and qualifying references were reviewed in full text. Data were extracted using a standardized questionnaire. COX‐2 gene expression analysis was conducted on samples of myometrium and decidua from women at term (after 37 weeks) and preterm (below 37 weeks) of gestation, with and without labour using reverse transcriptase‐polymerase chain reaction technique (RT‐PCR).ResultsThirty‐five studies were included in the review. Eight studies on fetal membranes (amnion and chorion) reported significantly higher COX expression or activity with labour. However, in the myometrium and decidua, which are the most relevant therapeutic target tissues for uterine contraction, nine reported no labour‐associated differences COX expression. In the myometrium, four studies reported higher levels of COX mRNA with labour, but did not consider protein expression. Our data show spatial (upper versus lower segment) and temporal (labour versus non‐labour) demonstrate differences in COX‐2 expression; at term, expression of COX‐2 is higher in lower segment myometrium and decidua samples from women in term labour (n=16) compared to term non‐labour (n=18), but no significant differences were observed for upper segment samples. Similarly, COX‐2 expression was higher in lower segment preterm labourThis 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 Residues in the Fifth Transmembrane‐Spanning Domain of the D2‐like Dopamine Receptors that Engender Signaling Bias
Authors: Soto Marta Sanchez , Willette Blair K. A. , Gonye Elizabeth C. , Moore Annah M. , Chun Lani S. , Moritz Amy E. , Verma Ravi Kumar , Yano Hideaki , Shi Lei , Free R. Benjamin , Sibley David R. 
Published: June 2021

The D2 dopamine (DA) receptor (D2R) signals through a variety of second messenger pathways making it difficult to discern which of these are linked to specific effects of D2R‐targeted drugs; however, this complexity provides a unique opportunity to develop pathway‐selective therapeutics. Structure‐activity analyses using analogs derived from our previously described D2R G protein‐biased agonist, MLS1547, coupled with molecular modeling led to a structural model for biased signaling that entails a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop of the D2R. In the current study, we used mutagenesis to investigate the role of these residues in regulating signaling by the D2‐like receptors (D2R, D3R, and D4R), and the β2‐adrenergic receptor (B2R). We constructed point mutations at I184, V190 and F189 of the D2R, and at the aligned residues for F189 (i.e., position 5.38) within the D3R, D4R, and B2R, and studied their effects on G protein‐mediated signaling and β‐arrestin recruitment using BRET‐based biosensors and in vitro signaling assays. The D2R point mutations I184A and V190A produced a small change in the potency of DA for stimulating β‐arrestin recruitment or G protein activation. Strikingly, the F189A mutation ablated the ability of DA and other D2R agonists to recruit β‐arrestin while G protein‐signaling efficacy was maintained. Further, the D2R F189A mutant was unable to directly interact with GRK2 and failed to internalize from the cell surface following DA stimulation. In addition, we found that mutating the residues in the D3R, D4R, and B2R at position 5.38 to Ala resulted in parallel findings (i.e., loss of agonist‐stimulated β‐arrestin recruitment, but minimal to no effects on G protein‐mediated signaling). These data demonstrate that the D2R F189A mutant, and similarly mutated D3R, D4R, and B2R, are highly biased towards G protein‐mediated signaling and suggest that the presence of a Phe or Tyr residue at this position (5.38) is important for stabilizing an activated state for recruiting β‐arrestin. Conformational changes propagated through TM5 might thus act as a molecular switch for receptor signaling via β‐arrestin recruitment. These results may have implications for the design of novel signaling‐biased compounds for the treatment of GPCR‐related disorders.Support or Funding InformationNINDS IRPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transporter Roles in the Pharmacokinetics and Tissue Distribution of Voxilaprevir, a Pan‐genotypic HCV NS3/4A Protease Inhibitor
Authors: Wang Yujin , Yang Chris , Hao Jia , MacLennan Kelly , Wong Belinda , Mwangi Judy , Murray Bernard , Liang Xiaomin , Lai Yurong , Taylor James G. 
Published: June 2021

IntroductionVoxilaprevir (VOX; GS‐9857) is a pan‐genotypic hepatitis C virus nonstructural (NS) protein 3/4A protease inhibitor (HCV‐PI) with potent antiviral activity against HCV genotypes 1 to 6 and an enhanced resistance profile over previous HCV‐PIs1. VOX is a component of Vosevi™, a fixed‐dose combination of three agents (SOF/VEL/VOX) and is approved for treatment of HCV. In vitro and in vivo assessments were conducted to determine the roles of drug transporters in the pharmacokinetics (PK) and preferential tissue distribution of VOX.MethodsVoxilaprevir metabolic stability was assessed in vitro in hepatic microsomal fraction and cryopreserved hepatocytes from the rat, dog, monkey and human. Permeability and efflux transport were characterized in Caco‐2 cells and Pgp‐ or BCRP‐overexpressing MDCKII cells. The uptake kinetics of VOX was characterized in rat and human hepatocytes. VOX PK and distribution into liver was determined following a single intravenous (IV) administration in rat, dog, and monkey. VOX elimination was evaluated in bile‐duct cannulated rat following a single IV dose. The distribution of VOX in select tissues (liver, kidney, & lung) was assessed with and without rifampicin pretreatment following IV administration in rat. PK following single ascending oral administration of VOX was also evaluated in rat.ResultsVOX was metabolically stable in hepatic microsomal fractions across all species tested, except for the monkey. In the rat, VOX was mainly eliminated via biliary excretion as unchanged parent drug (70% of dose), and to a lesser extent as its oxidated metabolites. Liver uptake of VOX was observed across the nonclinical species tested ‐ liver to plasma concentration ratio was 970, 34, and 41 in the rat, dog, and monkey, respectively. VOX was found to be a substrate of efflux transporters P‐gp and BCRP, and also a substrate of hepatic uptake transporters (OATPs). VOX displayed a non‐linear and saturable OATP‐mediated hepatic uptake kinetics in vitro. In the rat, VOX shown substantially higher liver concentration than reference tissues (kidney and lung) with and without rifampicin pretreatment. Non‐linear PK was also observed following ascending oral doses of VOX in rat.ConclusionsTaken together, these studies demonstrate the contribution of drug transporters in the PK and preferential liver distribution of VOX; which was also corroborated in its clinical PK and efficacy studies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compound 21 (C21), a Selective Angiotensin Type 2 (AT2) Receptor Agonist Attenuates Bleomycin induced Alveolar Epithelial Cell Death
Authors: Nalbandyan Armen , Shenoy Anitha , Steckelings Ulrike Muscha , Katovich Michael , Shenoy Vinayak 
Published: June 2021

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by excessive deposition of collagen‐rich matrix, which progressively leads to respiratory failure and death. Patients with IPF have a reduced survival rate and the disease remains refractory to current therapies. Hence, identifying novel targets and developing effective drugs are of paramount importance. Accumulating evidence indicate that death of alveolar epithelial cells (AEC) is a key event for the initiation of IPF. Bleomycin, a well‐known fibrotic agent is a potent inducer of apoptotic cell death in AEC through synthesis of Angiotensin II (Ang II). While the apoptotic actions of Ang II on AEC are mediated via the angiotensin type 1 (AT1) receptor, the functional role of angiotensin type 2 (AT2) receptor in AEC has not yet been characterized. We hypothesized that stimulation of the angiotensin type 2 (AT2) receptor by a non‐peptide agonist, Compound 21 (C21) protects against bleomycin‐induced alveolar epithelial cell death.MethodsIn this study, we investigated the effects of C21, the selective AT2 receptor agonist against bleomycin‐induced death of human pulmonary alveolar epithelial cells (A549). The MTT (3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide) assay was performed to determine cell viability of A549.ResultsWe first determined the concentration of bleomycin required for the induction of cell death in cultured A549 cells. A dose‐dependent induction of cell death was obtained with different concentration of bleomycin (μg/mL: % cell death; 1: 30%; 10:42% and 20:61%). Since we observed optimal cell death with 10 μg/mL concentration of bleomycin, we selected this dose for subsequent experiments. Next, we evaluated the cell‐protective effects of C21 at different drug doses (0.001, 0.005, 0.01, 0.05 and 0.1 μM). C21 treatment of bleomycin‐challenged A549 cells (10 μg/mL) produced a dose‐dependent decrease in cell death (μM: % cell death; 0.001:31%; 0.005:29%; 0.01:16%; 0.05:14% and 0.1:9%) as analyzed by the MTT assay. We also observed that treatment of control A549 cells with the above mentioned doses of C21 had little effect on cell proliferation or cell death.ConclusionOur results suggest that C21, the selective AT2 receptor agonist protects against bleomycin‐induced alveolar epithelial cell death. Further studies are warranted to elucidate the mechanisms underlying the cytoprotective effects of C21.Support or Funding InformationAmerican Heart Association Grant (SDG12080302) awarded to Vinayak ShenoyStart‐up Grant from California Health Sciences UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery, characterization, and preclinical development of a Kir4.1 (KCNJ10) inhibitor for the treatment of hypertension
Authors: Kharade Sujay V. , Kurata Haruto , Figueroa Eric , Blobaum Anna Louise , Kramer Meghan , Lindsley Craig , Hopkins Corey R. , Denton Jerod S. 
Published: June 2021

Kir4.1 (encoded by KCNJ10) inward rectifier potassium channels are emerging as key regulators of sodium reabsorption in the distal nephron of the kidney. Kir4.1 is expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use. These human validation data and recent animal studies suggest that Kir4.1 might represent a novel diuretic target for the management of hypertension through inhibition of distal nephron sodium reabsorption. The aim of this study was to develop a selective small‐molecule inhibitor for evaluating the therapeutic potential of renal Kir4.1 channels. From a high‐throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of ~0.9 μM and heterotetrameric Kir4.1/5.1 with an IC50 of ~ 9 μM. Lead optimization failed to identify analogs with improved potency toward Kir4.1. The selectivity screening against other Kir channels revealed that VU992 does not block Kir1.1, Kir2.x, Kir6.1/SUR1 and Kir7.1 channels. The voltage dependent unblock suggested that VU992 probably binds in the pore of the Kir4.1 channel. Computational docking analysis using a Kir4.1 homology model revealed several possible interacting residues in the pore. Experimental binding site analysis using mutagenesis and patch clamp electrophysiology suggested that the pore lining glutamic acid residue at 158 (E158) is crucial for the binding of VU992 to the Kir4.1. Mutating E158 to corresponding asparagine residue in Kir1.1 (E158N) abolished the VU992 block completely. Pharmacokinetic studies in rats showed that VU992 does not cross the blood brain barrier and therefore should not inhibit Kir4.1 channels expressed in the CNS. Finally, oral administration of VU992 dose‐dependently increased urine output and Na+ and K+ loss in volume‐loaded, freely behaving rats. This study shows proof‐of‐concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. Its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron.Support or Funding InformationNIH 5R21 NS073097‐01S1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.