bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Alcohol on Innate Immune Responses of BV‐2 Microglial Cell Line without Type 7 Adenylyl Cyclase
Authors: Hu Yawen , Yoshimura Masami
Published: June 2021

Alcohol consumption affects immune cell functions and induces neuroinflammation. The mechanism of ethanol's action on the immune system however, are not fully understood. Cyclic AMP (cAMP) is known for its immunosuppressive effects and also is required for proper development of the immune system. Previous work in our laboratory has demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform‐specific manner. Type 7 AC (AC7) is most enhanced by ethanol, and is highly expressed in immune cells including macrophages. Therefore, we hypothesize that AC7 plays an important role in alcohol's effects on innate immune functions of microglia and on neuroinflammation through a cAMP pathway. To test this hypothesis, we generated AC7 knockout (KO) cell lines from the mouse BV‐2 microglia cell line by CRISPR‐Cas9 mediated genome editing. A cAMP accumulation assay was used to confirm AC7 KO. The effects of ethanol on innate immune responses of BV‐2 cells with or without AC7 were examined by measuring phagocytosis and oxidative bursts via flow cytometry.

The cAMP accumulation assay on wild type (WT) BV‐2 cells showed enhancement of cAMP production in the presence of prostaglandin‐E1 (PGE1). cAMP production was further enhanced by addition of ethanol, as well as an AC7 specific activator, sphingosine‐1‐phosphate (S1P). cAMP production in AC7 KO cells was drastically decreased and did not respond to either ethanol or S1P. The results suggest that AC7 is the dominant form of AC in the microglia. Phagocytic activity measured using fluorescently labeled bacteria was significantly suppressed in the presence of isopropanol (Iso) in WT BV‐2 cells, while Iso did not significantly change the phagocytic activity of AC7 KO cells. Ethanol further reduced phagocytic activity of WT cells in the presence of Iso. Ethanol alone did not suppress phagocytosis in WT BV‐2 cells, but significantly inhibited phagocytosis in AC7 KO cells. This latter difference in response was unexpected and may indicate that AC7 can protect microglia from ethanol‐induced inhibition of phagocytosis. The production of reactive oxygen species was measured using dihydrorhodamine 123. We did not observe any notable differences in oxidative burst between WT and AC7 KO BV‐2 cells. The results indicated that AC7 elimination altered ethanol effects on some of the immune functions of BV‐2 cells, which is consistent with our hypothesis. We plan to further examine innate immune functions including bacterial killing, nitric oxide production, and proinflammatory cytokine expression in AC7 KO cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glutamic‐Oxaloacetic Transaminase Combined with Metabolic Therapy in a Mouse Model of Amyotrophic Lateral Sclerosis
Authors: Rogers Christopher Q. , Ramirez Melissa , Landon Carol S. , DeBlasi Janine M. , Koutnik Andrew P. , D'Agostino Dominic P.
Published: June 2021

Amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig's Disease is a progressive disease of neuronal degeneration in the spinal cord, brainstem and motor cortex resulting in impaired motor function and eventual demise. There is currently no widely accepted, effective treatment for this condition.

Glutamate (Glu) excitotoxicity is a process in which excessive Glu‐induced stimulation of neurons can lead to their damage and death. The drug riluzole (Rilutek) is the only medication approved by the FDA for ALS. The drug appears to slow the disease's progression in some people, perhaps by reducing CNS Glu that is often present in elevated levels in ALS patients.

Oxaloacetate (OX) is an anaplerotic molecule and initiation point for the activation of Kreb's Cycle. Previous work has demonstrated the ability of exogenous OX to reduce CNS Glu levels. Glutamic‐oxaloacetic transaminase (GOT) is an endogenous enzyme that catalyzes the following reaction: Glutamate + Oxaloacetate ↔ Alpha‐Ketoglutarate + Aspartate. GOT with OX has been shown to increase the removal of Glu from the blood and the CNS, thereby reducing excitotoxicity and improving neuronal health.

A novel metabolic therapy known as the Deanna Protocol (DP) has been used effectively in ALS patients and mice. It consists of dietary supplements (arginine, alpha‐ketoglutarate, medium‐chain triglyceride oil, phenibut and coenzyme Q‐10) to support mitochondria and the Kreb's Cycle.

Here we investigate the DP alone or in conjunction with GOT and OX as a potential therapy. Twenty ALS mice were randomly separated into four groups. Mice were singly‐housed. The control group received standard chow mash. The DP group received DP mixed with standard chow mash. The GOT/OX group received injections of GOT 3X/wk and OX mixed with standard chow mash. The DP + GOT/OX received injections of GOT 3X/wk and OX mixed in a DP‐containing chow mash.

Compared to the control group, the three treatment groups each had significantly longer survival times, improved motor performance, and better neurological scores. The treatments were neither additive nor synergistic, suggesting unique therapeutic effects derived from each treatment. These findings suggest that the DP and/or GOT/OX treatment may have therapeutic application in patients with ALS and perhaps other neurologic disorders.

Support or Funding Information

This study was supported by Winning the Fight against Neurodegenerative Diseases grant #111990. Oxaloacetate kindly provided by Alan Cash.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Lipocalin‐2 (LCN2) and High Mobility Box Group 1 (HMGB1) in Acetaminophen‐induced Acute Liver Failure
Authors: Reed E'Joven D. , Shashidharamurthy Rangaiah , Bhave Vishakha S.
Published: June 2021

Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF). Following APAP overdose, toxicity is initiated by the conversion of APAP to its reactive metabolic N‐acetyl‐p‐benzoquinone imine (NAPQI) by CYP2E1/CYP3A4. NAPQI binds covalently to mitochondrial proteins forming 3‐(cysteine‐S‐yl)‐acetaminophen (APAP‐Cys). This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. Lipocalin‐2 (LCN2), is an acute‐phase innate immune protein, upregulated during tissue injury in various organs including the liver. Recently we have demonstrated that LCN2 is pro‐inflammatory and elevated during APAP overdose in mouse model. Interestingly, LCN2 knockout (LCN2 KO) mice exhibit protection from APAP overdose compared to its wild type (WT) littermates. During APAP overdose there is necrosis of hepatocytes, these hepatocytes release Damage Associated Molecular Patterns (DAMPS) that further exacerbate the inflammatory process. One such DAMP is High Mobility Group Box 1 (HMGB1). However, the role of LCN2 and HMGB1 in APAP overdose has not yet been investigated.

Present study investigates the molecular mechanism of LCN2 during the progression of acute liver injury in APAP overdose model. We hypothesize that during APAP induced ALF, LCN2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and LCN2 KO mice will be utilized to test our hypothesis. HepG2 cells will be treated with recombinant LCN2 to investigate whether exogenous LCN2 can cause induction and translocation of HMGB1. Translocation of HMGB1 will be assessed by immunofluorescence staining. Serum and tissues samples of WT and LCN2 KO mice subjected to APAP overdose will be assessed for expression of HMGB1. APAP‐cys formation will be measured in WT and LCN2 KO using the High‐Performance Liquid Chromatography method to ensure comparable bioactivation based injury between the WT and LCN2 KO mice. Lastly, histopathology will be performed on liver samples from both groups to assess liver pathology. It is anticipated that LCN2 mediates progression of liver injury in APAP overdose via HMGB1.

Support or Funding Information

Philadelphia College of Osteopathic Medicine‐Division of Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histone Deacetylase 3 Promotes Liver Regeneration and Liver Cancer Cells Proliferation through Signal Transducer and Activator of Transcription 3 Signaling Pathway
Authors: Lu Xufeng , Zhuang Xiang , Zhu Yongjie , Cao Xiaoyue , Shao Mingyang , Xu Qing , Zhou Yongjie , Wu Zhenru , Ji Hongjie , Lu Qing Richard , Yang Jiayin , Shi Yujun , Liao Jiayu , Zeng Yong , Bu Hong
Published: June 2021

Histone deacetylase 3 (HDAC3) plays pivotal roles in cell cycle regulation and is often aberrantly expressed in various cancers including hepatocellular carcinoma (HCC), but little is known about its role in liver regeneration and liver cancer cells proliferation. Using an inducible hepatocyte‐selective HDAC3 knockout mouse, we find that lack of HDAC3 dramatically impaired liver regeneration and blocked hepatocyte proliferation in the G1 phase entry. HDAC3 inactivation robustly disrupted the signal transducer and activator of transcription 3 (STAT3) cascade. HDAC3 silencing impaired the ac‐STAT3‐to‐p‐STAT3 transition in the cytoplasm, leading to the subsequent breakdown of STAT3 signaling. Furthermore, over‐expressed HDAC3 was further associated with increased tumor growth and a poor prognosis in HCC patients. Inhibition of HDAC3 expression reduced liver cancer cells growth and inhibited xenograft tumor growth. Our results suggest that HDAC3 is an important regulator of STAT3‐dependent cell proliferation in liver regeneration and cancer. These findings provide new insights into the HDAC3‐STAT3 pathway in liver pathophysiological processes.

Support or Funding Information

This work was supported by grants from the Natural Science Foundation of China (NO. 81472303), the National Key Clinical Project, and the Major Science and Technology Project of Science & Technology Department of Sichuan Province (2017SZ0003).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heavy Baristas, Heavy Orders: Customers Order More Calories from Overweight Baristas
Authors: Wansink Brian , Junghans Astrid
Published: June 2021

Purchasing coffee is a daily ritual for many consumers. How might various characteristics of the barista influence what a customer decides to order? If baristas do have a small calorie‐biasing influence on an order, when repeated hundreds of times, could calories become kilograms? 1,010 customers were observed as they ordered at 34 different Starbucks locations. Customer and barista characteristics, customer orders, and the calories in these orders were coded. Analyses tested the effect of barista weight and gender on the likelihood that consumers would purchase add‐ons to their order, as well as the calories in these items. Customers ordering from overweight baristas appeared to be twice as likely to order food with their drink and to order extra add‐ins to their drink. While the overall calories in the drinks were not different, the total calories ordered (food calories plus drink calories) was greater for those who ordered from overweight baristas compared to normal weight ones. The barista's body size appears to be related to the number of calories ordered by consumers. This underscores the wide range of primes and cues that exist in restaurants that can unknowingly bias what and how much one orders. Pre‐committing to an order before arriving at a restaurant will reduce the influence of environmental primes and cues.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
First Seen First Bought: Supermarket Aisles Order and Spending
Authors: Wansink Brian , Stein Kate , Lutz Richard
Published: June 2021

In grocery stores, product location may be key to more persuasive marketing and increased sales; we investigated the links between aisle location, likelihood of aisle entry, and purchase incidence. We observed the behaviors of 328 shoppers at four different supermarkets, recording how often they entered each aisle and how many items they selected from that aisle. We found that aisle content and amount of time spent in the store determine the likelihood of aisle entrance. As a shopping trip continues, a number of factors may drive a shopper into greater selectivity in aisle entrance: impatience, product assortment in that aisle, and time pressures. Shoppers who enter an aisle later in their shopping trip will be more likely to select a product, yet they make fewer purchases in that aisle than in aisles they shopped earlier in their trip. Diminished motivation levels and decreased enthusiasm may prompt this observed effect.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High fat diet risk factor for gingival Immunosuppression and cancer
Authors: Kaur Kawaljit , Eibl Guido Anahid , Jewett Anahid
Published: June 2021

Objective

High fat diet leading to obesity has been associated with many diseases including cancer. We determined the effect of high fat diet and obesity in the phenotype and function of immune cell infiltrates within different tissues including gingival tissues of mice with wild type and targeted KRAS mutations in the pancreas.

Experimental methods

Conditional LSL‐KRASG12D and p48‐Cre or PDX‐1‐Cre mice were bred and their offsprings were used for the studies. Mice with both the KrasG12D and Cre allele were designated as mutant mice (KC). Mice with neither the KrasG12D nor the Cre allele were used as wildtype (WT). Mice were fed either a high fat, high calorie diet (HFCD) or a control diet (CD) for 4 to 6 months before they were used for experiments. NK cytotoxicity was determined by 51Cr release assay and cytokine secretion by ELISA.

Results

The Following profiles were obtained for NK cell cytotoxicity and secretion of IFN‐g from NK cells purified from spleen of KC or WT mice fed either with CD or HFCD (WT(CD)>WT(HFCD)>KC(CD)>KC(HFCD). Immune cells from blood and pancreas exhibited similar pattern of cytotoxicity to those obtained from the spleen but inverse profile of IFN‐g and IL‐6 secretion (WT(CD)<WT(HFCD)<KC(CD)<KC(HFCD) was seen. Mice fed with HFCD when compared to CD had much less NK cells both in WT and KC mice in pancreas, adipose and gingiva.

Conclusion

Mice fed with HFCD exhibited higher inflammatory index when compared to those fed with CD both in WT and KC mice. In addition, numbers and the cytotoxicity of NK cells were severely decreased in pancreas and gingiva of WT and KC mice fed with HFCD. High fat diet exacerbates the progression of neoplastic lesions in KC mice and could be responsible for the oral mucosa pathologies due to decrease function and numbers of key immune effectors.

Support or Funding Information

P01 CA163200; PAPD and XO club

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing Standardized Patient Encounter in Pharmacology Education of Undergraduate Medical Students
Authors: Athauda Gagani , Ottolenghi Joseph , Lupi Carla , Kashan Sanaz , Gray Elizabeth , Stumbar Sarah
Published: June 2021

Noting a gap in the curriculum, students and faculty at Florida International University (FIU) Herbert Wertheim College of Medicine (HWCOM) created a pre‐recorded lecture and in‐class, interactive Standardized Patient (SP) case on contraceptive pharmacology and risk communication. The lecture focused on mechanisms of action, bioavailability, drug‐drug interaction, effectiveness, and major vascular risks of combined hormonal and progestin‐only contraceptives. The in‐class session integrated knowledge of risks and effectiveness of contraception and basic clinical skills with risk communication surrounding contraceptive counseling.

Students watched a 20 minute pre‐recorded lecture before attending a 50‐minute intermediate‐sized group session led by an instructor and involving a SP. Students anonymously and voluntarily completed a 5 question session evaluation, rating their perceived learning impact of module and class session. Students learning was assessed with 5 multiple‐choice questions on the students' pharmacology final exam, taken a week later.

Overall, students agreed that the recording helped to prepare them for the session, and that the standardized patient exercise allowed them to apply and solidify their learning. The class average performance on the relevant final exam questions was 77.6%, with better performance on lower order questions derived from the online module. All questions had robust discrimination indices.

The lecture and in‐class SP case addressed gaps in the curriculum and provided students the opportunity to integrate multiple domains of learning. Students' perception of the materials was positive and students demonstrated adequate learning. Further work to improve performance at this level and build on learning is underway.

Support or Funding Information

This study was not supported by a grant and has been approved by FIU IRB.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Medical Pharmacology Board Scores Increase With Flipped Classroom and Video Lectures
Authors: Prancan Arthur , Ali Kamilah
Published: June 2021

Medical Pharmacology, in the second year at Touro College of Osteopathic Medicine in Harlem, New York, beginning in Fall 2008, was offered as a didactic lecture and examination program using a syllabus with course expectations, topics, class schedule and learning objectives as a guide for students. Content was offered in class with PowerPoint presentations, 50 minutes at a time. Attendance varied between 20% and 60% of the class. COMLEX‐USA Level 1 discipline mean scores in pharmacology ranged from 462.22 to 506.95 as reported by NBOME in the period 2010 through 2012. The National Mean covering this period was 515.74. The First Pass rate averaged 82.82% against the National rate which averaged 89.69%

In Fall 2012, the course content delivery and access was changed to video recordings of the lectures by faculty. Students accessed course content via a server which housed a library of course topics on video, still according to the syllabus. No classroom lectures were offered, but students were invited to participate in quiz and discussion sessions in the classroom, one hour per week per course. Classroom time was decreased to one‐third. Students now preview the videos and actively participate in the quiz sessions, often covering 2–3 topics in one hour. Discussion of topics among students is encouraged and often becomes very animated and dynamic. Students come to class prepared for quiz and discussion, receive minor credit for the quiz and leave aligned in their understanding of topics. Class attendance is typically 85% and may reach 100%. COMLEX‐USA Level 1 mean discipline scores in pharmacology have improved from 517.56 in 2013 to 600.02 in 2018 against an average National score in Pharmacology of 542.21 over the same period. The First Pass rate has increased from 87.29% in 2013 to a rate of 97.73% in 2018. The mean National First Pass rate for this period is 92.91%

The faculty at this medical college are able to provide consistent and regularly improved content presentations via the video lectures while freeing time for scholarly activities. Students manage their study efficiently during time which is free of scheduled lectures and they achieve board scores in the top half of the national scale.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Predicting Impulsivity and Compulsivity in Mice Using a Rapid Drug Abuse Liability Battery (RDALB) Assessing Hyperneophagia, Locomotor Reactivity, and Novelty Preference
Authors: Hyatt William , Berquist Michael , Russell Lauren , Fantegrossi William
Published: June 2021

Impulsivity and compulsivity are two major biobehavioral contributors to addiction in human and animal models. In preclinical research, assays typically used to measure these variables are often labor intensive and require an extensive training history, so developing inexpensive, high‐throughput procedures that are predictive of drug abuse vulnerability would be a significant advancement. The data presented here will describe a combined battery that concurrently measures baseline hyperneophagia, locomotor reactivity, and novelty preference in a 1‐hour session. All experiments took place in a 3‐compartment spatial conditioning chamber, comprised of two large chambers and a connecting hallway. Video was collected via a cell phone camera and analyzed using Behavior Cloud software that quantified locomotor activity, with each compartment analyzed separately as discrete zones. In the present experiments, 48 drug‐naïve mice were examined using the rapid drug abuse liability battery (RDALB) to profile animals into high‐ and low‐ impulsivity and compulsivity groups, which is hypothesized to inform abuse liability risk. Animals were initially placed in one compartment with the door closed for a 30 minute habituation period. This habituation period was also interpreted as ‘inescapable novelty’ and any locomotor activity observed was classified as locomotor reactivity, which is believed to predict impulsivity. After the habituation period the chamber door was raised, and animals were free to explore all three sections for a second 30 minute period. The time spent in the novel compartment, versus the time spent in the initial compartment, was calculated as novelty preference, which is believed to predict compulsive behaviors. Hyperneophagia was measured as the latency to drink a novel milk solution after emergence from the first chamber. Large individual differences were found between animals within each assay, allowing for separation into discrete phenotypes ‐‐ for instance, the latency for animals to drink milk ranged from 7 seconds to over 21 minutes. No correlations were observed between different behavioral outcomes, indicating that the RDALB is likely measuring distinct impulsive and compulsive phenotypes. Future oral self‐administration experiments are planned to validate the predictive validity of the RDALB.

Support or Funding Information

These studies are supported by DA039195, DA022981 and the UAMS Center for Translational Neuroscience.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dopamine Cell Loss within the Nigrostriatal Pathway Due to Oxidative Stress from Chronic Methylphenidate
Authors: Oakes Hannah Virginia , Hall Alexis , Ensley Tucker , Medlock Kristen , Ketchem Shannon , Pond Brooks
Published: June 2021

Attention deficit hyperactivity disorder (ADHD) is a neurobehavioral disorder that affects 11% of children in the U.S. alone. Methylphenidate (MPH) is the most commonly prescribed drug for the treatment of ADHD. MPH acts by blocking dopamine (DA) transporters and norepinephrine (NE) transporters, preventing the reuptake of these catecholamines following release. Many children receive MPH from childhood to early adulthood; yet, most of the scientific literature focuses on understanding short‐term consequences of MPH. As such, it is extremely important to examine the long‐term consequences of MPH exposure. Previous research has shown that long‐term exposure to MPH causes dopaminergic neurons within the nigrostriatal pathway to be more sensitive to the Parkinsonian toxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). We hypothesize that oxidative stress caused by the spontaneous oxidation of the excess DA in the synaptic cleft renders dopaminergic neurons within the nigrostriatal pathway to be more sensitive to MPTP.

To investigate this, adolescent male Swiss‐Webster mice received intraperitoneal (IP) injections of saline, 1, or 10 mg/kg MPH for 12 weeks. After 12 weeks, all animals received a drug washout period of 7 days and then half of each group was treated with MPTP (4 × 20mg/kg, every 2 hours), while the other half was administered 4 injections of sterile saline. Seven days after the MPTP injection, mice were sacrificed and the striatum (STR), substantia nigra (SN) and the occipital cortex (OCtx) were microdissected and flash‐frozen. Oxidative stress related to increased dopamine levels was examined using a glutathione assay to measure glutathione content and near‐infrared fluorescence to measure free and protein‐bound ortho‐quinones.

It appears that long‐term exposure to MPH sensitizes dopaminergic neurons within the nigrostriatal pathway to oxidative stress, rendering them vulnerable to further insults, such as MPTP exposure. As such, these studies provide insight into the risks of long‐term psychostimulant exposure.

Support or Funding Information

American Foundation of Pharmaceutical Education

East Tennessee State University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlation between neuroanatomical and respiratory functional changes in a Parkinson's disease model.
Authors: de Araujo Fernandes Silvio , Carvalho Karin , Moreira Thiago Santos , Takakura Ana Carolina
Published: June 2021

Parkinson's disease (PD) is a disorder characterized by motor and non‐motor symptoms associated with loss of dopaminergic neurons in the compact part of the substantia nigra (SNc). However, other brainstem structures may also be degenerated, contributing to the appearance of non‐motor abnormalities, as breathing disorders that have a negative impact on the life's quality of those individuals. The aim of this study was to perform a temporal correlation between respiratory and neuroanatomical changes in an experimental model of PD. Adult male Wistar rats, with bilateral injections of 6‐hydroxy‐dopamine (6‐OHDA, 24 μg/μl) or vehicle into the striatum were used. At 30, 40 and 60 days after PD induction, the animals had functional and neuroanatomical parameters assessed by whole body plethysmography and immunohistochemical analysis. The results showed that in this model there are more than 70% of reduction in the dopaminergic neurons of the SNc and a reduction in the resting respiratory frequency (40 days: 88 ± 2 and 60 days: 81 ± 2, vs. vehicle 113 ± 2, bpm) and ventilation (40 days: 643 ± 29 and 60 days: 377 ± 21, vs. vehicle 877 ± 48 ml/kg/min) from 40 days after PD‐induction. A reduction in the hypercapnia‐induced respiratory frequency (fR: 40 days: 115 ± 4 and 60 days: 120 ± 3, vs. vehicle 131 ± 2 bpm) and ventilation (VE: 40 days: 1138 ± 96 and 60 days: 701 ± 66, vs. vehicle 1414 ± 102 ml/kg/min) was also observed in the same period. The neuroanatomical results showed that there was a reduction in the number of Phox2b neurons from 30 days after the injury in the nucleus of the solitary tract (NTS: 46%) and retrotrapezoid nucleus (RTN: 55%) and a reduction in the density of glial fibrillary acidic protein (GFAP) in the region of the rostral ventral respiratory group (rVRG: 39.3%). At 40 days, a reduction in the density of receptors for neurokinin‐1 (NK1R) was also observed in the pre‐Botzinger Complex (preBotC: 25%) and in the rVRG (38%) and in the density of GFAP in the RTN region (40.7%). At 60 days, the reduction in the densitity of GFAP was also present in the regions of preBotC (51.9%) and NTS (47.4%). The present results show that the neuroanatomical changes start before functional respiratory deficits and can be a cause for respiratory problems observed in PD.

Support or Funding Information

FAPESP, CNPq and CAPES.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Involvement of CRF1 receptors in bed nucleus of stria terminalis (BNST) on baroreflex responses in chronically stressed rats
Authors: de Oliveira Leandro Augusto , de Souza Lucas Gomes , Benini Ricardo , Crestani Carlos Cesar
Published: June 2021

INTRODUCTION

Studies suggest the involvement of CRF receptors in bed nucleus of the stria terminalis (BNST) on cardiovascular responses in acute stress. Data from our group had demonstrated that the expression of CRF1 receptors within the BNST is decreased in animals exposed to chronic variable stress (CVS). Nevertheless, an involvement of BNST CRF neurotransmission on cardiovascular changes evoked by CVS has never been investigated.

AIM

To evaluate the involvement of CRF1 receptor in the BNST on baroreflex changes evoked by CVS in rats.

METHODOLOGY

Male Wistar rats (250g) were submitted to stereotaxic surgery for implantation of cannulas into the BNST. After 4 days of recovery, the animals were exposed to a CVS protocol for 10 days. After the last session, the rats had the femoral artery and vein cannulated for cardiovascular record and drug infusion, respectively. Twenty‐four hours later, the experiments were performed. Baroreflex function was assessed by evoking changes on arterial pressure throughout intravenous infusions of phenylephrine and sodium nitroprusside, which evoke pressor and depressor effects, respectively. Baroreflex activity was evaluated in both control rats and animals subjected to ECV protocol before and after bilateral microinjection of the selective CRF1 antagonist CP376395 (5nmol/100nL) into the BNST.

RESULTS

Comparison of values before BNST treatment revealed that ECV decreased the bradycardiac response evoked by blood pressure increase (−48±10 vs −114±23 bpm, P<0.03). Moreover, bilateral microinjection of CP376395 into the BNST decreased reflex bradycardia in control group (−51±13 vs −114±23 bpm, P<0.02), but not in animals subjected to ECV (−51±10 vs −48±10 bpm, P>0.05). The tachycardia evoked by blood pressure decrease was also decreased following ECV (57±5 vs 97±11 bpm, P<0.05). The BNST treatment with CP376395 decreased the tachycardiac response in control animals (50±9 vs 97±11 bpm, P<0.001), but without affecting this response in CVS rats (52±5 vs 57±5 bpm, P>0.05).

CONCLUSION

The impairment of baroreflex function evoked by CVS is related to decreased control of this reflex cardiovascular by CRF1 receptor in the BNST.

Support or Funding Information

FINANCIAL SUPPORT: FAPESP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial GPCRs Activate p38 MAPK Inflammatory Signaling Via Non‐canonical TAB1, 2 and 3‐dependent Pathways
Authors: Lin Ying , Narala Rachan , Grimsey Neil , Trejo JoAnn
Published: June 2021

Vasculature disease is a leading cause of morbidity and mortality worldwide. A hallmark of vascular inflammation is endothelial cell dysfunction, which is induced by vascular inflammatory mediators, many of which act through G protein‐coupled receptors (GPCRs). We previously showed that thrombin stimulation of endothelial protease‐activated receptor‐1 (PAR1) activates p38 MAPK through a non‐canonical pathway mediated by TAB1 (transforming growth factor‐β‐activated kinase binding protein‐1). TAB1 binds directly to p38α inducing a conformational change resulting in autophosphorylation and activation bypassing the requirement for upstream MAP2Ks. We also found that non‐canonical p38 activation induced by thrombin/PAR1 promotes endothelial barrier permeability in vitro and further showed that p38 is required for PAR1‐stimulated vascular leakage in vivo. While non‐canonical p38 activation induced by PAR1 is established, it remains unclear if other endothelial GPCRs also promote p38 activation through a TAB1‐dependent pathway and was examined. Here we report that multiple GPCRs agonists including histamine, PGE2, ADP and thrombin induce p38 activation via the non‐canonical TAB‐dependent pathway to promote cytokine production. We assessed GPCR‐induced p38 activation in macrovascular and microvascular endothelial cell lines including HUVEC, EA.hy926 and HDMECs. Using immunoblotting and qPCR, the expression of p38, TAB1, 2, 3 and MKK3,6 were quantified and appear to be comparable across the endothelial cell lines. To determine if p38 activation induced by GPCRs is mediated by autophosphorylation, we used the p38α/β SB203580 inhibitor which blocks p38 autophosphorylation but not phosphorylation mediated by upstream MAP2Ks. In HUVEC, EA.hy926 and HDMEC pretreated with SB203580, all four agonists including histamine, PGE2, ADP and thrombin failed to promote p38 phosphorylation, suggesting that endothelial GPCR‐induced p38 activation occurs through autophosphorylation. Next, we used combinations of TAB1, 2 and 3‐specific siRNAs to deplete endothelial cells of TAB proteins and found that all GPCR agonists require TAB expression to induce maximal p38 activation in endothelial cells. In addition, endothelial GPCR‐induced cytokine production was also dependent on p38 signaling and TAB expression. Taken together, these data support a critical role for TAB1‐dependent activation of p38 MAPK induced by endothelial GPCRs in inflammatory signaling.

Support or Funding Information

This work is funded by NIH/NIGMS R01 GM090689, R01 GM116597 and UC Tobacco‐related Disease Research Program High Impact Pilot Award 26IP‐0050

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ellagic Acid exerts anti‐inflammatory actions via disruption of store‐operated Ca entry (SOCE) pathway activators and coupling mediators[Office1]
Authors: Murphy Matthew , Quin Xia , Mathias Clinton , Thomas David , Bose Diptiman
Published: June 2021

Pharmacological agents that can modulate Ca2+ homeostasis are being extensively explored as potential anti‐inflammatory agents. Ellagic acid (EA), a polyphenolic compound found in many fruits and plant extracts, has been known to possess anti‐inflammatory properties. However, the mechanism of action behind its anti‐inflammatory action is unclear. We studied the effects of EA on Ca2+ handling, in particular, store‐operated Ca2+ entry (SOCE), a process critical to proper T cell function. EA is thought to modulate Ca2+ homeostasis by enhancing sarcoplasmic endoplasmic reticulum ATPase pump (SERCA) activity, however effects of EA on Ca2+ signaling is not clearly understood. We demonstrate that acute addition of EA induced Ca2+ release (EC50= 63 μM). A 12 h incubation of Jurkat T cells with EA decreased the ATP‐induced Ca2+ release and diminished the SOCE‐mediated Ca2+ influx in a dose dependent manner (IC50= 23 μM). The inhibition of SOCE was also confirmed by reduced Mn2+ entry rates in the EA‐treated cells. The two important components of SOCE are the endoplasmic reticulum (ER)‐Ca2+ sensor Stromal Interaction Molecule (STIM), and highly‐Ca2+ selective plasma membrane (PM) Orai channels. EA treatment attenuated the ATP‐induced Ca2+ influx in EA‐treated HEK293 cells transiently transfected with SOCE channel Orai1‐myc and ER‐sensor stromal interaction molecule (STIM1) (HEKSTIM/Orai). Furthermore, EA treatment interfered with the Orai1 and STIM1 coupling by disrupting STIM1 puncta formation in the HEKSTIM/Orai cells. Since expression of certain cytokines is directly dependent on Ca2+ influx, we measured the levels of IL‐2 and INFγ in EA‐treated Jurkat cells. Indeed, EA‐treated resulted in a significant decrease in IL‐2 and INFγ expression. The activity of nuclear factor of activated T‐cell (NFAT), a transcriptional factor has been shown to be dependent on SOCE mediated Ca2+ influx. Prolong exposure to EA decreased the translocation of nuclear factor of activated T‐cell in stimulated T cells. Hence, by inhibiting SOCE mediated Ca2+ influx, EA decreased downstream activation of pro‐inflammatory mediators. These observations suggest a novel target to EA‐mediated effects and provide insight into the mechanisms underlying EA mediated anti‐inflammatory effects.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Synaptic transmission: Quantitative analysis of Gβγ specificity to adrenergic α2a receptor and SNARE.
Authors: Yim Yun Young , Betke Katherine , McDonald W. Hayes , Gilsbach Ralf , Chen Yunjia , Hyde Karren , Wang Qin , Hein Lutz , Schey Kevin , Hamm Heidi
Published: June 2021

Modulation of neurotransmitter exocytosis by activated Gi/o coupled G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ interaction with soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE). There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR and the specificity to effectors in vivo are not known. Presynaptic α2a‐adrenergic receptors (α2aARs) in both adrenergic (auto α2aARs) and non‐adrenergic neurons (hetero α2aARs) inhibit neurotransmitter release and affect various physiological functions such as anesthetic sparing and working memory enhancement. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits to detect neuronal Gβ and Gγ subunits, several mouse models including transgenic Flag‐α2aARs, knock‐in HA‐α2aARs, and other biochemical techniques such as co‐immunoprecipitation, we investigate the specificity of Gβ and Gγ subunits to α2aARs in both adrenergic(auto α2aARs) and non‐adrenergic neurons (hetero α2aARs), and SNARE in presence of epinephrine. Gβ2, Gγ2, Gγ3, and Gγ4 preferentially interact with activated auto α2aARs while Gβ4 and Gγ12 preferentially interact with activated hetero α2aARs. We also detect a subset of Gβ and Gγ subunits interacting with SNARE upon α2aARs activation. Further understanding of Gβγ specificity on its downstream signaling, especially Gβγ‐SNARE interaction, offers new insights into the normal functioning of the brain and lead to an identification of potential pathophysiological states in which the Gβγ‐SNARE interaction may be dysregulated. These studies yield additional insights into Gi/o‐coupled GPCR‐mediated regulation of exocytosis.

Support or Funding Information

This work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32 GM07628).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Inhibitors of Cyclooxygenase‐2 and Soluble Epoxide Hydrolase: Studies of Binding Modes at the Active Sites and Time‐dependency of Inhibition, and Development of Water‐soluble Prodrugs
Authors: Hwang Sung Hee , Gaieb Zied , Morisseau Christophe , Yang Jun , Wagner Karen , Gilson Michael K. , Hammock Bruce D.
Published: June 2021

In recent years, there has been increasing interest in designing multi‐target agents that improve efficacy and safety compared to their single target counterparts.

Cyclooxygenase‐2 (COX‐2) selective inhibitors (coxibs) are effective drugs for treating inflammation, pain, and cancer. However, their mechanism‐based side effects limit their use. Meanwhile, soluble epoxide hydrolase (sEH) is involved in the metabolism of fatty acid epoxides that are obtained by cytochrome P450 (CYP) from arachidonic acid. These CYP metabolites, epoxyeicosatrienoic acids (EETs), are anti‐hypertensive, anti‐inflammatory, analgesic and organ‐protective. Therefore, co‐inhibition of both enzymes with a combination treatment is synergistic and is maximized by combining both actions into a single molecule, such as PTUPB. These dual inhibitors showed superior efficacy in several rodent models of inflammatory pain, metabolic syndrome, and cancer.

We found that they inhibit COX‐2 tightly and in a time‐dependent manner (25‐fold more potent) and EETs moderately inhibit COX‐2. Computational studies of their binding modes at the active site of enzymes using molecular docking confirm these results. In addition to these studies, water‐soluble prodrugs of these dual inhibitors were developed for their use in animal studies.

Support or Funding Information

This work was supported in part by NIEHS grant R01ES02710, NIEHS Superfund Basic Research Program grant P42 ES04699, National Institute of Neurological Disorders and Stroke (NINDS) U54 NS079202. NIH 5T32DC008072‐05.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Soluble Epoxide Hydrolase Attenuates Food Allergen‐induced Eosinophil Recruitment and Gastrointestinal Inflammation
Authors: Rao Savita , Bastan Idil , Ge Xiao Na , Dileepan Mythili , Greenberg Yana G. , Guedes Alonso G. , Hwang Sung He , Hammock Bruce D. , Washabau Robert J. , Sriramarao P
Published: June 2021

The prevalence of food allergies is on the rise, affecting nearly 4–6% of the population in the US and impacting their daily life in various ways. Although present in the normal gastrointestinal (GI) mucosa under homeostatic conditions, eosinophils are recruited to the intestinal mucosa in increased numbers in food allergy and contribute to inflammation. Soluble epoxide hydrolase (sEH) is known to play a pro‐inflammatory role during inflammation by metabolizing anti‐inflammatory epoxyeicosatrienoic acids (EETs) to pro‐inflammatory diols. In the current study, the role of sEH in a murine model of food allergy was investigated and the potential therapeutic effect of a highly selective sEH inhibitor (t‐TUCB) was evaluated. Oral exposure of mice on a soy‐free diet to soy protein isolate (SPI) induced expression of intestinal sEH, increased circulating total and antigen‐specific IgE levels and caused significant weight loss. Additionally, SPI induced GI inflammation characterized by increased recruitment of eosinophils and mast cells, elevated eotaxin‐1 levels, mucus hypersecretion and decreased epithelial junction protein expression. Administration of t‐TUCB to SPI‐challenged mice not only inhibited IgE levels and prevented SPI‐induced weight loss but also significantly decreased eosinophilia, mast cell recruitment and mucus secretion relative to untreated mice while SPI‐induced loss of junction protein expression was prevented to variable levels. Inflammatory mediators such as TNF‐α and eotaxin‐1 were found to induce sEH expression in eosinophils. Treatment of eosinophils with t‐TUCB significantly inhibited eosinophil migration, an effect which was mirrored by treatment with 11,12‐EET, by inhibiting intracellular signaling events such as ERK (1/2) activation and eotaxin‐1‐induced calcium flux. These studies suggest that sEH induced by soy proteins promotes allergic responses including eosinophilic GI inflammation and that inhibition of sEH can attenuate these allergen‐induced responses.

Support or Funding Information

Support was provided in part by National Institute of Environmental Health Sciences and National Institute of Diabetes and Digestive and Kidney Diseases to B.D.H. and by the University of Minnesota College of Veterinary Medicine Research Office, Minnesota Agricultural Experiment Station, and General Ag Research Funds to R.J.W.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological inhibition of soluble epoxide hydrolase promotes brown adipogenesis
Authors: Overby Haley , Kearns Jamie , Hildreth Kelsey , Chahed Samah , Kodani Sean , Morisseau Christophe , Hammock Bruce , Wang Shu , Bettaieb Ahmed , Zhao Ling
Published: June 2021

Brown adipose tissue (BAT) has emerged as a novel target for obesity treatment and prevention. In contrast to white adipocytes, brown adipocytes are responsible for nonshivering thermogenesis, in which ATP synthesis is uncoupled from respiration via uncoupling protein 1 (UCP1) leading to heat production. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. EpFAs are produced by cytochrome P‐450 epoxygenases from polyunsaturated fatty acids, such as arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid (DHA). Many EpFAs are autocrine/paracrine lipid signaling molecules that play important roles in physiological processes, including inflammation, vascular dilation, and cell growth and differentiation. Thus, sEH has become a pharmacological target and small and potent sEH inhibitors that stabilize endogenous EpFAs have been shown to be beneficial for many chronic diseases. The objectives of our studies are to study the regulation of sEH expression and the effects of sEH inhibitors on brown adipogenesis. We found that sEH expression was increased during differentiation of both murine and human brown adipocytes. sEH mRNA levels were significantly increased in the interscapular BAT, but not in the epididymal white adipose tissue (WAT), of diet‐induced obese mice. Moreover, sEH pharmacological inhibition using trans‐4‐{4‐[3‐(4‐trifluoromethoxy‐phenyl)‐ureido]‐cyclohexyloxy}‐benzoic acid) (t‐TUCB) and trans‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxy]‐benzoic acid (t‐AUCB) dose‐dependently promoted brown adipocyte differentiation, possibly through activating PPAR gamma and alpha. In agreement with these findings, our studies further demonstrated higher UCP1 and PRDM16 protein expression in the interscapular BAT under basal conditions in the sEH knockout (KO) mice compared to the wild type (WT) controls. Moreover, the increases in mRNA expression of brown marker genes in response to cold exposure were enhanced in the interscapular BAT and subcutaneous WAT of the sEH KO mice compared to the WT controls. sEH deficiency further enhanced the induction of brown marker mRNA expression by t‐TUCB and 19, 20‐EDP, the most abundant EpFA derived from DHA. Together, our findings suggest that sEH may regulate brown adipogenesis and its inhibition could be novel therapeutic strategies to increase BAT development and thermogenesis.

Support or Funding Information

The work was partially supported by NIH R15AT008733 (to L.Z. and S.W.), NIH K99DK100736 (to A.B.), R00DK100736 (to A.B.), R01ES002710 (to B.D.H), P42ES004699 (to B.D.H), and T32GM099608 (to S.D.K).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.