bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploring a competitive method of bias analysis on the kappa opioid receptor
Authors: Stahl Edward , Zhou Lei , Ehlert Frederick , Bohn Laura 
Published: June 2021

It has been shown that G protein‐coupled receptors may adopt multiple active states and that some agonists are able to distinguish between different active receptor states. This ability is described as the bias of an agonist for one signaling pathway over another. The recognition that this potential was available to agonists has led to a search for agonists that exhibit bias. As this search continues, it is likely that different degrees of bias will be uncovered. One example of this is extreme bias where an agonist produces the selective stimulation of a subset of signaling pathways while not stimulating other signaling pathways know to be downstream of the same receptor. Although this outcome would be expected as more biased ligands are discovered, the currently available methods of bias analysis are not able to accommodate extreme bias. For this reason, we have constructed a competitive mechanism of bias analysis. In the studies presented here, we employ our competitive method to analyze both simulated studies and experimental evidence from the kappa opioid receptor. Our results demonstrate that this method produces highly accurate estimates of the bias of several test ligands. From these studies, we conclude that our competitive method provides a useful tool for studying biased agonists. Specifically, this method is well suited for the specific subset of cases where an agonist exhibits extreme bias and for cases where the partial nature of an agonist in a system is not sufficient to achieve robust stimulation.Grant Funding Source: Supported by NIDA grant R01 DA031927(LMB)
Lorcaserin (Belviq®) Reduces the Discriminative Stimulus and Reinforcing Effects of Cocaine in Rhesus Monkeys
Authors: Collins Gregory , Gerak Lisa , France Charles 
Published: June 2021

Drug abuse and obesity are serious public health problems, and mounting evidence suggests that dopamine and serotonin systems are similarly involved in regulating the consumption of food and drugs. Lorcaserin, a serotonin 2C receptor agonist, was recently approved by the US FDA to help obese individuals lose weight and it might also be effective at reducing cocaine abuse. The current studies sought to (1) characterize the pharmacokinetic and behavioral profile of lorcaserin (I.G.), and (2) determine the effectiveness of lorcaserin to alter the discriminative stimulus and reinforcing effects of cocaine (I.V.) in rhesus monkeys. Administered acutely, lorcaserin dose‐dependently increased the occurrence of yawning and decreased spontaneous activity and operant responding for food. These effects occurred within 30‐60 min of administration and began to dissipate by 240 min, which closely matched plasma levels of lorcaserin. In monkeys discriminating cocaine from saline, lorcaserin alone did not increase cocaine‐appropriate responding. When administered prior to cocaine, lorcaserin shifted the cocaine dose‐response curve to the right and down. Lorcaserin also dose‐dependently decreased the rate of cocaine self‐administration with a dose of 3.2 mg/kg lorcaserin decreasing responding by 50% for the duration of the 14 day treatment. Together, these results provide clear evidence that lorcaserin effectively blunts the discriminative stimulus and reinforcing effects of cocaine following acute and repeated administration suggesting that it might have clinical utility for treating cocaine abuse.Supported by NIH/NIDA grants U01DA034992 and K05DA017918 (CPF).
CO‐ULTRAMICRONIZED PEALUT: A NEW THERAPEUTIC TREATMENT IN AUTISM SPECTRUM DISORDER
Authors: Crupi Rosalia , impellizzeri Daniela , Bruschetta Giuseppe , Cordaro Marika , Esposito Emanuela , Cuzzocrea Salvatore 
Published: June 2021

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by neurological deficits, especially as related to cognitive function. Brain inflammation can be a key element in the pathogenesis of neuropsychiatric disorders, such as ASD. N‐palmitoylethanolamide (PEA) is a parent molecule of ALIAmides, known for its anti‐inflammatory, analgesic and neuroprotective properties. Flavonoids, such as luteolin, possess neuroprotective actions in central nervous pathophysiological conditions. Association of PEA with Luteolin (co‐ultra PEALut) displays neuroprotective and anti‐inflammatory actions in CNS pathologies which are superior when compared to each molecule alone.Our goal was to evaluate the effects evoked by PEALut in the management of inflammatory events associated with ASD using established experimental model. A multidisciplinary approach was employed to study: behavioral, neuroinflammatory, neurogenesis and neuroplasticity alterations. On P14, C57BL/6 mice were injected with 400 mg/kg sodium valproate. Two different set of experiments were conducted. In the first, on P15 mice were administered with PEALut and on P30 the behavioral and neuroinflammatory studies were assessed. In the second, at P120, mice started treatment with PEALut (2 weeks) and then were sacrified for neurogenesis and neuroplasticity investigations. Our study showed that PEALut ameliorated aggressive behavior and improved spatial learning memory in the autistic mice. Moreover PEALut increases neurogenesis and neuroplasticity in mice hippocampus.
A Pre‐Assembled GPCR‐Signalosome Reveals Unappreciated Ligand Sensitivity and Mediates Unique Whole Cell Responses
Authors: Halls Michelle 
Published: June 2021

Cells endogenously express many different receptors that activate the same signaling pathways but with diverse physiological outcomes. This suggests considerable organization and regulation of intracellular signaling, achieved by the assembly of multimeric protein complexes with diverse functions. The use of high‐resolution technologies has opened a new window of sensitivity to measure sub‐cellular signal organization by G protein‐coupled receptors (GPCRs). We previously discovered a novel signaling complex (signalosome) organized around the relaxin receptor RXFP1 that facilitates responses to femtomolar (fM) relaxin (Halls & Cooper 2010 EMBO J). We now report that this is a previously undetected phenomenon common to many GPCRs including the prototypical β2‐adrenoceptor.We show that constitutive assembly of the β2‐signalosome is dependent on C‐terminal helix 8 and involves multiple signaling and scaffolding proteins. Activation by fM concentrations of ligand causes a sustained increase in cAMP, which is abolished following mutation of the orthosteric binding site. β2‐signalosome activation is dependent on constitutive receptor recycling, and prolonged stimulation increases receptors at the cell membrane and decreases trafficking to lysosomes, suggesting a role in sensing the extracellular environment. In addition to sustained cAMP, activation of the β2‐signalosome generates sustained nuclear ERK, increases global gene transcription and has unique effects at the proteomic level.The discovery of GPCR‐signalosomes that respond to very low concentrations of ligand has immediate relevance to our current understanding of GPCR function, and opens a new window for the therapeutic targeting of GPCRs.
Delavirdine is Substrate of Bcrp and P‐gp
Authors: Kefelegn Adey , Bednarczyk Dallas 
Published: June 2021

IntroductionDelavirdine is an antiretroviral drug characterized by a brain/plasma pharmacokinetic disequilibrium where the cerebrospinal fluid concentration, a surrogate for unbound brain concentration, in humans is approximately 5‐fold lower than corresponding unbound plasma concentrations (Rescriptor package insert). A previous report using Caco‐2 cells suggested that delavirdine is not a P‐gp substrate (Störmer, et al., 2002). Breast cancer resistance protein (Bcrp) was therefore investigated as a mechanism that could limit penetration of delavirdine into the CNS.Material and MethodsMDCK‐mBcrp and MDCK‐MDR1 cells were used to assess transport of delavirdine by each respective transporter. The bidirectional permeability of delavirdine was investigated as a function of time and in the absence or presence of inhibitor. Assay samples were then subject to liquid chromatography/mass spectrometry.ResultsDelavirdine demonstrated asymmetric flux by the MDCK‐mBcrp cells that was time dependent. Interestingly, time dependent asymmetric flux of delavirdine was also observed in the MDCK‐MDR1 cells. The asymmetric flux of delavirdine in both cell lines could further be brought to near unity in the presence of inhibitors of Bcrp and MDR1 (Ko143 and LY335979, respectively).ConclusionThe results established the asymmetric transport of delavirdine by P‐gp and mBcrp. The asymmetric transport of delavirdine by P‐gp and mBcrp was time dependent and inhibitable. Collectively, both transporters likely limit the CNS penetration of delavirdine observed in vivo.Störmer E, et al. Differential modulation of P‐glycoprotein expression and activity by non‐nucleoside HIV‐1 reverse transcriptase inhibitors in cell culture. Pharm Res. 19(7):1038‐45, 2002.
The Selection and Assessment of a Low‐Efflux MDCK Cell Line for use in a High‐Throughput Permeability Screening Assay
Authors: Boiselle Carri , Bednarczyk Dallas 
Published: June 2021

ObjectivesTo establish a low‐efflux Madin‐Darby canine kidney cell line for potential use in a high throughput permeability screening assay.MethodsMDCK cells were subcloned by limiting dilution. Individual MDCK colonies were isolated and clones were expanded to cell culture plates and assessed for efflux potential. The permeability characteristics and corresponding efflux ratios were measured using a standard bidirectional permeability assay conducted using 96‐well permeable support plates.Results72 clones were initially screened for efflux potential. 8 clones demonstrating the lowest level of efflux were further cultured for continued evaluation. The 8 clones were further reduced in number to 2 by comparison of efflux between clones, where clones demonstrating the lowest efflux were maintained. One clone, whose efflux did not increase with longitudinal culture time, was selected for use in a high‐throughput permeability screening assay. The final clone showed limited efflux of common P‐gp substrates: loperamide, quinidine, dipyridamole; BCRP substrates: prazosin, topotecan, imatinib; and an MRP‐2 substrate: valsartan. Despite the lack of transport of multiple probe substrates efflux was observed for some compounds such as vinblastine and saquinavir. The final clone was further tested using a diverse set of molecules.ConclusionsA low efflux MDCK subclone (MDCK‐LE) was identified. The MDCK‐LE cells demonstrate low efflux for many representative substrates of efflux transporters, though the cells are not entirely a “no efflux” cell line as demonstrated by the efflux of vinblastine and saquinavir, they do delineate the permeability of structurally diverse molecules over a broad permeability range.
Pharmacogenetic Analysis of Nucleotide Kinases that Metabolize the Antiviral Drug Tenofovir
Authors: Lade Julie , Hendrix Craig , Bumpus Namandjé 
Published: June 2021

Tenofovir (TFV) is a nucleotide analog used in HIV pre‐exposure prophylaxis, requiring two phosphorylation events for pharmacological activity. We aimed to determine the nucleotide kinases responsible for TFV activation in peripheral blood mononuclear cells (PBMC), vaginal tissue and colorectal tissue as this is currently unknown. In order to identify relevant kinases, siRNA knockdown was performed in PBMC and tissues, incubated with 10µM TFV and metabolites were measured using mass spectrometry. Adenylate kinase 2 (AK2) knockdown decreased TFV‐monophosphate production to 17 ± 1.4%, 9.5 ± 1.1% and 12 ± 1.5% of control in PBMC, vaginal tissue and colorectal tissue, respectively. Knockdown of muscle pyruvate kinase (PKM) decreased TFV‐diphosphate (TFV‐DP) production to 33 ± 5.8% and 27 ± 4.3% and knockdown of liver and red blood cell pyruvate kinase (PKLR) decreased TFV‐DP production to 78 ± 6.6% and 81 ± 7.4% of control in PBMC and vaginal tissue, respectively. Muscle creatine kinase (CKM) knockdown in colorectal tissue decreased TFV‐DP production to 8 ± 2.9% of control. Subsequently, clinical trial participants taking oral TFV were genotyped for variants in these 4 kinases and results were compared to pharmacokinetic data. Of the 148 previously unreported single nucleotide variants and deletions identified, bioinformatics tools predicted 10 AK2 variants (8% of 142 participants), 12 PKLR variants (7%) and 8 CKM variants (6%) to be deleterious. PKM variants were of unknown consequence. In conclusion, these data demonstrate AK2 to be a key contributor to TFV activation and genetic variation within the kinases AK2, PKM, PKLR and CKM may have a potential impact on TFV pharmacokinetics.Funding: R01 GM103853, UM1 AI068613 and UM1 AI106707
Prediction of the Magnitude of Induction of CYP3A Activity during Early Human Pregnancy
Authors: Zhang Zufei , Farooq Muhammad , Grepper Sue , Unadkat Jashvant 
Published: June 2021

BackgroundIn pregnant women, CYP3A activity increases by ~2 fold during the third trimester (T3) [1]. Due to logistical and ethical constraints, the magnitude of CYP3A induction during the first and second trimesters (T1 and T2, respectively) is unknown. We have shown that the combination of five pregnancy‐related hormones (PRHs), cortisol(C), estradiol (E2), progesterone (P), growth hormone (GH), and placental growth hormone (PGH), at their observed T3 plasma concentrations, induced CYP3A activity in human hepatocytes to an extent comparable to that observed in vivo. Amongst these PRHs, only C consistently and significantly induced CYP3A activity [2].ObjectivesUsing human hepatocytes: 1) confirm C is the major CYP3A inducer during T1 and T2; 2) predict the magnitude of induction of CYP3A activity during T1 and T2 .MethodsSandwich cultured human hepatocytes from 3 premenopausal donors were incubated for 72h with 1X or 10X T1, T2, or T3 plasma concentrations of PRHs (adjusted for depletion). CYP3A activity was measured by 1'‐OH‐midazolam formation.ResultsAt 1X T1, T2, or T3 concentrations: 1) C consistently and significantly induced CYP3A activity; 2) induction of CYP3A activity by C or PRHs was not significantly different.ConclusionsC is the major inducer of CYP3A activity during T1‐T3. Based on these data, we predict that the magnitude of CYP3A induction during T1 and T2 will be similar to that observed during T3. This prediction is consistent with the similar increase in oral clearance of indinavir (CYP3A cleared drug) observed during T2 and T3 [3].AcknowledgementSupported by NIH grant P01DA032507
Differential Regulation of Adaptive and Innate Immunity activated Human Mast Cell by Osteopontin
Authors: Ng Chun Wai , Lau Hang Yung Alaster 
Published: June 2021

Osteopontin (OPN), a RGD‐containing protein which has been shown to enhance inflammatory responses by modulating different functions of different cell types such as macrophages, dendritic cells and natural killer cells. However, OPN has also been shown to suppress inflammatory responses and is important in the healing process. To understand more about OPN, anti‐IgE and PGN activated human mast cells developed from human CD34+ stem cells were employed to study the role of this protein in the inflammatory responses. The release of histamine and cytokines (IL‐5, IL‐8 and TNF‐α) releases as well as cell adhesion were studied by spectro‐fluorescence assay, ELISA and CyQUANT assay respectively. OPN induced adhesion of human mast cells after anti‐IgE and PGN stimulation, and the adhesion was able to inhibit anti‐IgE‐induced IL‐5, IL‐8 and TNF‐α release while only PGN‐induced IL‐5 was enhanced by OPN. To investigate the interaction between OPN and human mast cells, cyclic RGD (cRGD) and neutralizing antibodies against integrin αVβ3 and CD44 were employed. cRGD was able to inhibit both anti‐IgE and PGN‐activated adhesion and adhesion‐mediated regulation of cytokines release. While only antibody against Integrin αVβ3 was able to reverse the anti‐IgE‐induced adhesion and the inhibition of anti‐IgE‐mediated cytokines release by OPN. In conclusion, OPN suppresses anti‐IgE‐induced inflammatory response by inhibiting the synthesis of chemokine (IL‐8) and both Th1 (TNF‐α) and Th2 (IL‐5) cytokine and the interaction is through the RGD domain in OPN and integrin αVβ3 expressed on human mast cells. On the other hand, OPN enhances Th2 (IL‐5) synthesis when human mast cells are stimulated by PGN.Research Support: RGC Grant 469310
Discovery of a Novel Enhydrazinone Ester (CEE‐1) That Inhibits the Degranulation of Both Mast Cells and Eosinophils
Authors: Ezeamuzie Charles , Edafiogho Ivan 
Published: June 2021

Allergen‐dependent activation of mast cells and eosinophils is a fundamental process in the pathophysiology of allergic diseases. We have previously reported that the novel enhydrazinone ester CEE‐1 (ethyl 4‐phenylhydrazinocyclohex‐3‐en‐2‐oxo‐6‐phenyl‐1‐oate) – possess potent anti‐inflammatory activity. We have now tested whether the compound also possess anti‐allergic effects using IgE‐dependent degranulation of rat mast cell line (RBL‐2H3) and the degranulation of isolated human blood eosinophils. RBL‐2H3 cells were passively sensitized with anti‐DNP IgE, pre‐treated with CEE‐1 for 10 min before being stimulated with the antigen, DNP‐BSA. Released β‐hexoseaminidase was determined as index of degranulation. Aliquots of purified human eosinophils, were similarly pre‐treated with CEE‐1 before stimulation with human C5a (30 nM) plus cytochalasin B (5 µg/ml) for 30 min. The amount of released granular eosinophil peroxidase (EPO) was determined by the O‐phenylenediamine method. Results showed that pre‐treatment of sensitized mast cells with CEE‐1 resulted in a strong concentration‐dependent inhibition of β‐hexoseaminidase release. The IC50 value was 4.5 µM, and at 30 μM inhibition was more than 80%. The compound also inhibited C5a‐stimulated EPO release from human eosinophils with even greater potency (IC50 = 0.1 μM). These results show that CEE‐1 possesses strong anti‐allergic properties exerted on both mast cells and eosinophils, and therefore has the potential to be a useful therapy for allergic diseases.Sponsored by Research Sector, Kuwait University, Grant # MR 03/09.
Adiponectin Modulates NF‐κB Mediated Cardioprotective Pathways after Acute High‐fat Feeding.
Authors: Haar Lauren , Luther Kristin , Rubinstein Jack , Ren Xiaoping , Jones WK 
Published: June 2021

Previous studies indicate that the consumption of a high fat diet (HFD) 24h‐2wks prior to the experience of ischemic injury can decrease the size of the infarcted area (normalized to the area at risk) by as much as 70% relative to control fed (CFD) animals.This protective effect is NF‐κB dependent and has also been shown to work via an autophagic priming mechanism to confer protection. The current study will address the hypothesis that adiponectin activates NF‐κB mediated signaling that confers a high fat induced protective effect.qRT‐PCR analysis of cardiac tissue from HFD animals indicated alterations in gene profiles associated with NF‐κB mediated carioprotection (HSP70, HIF1a, COX2). Cell experiments indicate that globular adiponectin activates NF‐kB activity (promoter luciferase‐based marker assay), and that siRNA knockdown of Adiponectin receptors reduces this activity. Serum levels of adiponectin were evaluated from high fat fed animals at cardioprotective time points, and show alterations in adiponectin and leptin levels that correspond to protective timelines. Finally, Adiponectin KO animals were HFD fed and assessed for response to ischemia/reperfusion injury. 24h HFD and CFD animals showed no change in infarct size after reversible 30m LAD occlusion. Ultimately this study provides a new hormonal target that may an upstream effecter of NF‐κB mediated protection induced by high fat diet. Better understanding this relationship will lead to the development of diet based therapeutics for cardiac pathology and new targets for the treatment of myocardial ischemic injury.
Cardiac Tissue Sensitization to the Sympathetic Nervous System in Renal Ischemia/Reperfusion induced Cardiac Hypertrophy
Authors: Panico Karine , Alvim Juliana , Carneiro‐Ramos Marcela 
Published: June 2021

ObjectivesHeart and kidney share functions of the conservation of homeostasis. Acute kidney injury occurs by an abrupt decline in renal function and can lead to the development of cardiac hypertrophy (CH), among other ways, by catecholamines release into circulation. This study aims to investigate possible modulation of sympathetic nervous system (SNS) in CH development through the cardiac beta‐adrenergic receptors pathway in CH‐induced ischemic renal injury model.MethodsC57BL/6 male mice (n=88) were subjected to surgical occlusion of left renal pedicle for 60min followed for reperfusion (REP) for 12 and 15 days. Another group of animals were treated with Propranolol (10 mg/kg/day) for 5 days after occlusion. Morphological and molecular parameters were used to evaluate kidney and CH status.ResultsRenal ischemia/reperfusion was able to induce CH after 12d. The left kidney mass was reduced (p<0.05) and an increase on Vimentin and urea levels were observed (p<0.001) and (p<0.05), respectively. Beta‐adrenergic receptors increased after 12days when compared to Sham group (p<0.05). Treatment with SNS inhibitor was able to improve the reduction of the left kidney mass.ConclusionCH was accompanied by an increase in levels of beta‐adrenergic receptor, suggesting an increase in cardiac sensitivity to SNS.
No Thermal Effect of GSM Electromagnetic Waves Exposure on Slow‐Type Skeletal Muscles in Female Rats
Authors: Ramadan Wiam , Darwich Walaa , El‐Dirani Zeinab , Khachfe Hassan , Moustafa ElSayed Mohammed , Estève Eric , H. Joumaa Wissam 
Published: June 2021

The concern from the increase of population exposure to electromagnetic fields from various sources has been addressed by the World Health Organization and other international organizations and institutions. The study of the effect of GSM‐signals on mammalian skeletal muscle is encouraging because muscles are extremely plastic and are capable of modifying their structure in coordination with the activity they develop. Our results show that 6 weeks of continuous exposure to GSM electromagnetic waves (900 MHz, Eeff= 25V/m) for 24 hours a day induce an increase by 15% in muscle mass. Such an increase in muscle mass observed could be related to modification in water content and/or in the proteins synthesis. In fact, 6 weeks of continuous electromagnetic waves exposure induces an increase by 42% and 32% in percentage of water content and were observed respectively. Moreover, no significant effect was observed in total testosterone level after GSM wave's exposure period (control: 2.01 ± 0.28 ng/ml, Exposed: 2.12 ± 0.31 ng/ml, n=20).These results suggest that continuous exposure to GSM relay antenna can induce a morphological modification in slow‐ type skeletal muscle of female rats which could have an effect on functional characteristics as well as Excitation‐Contraction Coupling mechanism.