bluuebunny/crossref_metadata_2025_split huggingface dataset:
Drug discovery of CLK1 inhibitors and their activity evaluation as anti‐influenza viral agents (654.11)
Authors: Liu Ai‐Lin , Du Guan‐Hua 
Published: June 2021

High mutation rates facilitate the generation of influenza viral escape mutants, rendering vaccines and drugs directed against virus‐encoded targets potentially ineffective, while drugs targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. CDC‐like kinase 1 (CLK1) in human host cells as the crucial protein in influenza A virus replication process, is expected to become a new target for anti‐influenza drugs. In this study, we expressed CLK1 protein, established CLK1 activity assay, carried out drug screening for CLK1 inhibitors combined with virtual screening, and demonstrated their anti‐influenza viral activities.The recombinant His6‐CLK1 protein was expressed in Sf9 cells by the Baculovirus Expression System. His6‐CLK1 inhibitor screening model was established by Kinase‐Glo assay. 80 compounds selected by structure‐based virtual screening were evaluated for their CLK1 inhibitory activities and their in vitro anti‐influenza virus activities were evaluated. Finally, the in vivo anti‐influenza effect of the strongest CLK1 inhibitor was evaluated using mice model.The in vitro experiment results showed that J10688 displayed both significant CLK1 inhibitory activity with IC50 value of 2.71uM and cytopathic effect reduction with EC50 value of 9.1 uM. The in vivo experiment results indicated that J10688 could notably increase the survival rate of the mice infected by influenza virus A/PR/8/34(H1N1) with at the dosages 10 mg/kg and 30 mg/kg, which has the same survival rate with oseltamivir at the dosage 10 mg/kg.In conclusion, the CLK1 may be a potential drug target for anti‐influenza drug discovery. The in vitro and in vivo activities of J10688 will lay important experiment foundation for further research and development.
SIRT1 upregulators from high‐throughput screening as anti‐proliferation and anti‐migration agents in vascular smooth muscle cells (654.2)
Authors: Li Li , Zhao Gang , Yang Hai‐Guang , Cao Qi‐Zhi , Gao Peng , Chen Hou‐zao , Zhang Zhu‐qin , Liu De‐Pei , Du Guan‐Hua 
Published: June 2021

Objective: To establish a cell‐based drug screening system for discovering up‐regulators of SIRT1 and confirmed their biological activities in rat aortic VSMCs. Methods and Results: A stable transfected with a human SIRT1‐promoter‐luciferase reporter gene construct cell line was obtained in 293A cells. Using Resveratrol as a positive control, a cell‐based SIRT1 up‐regulator high‐throughput screening (HTS) model was established in a 384‐well microplate format. Of 27,274 compounds, 86 compounds were identified to up‐regulate SIRT1 transcriptional activity in the cell‐based reporter assay. Furthermore, 10 compounds including a series of Resveratrol analogues showing low toxic in both VSMCs and HUVECs were demonstrated to not only stimulate the expression of SIRT1 on the transcriptional level in VSMCs, but also have anti‐proliferation and anti‐migration properties in PDGF‐BB (40 ng/ml)‐stimulated VSMCs. In addition, several natural compounds (Baicalin, Genistein, Chrysin, etc.) were identified to elevate SIRT1 transcriptional level, with the reported property of attenuating neointima formation following injury via suppression of VSMC proliferation, migration or both. Conclusion: A cell‐based drug screening model was established to screen SIRT1 up‐regulators. The active compounds originated from this HTS assay may be developed to drug candidates or lead compounds for new anti‐restenosis drugs.Grant Funding Source: This work was supported by the National Natural Science Foundation of China (No. 81102444).
A novel, potent, orally‐bioavailable nociceptin‐1 receptor antagonist reduces ethanol self‐administration and ethanol‐seeking in animal models of alcohol addiction (656.2)
Authors: Rorick‐Kehn Linda , Ciccocioppo Roberto , Adams Benjamin , Katner Jason , Perry Kenneth , Witkin Jeffrey , Toledo Miguel , Diaz Nuria , Lafuente Celia , Jimenez Alma , Benito Ana , Martinez‐Grau Maria , Pedregal Concepcion , Statnick Michael 
Published: June 2021

Background: The receptor for nociceptin/orphanin‐FQ (NOP) is localized in the mesolimbic reward pathway and has been suggested to play a role in addiction and motivated behavior. We characterize here a novel, potent, orally‐bioavailable NOP antagonist in several ethanol self‐administration models.Methods: Compound 1 was evaluated in homecage ethanol self‐administration models, progressive ratio operant self‐administration, stress‐induced reinstatement to ethanol‐seeking, and in vivo microdialysis.Results: Compound 1 dose‐dependently reduced homecage ethanol self‐administration in both P and msP rats, without affecting food or water intake. Compound 1 also attenuated progressive ratio operant responding and breakpoints in P rats. Stress‐induced reinstatement of ethanol‐seeking in msP rats was completely blocked by compound 1. Furthermore, compound 1 blocked ethanol‐induced dopamine release in the nucleus accumbens.Conclusions: Compound 1 demonstrates efficacy in attenuating ethanol self‐administration and stress‐induced ethanol‐seeking in two different lines of rats that exhibit excessive ethanol consumption. Compound 1 also blocks the motivation to consume ethanol and ethanol‐stimulated increases in extracellular dopamine levels in the nucleus accumbens, suggesting potential therapeutic utility of NOP receptor antagonists in treating alcohol addiction.
β‐Cyclodextrin‐complexed carvacrol produces antinociceptive effect superior to that of carvacrol in orofacial pain models (657.15)
Authors: Barreto Rosana , Guimarães Adriana , Menezes Paula , Araújo Adriano , Quintans Jullyana , Barreto André , Guedes Renan , Quintans‐Junior Lucindo 
Published: June 2021

The aim of this study was to evaluate the antinociceptive effect of carvacrol/ β‐cyclodextrin complex (CARV/ β‐CD) and carvacrol (CARV) isolated, a monoterpene phenol, in formalin‐, capsaicin‐ and glutamate‐induced orofacial nociception on mice. Male mice were pretreated with CARV/ β‐CD (10 or 20 mg/kg, p.o.),CARV (10 or 20 mg/kg, p.o.), morphine (5 mg/kg, i.p.) or vehicle (distilled water), 1h before painful orofacial test (‐formalin (20 μl, 2%), ‐capsaicin (20 μl, 2.5 µg) or ‐glutamate (40 ìl, 25 ìM) induced by injection into the right upper lip). Experimental protocols were approved by the Animal Care and Use Committee at the Federal University of Sergipe (CEPA/UFS # 18/10). Our results demonstrated that acute treatment with complex CARV/ β‐CD was effective in reducing nociceptive face‐rubbing behavior in both phases on formalin test (p<0.01 and p<0.01or p<0.001) whereas isolated CARV produced similar effect did so only in higher dose (p<0.05) in second phase. Also produced an improvement significant antinociceptive effect at all doses in the capsaicin‐ (p<0.001or p<0.001) and glutamate‐ (p<0.01 or p<0.001) tests when compared with isolated monoterpene (CARV). Such results were unlikely to be provoked by motor abnormality. Our results provide evidence to propose that the complex with β‐CD improve analgesic profile of carvacrol. CARV/ β‐ CD might represent important tool for management of orofacial painful disorders.Grant Funding Source: FAPITEC/SE; CNPQ (Brazil).
μ opioid receptor activity is regulated by endothelin converting enzyme‐2 (659.5)
Authors: Bobeck Erin , Gupta Achla , Fujita Wakako , Gomes Ivone , Devi Lakshmi 
Published: June 2021

Regulation of neuropeptide receptors via endocytosis and recycling has been studied extensively, however very few studies have evaluated the fate of the internalized neuropeptide. It is generally thought that the internalized peptide is hydrolyzed allowing for recycling of the receptor. Previously we have shown that endothelin‐converting enzyme‐2 (ECE‐2), a member of the neprilysin family of metallopeptidases, is important for opioid peptide processing in vitro and opioid actions in vivo. In the current study, we directly examined the involvement of ECE‐2 in modulating µ opioid receptor (µOR) recycling and analgesia using a selective ECE‐2 inhibitor. In cells stably expressing µORs and ECE‐2, receptor endocytosis is not altered by inhibition of ECE‐2, but recycling is significantly impaired. Recycling was only blocked following administration of opioid peptides (i.e. dynorphin B) which are substrates of ECE‐2, but not for other opioid peptides (i.e. Leu‐enkephalin, dynorphin A8). Similar results were found in dorsal root ganglion cells that endogenously express µORs and ECE‐2. Finally, we show that ECE‐2 inhibition decreases stress‐induced analgesia in wild‐type mice. Interestingly, this is not seen in animals lacking δOR suggesting the involvement of µOR in this process. Taken together these data support a role for ECE‐2 in modulating the endogenous opioid peptide/receptor system.Grant Funding Source: Supported by NS 26880
Effects of nicotinic acetylcholine receptor positive allosteric modulator in a neuropathic pain model (661.10)
Authors: Rahman Shafiqur , Abbas Muzaffar 
Published: June 2021

Neuropathic pain is a chronic neurological disorder characterized by hyperalgesia and allodynia. Recent evidence indicates that activation of immune system plays a critical role in the pathophysiology of neuropathic pain. Moreover, pro‐inflammatory cytokines, including TNF‐alpha has a significant role in initiation and maintenance of neuropathic pain involving alpha7 nicotinic acetylcholine receptors (nAChRs). The objective of the present study was to determine the effect of TQS, an alpha7 nAChR positive allosteric modulator, on lipopolysaccharide (LPS)‐induced neuropathic pain using hot plate, a widely used technique for hyperalgesia assessment in male mice. In addition, we have investigated the effect of TQS (4 mg/kg, i.p) on tumor necrosis factor‐α (TNFα) using ELISA. Acute administration of LPS (1.0 mg/kg, i.p.) increased (p<0.05) the sensitivity to pain (hyperalgesia) after 24 hours. Pretreatment of TQS (1 or 4 mg/kg, i.p.) decreased the LPS induced hyperalgesic response in a dose‐dependent manner. The effect with TQS (4 mg/kg, i.p) was significantly different from control (p<0.01). TQS (4 mg/kg, i.p) also reduced TNFα level in serum. These results suggest that TQS reduces LPS‐induced neuropathic pain by targeting alpha7 nAChR allosteric site and associated nicotinic cholinergic mechanisms.Grant Funding Source: Supported by Fulbright Foundation USA
Optogenetic and pharmacologic dissection of G‐protein and arrestin dependent signaling mechanisms of β2 adrenergic receptors (662.3)
Authors: Siuda Edward , Bruchas Michael 
Published: June 2021

β‐adrenergic receptors (βARs) are GPCRs that signal in a canonical Gαs‐mediated fashion. It is also clear that βARs signal independently of G‐proteins in a manner referred to as functional selectivity. While βARs activate downstream effectors through G‐protein‐dependent and independent mechanisms, it is unclear whether βAR functional selectivity occurs in the CNS in endogenous systems, and what role this plays in behavior. Here we report that recruitment of noradrenergic inputs and activation of β2‐adrenergic signaling in the basolateral amygdala (BLA) induces anxiety‐like behavior in mice. However, it is unclear how signaling to G‐proteins, or arrestin alters downstream behavioral phenotypes within the BLA complex in affective behaviors. To elucidate the roles of G‐protein and arrestin dependent intracellular signaling we first developed novel “functionally selective” optically sensitive GPCRs. We then used pharmacological methods and in vitro optogenetic techniques to characterize the temporal kinetics of cAMP and MAPK activity. Finally, we combined behavioral pharmacology and in vivo optogenetic techniques to express cell‐type‐specific, optically sensitive, wild type and functionally selective mutants to examine the effects of G‐protein and arrestin dependent signaling on anxiety behavior. Together, these tools and findings have use in dissecting neural circuits in anxiety, and the role of biased GPCR signaling in vivo.Grant Funding Source: Supported by R21‐DA035144, EUREKA Grant R01‐DA037152, TR01‐NS081707‐01; NIDA (MRB)
Exogenous diacylglycerol restores arteriolar myogenic constriction following candesartan (664.10)
Authors: Hong Kwangseok , Meininger Gerald , Hill Michael 
Published: June 2021

Growing evidence supports the Gq/11 protein‐coupled angiotensin II type I receptor (AT1R) acting as a mechanosensor and contributing to pressure‐induced vasoconstriction of small arteries. While mechanical activation of the AT1R does not involve its classical agonist, angiotensin II, the exact signaling mechanisms underlying this mode of activation are yet to be delineated. Our recent studies have shown that pressure‐induced changes in global smooth muscle [Ca2+]I are similar in the presence or absence of the AT1R inverse agonist, candesartan, implicating involvement of other second messenger‐mediated pathways. The aim of the present study was to further consider the generation of second messengers in response to AT1R activation and their role in myogenic vasoconstriction. Specifically, the diacylglycerol analog 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) was applied to isolated and pressurized rat cremaster arterioles in the presence and absence of candesartan. In the absence of candesartan OAG appeared to potentiate pressure‐induced vasoconstriction (e.g. control vs. OAG 10 µM; 44.2 ± 1.2 vs. 40.2 ± 1.5 µm). Candesartan (10 µM) alone showed significant inhibitory effects on steady‐state myogenic tone (control vs. candesartan at 70 mmHg; 50.3 ± 3.8 vs. 76.7 ± 8.2 µm; p < .05). This inhibition of myogenic reactivity was reversed in the presence of exogenous OAG (control vs. OAG 10 µM + candesartan at intraluminal pressures over the range 50 ‒ 110 mmHg; p > .05). Collectively, the data are consistent with mechanical activation of the AT1R resulting in generation of diacylglycerol and vasoconstriction that may be related to PKC‐dependent Ca2+ sensitization or actin polymerization for pressure‐induced vasoconstriction.Grant Funding Source: Supported by NIH HL 092241 (MAH), HL 095486 (GAM)
Early involvement of oxidative stress in the progression of chronic kidney disease induced by an acute kidney injury episode (667.6)
Authors: Rodríguez Roxana , Benítez Kenia , Barrera Jonatán , Pérez Rosalba , Rocha Leslie , Gamba Gerardo , Bobadilla Norma A 
Published: June 2021

Recently, we showed that CKD induced by an AKI episode in the rat is associated with podocyte injury and aberrant tubular epithelium proliferation. This study was designed to evaluate the temporal influence of oxidative stress in this model of renal injury. Forty‐seven Wistar male rats that were divided into two groups: sham operated (S) and rats underwent to renal bilateral ischemia for 45 minutes (I). The animals were followed since 1 to 4 months after ischemia and at least six rats were studied and sacrificed each 30 days. At the end of each experimental period, mean arterial pressure (MAP), renal blood flow (RBF) and creatinine clearance (CCr), as well as, urinary protein (UProt) and peroxide excretion (UH2O2) were measured.Rats underwent to ischemia exhibited a significant increase in UProt that returned to normal values after 1 month, then, a progressive increment was observed, reaching a significant difference after 4 months. In spite of the absence in MAP or renal function alterations a significant increase in UH2O2 was observed since 24‐h after ischemia and the values did not return to basal level along the study.Our results suggest that oxidative stress contributes to renal progression injury since a very early stage and pointed out the importance of studying the effect of anti‐oxidants just when ischemic insult has happened and determining their impact in the progression to CKD.Grant Funding Source: National council of science and technology of Mexico. grant to NAB 181267
Recording of intracranial pressure in conscious rats via telemetry (668.2)
Authors: Guild Sarah‐Jane , Budgett David , Malpas Simon 
Published: June 2021

Measurement of intracranial pressure (ICP) is a critical diagnostic measure in treating head trauma, intracerebral hemorrhage and hydrocephalus. The pressure values are generally between 5‐15 mmHg with >20 mmHg considered abnormal. There is a scarcity of animal models of chronic ICP measurement in part associated with the difficulty of measuring ICP chronically with good accuracy. Previous telemetry devices utilizing fluid filled catheters have proved problematic due to movement artefacts. The purpose of this study was to develop the methodology to record ICP in the conscious rat using a solid state pressure sensor by telemetry.We implanted TRM54PP telemetry devices which incorporate solid state 2 Fr (0.6 mm) sensors in rats. Arterial pressure (AP) was measured via the abdominal aorta and the other pressure catheter (25cm long) was tunnelled subcutaneously to the skull and inserted thru the dura to lie against the brain tissue for the measurement of ICP.All rats appeared healthy and recovered well from surgery. ICP and AP were successfully recorded in conscious rats for up to 4 weeks post‐surgery. When rats were resting quietly, a strong respiratory rhythm was seen in both signals. Postural changes during head up and head down tilt tests resulted in reversible decreases and increases of ICP as expected. No visible damaged from the ICP catheter was found on the brain surface at post‐mortem. The stability and fidelity of the signals were excellent over time. In a second group of animals, basal cistern injection of kaolin was used to increase ICP as a model of communicating hydrocephalus.Grant Funding Source: Supported by the Neurological Foundation of New Zealand
Monocytes/macrophages are recruited from post‐capillary venules during microvascular arteriogenesis (670.7)
Authors: Bruce Anthony , Kelly‐Goss Molly , Meisner Joshua , Price Richard , Peirce Shayn 
Published: June 2021

It has previously been shown that circulating monocytes are essential to arteriogenesis in collateral arteries/arterioles following elevated shear stress induced by occlusion. Once recruited to the stimulated collateral vessel, these cells promote smooth muscle cell and endothelial cell proliferation through secreted cytokines. However, questions remain regarding the route by which circulating monocytes extravasate (egress from arteriole vs. venule) during microvascular arteriogenesis. We utilize confocal microscopy to evaluate recruitment of CX3CR1‐GFP positive monocytes/macrophages to collateral arterioles and post‐capillary venules at multiple time points following feeder arteriole ligation in the mouse spinotrapezius. We find arterioles and venules in ligated muscles to have significantly increased CX3CR1‐GFP cell recruitment compared to sham‐operated muscles. However, these increases occur at different times depending on microvessel type. We document a shift in the position of CX3CR1‐GFP high cells from venule‐associated to arteriole‐associated over the course of three days. We find that accumulation of adhered monocytes occurs only in venular lumina for all time points examined. Our results indicate that monocytes recruited during microvascular arteriogenesis extravasate from post‐capillary venules and that macrophages recruited to remodeling collateral arterioles express high levels of CX3CR1.Grant Funding Source: NIH‐HL082838‐02
Constitutive activation of α‐adrenoreceptors with advanced age impairs rapid onset vasodilation: key role for feed arteries (674.6)
Authors: Sinkler Shenghua , Segal Steven 
Published: June 2021

Rapid onset vasodilation (ROV) is depressed in second‐order arterioles of the mouse gluteus maximus muscle (GM) during advanced age (PMC2911225). However the extent to which aging impairs ROV is unknown. Therefore, we tested the hypothesis that advanced age attenuates ROV throughout microvascular resistance networks. In Young and Old male C57BL/6 mice (4 and 24 mo; n=6/group), the inferior gluteal motor nerve was stimulated (0.1ms, 30V, 100 Hz) to evoke single tetanic contractions. Within 1 s following 500 ms contraction, ROV was manifested in first (1A)‐, second (2A)‐ and third (3A)‐order arterioles and ascended into feed arteries (FA); peak dilations occurred at ~ 4s in all branches. Thus ROV was coordinated both temporally and spatially. Relative to respective maximal diameters, peak dilation increased with vessel branch order (P<0.05) yet was attenuated consistently (P<0.05) in Old vs. Young: FA (25±3 vs. 34±5%) < 1A (46±7 vs. 64±4%) < 2A (61±7 vs. 72±5%) < 3A (63±4 vs. 77±6%). Inhibition of α‐adrenoreceptors (αAR) with phentolamine (1 µM) improved dilations in Old (P<0.05) an additional 20±5% in FA, 7±2% in 1A, 16±3% in 2A and 12±3% in 3A. Thus, constitutive activation of αAR with advanced age impairs ROV with relatively greatest effect in FA. This consequence of advanced age will restrict total muscle blood flow upon the onset of muscle contraction and can thereby impede transitioning from rest to physical activity.Grant Funding Source: Supported by NIH R01 HL086483
Aging increases the amplitude of inward‐rectifier K+ channel currents in murine resistance artery smooth muscle cells (677.8)
Authors: Hayoz Sebastien , Pettis Jessica , Bradley Vanessa , Segal Steven , Jackson William 
Published: June 2021

Elevating extracellular K+ ([K+]o) dilates resistance arteries by activating smooth muscle cell (SMC) inward‐rectifier K+ channels (KIR). The rise in [K+]o of exercising skeletal muscle can thereby contribute to functional hyperemia. As muscle blood flow is diminished with aging, we tested the hypotheses that aging impairs K+‐induced activation of KIR in SMCs of resistance arteries. Superior epigastric arteries (SEAs) were dissected from abdominal skeletal muscle of Young (Y; 蠅3 mo) or Old (O; 蠅24 mo) male C57BL/6 mice and either dissociated to yield SMCs for perforated patch clamp recording or cannulated and pressurized for myography. Elevation of [K+]o from 5 to 15 mM activated Ba2+ (100 μM)‐sensitive inward K+ currents between ‐120 and ‐60 mV that were smaller in SMCs from Y than in O: at ‐120 mV, KIR current density was ‐0.2±0.3 pA/pF in Y vs ‐0.8±0.2 pA/pF in O; n=26; p<0.05. In contrast, elevating [K+]o from 5 to 15 mM evoked similar dilation in SEAs of Y (61±6%; n=12) and O (60±7%; n=13); responses in both were abolished by 100 μM [Ba2+]o (n=5). Thus, aging does not impair KIR currents in SMCs of resistance arteries and their dilation via KIR activation is maintained. These findings indicate that compensatory adaptations with aging maintain SEA function in the presence of increased KIR activity.Grant Funding Source: Supported by NIH R01 HL086483 and R25 HL103156