bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro determination of bioactive fraction of blueberry for type 2 diabetes prevention (647.17)
Authors: Kang Bouhee , Racicot Kenneth , Pilkenton Sarah , Apostolidis Emmanouil
Published: June 2021

The present study investigates the carbohydrate hydrolysis enzyme inhibitory activity of blueberries and if this activity is proanthocaynidin (PAC) or low molecular weight phenolic (LMP) dependent. Freeze‐dried blueberry powder was extracted using 70% acetone solution (BAE) and in deionized water (BWE). Sugars were removed from both extracts using C18 cartridges. BAE resulted to higher PAC and total phenolic content (TPC), so BAE was used for the remaining experiment. LMP and PAC were separated from BAE using a LH‐20 column by washing using 30%methanol solution (LMP recovery) and eluting using 70% acetone solution (PAC recovery). PAC contents were determined as 0.86 mg/mL, 0.56 mg/g, 0.11 mg/g and 0.61 mg/mL for BAE, BWE, LMP and PAC, respectively. The TPC of BAE, BWE, LMP and PAC were determined to be 3.88 mg/mL, 2.64 mg/mL, 1.90 mg/mL and 1.85 mg/mL, respectively. Evaluation of rat α‐glucosidase inhibitory activity, revealed that LMP (IC50 0.24 mg/mL TPC basis) had the highest inhibitory activity followed by BAE (IC50 0.39 mg/mL TPC basis), PAC (IC50 0.92 mg/mL TPC basis). Evaluation of specific maltase and sucrase inhibitory activities confirmed same trend as with observed rat α‐glucosidase inhibitory activities (LMP>BAE>PAC). Our findings suggest that blueberry extract has α‐glucosidase inhibitory activity, which is low molecular weight phenolic dependent.

Grant Funding Source: Supported by US Army Natick Soldier Research Engineering Center
Effects of fermented aqueous extracts from leaves of Ligularia fischeri on hepatotoxicity in D‐galactosamine‐induced rats (647.26)
Authors: Yu Keun‐Hyung , Lee Sun‐Yeop , Yang Hyun‐Mo , Kam Young‐Ahn , Lee Soo‐Ung , Chae Seoung‐Wan , Lee Yong‐Jin
Published: June 2021

Ligularia fischeri herb can be used for the treatment of jaundice and hepatic failure. This study was conducted to identify antioxidant activity and anti‐hepatotoxicity effects of fermented aqueous extracts from the leaves of Ligularia fischeri (LA). The contents of total phenolic compounds and caffeoylquinic acid (CQA) of fermented LA (LAF) significantly increased, compared to those of LA. The ABTS and DPPH scavenging activities of LAF were significantly elevated. Male Sprague‐Dawley rats were used as a D‐galactosamine (D‐GalN) animal model. The activities of GGT, AST, ALT and LDH were diminished in D‐GalN‐exposed and LAF‐treated animal groups (D‐GalN/LAF) relative to those in D‐GalN‐alone‐treated groups (D‐GalN). The HDL‐cholesterol levels were enhanced in D‐GalN/LAF‐treated groups, whereas the levels of LDL‐cholesterol and triglyceride declined. In addition, the SOD activity of liver tissues increased in the D‐GalN/LAF‐treated groups, but their MDA contents were reduced in these groups. Histopathological observations revealed that there were apoptotic cells appeared and mild portal inflammation occurred in liver tissues of D‐GalN/LAF‐treated groups. These results demonstrate that LAF may improve plasma lipid profile and alleviate hepatic damage by D‐GalN.
Clinical trial for improvement in metabolic syndrome by Korean Chungkookjang (647.27)
Authors: Byun Moon‐Sun , Yu Ok‐Kyeong , Kim Hyun‐Suk , Park Tae‐Sun , Cha Youn‐Soo
Published: June 2021

In this study, we investigated the effect of Korean trational Chungkookjang compared to placebo on variables of metabolic syndrome in overweight/obese subjects. This double‐blind randomized controlled crossover trial was conducted on 120 overweight/obese subjects; aged 19‐29 years. Each group received either 35 g of Chungkookjang (CK; n = 60) or placebo (PC; n = 60) on a regular daily basis for 12 weeks. After 12‐weeks period, the groups was crossed over. The anthropometric measures, blood pressure, glucose parameter, lipid profile, adipocytokine, and carnitine assay was determined at baseline and 12 week. Also, safety was assessded by measuring total bilirubin, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, total protein, albumin, blood urea nitrogen, creatinine, and creatine kinase at baseline and 12 weeks. 24‐hour dietary recalls were collected at the baseline and at the end of the trial. Bodyweight, bodyweight gain, serum insulin , leptin level and energy intake were reduced in the CK group. These results suggested that the supplementation of Chungkookjang may contribute to the reduction of risk factor for metabolic syndrome caused by obesity.

Grant Funding Source: Korean Institute of Planning & Evaluation for Technology in Food, Agriculture Forestry & Fisheries
Influences of fermentation period on anticancer effects of Shuidouchi in MCF‐7 human breast adenocarcinoma cells (647.3)
Authors: Zhao Xin , Qian Yu , Li Guijie , Song Jia‐Le , Wang Qiang , Sun Peng
Published: June 2021

Shuidouchi (SDC) is a kind of short time‐fermented soybean food. Based on the SDC made through different fermenting time, the relationship between physicochemical properties and in vitro anticancer effects were evaluated by pH, acidity, total viable count, amino and ammonia type nitrogens, activity of γ‐GTP, sensory evaluation, MTT and RT‐PCR analysis. As the fermentation time increased, the pH and the total viable count increased in SDC. SDC fermented for 48 hour demonstrated the highest content of acidity and γ‐GTP activity. The ammonium nitrogen and amino acid nitrogen contents of 48 h fermented SDC were a little lower than 60 h fermented SDC, but much higher than 24 h fermented SDC. The 48 h fermented SDC also appeared the highest content of daidzein and genistein. At 2 mg/mL concentration, the growth inhibitory rate of 48 h fermented SDC was 78.8% higher than that of sea 24 h and 60 h fermented SDC in MCF‐7 human breast adenocarcinoma cells. The low concentration (1 mg/mL) of 24 h, 48 h and 60 h fermented SDC showed the similar growth inhibitory effect to 2 mg/mL SDC treatment at 22.5%, 43.5% and 35.2%, respectively. 2 mg/mL of 48 h fermented SDC treated MCF‐7 cells increased the Bax, caspase3,9 genes, decreased the Bcl‐2 gene compared with the other periods fermented SDC. From the results, we concluded that the 48 h fermented SDCZ showed the best physicochemical quality and in vitro anticancer effects.
Synbiotic‐induced alterations in fecal microbiota are associated with increased ANGPTL4 levels in healthy overweight adults (647.46)
Authors: Pham Priscilla , Shen Qing , Newton Samantha , Vattem Dhiraj , Maitin Vatsala
Published: June 2021

Gut bacteria may influence diet‐related obesity, partly via ANGPTL4, a circulating lipoprotein lipase (LPL) inhibitor that regulates fat‐storage in the adipose. Modulating gut flora for stimulatory activity towards ANGPTL4 may thus serve a protective function. We have previously shown that bioactives from Bifidobacterium sp. increased the levels of enterocytic ANGPTL4 in vitro. The goal of the present study was to investigate whether a synbiotic formulation containing probiotic Bifidobacterium sp. along with prebiotics inulin and galactooligosacharides can enhance ANGPTL4 in human subjects, by potentially enriching Bifidobacterium in their gut. 57 healthy overweight adults participated in this 12‐week parallel, double‐blind, placebo‐controlled pilot study, by adding the synbiotic or placebo to their habitual diet. Fecal samples, plasma samples, colonocytes and peripheral blood mononuclear cells (PBMCs) were collected from subjects at 0 and 12‐weeks for assessing changes in fecal microbiota, ANGPTL4 gene‐expression and plasma ANGPTL4. The synbiotic group demonstrated significant increases in Bifidobacterium numbers, ANGPTL4 gene‐expression in PBMCs, and plasma ANGPTL4 levels compared to placebo. Also, a significant positive correlation was seen between Bifidobacterium numbers and plasma ANGPTL4 levels in the synbiotic group. Bifidogenic diets may thus lower LPL‐mediated fat‐storage.
Preventive activity of Ajuga pantantha Hand‐Mazz on reserpine‐induced experimental gastric ulcer in Kunming mice (647.8)
Authors: Li Guijie , Zhao Xin , Song Jia‐Le , Sun Peng , Qian Yu , Wang Qiang
Published: June 2021

The aim of this study was to investigate the gastric ulcer preventive activity of aqueous extract from Ajuga pantantha Hand‐Mazz (AHMAE) on reserpine‐induced experimental gastric ulcer in Kunming mice. The mice were divided into the following five groups: normal, reserpine, 250 mg/kg and 500 mg/kg AHMAE and ranitidine. The mice were first treated with AHMAE for 28 days, and the gastric ulcer was induced by reserpine(10 mg/kg) via a intraperitoneal injection at day 29. Ranitidine (50 mg/kg) used as a positive control. Following intraperitoneal injection 24 h later, the area of gastric ulcer, gastric juice volume, gastric juice acidity, serum levels of MOT, SS, SP and VIP, serum pro‐inflammatory cytokines (IL‐6, IL‐12, TNF‐α and IFN‐γ), iNOS and COX‐2 gene expressions were analyzed. Following pretreatment with 250 and 500 mg/kg AHMAE significantly reduced the reserpine‐induced gastric ulceration. AHMAE also increased the serum SS and VIP and decreased the MOT and SP levels in serum. In addition, AHMAE significantly reduced the serum levels of pro‐inflammatory cytokines of IL‐6, 12, TNF‐α and IFN‐γ compared to control group. AHMAE also decreased the expression of iNOS and COX‐2. These results suggest that AHMAE showed a good gastric ulcer preventive effect.

Grant Funding Source: Supported by the China Scholarship Council (No. [2012]5030) for fellowship
Dystrophin expression in myocardium in mice submitted “Two‐Hit” model of sepsis (648.1)
Authors: Figueiredo Maria José , Celes Mara Rúbia , Nascimento Daniele , Pulici Erica , Freitas Ana Caroline , Cunha Fernando , Alves Filho José Carlos , Ramos Simone
Published: June 2021

This study was designed to analyse the morphological changes and dystrophin expression on heart of animals undergoing sepsis induced by cecal ligation and puncture (CLP) followed by pneumonia. Male C57/Bl6 mice were subjected to sham or moderated septic injury induced by CLP. Three days post‐CLP, surviving mice were subjected to a second hit with Pseudomonas aeruginosa (Pa). Twenty‐four hours after pneumonia induction we evaluated the bacterial count and cytokines levels in BAL and serum and the morphology and dystrophin expression in the heart of animals. We observed an increased number of bacteria in the BAL of Sham+Pa and CLP+Pa groups but not in the serum of mice. Sham+Pa and CLP+Pa animals presented significantly increased levels of IL‐6 and KC in both BAL and serum. Myocardium of CLP+Pa mice presented increased foci of myocytolysis and tumefaction of cardiac cells as compared to CLP+Sal myocardium. Consonantly, reduced amounts of dystrophin were detected in CLP+Sal myocardium as compared to controls; however, dystrophin loss was more pronounced in myocardium of septic mice subjected “two‐hit”. Mortality was severely increased after second hit as compared with mice receiving CLP or pneumonia alone, suggesting that second hit worsen the survival.This is the first study evaluating dystrophin loss/disruption in two‐hit sepsis model (that more closely mimics the clinical scenario secondary infection). Additional studies are needed to further elucidate these myocardial structural changes in sepsis given the continued impact of cardiovascular involvement and the risks of secondary infection on mortality.
The role of frataxin in doxorubicin‐mediated cardiac hypertrophy (648.6)
Authors: Mouli Shravanthi , Nanayakkara Gayani , Fu Rain , Berlin Avery , Alasmari Abdul , Arnold Robert , Amin Rajesh
Published: June 2021

Heart failure is the major fatal adverse effect associated with cancer chemotherapeutic, doxorubicin (DOX). We and others have found that the progression towards DOX mediated heart failure involves myocardial energy dysregulation and the development of cardiac hypertrophy. Hypothesis: We hypothesize that Frataxin (FXN) is a key player in the development and progression of DOX mediated cardiac hypertrophy. Results: Recently, we observed that DOX (2µM) treatment in H9C2 cardiomyocytes results in the reduction of the active version of the mitochondrial protein FXN which is a major player in the iron sulfur cluster biogenesis. This deficiency in the active form of FXN resulted in an increase in mitochondrial iron accumulation and consequent reduction in activities of aconitase and the electron transferring complexes in mitochondria thus greatly affecting oxidative phosphorylation leading to reduced measured levels of ATP. Further the increase in mitochondrial iron accumulation lead to an increase in production of mitochondrial reactive oxygen species (ROS) and oxidative stress and helps further explain the subsequent cardiac hypertrophy. In order to verify that restoration of FXN could prevent against myocardial energy dysregulation and cardiac hypertrophy, we created FXN over expressing and knock out cell lines and found that over expression promoted iron homeostasis in the mitochondria which lead to a significant improvement in oxidative phosphorylation, substantial decrease in ROS production in the mitochondria and protected against the development of DOX mediated cardiac hypertrophy. Conclusion: Over expression of FXN in the mitochondria is protective against DOX mediated myocardial energy dysregulation and subsequent cardiac hypertrophy.

Grant Funding Source: Auburn university Internal grant program
Effects of LY294002 on the dystrophin expression in myocardium in mice submitted to cecal ligation and puncture (648.9)
Authors: Campos Pulici Erica , Celes Mara , Figueiredo Maria , Freitas Ana , Ramos Simone
Published: June 2021

The cell signaling pathways related to PI3K play an important role in the pathogenesis of diseases with inflammatory component, such as cancer, myocardial infarction, atherosclerosis, ischemia‐reperfusion injury and sepsis. Recent evidence indicates that the PI3K/Akt signaling pathway may represent a compensatory mechanism in order to limit the pro‐apoptotic and pro‐inflammatory events in response to sepsis. This pathway has also been reported as a regulator of cardiomyocyte contractility, which could be related to expression of the structural glycoproteins, mainly dystrophin that confers structural stability to the myofiber sarcolemma and transmits force between sarcomeres and cell membrane to the extracellular matrix. Severe sepsis was induced by cecal ligation and puncture (CLP) in mice and the LY294002 (5mg/kg) was used 1 hour before surgery to inhibit the PI3K/Akt signaling. The survival rate was higher in CLP plus LY294002 group as compared to only CLP and controls. Histopathologycal analysis showed foci of disorganized miofibrils and increased membrane permeability, more evident in CLP plus LY294002 group as compared to only CLP and controls. We observed decreased myocardial dystrophin expression in septic mice as compared to controls, but this dysthophin loss was more pronounced in myocardium from septic mice treated with LY294002. The initial evidences suggest that PI3K/AKT signaling may be involved in these myocardial structural changes and could be implicated in sepsis‐induced cardiac depression and represents an innovative approach to study the intrinsic mechanism of cardiac abnormalities caused by sepsis, which can provide highly significant contribution in this specific area of knowledge.

Grant Funding Source: FAPESP
Dermal regulatory T cells display distinct migratory behavior that is modulated during adaptive and innate inflammation (650.13)
Authors: Chow Zachary , Mueller Scott , Deane James , Hickey Michael
Published: June 2021

Regulatory T cells (Tregs) are important in controlling skin inflammation, an effect dependent on their ability to home to this organ. However, little is known regarding their behavior in the skin. Here we used multiphoton imaging in Foxp3‐GFP mice to examine the behavior of endogenous Tregs in skin. While Tregs were readily detectable in the uninflamed dermis, most were non‐motile. Induction of contact sensitivity increased the proportion of motile Tregs, as well as inducing Treg recruitment. This response was significantly blunted in mice challenged with an irrelevant hapten, and by inhibition of effector cell recruitment, indicating a role for T cell‐dependent inflammation in induction of Treg migration. Moreover, induction of Treg migration was inhibited by local injection of a CCR4 antagonist, indicating a role for CCR4 in this response. Exposure of naïve mice to hapten also induced an increase in the proportion of migratory Tregs, demonstrating that innate signals can also induce Treg migration. Simultaneous examination of the migration of CD4+ effector cells and Tregs in the same region of uninflamed skin demonstrated that effector cells behaved differently, being uniformly highly migratory. These findings indicate that Treg behavior in skin differs from that of CD4+ effector cells, in that only a low proportion of Tregs are migratory under resting conditions. However, during inflammation the proportion of migratory Tregs increases, raising the possibility that this response may be important in controlling skin inflammation.
Friedreich ataxia: metal dysmetabolism in dorsal root ganglia (651.1)
Authors: Mazurkiewicz Joseph , Ramirez R Liane , Koeppen Arnulf
Published: June 2021

Friedreich ataxia (FA) is caused by an inherited deficiency of frataxin, a small nuclear genome‐encoded mitochondrial protein and leads to Fe related mitochondrial dysfunction. Atrophy of large dorsal root ganglion (DRG) neurons, hyperplasia of satellite cells, and absorption of dying neurons into residual nodules are hallmarks of FA. Dysregulation of antioxidant defenses due to over production of reactive oxygen species by transition metals (e.g. Zn and Fe) has been implicated in FA pathology. Tissue levels of Fe and Zn in normal and FA DRG did not differ significantly, however, the Zn level found, ~90.24 μM, is much higher than needed for normal function of Zn‐dependent proteins and the disturbance of Zn buffering could lead to mitochondrial damage and cell death. The Zn‐transporter, Zip14 (neurons & satellite cells), metallothionein MT1/2 (Zn; satellite cells), metallothionein 3, MT3 (Zn; neurons), and ferritin (Fe) were used as surrogates for Zn and Fe. Major shifts in Zip14 and MT1/2 immunofluorescence between neurons and satellite cells in FA DRG suggests that the metals released from dying neurons move into the proliferating satellite cells. In contrast to Zn, sequestration of Fe in satellite cells may represent a protective mechanism. Changes in the expression and cellular localization of metal handling proteins link metal dysmetabolism with the pathogenesis of the DRG lesion in FA.

Grant Funding Source: Supported by grant from National Institutes of Health (R01‐NS069454), Bethesda, MD, USA
Traumatic brain injury augments disease pathology in a mouse model of amyotrophic lateral sclerosis and wild type mice (651.11)
Authors: Evans Teresa , Jaramillo Carlos , Van Remmen Holly
Published: June 2021

Traumatic Brain Injury (TBI) is a known risk factor for ALS. The goal of this study is to elucidate the mechanism linking TBI to motor neuron disease, by testing the hypothesis that TBI will accelerate disease progression in an animal model of ALS. We used a well‐characterized mouse model of familial ALS (G93A SOD1) to study the effect of TBI on ALS progression. Mice were subjected to a closed head traumatic brain injury and magnetic resonance imaging (MRI) was used 3 days after injury to characterize structural central nervous system pathology and the severity of brain injury. Histological techniques showed astrocyte infiltration (GFAP) and edema (Nissl) following mild TBI in brain and spinal cord of wildtype (WT) and transgenic mice (TG). Our results indicate that TBI leads to early reductions in grip strength and rotarod performance. Further, muscle denervation is potentiated as is shown via electromyography abnormalities. We have also characterized the effect of TBI on disease related weight loss and overall disease score in G93A mice. Our results are the first to show that TBI, in an animal model of ALS, results in significantly increased muscle denervation and brain and spinal cord pathology.

Grant Funding Source: This work is supported by an individual NRSA fellowship grant, 1F31NS080508‐01
Effects of HIV‐1 Tat and morphine on the biophysical properties of sodium channels (653.5)
Authors: Fitting Sylvia , Ngwainmbi Joy , Knapp Pamela , Hauser Kurt , Akbarali Hamid
Published: June 2021

HIV‐infection continues to be a major clinical manifestation in the combination antiretroviral therapy era (cART), that is enhanced in opioid‐drug abusers. The gastrointestinal (GI) mucosa plays a major role in HIV‐infection as HIV is mostly transmitted across mucosal surfaces. The enteric nervous system (ENS) controls several GI processes including motility and secretion which are specifically affected in HIV‐infected patients. Opioids directly affect the ENS, causing severe constipation. Because the virus does not infect neurons, it is suggested that viral toxins, such as Tat, modulate neuronal function in HIV‐1‐infected patients. Tat has been shown to increase neuronal excitability but not much is known about effects of Tat on enteric neurons in combination with drug abuse. Recent experimental studies in our lab have shown that Tat increases enteric neuronal excitability by shifting the voltage‐dependence of activation of sodium channels to more negative potentials. In contrast, morphine alone decreases the availability of sodium channels in enteric neurons reducing excitability. Voltage gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in neurons. In order to determine the effects of Tat (100 nM) in combination with morphine (0.3µM, 3µM) on the voltage dependence of steady‐state activation/ inactivation of sodium channels, we used a double‐pulse protocol in which a variable conditioning pulse was applied from −100mV to +50mV in 10mV steps for 50 ms followed by a test pulse. In Tat‐treated neurons, the sensitivity to morphine was significantly enhanced. The V0.5 for steady‐state inactivation was shifted in Tat‐treated neurons at 0.3 uM morphine with 50% decrease in maximal availability at ‐100 mV. Morphine did not alter the steady‐state activation. Taken together, data suggest that Tat increases the sensitivity to morphine in enteric neurons that may exacerbate the deleterious effects of morphine on GI motility.

Grant Funding Source: Supported by NIDA R01 DA018633, K02 DA027374, K99 DA033878, DK046367.
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).
Identification of novel CFTR activator and its application to inducing chloride transport at the mouse ocular surface (654.7)
Authors: Park Jinhong , Seo Yohan , Verkman Alan , Namkung Wan
Published: June 2021

Impaired cystic fibrosis transmembrane conductance regulator (CFTR) function can lead to cystic fibrosis (CF). We developed a high‐throughput screen to identify small‐molecule activators of human airway epithelial Ca2+‐activated Cl‐ channels (CaCCs) for CF therapy. Unexpectedly, screening of ~110,000 synthetic small molecules revealed an amino‐carbonitrile‐pyrazole, Cact‐A1, that activated CFTR but not CaCC Cl‐ conductance. Cact‐A1 produced large and sustained CFTR Cl‐ currents in CFTR‐expressing Fisher rat thyroid (FRT) cells and in primary cultures of human bronchial epithelial (HBE) cells, without increasing intracellular cAMP and in the absence of a cAMP agonist. To investigate possible application of Cact‐A1 for dry eye, we studied that the effect of Cact‐A1 on the chloride transport at the mouse ocular surface in vivo. Open‐circuit potential differences across the ocular surface were measured in anesthetized mice. Application of Cact‐A1 on the mouse ocular surface induced further sustained hyperpolarization of ocular surface PD and it was inhibited by specific CFTR channel inhibitor CFTRinh‐172. The novel CFTR activator, Cact‐A1, identified in this study may be useful for drug therapy of dry eye.
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
Smooth muscle‐dependent vasodilation is impaired in immature arterialized collateral capillaries (677.17)
Authors: Hellstrom Sara , Cutts Joshua , Cardinal Trevor
Published: June 2021

Native collateral vessels creates a natural bypass for blood flow around an arterial occlusion, potentially mitigating the effects of ischemic diseases, such as peripheral arterial occlusive disease in which arterial stenosis restricts blood flow to the limbs. Unfortunately, not all patients have robust collateral networks; however, animal models lacking native collaterals demonstrate the ability to adapt to the ischemic environment through arterialization of collateral capillaries, reperfusing the ischemic zone. Reactivity is important for normal muscle function in these developed vessels because increased metabolic demand is only satisfied when blood vessels can dilate to increase flow. Understanding cellular mechanisms behind impaired vasodilation is key to identifying potential therapeutic targets for improving reactivity. To assess vascular reactivity, the lateral cranial spinotrapezius feed artery was ligated 7 days prior to the application of endothelial‐dependent (acetylcholine, ACh) and endothelial‐independent (sodium nitroprusside, SNP) vasodilators. Although arterialized capillaries dilated significantly in response to ACh and SNP, dilation was impaired when compared to equivalent‐diameter terminal arterials on the sham side (19.0 ± 3.6% vs. 69.5 ± 14.0% and 20.0 ± 4.9% vs. 95.7 ± 9.2%, respectively). This impairment can be attributed to smooth muscle cell dysfunction because the responses to ACh and SNP are similar. To determine if other smooth muscle‐dependent vasodilatory pathways are also impaired, hydrogen sulfide and papaverine were applied using the same methods (31.3 ± 16.6% vs. 97.1 ± 16.2% and 49.0 ± 8.8% vs. 86.3 ± 6.1%). Future work will further characterize the signaling cascades described herein.