bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex‐based alteration of relative importance of EDRFs in modulating vascular reactivity in Zucker diabetic fatty rats (1051.15)
Authors: Shaligram Sonali , Han Xiaoyuan , Zhang Rui , Anderson Leigh , Rahimian Roshanak
Published: June 2021

Little is known about interaction of sex and diabetes in vasculature. Our study investigates the effects of type 2 diabetes on endothelium‐dependent and ‐independent relaxations. Also, if there are sex‐based changes in relative contributions of endothelium derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries (MA) from ZDF rats. Relaxation responses to acetylcholine (ACh) in MA pre‐contracted with phenylephrine (PE) were obtained before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L‐NAME (nitric oxide synthase inhibitor) or barium chloride (Kir blocker) plus ouabain (Na+‐K+‐ATPase inhibitor). Vascular responses to sodium nitroprusside (SNP) were also measured in MA. ACh‐induced relaxations were significantly impaired in MA of diabetic rats, regardless of sex. In diabetic females, the relative importance of endothelium derived hyperpolarizing factor (EDHF) in relaxation to ACh was reduced, while in diabetic males, role of nitric oxide (NO) was reduced. Interestingly, relaxation to SNP was enhanced in diabetic animals, irrespective of sex. In summary, the relative importance of NO and EDHF in regulating vascular tone of rat MA is altered in type 2 diabetes with respect to sex. Furthermore, increased smooth muscle sensitivity to NO may be an attempt to compensate for impaired endothelial function in both diabetic male and female rats.

Grant Funding Source: Supported by NIDCR
Increased basal and adenosine‐mediated coronary flow in ex vivo hearts from type I diabetic mice (1051.16)
Authors: Labazi Hicham , Zhou Xueping , Teng Bunyen , Mustafa S. Jamal
Published: June 2021

Diabetes is known to cause morphological and functional changes in the cardiovascular system, resulting in abnormal responses to various stimuli such as adenosine. Our aim was to examine the functional changes in coronary flow (CF) to adenosine receptor (AR) agonists resulting from type I diabetes. Diabetes was induced in mice using streptozotocin (STZ, 10mg/Kg; 8 weeks) and concentration response curves to the non‐selective AR agonist NECA and to the A2A AR selective agonist (CGS‐21680; CGS) were performed in isolated perfused hearts from STZ and vehicle (C) group. Diabetes resulted in a decrease in heart weight (0.093g ± 0.002 in STZ vs. 0.117g ± 0.004 in C; p<0.05), an increase in blood glucose level (510.8mg/dL ± 22.2 in STZ vs. 140.9mg/dL ± 9.4 in C; p<0.05) and an increase in the basal coronary flow (21.6 ± 1.5 in STZ vs. 16.1 ± 0.6 in C; p<0.05). Both NECA and CGS induced a concentration‐dependent increase in CF (ml.min‐1.mg‐1) which was significantly increased in STZ compared to C (NECA: Emax 39.8 ± 1.8 in STZ vs. 28.2 ± 0.7 in C; p<0.05) (CGS; Emax 38.8 ± 1 in STZ vs. 28.3 ± 1.8 in C; p<0.05). No significant differences were observed between the groups for changes in both heart rate and left ventricular diastolic pressure to both agonists. Our data suggest that STZ‐induced diabetes resulted in an increase in basal CF as well as increased vasodilatory response to adenosine agonists, probably by upregulating A2A receptor signaling.

Grant Funding Source: Supported by HL 094447 and HL 027339
DPP‐4 inhibitor linagliptin restores endothelium‐dependent relaxation in small mesenteric artery from type‐1 diabetic rats (1051.4)
Authors: Salheen Salheen , Woodman Owen , Mather Amanda , Panchapakesan Usha , Pollock Carol
Published: June 2021

Dipeptidyl peptidase‐4 (DPP‐4) inhibitors which are used to treat hyperglycaemia in type 2 diabetes may exert beneficial cardiovascular effects independently of their glucose lowering action. We hypothesized that linagliptin (LNG) treatment (4 weeks, 2 mg/kg per day orally) would improve relaxation in mesenteric arteries (MA) from STZ‐induced diabetic rats. In MA from diabetic rats there was a significant increase in superoxide levels, assayed by lucigenin chemiluminescence (diabetes, 1448± 108 counts/mg versus normal 644±380 counts/mg, n=7‐9, p<0.01). LNG does not reduce high blood glucose levels in diabetic rats (diabetes, 25±2.2 mM versus diabetes+ LNG, 27±1.7 mM). Acetylcholine (ACh)‐induced relaxation of MA was assessed using wire myography (pEC50=7.26±0.13, n = 8). Diabetes significantly reduced the sensitivity to ACh and treatment with LNG improved endothelial dysfunction (pEC50, diabetic 6.15±0.21 versus diabetic+ LNG, 6.84±0.13, n=8‐10, p<0.05). Diabetes impaired the contribution of both NO and EDHF to ACh‐induced relaxation and LNG treatment significantly enhanced the contribution of both relaxing factors. Our results indicate that the treatment of diabetic rats with LNG significantly reduced vascular superoxide levels and preserved both NO and EDHF mediated relaxation indicating that LNG can improve endothelial function in diabetes independently of any glucose lowering activity.
Protein and gene expression of angiotensin II receptors (AT1 and AT2) in aorta of diabetic and hypertensive rats (1051.6)
Authors: Romero Rodrigo , Reséndiz‐Albor Aldo , Rodriguez Jessica , Sanchez‐Muñoz Fausto , Hong Enrique , Villafaña Santiago
Published: June 2021

Diabetes and hypertension have been associated with cardiovascular disease and stroke. Some reports have been related the coexistence of hypertension and diabetes with increases in the risk of developing vascular complications. Recently some studies have shown contradictory results suggesting that in early stages of diabetes and hypertension exist a reduced functional response to vasoconstrictor agents like angiotensin II (Ang II) which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of receptors AT1 and AT2 in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes the protein expression was obtained by western blot and the mRNA expression by RT‐PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1receptors than normotensive rats while the AT2 expression was unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT2 receptors and these changes could be associated with the reduction in the response to Ang II in early stage of diabetes.

Grant Funding Source: CONACYT CB‐2012‐01‐183660/COFAA
Expression of the orphan receptors GPR22, GPR162, GPR27 and GPR17 in cardiovascular tissues of diabetic rats (1051.7)
Authors: Ruiz H Armando , Hong Enrique , Sanchez‐Muñoz Fausto , Villafaña Santiago
Published: June 2021

Diabetes mellitus is a chronic degenerative disease characterized by high blood glucose levels as a result of problems in the action or secretion of insulin. Although there exist treatments for this pathology, it has been associated with a high mortality. For this reason, it is important to try to identify new pathways that could be involved in the diabetes. Recently, a new class of receptors has been reported, called orphan receptors because the associated ligand and signaling pathways are unknown, these receptors have been associated with certain pathologies. Therefore, the aim of this work was to study the expression of the orphan receptor GPR22, GPR162, GPR27 and GPR17 in heart, aorta, brain and kidney of diabetic rats. We used male rats of Wistar strain with 10‐12 weeks of age. Diabetes was induced by a single dose of streptozotocin (60 mg/kg i.p.). After 4 weeks, the tissue was obtained and the expression of the mRNA was measured by RT‐PCR. Our results showed that the orphan receptors are expressed in a different way in heart, kidney, brain and aorta of diabetic and non‐diabetic rats. In conclusion, we suggest that orphan receptors could be involved in the development of diabetes.

Grant Funding Source: CONACYT CB‐2012‐01‐183660/COFAA
Altered irinotecan pharmacokinetics in diet‐induced obesity (1052.6)
Authors: Shah Pranav , Gandhi Adarsh , Ghose Romi
Published: June 2021

Irinotecan (CPT‐11) is a topoisomerase I inhibitor which is highly effective in treatment of variety of cancers. It was found that patients with a BMI>25 were twice as much susceptible to developing irinotecan‐induced liver toxicity than patients with BMI<25. CPT‐11 is metabolized to SN‐38, which then undergoes glucuronidation by uridine glucuronosyl transferase (Ugt)1a1 to form SN‐38 glucuronide (SN‐38G). Excess accumulation of the toxic metabolite SN‐38 is known to cause fatal diarrhea in cancer patients. We hypothesize that reduction in Ugt1a1‐mediated SN‐38 glucuronidation causes SN‐38 accumulation leading to liver toxicity in obesity. Metabolism studies in liver S9 fractions showed that the rate of formation of SN‐38G was 2‐fold lower in the diet‐induced obese (DIO) mice compared to the lean controls. Pharmacokinetic studies showed plasma exposure of SN‐38 increased while that of SN‐38G decreased by 2‐folds in the DIO mice as compared to the lean controls. This corresponded with reduced expression of Ugt1a1 in DIO mice livers. We also observed significantly higher mRNA and serum levels of TNFα in the DIO mice as compared to the lean mice. Higher TNFα levels are known to be associated with liver toxicity. Thus, CPT‐11 dosage should be closely monitored for effective and safe chemotherapy in obese patients who are at a higher risk of developing liver toxicity.
A corneal penetrating drug delivery system based on elastin‐like polypeptide (1053.4)
Authors: Bidwell Gene , Liu Huiling , Robinson Grant , Marquart Mary , George Eric
Published: June 2021

The corneal epithelium is connected by tight junctions and serves as a protective layer, but its resistance to penetration also presents a challenge to drug delivery. Upon injury, the cornea is susceptible to infection and/or neovascularization (NV), both of which can threaten sight. In the case of corneal bacterial infection, delivery of antibiotics and the development of antibiotic resistance are significant hurdles to effective therapy. In the case of corneal NV, there are no good treatments beyond removal of the insulting agent and resting of the eye. The goal of this study was to generate an effective and non‐toxic protein‐based delivery system for targeting therapeutic agents through the corneal epithelium. This drug delivery system utilized a biopolymer called elastin‐like polypeptide (ELP), an engineered protein that is easily purified and easily modified to carry drugs or therapeutic proteins. To facilitate delivery to the corneal stroma, we fused cell penetrating peptides (CPPs) to ELP. Addition of CPPs enhanced the total uptake and retention of ELP as much as 3‐fold in healthy rabbit corneas and in a rabbit burn model of corneal NV. The CPP also mediated penetration of ELP beyond the epithelium and basal lamina into the stromal layer. Our results demonstrate that CPP‐ELPs are a powerful platform for corneal drug delivery, and we describe their use for delivery of antibacterial and antiangiogenic agents.

Grant Funding Source: Supported by an Intramural grant to GLB from U of MS Med Center and NIH grant 1K99HL116774‐01 to EMG
A platform‐based approach for peptide drug discovery: generating probes to understand and treat itch (1054.1)
Authors: Doyle Jamie , Kumar Krishna , Dong Xinzhong , Kopin Alan
Published: June 2021

There is an unmet medical need for novel strategies to treat pruritus (itch). Recently a family of Mas‐related G protein‐coupled receptors (Mrgprs) were cloned and shown to be expressed in primary sensory neurons. Activation of MrgprX1 by the endogenous peptide BAM8‐22 results in histamine independent itch. MrgprX1 is also the target of chloroquine induced itch, a major side effect of this frequently used antimalarial drug. Pharmacological modulators of MrgprX1 are needed as tools to dissect the role of this receptor in mediating pruritus and as candidate therapeutics to combat itch. To define new ligands for MrgprX1, we utilized membrane tethered ligand (MTL) technology. MTLs are recombinant proteins comprised of an extracellular peptide ligand, a linker sequence, and an anchoring transmembrane domain. As MTLs, both BAM8‐22 and γMSH (another peptide ligand of MrgprX1) showed agonist activity. Because of the recombinant nature of MTLs, peptide variants can be rapidly generated and functionally assessed. Using alanine scanning of the two MTLs, we defined specific amino acid positions critical for activity at MrgprX1. With the knowledge gained from these studies, we have begun designing MrgprX1 membrane tethered peptide antagonists. Prior success in converting optimized recombinant MTLs to synthetic lipid anchored peptides provides a roadmap for developing MrgprX1 antagonists as therapeutics for pruritus.
Evaluation of functional activity and aggregation properties of hemopressin (1054.2)
Authors: Nayak Surendra , Song Benben , Kibler Patrick , Adem Amina , Endsley Aaron , Jambunathan Kalyani , Galande Amit
Published: June 2021

Hemopressin is a naturally occurring nonapeptide derived from the α‐chain of hemoglobin that acts as an inverse agonist for the type‐1 cannabinoid (CB1) receptor. Based on its in vivo activity, hemopressin is considered to have potential therapeutic applications in hypertension, pain, and obesity. We tested the hemopressin peptide for CB1 receptor agonist and antagonist activity in cAMP and β‐arrestin recruitment assay and found it to be inactive. The lack of reproducibility of CB1 receptor antagonist/ inverse agonist activity of hemopressin has been attributed to formation of aggregates. Hemopressin was previously reported to convert into amyloid‐like fibrils under aqueous conditions. However, the amino acid residues that modulate the aggregation propensity of hemopressin are not known. We have synthesized 25 different analogs of hemopressin and analyzed their aggregation properties using the principle of dynamic light scattering. We identified four conservative changes in the peptide sequence (Val2→DVal2, Asn3→Gln3, Leu7→Npg7, and C‐OH→C‐NH2) that lower the aggregation propensity of hemopressin. The results indicate that hemopressin aggregation is cooperative in nature and involves contribution from multiple amino acids within the peptide chain. The analogs are being evaluated for their functional activity in CB1 receptor assays. The structure‐function studies of hemopressin would further help understand its aggregation property and explore its potential therapeutic applications.

Grant Funding Source: Supported by the National Institute on Drug Abuse of the NIH under Award Number R21 DA034422.
The impact of immune modulators from the group of substituted thiadiazines on the severity of stress reaction (1054.7)
Authors: Sarapultsev Alexey , Chupakhin Oleg , Sarapultsev Petr , Mukhlynina Elena , Sarapultsev German , Medvedeva Svetlana , Danilova Irina
Published: June 2021

Central mode stress relieving effect of immune modulators from the group of substituted thiadiazines.

Stress reaction is characterized by a number of changes on the part of immune system in the form of high concentration of neutrophils, decreasing aggregate amount of lymphocytes, as well as appearance of catecholamine block of pancreas α‐receptors, which at the early stages of stress causes the so‐called «strain diabetes», and at its final stage (stage of exhaustion), progress of typical «steroid diabetes».

Previous studies demonstrated that the main effect mechanism of compounds from the group of substituted thiadiazines is their capability to modulate inflammation process. The goal of the present research was the evaluation of their effect on the course of stress reaction. The experiment was conducted by the pattern of 6‐hour and 48‐hour immobilization stress with rats. The animals got injection of the compounds, dozed 40 mg/kg. Histological study and biochemical analysis has been carried out at the 6 and 48 hours of the experiment.

On the background of compounds introduction, we detected lowering level of corticosterone, changes in the histomorphology of lymphopoiesis organs, as well as increase in the number of lymphocyte and monocyte series in the setting of decreasing number of eosinophils in the blood. The conducted research allowed to assume the existence of central effect mechanism of immune modulators from the group of substituted thiadiazines.

Grant Funding Source: Supported by grant 12‐M‐34‐2064
Fyn kinase regulates microglial neuroinflammatory responses in cell culture and animal models of Parkinson’s disease (1055.7)
Authors: Panicker Nikhil , Jin Huajun , Saminathan Hariharan , Kanthasamy Kavin , Anantram Vellareddy , Kanthasamy Anumantha , Kanthasamy Arthi
Published: June 2021

Sustained neuroinflammation is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson’s disease (PD). Resident brain microglia mediate chronic neuroinflammation through the production of proinflammatory cytokines and chemokines. Identifying the key molecular signaling events perpetuating microglial activation could unravel novel mechanisms underlying glial‐neuronal interactions that contribute to persistent inflammation and progressive neurodegeneration in PD. In the present study, we examined the role that Fyn, a non‐receptor tyrosine kinase, plays in microglial activation in cell culture and animal models of PD. First, we show that the known inflammogen LPS (lipopolysaccharide) rapidly activated Fyn kinase, as measured by Fyn‐Y416 phosphorylation and Fyn kinase assays in the BV2 microglial cell line as well as in primary microglia. Notably, immunocytochemical studies revealed that activated Fyn is preferentially localized to the microglial membrane periphery. Furthermore, stimulation of microglia with LPS induced phosphorylation of PKCδ at tyrosine 311. PKCδ Y311 phosphorylation was diminished in Fyn Knockout (Fyn KO) microglia, indicating that Fyn mediates this event. Functional studies revealed that the LPS‐induced production of cytokines, chemokines and nitrite were significantly attenuated in microglia isolated from Fyn KO and PKCδ KO primary microglia, when compared to wild type microglia. Furthermore, Fyn was found to be essential for LPS‐induced MAP kinase phosphorylation and activation of the NFκB pathway. Next, we extended our studies to LPS and MPTP‐induced animal models of neuroinflammation. We observed attenuated microglial proliferation and reduced production of the striatal TNFα, IL‐6 and IL‐12 in Fyn knockout mice. Collectively, these findings suggest that Fyn‐PKCδ signaling plays a critical role in mediating neuroinflammatory responses in PD

Grant Funding Source: Supported by NIH grants NS65167 and NS078247
Evaluation of anti‐inflammatory effect of analogs of L‐carnitine in a model of chronic inflammation in vivo (1056.1)
Authors: Mendoza‐Rivera Brissa , Reyes‐Esparza Jorge , De La Cruz Cordero Ricardo , Rodriguez‐Fragoso Lourdes
Published: June 2021

In this project we developed a model of chronic inflammation induced by subcutaneous implantation of cotton tassel to induce granuloma formation and evaluate drug efficacy of 3 analogs of L‐carnitine. Chronic granulomatous inflammation is a specific type of chronic inflammatory reaction, characterized by the accumulation of modified macrophages (epitheloid cells). Wistar male rats with granuloma were treated with the three analogs at a dose of 2mg/kg. Different serum interleukins were determined and histological analysis of thymus, spleen and granuloma was made. The analysis shows a reduced level of pro‐inflammatory interleukins (Il‐6 and TNF‐α) in animals treated with analogs having a greater effect the analog 2 and 3. Histological analysis showed the presence of epithelioid cells and giant cells in animals treated with L‐carnitine and analog 1, whereas animals treated with analogue 2 and 3 showed leukocyte infiltration. With regard the thymus, only was seen an increase in medulla area with the analog 2. While in the spleen was observed a reduction of medulla area with the analogs 2 and 3. In conclusion the analogs had anti‐inflammatory and anti‐granuloma activity, but they had different efficacy.

Grant Funding Source: CONACYT
TLR4‐p38 pathway as a mechanism of APOE‐modulated neuroinflammation in Alzheimer disease (1056.6)
Authors: LaDu Mary , Wohlgenant Susan , Van Eldik Linda , Tai Leon
Published: June 2021

Soluble oligomeric Aβ (oAβ) is a neurotoxic aggregate thought to be a proximal cause of Alzheimer disease (AD). Chronic inflammation of glial cells induced by oAβ‐induced likely results in both direct neurotoxicity and decreased phagocytic clearance of Aβ and cellular debris. The strongest genetic risk factor for AD is the ε4 allele of the APOE gene. As APOE4 is associated with a proinflammatory response compared to APOE3, we determined the APOE‐specific neuroinflammatory phenotype in novel EFAD transgenic mice, which overexpress human Aβ42 and apolipoprotein E (apoE). Overall, our data are consistent with an increased pro‐inflammatory, anti‐phagocytic (M1) and reduced anti‐inflammatory, repair, (M2) response in E4FAD compared to E3FAD mice. To determine the mechanism underlying these effects and test our hypothesis that TLR4 signaling mediates Aβ‐induced APOE‐modulated neuroinflammation, a primary glial screen was developed. LPS‐ and oAβ‐induced secretion of TNF‐α is concentration dependent and higher with apoE4 than apoE3 in vitro. Importantly, TLR4 antagonists (LPS‐RS, IAXO series), and an inhibitor of p38α (a down‐stream target of TLR4) prevented oAβ42‐induced TNF‐α secretion. Thus, TLR4 signaling represents a novel pathway for APOE4‐specific neuroinflammation.
In vivo immunomodulatory effect of S22‐Gda (1056.9)
Authors: Jang Yeonjeong , Kim Jiyun , Lee Chunggi , Pyo Suhkneung
Published: June 2021

S22‐Gda is a purified extract from mixture of Celosia argentea Linne, Allium sativum L. and Plantago asiatica. Many herbal extract mixtures have been known to have various biological activities including stimulation of immune response. However, the biological effects of S22‐Gda have not been addressed. In the present study, we examined immunomodulatory effect of S22‐Gda on peritoneal macrophages and splenic immune cells from six week‐old mice that were fed either a control or oral administration 50 mg/kg of S22‐Gda for 14 days. The results from MTT assay showed that S22‐Gda enhanced macrophages‐ or NK cells‐mediated cytotoxicity to tumor cells. In addition, S22‐Gda significantly increased phagocytosis capacity of peritoneal macrophage. The colorimetric NO assay revealed that NO production was not increased by S22‐Gda in peritoneal macrophage. However, the results from ELISA assay demonstrate that S22‐Gda significantly increased the production of IL‐6, TNF‐α and IFN‐α but not INF‐β in peritoneal macrophage. Overall, our results suggest that S22‐Gda may be a potential immunotherapeutic agent by stimulating macrophages and splenic immune cells functions.
Arachidonic acid induced calcium signaling at acidic pH (1057.2)
Authors: Moss Nicholas , Hou Bing , Young Richard , Bruns Alexander , Wilson Lesley , Beech David
Published: June 2021

Arachidonic acid (AA) induces calcium signalling and downstream functional responses. It is often present in pathophysiological environments where there is also acidity, as in ischaemic tissues and tumours. The aim of our study was to investigate the relevance of the acidic environment to AA‐evoked responses. In intracellular Ca2+ measurement experiments, AA induced a small Ca2+ influx at pH 7.4 in human umbilical vein endothelial cells which was potentiated by almost an order of magnitude at pH 6.8. The response at pH 6.8 was not dependent on the AA‐activated channels TRPV4 or Orai3. Unexpectedly, inhibitor antibodies targeted to TRPC4 or TRPC5 suppressed the response. Furthermore, TRPC5 channels stably expressed in HEK293 cells were activated by AA at pH 6.8 but not 7.4. The data suggest that mild acidosis strongly potentiates the contribution of TRPC channels to AA responses.

Grant Funding Source: Supported by the British Heart Foundation
Technical and knowledge‐based outcomes following a one‐week high school research program in toxicology and environmental health sciences (1058.1)
Authors: Guo Grace , Aleksunes Lauren , Moscovitz Jamie , Venosa Alessandro , Gow Andrew , Laskin Debra
Published: June 2021

As an applied science, students are often not exposed to the fields of toxicology and environmental health sciences until their undergraduate or graduate studies. The purpose of this summer program was to introduce rising sophomore, junior, and senior high school students to the concepts of toxicology and environmental health sciences, provide hands‐on laboratory training, and highlight various careers in toxicology and environmental sciences, as well as biomedical fields. Two groups of 23 students (n=46 total) participated in a non‐residential, one‐week summer program at the Rutgers University that consisted of didactic (70%) and laboratory (30%) activities organized by toxicology graduate students and faculty instructors. Activities focused on dose‐response relationships, experimental design, clinical toxicology, histology and pathology, epidemiology, and genetics. Pre‐ and post‐training surveys aimed to assess student motivation to participate in the program, research and technical abilities, and basic toxicology and biomedical knowledge. Students informed that the greatest motivating factors to participate in this program included an opportunity to explore their interest in science and gain hands‐on laboratory experience. Self‐assessment surveys revealed improved presentation skills (24%), laboratory proficiency including DNA isolation (49%) and cell culture (31%) as well as assessment of patient clinical findings (32%). Using a true/false exam, student’s knowledge of key toxicology concepts increased from the beginning (67% correct responses) to the end of the program (81% correct responses). The genetics, histology, and pathology labs received the highest ratings. A one‐week summer laboratory and didactic program engages high school students in toxicology and environmental health sciences and improves laboratory and presentation skills as well as increases knowledge of overall biomedical concepts.
Mechanisms underlying behavioral tolerance following repeated 5‐HT2CR agonist administration in vivo (1059.11)
Authors: Cooper Debra , Davis‐Reyes Brionna , Bubar Marcy , Cunningham Kathryn
Published: June 2021

The serotonin 5‐HT2C receptor (5‐HT2CR) is known to play a role in several psychiatric disorders. Repeated treatment with 5‐HT2CR agonists, however, may induce tolerance to the therapeutic effects by altering receptor responsiveness. The present study explores the molecular mechanisms underlying tolerance to 5‐HT2CR agonist‐induced hypomotility following repeated administration. Male, Sprague‐Dawley rats were treated daily for seven days with saline or the preferential 5‐HT2CR agonist MK 212 (2 mg/kg, IP, BID). Administration of MK 212 (2mg/kg, IP) 24‐hrs following the 7‐day regimen induced hypomotility in saline treated rats, which was blunted following repeated MK 212 treatment, suggesting tolerance developed to the 5‐HT2CR agonist. This loss of 5‐HT2CR responsiveness was not associated with changes in crude membrane‐bound 5‐HT2CR protein expression in the ventral tegmental area, nucleus accumbens or prefrontal cortex. More detailed assessments of 5‐HT2CR expression, subcellular localization and trafficking are being explored in a separate group of rats treated daily for seven days with the more selective 5‐HT2CR agonist lorcaserin (Belviq®), the first‐in‐class selective 5‐HT2CR agonist approved by the FDA for obesity. Mechanisms identified in this study will be important in addressing tolerance to the therapeutic effects of 5‐HT2CR agonists in clinical use.

Grant Funding Source: Supported by: DA033935, DA020087, DA07287, DA13595, DA15259, GM069285
Development of novel serotonin 7‐targeting compounds based on the 2‐dimethylaminotetralin scaffold (1059.13)
Authors: Canal Clinton , Felsing Daniel , Zhu Wanying , Liu Yue , Cordova‐Sintjago Tania , Booth Raymond
Published: June 2021

The serotonin (5‐HT) 7 G protein‐coupled receptor (GPCR) is the most recent 5‐HT receptor cloned, and is expressed in both the periphery and the CNS. Reports propose 5‐HT7 receptors are involved in a wide range of CNS functions, including modulation of circadian rhythms, sleep, pain, body temperature, and cognition. 5‐HT7 receptors couple to Gαs, leading to activation of adenylyl cyclases and production of cAMP. Due to paucity of available, selective 5‐HT7 ligands, the complexity of CNS processes and to a limited number of studies examining 5‐HT7, however, there is no consensus about 5‐HT7 receptor CNS neuropharmacological function. We have begun an iterative 5‐HT7 receptor structure‐based ligand synthesis program to develop novel 5‐HT7‐targeting phenylaminotetralin (PAT) compounds. A molecular model of the 5‐HT7 receptor was built by homology to the recently reported structure of the 5‐HT1B GPCR, and computational ligand docking studies were undertaken. Computational results were validated by experimental results of ligand affinity (Ki) at agonist‐labeled human 5‐HT7 receptors in membranes prepared from HEK cells stably expressing a high density of receptors (~7 pmol/mg protein). A number of PAT ligands had Ki 蠄 7 nM at 5‐HT7 receptors. PAT ligands with Ki less than 50 nM at 5‐HT7 receptors were assessed for 5‐HT7 receptor‐mediated cAMP formation in the 5‐HT7 stable cells; the PATs assessed behaved as inverse agonists or partial agonists with IC/EC50 values similar to their Ki values. PAT affinities were also determined at human 5‐HT2A, 2B, and 2C, histamine H1, α1A, 1B, 2A, 2B, and 2C adrenergic GPCRs. In translational studies, none of the PAT compounds tested were active in the hot‐plate analgesia assay in C57Bl/6J mice after peripheral administration, including PATs known to cross the blood‐brain barrier. Additional medicinal chemical syntheses, guided by results from molecular modeling and ligand docking studies are underway, as are in vitro and in vivo neuropharmacology studies.

Grant Funding Source: Supported by NIH R01 DA030989 and MH081193
A translational approach to nicotine dependence: interrogating the Neuregulin‐ErbB4 pathway in mouse and man (1059.6)
Authors: Turner Jill
Published: June 2021

Addiction to nicotine and ability to quit smoking are influenced by genetic factors. Recently, we published data using functional genomic approaches (chromatin immunoprecipitation and whole genome sequencing) to identify novel CREB targets following chronic nicotine administration and withdrawal (Mol Psychiatry 2013). We observed in rodents that nicotine withdrawal modulates CREB binding to the gene for Neuregulin 3 (NRG3). Further studies in humans indicated that SNPs across NRG3 were significantly associated with prospective smoking cessation. NRG3 is a neural‐enriched member of the EGF family, and a specific ligand for the receptor tyrosine kinase ErbB4, which is also up‐regulated following nicotine treatment and withdrawal. Our findings, along with a recent study identifying associations of ErbB4 with smoking status (Loukola 2013), strongly implicate this pathway in the molecular mechanisms underlying nicotine dependence and smoking cessation. Interestingly, in addition to its relevance in smoking cessation, this pathway has also been described in the pathophysiology of schizophrenia, suggesting a common molecular link between these two highly co‐morbid diseases. Ongoing experiments examining molecular changes downstream of NRG3/ErbB4 signaling indicate that alterations in NMDAR signaling may be influencing both smoking behavior as well as schizophrenia.

Grant Funding Source: Supported by 1‐K99‐DA032681‐01 and1‐R00‐DA032681‐02.
Hyperbaric oxygen suppresses paclitaxel‐induced cold allodynia in rats (1061.2)
Authors: Zhang Yangmiao , Nelson Jordan , Shirachi Donald , Quock Raymond
Published: June 2021

HBO2 treatment can relieve chronic pain (Thompson et al., Neurosci Res 66:279, 2010). Previously, we reported that HBO2 suppressed PAC‐induced mechanical allodynia (Zhang et al., FASEB J 26:662.9, 2012). The aim of this study was to determine whether HBO2 treatment could also reduce PAC‐induced cold allodynia. Male Sprague Dawley rats, 250‐350 g, were injected intraperitoneally with either 1.0 mg/kg PAC or vehicle on days 0, 2, 4 and 6. On day 7, rats were treated with 60‐min HBO2 at 3.5 atmospheres absolute (ATA) or room air for either one or four days. Cold allodynia was assessed every other day by applying 50 µL acetone to each hind paw. Allodynia was scored using a 0‐3 point scale. Results showed that PAC‐treated rats developed cold allodynia in response to acetone. A single 60‐min HBO2 treatment at 3.5 ATA did not produce obvious recovery from allodynia. However, allodynia was suppressed in rats that underwent four daily 60‐min HBO2 treatments. These results indicate that, while one HBO2 treatment is not sufficient to reduce PAC‐induced cold allodynia, repeated (4×) HBO2 treatment can suppress the development of PAC‐induced cold allodynia.

Grant Funding Source: Supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship.
A novel class of allosteric ligands modulating TRPA1: discovery of a switch in agonist‐antagonist function (1061.7)
Authors: Herz Jeffery , Zhu Yingmin , Lu Yungang , Kesicki Edward , Thomsen William , Zhu Michael
Published: June 2021

TRPA1 functions as a ligand‐gated cation channel in sensory neurons and is an important drug discovery target for identification of novel analgesics. We report identification of a novel structural class of ligands reversibly modulating TRPA1 channel function. Using intracellular calcium measurement and whole cell patch clamp recording of HEK293 cells expressing human TRPA1, we characterized the structure‐activity relationships among a group of amino‐triarylmethane analogs. A minimal structural change switched a full agonist to antagonist and small changes in side chain substituents in the same ring greatly altered affinity among antagonists. The agonists, ALGX‐2014 and ALGX‐2015, only elicited TRPA1 currents when applied extracellularly. Their stimulatory effect was blocked by TRPA1 antagonists and unaffected by substituting the three cysteine residues of TRPA1 critical for covalent stimulation by electrophilic agonists, such as AITC and PGJ2, with alanines, suggesting channel activation occurs in a noncovalent manner. This novel class of allosteric agonists and antagonist ligands also interact with other well‐defined TRPA1 ligands and results suggest a highly conserved binding site among TRPA1 and other members of the thermo‐TRP family which may reside in the channel domain.

Grant Funding Source: Supported by NIDA grant (DA031516) to JMH and NIDDK grant (DK081654) to MXZ
Dietary supplements influence activity of acid‐sensing ion channels (1061.8)
Authors: Agharkar Amruta , Gonzales Eric
Published: June 2021

Dietary supplements and nutraceuticals have been the focus of research to identify novel therapeutics for a variety of pathologies, including the prevention of long‐term consequences of stroke and reducing pain. Ion channels offer a growing group of molecular targets for treatment, which include the acid‐sensing ion channels (ASICs). Acid‐sensing ion channels (ASICs) are sodium channels that are sensitive to changes in extracellular pH, specifically those changes following injury and ischemia. These channels are expressed most prominently in peripheral and central nervous system. Their role in physiology is yet to be fully understood, but these channels have been implicated in pain sensation and centrally in the neurodegeneration following ischemic stroke. We identified an over‐the‐counter dietary supplement (DS) that shares similarity to guanidine compounds that selectively modulate acid‐sensing ion channels. Thus, we hypothesize that this dietary supplement inhibits channel activity in acid‐sensing ion channels. Our preliminary data show that DS decreased the ASIC1a pH sensitivity by shifting the observed proton activation profile to the right. Furthermore, we observed a change in the Hill coefficient of the DS influenced ASIC1a steady‐state desensitization profile. Future experiments will focus on determining the influence of DS on other acid‐sensing ion channel subtypes and identifying the DS binding site with the protein structure.

Grant Funding Source: American Heart Association 12BGIA8820001
Claudin‐1 as a target for modulation of epidermal barriers (1062.6)
Authors: Nakajima Misaki , Yamashita Mayo , Watari Akihiro , Yagi Kiyohito , Fukasawa Masayoshi , Kondoh Masuo
Published: June 2021

Epithelium plays pivotal roles in barriers that separate the inside of the body from the outside environment. Passage across epithelial barriers is the first step in drug absorption. Epidermal absorption is an ideal route for drug administration because it is non‐invasive and easily controlled. Claudin‐1 (CLDN1) seals tight junctions in the stratum granulosum, but whether modulation of CLDN1 enhances epidermal permeability is unclear. We investigated the effect of an anti‐CLDN1 antibody on the epidermal epithelial barrier by using human epidermal keratinocytes (NHEK), a popular in vitro model of human epithelial cell sheets. Immunoblot and FACS analyses showed that CLDN1 was expressed on NHEK cell membranes. Treatment of NHEK monolayers with anti‐CLDN1 antibody decreased the integrity of tight junctions between adjacent cells and enhanced the permeation of solutes across the cell sheets. Furthermore, treatment of Caco‐2 cell monolayers, a popular model of the human intestinal barrier that expresses CLDN1 and CLDN4, with anti‐CLDN4, but not treatment with anti‐CLDN1, decreased the integrity of tight junctions. These findings suggest that CLND1 may be a target for specific modulation of epidermal barrier.

Grant Funding Source: supported by the Ministry of Health, Labor, and Welfare of Japan
Role of renal biotransformation in 3,4,5‐trichloroaniline nephrotoxicity in vitro (1063.1)
Authors: Rankin Gary , Anestis Dianne , Racine Christopher
Published: June 2021

Chlorinated anilines are commonly used in the production of agricultural products. The purpose of this study was to explore the role of renal biotransformation in 3,4,5‐trichloroaniline (3,4,5‐TCA) nephrotoxicity using an in vitro model. Isolated renal cortical cells (IRCC; 3 ml: 4.0 X 106 cells/ml) from male Fischer 344 rats were treated with 0.5 mM 3,4,5‐TCA or DMSO (vehicle control) for 120 minutes. Cytotoxicity was determined using lactate dehydrogenase (LDH) release assays. In some experiments, cells were pretreated with either an antioxidant [ascorbate (2.0 mM), glutathione (1.0 mM), N‐acetyl‐L‐cysteine (2.0 mM) or alpha‐tocopherol (1.0 mM)], a flavin monooxygenase (FMO) inhibitor [methimazole (1.0 mM), or N‐octylamine (1.0)], a cytochrome P450 (CYP) inhibitor [metyrapone (1.0 mM) or piperonyl butoxide (PIBX; 1.0 mM)], a cyclooxygenase inhibitor [indomethacin (1.0 mM)] or a peroxidase inhibitor (mercaptosuccinate, 0.1 mM). 3,4,5‐TCA nephrotoxicity was not attenuated by pretreatment with methimazole or N‐octylamine, suggesting that FMO mediated oxidation doesn’t play a significant bioactivating role in 3,4,5‐TCA nephrotoxicity. Pretreatment with indomethacin or mercaptosuccinate significantly attenuated toxicity. The CYP inhibitor PiBx, but not metyrapone, also significantly attenuated 3,4,5‐TCA toxicity. All antioxidant pretreatments significantly attenuated 3,4,5‐TCA nephrotoxicity. These results suggest that free radicals play a role in 3,4,5‐TCA nephrotoxicity in vitro, and that these species arise from biotransformation of 3,4,5‐TCA by cyclooxygenase and peroxidase activity and/or via CYP isozymes inhibited by PiBx but not metyrapone.
Anemia in adenine‐induced chronic renal failure and the influence of treatment with gum acacia thereon (1063.4)
Authors: Ali Badreldin , Al Z'aabi Mohammed , Ramkumar A. , Nemmar A.
Published: June 2021

Anemia is an early complication of human chronic kidney disease (CKD) and chronic renal failure (CRF) in animals. We investigated the effect of induction of CRF in rats by feeding them adenine (0.75%, w/w, 4 weeks) on erythrocyte count (EC), hematocrit (PCV) and hemoglobin (Hb) concentration, as well as on the activity of L‐γ‐glutamyl transferase (GGT) and iron (Fe), transferrin (Tf), ferritin (F), total iron binding capacity (TIBC) / unsaturated iron binding capacity (UIBC) and hepcidin (Hp) in serum and erythropoietin (Epo) in renal tissue. Renal damage was assessed histopathologically, and also by measuring the serum concentrations of the uremic toxin indoxyl sulfate (IS), creatinine, and urea, and by creatinine clearance. We also assessed the influence of concomitant treatment with gum acacia (GA) in the drinking water (15%, w/v, 4 weeks) on the above analytes. Adenine ‐ induced CRF, accompanied by significant decreases (P < 0.05) in EC, PCV, and Hb, and in the serum concentrations of Fe, Tf, TIBC, UIBC and Epo. It also increased Hp and F levels. GA significantly ameliorated these changes in the adenine‐treated rats. A general improvement in the renal status of rats with CRF after GA is shown due to its anti‐inflammatory and anti‐oxidant actions, and reduction of the uremic toxin IS, which is known to suppress Epo production, and this may be the basis of its ameliorative actions on the indices of anemia studied.

Grant Funding Source: Supported by the Research Council of Oman
The human testis as a model of retinoic acid formation (1064.10)
Authors: Arnold Samuel , Kent Travis , Schlatt Stefan , Prasad Bhagwat Prasad , Haenisch Michael , Muller Chip , Hogarth Cathryn , Griswold Michael , Paik Jisun , Walsh Thomas , Amory John , Isoherranen Nina
Published: June 2021

Retinoic acid (RA), the active form of vitamin A (retinol), is indispensable for maintaining many essential biological processes. RA formation depends on a complex network of enzymes. The rate limiting step is irreversible formation of RA by aldehyde dehydrogenase1A (ALDH1A1‐1A3). However, the specific roles, tissue expression, and localization of these enzymes are not well understood.

While RA is necessary for spermatogenesis in animal models, the source of intratesticular RA is unknown. Sertoli cell specific ALDH1A knockouts have demonstrated intratesticular RA formation is required for male fertility. Due to the requirement for intratesticular RA formation and presence of mRNA for all three ALDH1A enzymes in the human testis, the human testis was chosen as a model of the biochemistry of RA formation.

In order to model RA formation, a novel LC‐MS/MS based peptide quantification method was developed to quantify ALDH1A in a cohort of 18 men. Each ALDH1A concentration was used to predict the velocity of RA formation in each donor. The accuracy of our predictions was confirmed by measuring intrinsic RA formation and RA concentrations in the same samples. Additionally, weidentified a distinct localization pattern for ALDH1A enzymes in the human testis using immunohistochemistry.

In conclusion, these novel techniques were successfully used to model RA formation in human testicular tissue. In the future, RA formation in other tissues can be investigated using the assays developed.

Grant Funding Source: Supported by NIH/NICHD grant U54 HD042454 and TL1TR000422
Activation of vascular Toll‐like receptor 3 induces phosphorylation of caldesmon via ERK1/2 pathway (1065.12)
Authors: Carrillo‐Sepulveda Maria Alicia , Hardigan Trevor , Webb R. Clinton
Published: June 2021

TLR3, a pattern‐recognition receptor of the innate immune system, is constitutively expressed in vascular smooth muscle cells (VSMC) and has been showed to be functional in the vascular system; however its role in contraction remains unknown. Caldesmon (CaD) is a potent and versatile regulatory protein that play crucial role in contractility of smooth muscle. We hypothesized that TLR3 contributes to contractile events in VSMC by phosphorylation of CaD in Ser789. Phosphorylation of myosin light chain (pMLC), marker of contraction, increased by 65% in mouse C57bl/6 aortic smooth muscle cells (ASMC) stimulated with TLR3 agonist polyinosine polycytidylic acid (Poly I:C) (10 μg/mL, 60 min). This effect was accompanied by increased phosphorylation of CaD on Ser789 (3 fold versus control unstimulated ASMC) as well as significantly activation of mitogen‐activated protein/extracellular signal‐regulated kinase (ERK1/2). Pre‐treatment with PD98059 (50μM, 30 min), ERK1/2 inhibitor, abolished Poly I:C‐mediated phosphorylation of CaD. Interestingly, chronic treatment with Poly I:C for 24 hours downregulated by 40% TLR3 expression in ASMC. The present results showed that activation of TLR3 in VSMC causes ERK1/2‐mediated phosphorylation of CaD, thereby contributing to augmented contraction and consequently to development of vascular dysfunction. Our data provide the first functional assessment of the role of TLR3 in vascular contractile events, suggesting TLR3 as a potential new therapeutic target in vascular dysfunction.

Grant Funding Source: AHA 13POST14690026
Contribution of nuclear factor E2‐related factor 2 to the atherogenic phenotype transition in coronary arterial smooth muscle cells lacking CD38 gene (1065.16)
Authors: Xu Ming , Li Xiaoxue , Wang Mi , Zhang Yang , Li Pin‐lan
Published: June 2021

Recent studies have reported that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle (SMCs) upon atherogenic stimulations. However, the molecular mechanisms mediating this SMC phenotypic change remain unknown. In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38‐/‐ mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin and α‐SMA and increased the expression of SMC dedifferentiation marker vimentin, which was accompanied by collagen I accumulation and cell proliferation. This phenotypic change was enhanced by 7‐ketocholesterol (7‐Ket), an atherogenic stimulus in CAMs from CD38‐/‐ mice. We further found that the CD38 deficiency decreased the protein level of nuclear factor E2‐related factor 2 (Nrf2) and inhibited its activity. Given that Nrf2 is a transcription factor that responds to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity. It was demonstrated that in CD38‐/‐ CAMs 7‐Ket failed to produce O2‐., while in CD38+/+ CAMs 7‐Ket‐induced O2‐. production was substantially attenuated by Nox4 gene silencing. In addition, 7‐Ket‐induced increase in Nrf2 activity in CD38+/+ CAMs was significantly inhibited by silencing Nox4 gene. Finally, we demonstrated that both Nrf2 gene silencing and CD38 deletion had similar effect to increase CAM dedifferentiation upon 7‐Ket stimulation. In contrast, the overexpression of Nrf2 gene markedly abolished 7‐Ket‐induced dedifferentiation in CD38‐/‐ CAMs. These results suggest that CD38 activity is required for 7‐Ket‐induced O2‐. production, which increases Nrf2 activity maintaining CAMs in a differentiated status. When CD38 gene expression and function are deficient, the Nrf2 activity is suppressed thereby leading to dedifferentiation of CAMs.

Grant Funding Source: HL057244, HL091464 and HL075316