bluuebunny/crossref_metadata_2025 huggingface dataset:
Th1 Inflammatory Cytokines Induce Corticosteroid Resistance in Pediatric Airway Smooth Muscle Cells
Authors: Jackson Devine , Walum Josh , Britt Rodney Deon
Published: June 2021

Introduction

Steroid‐based therapies are anti‐inflammatory and commonly used to manage asthmatic symptoms in pediatric patients. Although manageable for most children, some patients with moderate‐severe asthma require administrations of high doses of steroids, which is relatively ineffective in limiting asthmatic symptoms. Airway smooth muscle (ASM) plays a central role by contributing to airway inflammation, remodeling, and hyperreactivity in asthma. ASM contributes to airway inflammation during responses to allergen exposures (Th2 inflammation) and infection (Th1 inflammation). Previous studies have suggested that adult ASM exposed to Th1 cytokines, TNFα and IFNγ, reduce steroid sensitivity and efficacy. In the present study, we examined the effects of TNFα and IFNγ on steroid sensitivity in pediatric ASM.

Methods and Results

Human ASM was isolated from trachea of patients (1–10 years old) undergoing tracheal reconstruction surgery (approved by NCH IRB). Cultured ASM were treated with vehicle (0.01% DMSO), 10 nM fluticasone propionate (FP), 10 ng/mL TNFα, and/or 25 ng/mL IFNγ for 24 h. Cells were harvested to measure mRNA levels of pro‐inflammatory genes (CCL5, CXCL10, p65) and glucocorticoid receptor target genes (FKBP5, GILZ) by qRT‐PCR. Media was collected to measure CCL5 levels by ELISA. Effects of TNFα or IFNγ alone on CCL5 and CXCL10 mRNA levels were inhibited by FP, however CCL5 and CXCL10 mRNA levels remained increased in cells treated with TNFα/IFNγ and FP. Similarly, TNFα‐induced CCL5 secretion was reduced by FP, but this anti‐inflammatory effect was absent in cells treated with TNFα/IFNγ. Transcription of glucocorticoid receptor target genes, FKBP5 and GILZ, were increased by FP, evidence of glucocorticoid receptor activity. In contrast to FKBP5, GILZ mRNA levels were attenuated by TNFα and IFNγ treatment, and blunted by TNFα/IFNγ. Treatment with TNFα/IFNγ substantially increased mRNA expression of the NFκB subunit, p65, an effect that remained in the presence of FP.

Conclusions

Together these findings suggest that TNFα/IFNγ induce steroid resistance in pediatric ASM. Although there is evidence of glucocorticoid receptor activity, we speculate that interactions between TNFα‐ and IFNγ‐mediated pathways promotes inflammation in the presence of steroids. Th1‐mediated inflammation may contribute to development of steroid resistance in children with severe asthma.

Support or Funding Information

This study is funded by National Institutes of Health R00 HL131682 (RDB).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating sex differences in miR‐106a expression in a mouse model of lung inflammation and asthma
Authors: Steckbeck Rachel Joy , Fuentes Nathalie , Weaver Ashley , Rivera Lidys , Nicoleau Marvin , Silveyra Patricia
Published: June 2021

MicroRNAs (miRNAs) are oligonucleotides involved in various processes in the cell, including gene expression regulation. MicroRNA‐106a is involved in transcription regulation of the gene coding for interleukin 10 (IL‐10), an anti‐inflammatory cytokine, and has been implicated in asthma exacerbation in mice. However, despite the well‐known sex differences in human asthma incidence, no studies have been done evaluating sex differences on the impact of miR‐106a in asthma. In this study, we hypothesized that there are sex differences in miR‐106a expression in the lungs of asthmatic mice, under normal conditions and in response to air pollution challenge. To test this hypothesis, both male and female mice were exposed to house dust mites (HDM) or phosphate‐buffered saline (control) for 5 weeks to trigger the asthma phenotype. Mice were subsequently subjected to ozone challenge (2ppm, 3 hours) to model asthma exacerbations. The asthma phenotype was confirmed by lung histology, lung function testing using a rodent ventilator, and expression of inflammatory markers by Real Time PCR. To measure miRNA expression, lungs were harvested, total RNA was extracted and converted to cDNA. We performed Real Time PCR with specific primers for miR‐106a and the small RNA U6 (normalization control) using the miRCURY LNA® RT® kit. Our results show sex differences in expression of miR‐106a upon HDM and ozone challenge. Ozone exposure increased miR‐106a expression in non‐asthmatic females and asthmatic males when compared to filtered air exposed mice (p<0.0001 and p<0.05, respectively). Male asthmatic mice had higher miR‐106a expression than non‐asthmatic male mice (p<0.0001). However, the interaction between ozone exposure and sex was significant in non‐asthmatic mice only. We conclude that the observed differences in miR‐106a expression may be a result of different lung immune cells recruited to the male and female asthmatic lung. Understanding the mechanisms behind these sex differences may lead to future development of novel treatments for asthma.

Support or Funding Information

Thanks to the American Physiological Society Undergraduate Summer Research Fellowship for allowing me to do this research and to the NIH grants HL133520 and HL141618 for supporting this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HIF‐1 alpha Mediates Sex‐specific miR‐30a Expression in Neonatal Hyperoxic Lung Injury: Implications for Bronchopulmonary Dysplasia
Authors: Zhang Yuhao , Dong Xiaoyu , Lingappan Krithika
Published: June 2021

Background

Bronchopulmonary Dysplasia (BPD) is characterized by an arrest in lung development with severe impairment of alveolar septation and vascular development. Premature male neonates are at a greater risk of developing bronchopulmonary dysplasia (BPD). The reasons underlying sexually dimorphic outcomes in premature neonates are not known. Neonatal female mice have improved alveolarization and pulmonary vascular development compared to male littermates in a murine model of BPD. Analysis of the pulmonary transcriptome suggests that miR‐30a, may modulate these sex‐specific differences in gene expression. HIF‐1α plays an important role in post‐natal lung development, especially in recovery from hyperoxic injury and acute lung injury. We hypothesized that differential sex‐specific expression of miR‐30a is mediated through HIF‐1α.

Methods

Male and female neonatal WT (C57BL6) mice were exposed to hyperoxia (P1–P5; saccular stage of lung development) and euthanized on P7 or P21 after recovery in normoxia. Human pulmonary microvascular endothelial cells (HPMECs; from 18–24 weeks gestation donors; 3 male and 3 female) were obtained from ScienCell (Carlsbad, California). They were cultured in endothelial cell media per protocol. Cells from passage from 3 to 6 were used for experiments. For hyperoxic exposure, cells were incubated in 95% O2 and 5% CO2 at 37 °C for upto 72 hours. Cells in normoxia were maintained in air and 5% CO2. miR‐30a and Dll4 (miR‐30a target) expression was measured. Hif‐1α binding to its targets genes (by ChIP‐qPCR) and expression was measured in vivo. To further establish potential regulation of miR‐30a by Hif‐1α, miR‐30a expression after Hif‐1α inhibition was measured in neonatal HPMECs.

Results

Expression of miR‐30a was increased and Dll4 decreased on PND21 in hyperoxia‐exposed neonatal female mice compared to males. On PND21, hyperoxia‐ exposed female mice showed a significant increase in Hif‐1α binding to target genes compared to room air controls and hyperoxia‐exposed male mice. In contrast to male mice, females did not show a decrease in Hif‐1α expression at PND21 after early hyperoxia exposure and this was accompanied by a significant decrease in Phd2 expression in female mice, which metabolizes Hif‐1α. Motif analysis revealed a hypoxia response element (HRE) [AG]CGTG site 428 base pairs upstream of miR‐30a. siRNA mediated loss of Hif‐1α expression in human pulmonary microvascular endothelial cells decreased miR‐30a and increased Dll4 mRNA and protein expression.

Conclusions

Hif‐1α mediated differential sex‐specific miR‐30a expression may contribute to protection from hyperoxic lung injury in female neonatal mice and contribute to the sex‐specific differences seen in BPD.

Support or Funding Information

K08 HL‐127103R03 HL‐141572

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Creatine‐Monohydrate on Cognitive Function in Healthy Male and Female Subjects
Authors: Monsanto Nathan , Hopkins Derek , Deschenes Ashni , Scott Laurie , Skelton Michele
Published: June 2021

The purpose of this study was to examine the effects of 4 weeks of creatine supplementation on cognitive function using the ImPACT test in healthy male and female subjects. In a randomized, double‐blind placebo‐controlled study, twenty‐five subjects were divided into two groups. One group received creatine while the other group received a placebo. Subjects completed a 5‐day loading phase (20 grams per day) followed by a 24‐day maintenance phase (5 grams per day). Cognitive function was assessed prior to supplementation and immediately after a 4‐week supplementation phase. The ImPACT test was used and provides 6 measures of cognitive function. There were no significant changes in any of the cognitive measures between groups. However, females in the creatine group scored significantly higher than males on the visual memory test. Preliminary analysis suggests that acute supplementation of creatine does not enhance cognition in healthy subjects. A gender difference was observed in the treatment group suggesting that females may respond differently than males with regard to cognitive benefits with creatine supplementation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anomalous Origin in the Left Circumflex Artery from the Right Coronary Artery
Authors: Kruse Amelia , Connell Rachel , Harley Jackson , Rehman Rafey , Heintschel Alyssa , Barremkala Malli
Published: June 2021

The typical branching pattern of the right coronary artery (RCA) consists of a marginal branch and a continuation as the posterior interventricular artery. The left coronary artery classically consists of an anterior interventricular artery and a circumflex branch, the latter of which courses around the left auricle and joins with the right coronary artery. According to past case reports, anomalies in this standard pattern are observed on angiograms with a 0.6% to 1.5% rate of occurrence (Dursunoğlu et al. 2007). Specific anomalies are found to occur at a lower rate, or are unreported due to insufficient studies and publications. Our team analyzed 23 human heart specimens (average age of 82 years) during routine cadaver dissection by undergraduate medical students and discovered an anomalous origin in the left circumflex artery (AOLCX). On the specified cadaver heart (71 year old male), an absence of the circumflex branch from the left coronary artery was noted; and upon further examination AOLCX was arising from a super‐dominant right coronary artery. After arising from the right proximal coronary artery, AOLCX wrapped around the posterior aspect of the aorta before arising anteriorly and circumflexing around the left auricle towards the posterior surface of the heart. The significance of this abnormality is that systolic compression of the AOLCX produced by the aorta and left atrium during daily activities may result in a transient reduction in coronary flow (Carboni). AOLCX from RCA has an incidence of 0.7 % (Roberts WC) and is associated with sudden death (Edwards, et al).

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insight into the Activin Receptor Assembly Mechanism
Authors: Goebel Erich J , Thompson Thomas
Published: June 2021

Ligands of the TGFB family, which include BMPs and activins have been implicated to play critical roles in development, cardiac disease, and aging. Upon reaching the cell surface, each ligand signals by forming a ternary complex with type I and type II serine‐threonine kinase receptors. For TGFB and BMP ligands, crystal structures of ternary complexes have revealed differences in receptor assembly, providing context for differential receptor preferences. However, structural information for how activin class ligands assemble both receptors has been lacking. Along this, we have resolved the ternary crystal structure of an activin class member, GDF11, in complex with the type II receptor, ActRIIB, and the type I receptor, Alk5. The structure reveals that receptor positioning is similar to the BMP class with no inter‐receptor interactions, however, the location of the type I receptor is shifted toward the ligand fingertips away from the dimer interface. Since Alk5 serves as a low affinity receptor for both TGFB ligands and GDF11, the first activin receptor complex structure also reveals that different mechanisms have evolved to bind the same type I receptor. While TGFB utilizes inter‐receptor interactions and a cooperative mechanism to bind Alk5, GDF11 cradles Alk5 to form additional contacts at the fingertip region, which are shown, through mutagenesis analysis, to play a role in type I receptor specificity within the activin class. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFB family, different mechanisms have evolved to regulate type I receptor binding and specificity and provides a molecular explanation for how the activin class accommodates low affinity type I interactions without the requirement of cooperative receptor interactions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enzyme Kinetic Characterization and Substrate Specificity of Schizophyllum commune Metacaspases
Authors: Nguyen Le Minh , Fox Kristin M.
Published: June 2021

Metacaspases are heterodimeric enzymes that belong to the family of cysteine‐dependent proteases, making them distantly related to metazoan caspases and potentially involved in programmed cell death in fungi, protozoa, and plants. Although metacaspases share structural characteristics with caspases, they lack aspartate specificity and instead show a preference for cleaving peptide bonds after arginine or lysine residues at P1. In our research, the five Type 1 Schizophyllum commune metacaspases 1–5 (ScMc1‐5) exhibit arginine and lysine specificity. However, the question of whether the enzymes show preference towards any specific amino acids that come before the cleavage site remains unanswered. To investigate the importance of substrate amino acid sequence for substrate specificity, we performed in‐vitro fluorescent activity assays comparing how ScMC1‐5 cleave various synthetic peptide substrates. Specifically, we examined the effect of amino acid size, charge and polarity on peptide cleavage. The results indicate that ScMC1‐5 demonstrate a preference to cleave more readily after arginine than lysine. They also exhibit significantly higher hydrolytic activity before small residues like glycine relative to larger residues like phenylalanine. Studying such differences in substrate specificity can be relevant for determining the types of proteins metacaspases cleave in vivo. In addition, we conducted enzymatic characterization to analyze ScMC1‐5 kinetics. ScMC1 showed a significantly larger Km, but the Vmax values for all the S. commune metacaspases were similar. Examining the differences among the metacaspases can further our understanding of their individual, physiological functions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Ketoreductases for the Chemoenzymatic Synthesis of a Stereotriad
Authors: Kumru Kaan
Published: June 2021

Polyketides are diverse natural products of high biological activity produced from complex molecular assembly lines called polyketide synthases (PKSs). Some of the most important bioactive molecules are polyketides, including geldanamycin, doxycycline, tetracycline, and erythromycin. Other polyketides have been shown to have antifungal, antiparasitic, immunosuppressive, and even anticancer properties. Of particular focus here is the ketoreductase (KR), one of the many possible domains within PKSs. The KR reduces carbonyl groups stereoselectively and controls the majority of stereocenters in the polyketide product. KRs have been used in a bottom‐up approach here, harnessing their stereoselective reductions to produce products that would traditionally be difficult and time‐consuming to synthesize. A diketide was first synthesized chemically and reduced with a tylosin KR from Streptomyces fradiae (TylKR2). This reduced diketide was then extended into a triketide through the Masamune C‐acylation reaction. Finally, TylKR2 and a mycolactone KR from a bacterial artificial chromosome (MycKR6) were found to reduce S‐(2‐acetamidoethyl)(4R,5R)‐5‐methoxy‐4‐methyl‐3‐oxoheptanethioate to S‐(2‐acetamidoethyl) (3S,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of TylKR2 and to S‐(2‐acetamidoethyl) (3R,4S,5R)‐3‐hydroxy‐5‐methoxy‐4‐methylheptanethioate in the case of MycKR6. The reductions were performed in vitro using the glucose dehydrogenase regeneration system with NADP+. In this way, each of the 3 stereocenters was set chemoenzymatically using KRs to form a stereotriad. With similar KRs, it is likely that all 8 possible stereoisomers could be synthesized. Ultimately, these di‐ and triketide building blocks could be extremely useful functional groups to experiment with in drug synthesis and may result in more potent or effective medicines. Additionally, this work could be extended into a methodology for setting as many stereocenters as desired ‐ possibly one day allowing for customizable polyketide synthesis in the same way oligonucleotides and polypeptides can be made today.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification, Isolation and Inhibition of Entamoeba histolytica's bifunctional alcohol/aldehyde dehydrogenase (EHADH2)
Authors: Gabrielle Matthew Lee , Espinosa Avelina
Published: June 2021

Entamoeba histolytica is a unicellular eukaryote that is the causative agent for amebiasis in humans. There are ~100,000 fatalities annually, distributed mostly in developing countries. Cyst‐contaminated food and water disproportionately present in conflict zones. Additionally, lack of resources, clean water, sanitation, and limited access to education and health contribute to the rate of disease worldwide. The current treatment is metronidazole which is effective but has toxic and neurological side effects. The bifunctional alcohol‐aldehyde dehydrogenase (EhADH2) has been suggested as a target for anti‐amebic inhibitors, because it is essential for E. histolytica trophozoite growth. Entamoeba spp use EhADH2 to convert (anaerobically) acetyl‐CoA to acetaldehyde and acetaldehyde to ethanol. Substrate and cofactor (NADH, Fe) cofactor binding sites could be targeted for inhibition. Cyclic carbinol molecules such as cyclobutyl and cyclopropyl inhibit E. histolytica growth by affecting EhADH2 activities. Pyrazoline molecules have also showed inhibitory effects on E. histolytica growth. Three series of pyrazoline inhibitors previously synthesized and preliminarily selected by trophozoite growth inhibition will be tested for their ability to inhibit EhADH2 bifunctional activities. Establishing the mechanism of action for anti‐amebic inhibitors through restriction of EhADH2 activity may provide an alternative treatment to current toxic drugs such as metronidazole.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ginger provides neuroprotection in experimental model of traumatic brain injury
Authors: Sahin Kazim , Kilic Ertugrul , Balcikanli Zeynep , Ates Nilay , Orhan Cemal , Tuzcu Mehmet , Juturu Vijaya
Published: June 2021

Ginger (Zingiberaceae family) containing gingerols, shogaols, zingerone and volatile oils active compounds, exerts anti‐inflammatory, antioxidant, antiplatelet, fat loss, prevention of cardiovascular disease and anticancer activity. In spite of these known effects, the underlying mechanisms in the signaling pathways that ginger protects the brain after brain trauma are largely unknown. Therefore, the objective of this study was to examine the administration of ginger extract (Gingever®) to improve histopathological results and to demonstrate neuroprotective activity with antioxidative and anti‐inflammatory properties following TBI. Brain injury was induced using a cold trauma model in male mice that were treated with ginger extract (50 mg/kg) or vehicle via intraperitoneal administration just after TBI. Mice were divided into two groups: TBI+vehicle group and TBI+ ginger extract group. Our results show that ginger administration decreased infarct volume by decreasing nuclear factor‐κB (NF‐κB), interleukin 1‐beta (IL‐1b), and glial fibrillary acidic protein (GFAP) and by increasing neurotrophic factor BDNF, growth‐associated protein‐43 (GAP‐43), neural cell adhesion molecule (ICAM) and nuclear factor erythroid 2‐related factor 2 (Nrf2) in the brain. Our findings suggested that ginger promotes neuroprotection following TBI by inhibiting the levels of inflammation, NFkB, and GFAP, and increasing GAP‐43 and Nrf2 levels.

Support or Funding Information

This research was funded by the OmniActive Health Technologies Inc, NJ, USA and partly supported by the Turkish Academy of Sciences (Ankara, Turkey, KS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of EGF‐R signaling by desmoglein 2 in pancreatic cancer cells
Authors: Hoffmann Jan , Schinner Camilla , Dietrich Niclas , Zeiler Julian , Spindler Volker
Published: June 2021

Desmogleins are cell adhesion molecules located both in desmosomes and extradesmosomal regions in the membrane with well‐established roles for intercellular adhesion and signaling. We have previously shown that loss of the most widespread desmoglein, Dsg2, increases malignant behavior and induces a non‐epithelial phenotype through dysregulation of Erk signaling in pancreatic cancer cell lines. Here, we further explored the interdependence of Dsg2 and EGFR/Erk signaling by using Dsg2 knockouts of the pancreatic cancer cell line AsPC‐1. Dsg2 knockout clones showed reduced cell adhesion, enhanced migration and augmented invasion in extracellular matrix. Dsg2 interacted with EGFR as demonstrated in immunoprecipitation experiments. However, upon serum depletion, the increased migratory capacity of Dsg2 knockouts was abrogated and not restored upon EGF treatment. This indicates that increased migration in response to Dsg2 loss is agonist‐dependent but independent of EGF. Furthermore, inhibition of EGFR tyrosin kinase activity using erlotinib was significantly less effective in reducing migration of knockouts compared to wildtype controls. In line with this, erlotinib treatment reduced ERK activity in wildtype controls but not in Dsg2 knockout cells and only partially reverted the non‐epithelial phenotype in response to Dsg2 loss. These data indicate that Dsg2 modulates the activity of EGFR signaling in an agonist‐dependent manner.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of acetazolamide on brain swelling and aquaporin‐4 expression in rats brain after ischemic stroke
Authors: Duan Hubin , Hao Chunyan , Duan Shengqiang , Wang suping , Gao Weiqing , Zhang Ce
Published: June 2021

Objective

To study the effect of acetazolamide(AZA) on cerebral edema and aquaporin‐ 4 (AQP4) expression after cerebral infarction in rats. To explore the relations between ischemic cerebral edema and AQP4 expression.

Methods

150 male SD rats were randomly divided into three groups: sham operation group, non‐intervention group, AZA treatment group. Each group was divided into five subgroups according to the time of cerebral ischemia (6h, 1d, 3d, 5d and 7d), and there were 10 rats in each subgroup . The rats model of cerebral infarction was adopted by means of bilateral carotid arteries ligation(2‐VO) method. The treatment group was given intraperitoneal injection of AZA, while the sham operation group and the non‐intervention group were injected the same amount of normal saline at the corresponding time point above. The cerebral edema was measured by dry‐wet weight method. The expression of AQP4 in cerebral infarction was detected by immunohistochemistry.

Results

The brain water content(BWC) and the expression of AQP4 in non‐intervention group and AZA treatment group began to increase at 6h after cerebral infarction, it reached the peak at 3d, and then decreased gradually.(P<0.05). The BWC and the expression of AQP4 in sham operation group had not change significantly at any time point above (P>0.05). At different time points, the BWC and the expression of AQP4 in non‐intervention group and AZA treatment group was higher than that in sham operation group.(P<0.05). The BWC and the expression of AQP4 in AZA treatment group was lower than that in non‐intervention group.(P<0.05). The expression of AQP4 in sham operation group had not change significantly at any time point above (P>0.05).

Conclusions

Acetazolamide can alleviate rats cerebral edema and the expression of AQP4 after rats cerebral infarction, and it may alleviate rats cerebral edema by reducing the expression of AQP4 in rats brain.

Support or Funding Information

This study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Foundation(2014M561207), the Shanxi Province Basic Research Program ( 2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province, The science and technology research projects of health department of Shanxi Province(2016D011119), The Teaching Reform Program of Shanxi Higher Education , The Education Reform Project of Postgraduate Students in Shanxi Province. The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Family of Imidazoline I2 Receptor Ligands Improves Behavior and Cognition in SAMP8 Mice
Authors: Escolano Carmen , Griñán‐Ferré Christian , Vasilopoulou Foetini , Abas Sonia , Rodriguez‐Arevalo Sergio , Bagan Andrea , Perez Belen , Callado Luis F , Garcia‐Sevilla Jesus A , Garcia‐Fuster M. Julia , Pallas Merce , Brea Jose M. , Loza Maria Isabel
Published: June 2021

Imidazoline I2 receptors (I2‐IR) are widely distributed in the central nervous system. I2‐IR are associated with human brain disorders and I2‐IR proteins are increased in the patients that suffered from Alzheimer's disease (AD). Since structural data for I2‐IR remains unknown, the discovery of selective I2‐IR ligands devoid of alfa2‐adrenoceptor affinity is likely to provide valuable tools to define the pharmacological characterization of these receptors.

We reported a new family of (2‐imidazoline‐4‐yl)phosphonates that showed, in radioligand binding studies, higher affinity and selectivity for I2‐IR than idazoxan and other standard I2‐IR ligands. New compounds exhibit good blood‐brain‐barrier properties (PAMPA assay). In vivo studies in mice showed that acute treatments with two of the new ligands induces hypothermia, significantly increases p‐FADD/FADD ratio, an index of cell survival, and reduced hippocampal p35 cleavage into neurotoxic p25, indicating a neuroprotective role.

Herein, we present the cognitive and molecular analysis of the treatment of female senescence accelerated mouse‐prone 8 mice (SAMP8) with two leads of our new family of I2‐IR ligands. In particular, 12 months‐old SAMP8 (n=26) were divided randomly into three groups (control, n=10), SAMP8 treated with I2‐IR ligands (compound 1, n= 8 and compound 2, n= 8). Compounds were administered through drinking water and concentration dose adjusted to reach a 5 mg/kg/day during 4 weeks. Behavior and cognitive effects were investigated through three well‐established tests, OFT, EPM and NORT, showing beneficial effects on treated animals. These results were supported by the assessment of key molecular markers for oxidative stress, inflammation indicators, synapsis and apoptosis, parameters related with cognitive decline and AD.

Further, in vitro selectivity and ADME‐TOX profiling (human and mice microsomal stability, cytotoxicity, cytochromes inhibition, Caco‐2 permeability, hERG inhibition) and pharmacokinetics will be presented for a lead candidate.

In summary, here we present the effectiveness of two new I2‐IR ligands in an in vivo model (SAMP8) for cognitive decline. Although different authors reported the relationship between I2‐ IR and cognitive decline, this study is the first experimental evidence that propose I2‐IR as a new therapeutic target to face AD, an unmet medical need.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Mechanism of Ligand‐Induced Activation of the Tie Family of Receptor Tyrosine Kinases
Authors: MADUKWE JERRY , FERGUSON KATHRYN
Published: June 2021

Tie1 and Tie2, members of the tyrosine kinase family with immunoglobulin and EGF homology domains, are receptor tyrosine kinases found primarily in endothelial cells with key roles in development and maintenance of the vasculature and in angiogenesis. They are attractive targets for therapeutic intervention in tumor angiogenesis, inflammation, and sepsis. Tie2 is regulated directly by the multimeric angiopoietin (Ang) ligands, with Ang1 being its primary activator. We have previously shown that the Tie2 extracellular region (ECR) forms strong dimers even in the absence of bound ligand and that dimerization is mediated by membrane‐proximal fibronectin type III (FNIII) domains that were omitted in previous structural studies as revealed in the 2.5‐Å resolution X‐ray crystal structure of the membrane‐proximal three Tie2 FNIII domains. However, it is not known whether the membrane proximal FNIII domains of Tie receptors mediate homo‐ and heteromeric interactions that are key in determining the signaling outcome of ligand binding to Tie2. We propose to elucidate the role of the membrane proximal FNIII domains of Tie2 receptors on ligand binding using purified proteins in biophysical and cell‐based experiments. We have successfully purified significant yields of Tie2 and Tie1 ECR lacking or containing the proximal FNIII domains. We are currently optimizing our yields of purified angiopoietin ligands of defined oligomeric states. The oligomeric states of the Ang ligands will be confirmed by size exclusion chromatography and small angle x‐ray scattering analysis. We will use surface plasmon resonance to assess binding of the Ang ligands to the Tie2 ECR variants. We will examine the size of the complex formed between the Ang ligands and Tie2 variants using size exclusion chromatography/multi angle laser light scattering analysis. In addition, we will determine a high‐resolution structure of the complete ECR of Tie2 with or without Ang ligands bound. Taken together, these studies will reveal the effect of the Tie2 extracellular proximal FNIII domain on ligand binding and complex formation. Finally, we will assess the importance of our biophysical experiments findings on cellular signaling using cell‐based assays. We will test the ability of Ang ligands with defined oligomeric states to activate Tie2. Phosphorylation status of Tie2 and downstream signaling molecules such as Akt will serve as a measure of Tie2 activation.

Support or Funding Information

NIH grant R01 CA214704

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Lymphatic‐Targeted Fluorescent Organic Nanoparticles to Label Cells in vitro
Authors: Petersen Chase Benson , Morris Kahlen , Svechkarev Denis , Mohs Aaron M. , Fink Darci M.
Published: June 2021

We have developed a new class of hyaluronic acid (HA)‐conjugated fluorescent organic nanoparticles. These conjugates are designed to specifically target lymphatic endothelial cells (LECs) based on the ligand‐receptor relationship between HA and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1). The lymphatic vasculature undergoes dramatic remodeling during inflammation, wound healing, and in the tumor microenvironment, and is a significant route of metastasis. The long term goal of this project is to develop new tools for pre‐clinical and clinical imaging of pathological lymphatic vessel remodeling signatures that are indicative of the presence of early malignancy. Our work here has focused on 10 kDa and 100 kDa HA‐dye conjugates; future translational experiments will test nanoparticles with a paramagnetic core. The primary objective here was to optimize the dosage for delivery of HA‐conjugated fluorescent dyes to immortalized mouse and primary human LECs for detection by fluorescence microscopy. This included identification of LYVE‐1 expressing experimental cell lines, analysis of cell death, and dose‐dependent HA‐dye in vitro labeling studies. The toxicity of the HA‐conjugated dye was determined using a caspase 3/7 glo luminescence assay that measured the amount of cleaved caspase 3/7 in HA‐treated mouse and human lymphatic cells. The dye was found to be non‐toxic to both the mouse and human lymphatic cells at all dilutions tested including the working staining dilution as compared to staurosporine control used to induce apoptosis. LYVE‐1 expression in human and mouse LECs was confirmed by western blotting and immunofluorescence microscopy techniques including epifluorescence and spinning disk confocal. Fluorescence microscopy analysis showed a dose‐dependent increase in HA‐conjugate dye labeling of LECs. We have shown that HA‐conjugated dyes are non‐toxic to both mouse and primary human LECs, and that this conjugate can experimentally label cells by fluorescence microscopy. Ongoing tubulogenesis and migration experiments are designed to determine if LEC function is impaired with the HA‐conjugate dyes bound. Co‐culture and corneal explant assays will confirm specificity of HA‐conjugate dye for LECs.

Support or Funding Information

In collaboration with Dr. Aaron M. Mohs and Denis Svechkarev at the University of Nebraska Medical Center and bio‐systems networks & translational research (BioSNTR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Estrogen Inhibits Colon Polyp Formation in a Benzo(a)Pyrene‐Induced Colon Cancer Rat Model
Authors: Harris Kenneth J. , Amos‐Landgraf James , Archibong Anthony E. , Adunyah Samuel E. , Ramesh Aramandla
Published: June 2021

Colorectal cancer (CRC) is the third most common diagnosed cancer and the third leading cause of cancer‐related deaths in the United States. The incidence and mortality rates of colorectal cancer are higher in men than in women. Epidemiological evidence show estrogen might influence the incidence of CRC in women by acting in a protective role. Whether estrogen acts via estrogen receptor beta (ERβ) to render its protective effect or through some other mechanism is not yet known. Using the Polyposis in Rat Colon (PIRC) animal model, we have demonstrated that male animals develop twice as many polyps in the colon than female animals when treated with the food toxicant, Benzo(a)pyrene [B(a)P]. In this study, to assess the potential protective role of estrogen against polyp formation in the colon, we treated female PIRC rats, depleted of ovarian steroids by ovariectomy (OVX), with B(a)P and measured polyp development. We found that B(a)P treatment of OVX‐female PIRC rats increased the polyp numbers when compared to control OVX‐female animals. Estrogen replacement in these B(a)P‐treated OVX PIRC rats significantly decreased total colon polyp numbers. These results suggest that estrogen does provide protection against B(a)P‐induced colon carcinogenesis.

Support or Funding Information

This research was funded by NIH grants 5R25GM059994‐3, U54CA163069‐04 and G12MD007586‐29.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactions of N, N‐ diethyl‐meta‐toluamide (DEET) and Novel Insect Repellents with Mammalian GABAA and Glycine Receptors
Authors: Grant Gariel G , Hall Adam C
Published: June 2021

Mosquito‐borne diseases such as malaria, dengue and yellow fever are responsible for millions of deaths annually. Repellents disrupt interactions between humans and insects, thus providing personal protection from bites through which diseases are transmitted. The most commonly used repellent today is N, N‐ diethyl‐meta‐toluamide, DEET. Despite its popularity, DEET is not effective against all species of mosquitoes, and there are widespread concerns about its possible toxicity to users. Consequently, there is a demand for new repellents which are safer, effective for longer durations, and more efficacious against a broader range of arthropods. The novel repellents tested in the following study fall under two classes of chemicals: acylpiperidines and carboxamides.

In the present electrophysiological investigation, the neurotoxicity of these novel repellents was explored relative to that of DEET, focusing on the effects of the repellents on inhibitory neurotransmitter receptors. Two‐electrode voltage clamp technique was employed with Xenopus laevis oocytes expressing mammalian GABAA (α1β3γ2S) and glycine (α1 homomer) receptors. Results indicated that high concentrations of DEET negatively modulated glycine receptor activity when co‐applied with submaximal glycine concentrations. 1 mM DEET inhibited glycine receptor activity by 48.4% ± 4.0% and 10 mM DEET produced a maximal inhibition of 80.4% ± 3.2%. Interestingly, most carboxamides (including DEET) inhibited the activity of glycine receptors, while the acylpiperidines enhanced the activity of glycine receptors, acting as positive modulators. Neither acylpiperidines nor carboxamides were found to inhibit the activity of GABAA receptors, with DEET and other carboxamides acting as weak agonists at high concentrations (>1 mM) to produce directly‐activated GABA currents. The EC50 for the activation of GABAA receptors by DEET was 4760.4 ± 50.6 μM, compared to a GABA EC50 of 42.9 ± 8.0 μM. These data suggest that DEET and the novel repellents have distinct effects on mammalian GABAA and glycine receptors. Furthermore, the results highlighted a stark difference between the two classes of chemicals, whereby acylpiperidines have the potential to result in less neurotoxicity relative to carboxamides. Like strychnine, carboxamides, including DEET, act as competitive antagonists at glycine receptors albeit with considerably less potency.

Support or Funding Information

Blakeslee and Holmes funding, Smith College

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Berberine attenuates the acute pancreatitis induced by Dibutyltin dichloride (DBTC) in albino Wistar rats
Authors: Putta Swathi , Qureshi Absar Ahmed , Kilari Eswar Kumar
Published: June 2021

Acute pancreatitis is a life threatening inflammatory disorder of pancreas. Currently, there is no effective pharmacological therapy available for this disorder and they does not showed any effect on root cause of the disease. The objective of the study was to evaluate the protective role of berberine in Dibutyltin dichloride (DBTC) induced acute pancreatitis in Wistar albino rats. The rats were divided into 4 groups and each carrying 6 rats. Group 1 served as control, Group 2 served as DBTC control (6mg/kg., i.p), Group 3 and 4 received 50mg/kg and 100mg/kg of berberine orally for a period of 28 days. Blood samples were collected at 1st, 3rd day and 28th day from all groups for the estimation of serum amylase and lipase. The DBTC reported to have effect on both exocrine and endocrine portion of the pancreas. The serum blood glucose, liver and kidney function markers (SGOT, SGPT, ALP, bilirubin, creatinine and protein); oxidative stress parameters such as SOD, CAT, GSH, Nitrite, MDA and Protein carbonyl content were estimated in both pancreatic and liver homogenates; inflammatory mediators (IL6, TNF α and CRP) were also estimated in both pancreatic and liver homogenates at the end of 28 days treatment period. The histopathological study was conducted for pancreas and liver. The treatment with berberine showed significant reduction of fasting blood glucose and liver kidney markers in DBTC induced pancreatitis in rats. The antioxidant enzymes SOD, CAT and GSH were found to be elevated and oxidative stress parameters (Nitrite, MDA and Protein carbonyl content) were found to be reduced with treatment with berberine in DBTC induced pancreatitis in rats. The berberine normalizes the elevated inflammatory cytokines with DBTC induction. The histopathology study indicating that berberine ameliorated the necrosis of both pancreatic acini and hepatocytes in DBTC induced pancreatitis in rats. The possible protective action against acute pancreatitis could be the action of berberine to counteract the oxidative damage caused by radicals or may attenuates the inflammatory infiltration induced by DBTC. Hence the berberine could be a potential source of traditional drug for acute pancreatitis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into the pharmacology of GPER/GPR30 and its involvement in gallstone formation
Authors: Arnatt Christopher , Sondergard Chelsea , Wang Helen , Wang David
Published: June 2021

The prevalence of gallstone disease is a major public health issue globally, and due to complex etiology of gallstones, there are a limited number of pharmaceuticals available. While the G protein‐coupled estrogen receptor (GPR30/GPER) has been shown to induce gallstone formation, little is known about its chemical biology and pharmacology. Building upon previous computational modeling of GPER, a novel series of antagonists were designed and synthesized. These novel compounds were assessed for their therapeutic value via calcium mobilization, cyclic AMP, and estrogen receptor a and β binding assays. From this series of compounds, one compound, SG‐1, exhibited superior antagonism and selectivity for GPER. The potential therapeutic value of SG‐1 was further validated in an in vivo mouse model of gallstone formation. Data revealed that SG‐1 treatment results in a dose‐dependent reduction in gallstone prevalence in estrogen‐treated ovariectomized mice.

Support or Funding Information

Supported by startup funds from Saint Louis University (C.A.) and this work was supported in part by research grants DK73917 and DK101793 (to D.W.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Piezo1 Channels Contribute to Flow‐Induced Intracellular Calcium Transients in Mouse Cortical Collecting Duct (CCD)
Authors: CARRISOZA‐GAYTAN ROLANDO , DALGHI MARIANELA G , APODACA GERARD L , KLEYMAN THOMAS R , SATLIN LISA M
Published: June 2021

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) (i) subjects principal (PC) and intercalated (IC) cells therein to biomechanical forces including hydrostatic pressure/circumferential stretch (CS), fluid shear stress (FSS) and drag/torque on PC cilia, and (ii) leads to a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) characterized by an immediate high amplitude increase to a peak value within ~10 s due to release of IP3‐sensitive internal Ca2+ stores coupled to extracellular Ca2+ entry at the basolateral membrane, followed by a decay to a plateau elevation in [Ca2+]i that exceeds baseline, sustained by luminal Ca2+ entry (Liu et al. AJP Renal Physiology 2003, 2005; 2007). The latter increase in [Ca2+]i is necessary for flow induced K+ secretion (FIKS) in the microperfused mammalian CCD. The identity of the basolateral Ca2+ entry path is unknown. We sought to examine whether Piezo channels, a family of mechanically‐activated Ca2+ permeable channels, contribute to this response. RNAscope detected single Piezo1 RNA molecules in mouse renal cortex. Immunofluorescence studies in mice expressing Piezo1‐tdTomato construct revealed robust basolateral and faint apical Piezo1 localization in both PC and IC. Flow‐induced [Ca2+]i transients, inferred from fluorescence intensity ratios (FIRs), were measured in microperfused mouse CCDs loaded with the Ca2+ indicator fura‐2 and then subject to low or high TFFRs, in the absence or presence of GsMTx4 (piezo‐blocker) or Yoda1 (piezo1‐activator). IC in CCDs from male and female mice preincubated with luminal or bath GsMTx4 (5 μM) failed to exhibit a flow‐induced increase in [Ca2+]i in response to an acute increase in TFFR. Addition of luminal or bath Yoda 1 (1 μM) to CCDs perfused at a low TFFR led to an increase in [Ca2+]i over 90 s in both PC and IC; addition of Yoda1 (10 μM) to the bath further increased [Ca2+]i. We speculate that mechanosensitive Piezo1 channels, expressed on the basolateral>apical membranes of PC and IC in the mouse CCD, contribute to the increase in [Ca2+]i triggered by an increase in TFFR.

Support or Funding Information

NIDDK: R01 DK38470, R01 DK51391 and P30 DK079307

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.