bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8+ T CELLS REGULATE LIVER INJURY IN OBESITY‐RELATED NONALCOHOLIC FATTY LIVER DISEASE
Authors: Kennedy Arion , Pacheco Cristina , Washington Mary Kay , Hasty Alyssa
Published: June 2021

Nonalcoholic Fatty Liver Disease (NAFLD) is ranked as one of the top causes of liver disease in Western countries. We found an elevation in CD8 staining in livers from obese patients with NASH or cirrhosis that positively correlated with an elevation in smooth muscle actin (SMA). To examine the role of CD8+ T cells in the development of NAFLD, we used an obese and hyperlipidemic mild NASH model, the low density lipoprotein receptor deficient (LDLR−/−) mice fed a western diet (WD). CD8+ T‐cells were elevated by 3.5‐fold in the livers of mild NASH mice. Similarly, hepatic CD8 gene expression was significantly increased, along with cytokines Tnf and Il10 as well as hepatic stellate cell associated markers. Isolated hepatic CD8+ T‐cells from the mild NASH mice, had increased secretion of IL‐10, TNFa, IL‐6, IL‐4 and IL‐2 compared to chow fed mice. Chronic treatment with antibody against CD8 led to significant reductions in the gene expression of Il10, Tnf, Il2, Il4, Acta2 and Tgfb. Depletion of CD8+ T cells also led to a reduction in the recruitment of macrophages to the liver. In contrast, adoptive transfer of hepatic CD8+ T cells from the mild NASH mice into chow fed mice significantly increased hepatic Acta2 expression. In co‐culture studies, isolated CD8+ T cells from mild NASH mice induced hepatic stellate cell activation. In a non‐obese choline deficient mouse model of aggressive NASH, depletion and knockdown of CD8 T cells did not impact liver inflammation or hepatic stellate cell activation. In conclusion, our findings suggest that mild NASH – induced by obese and hyperlipidemia, is characterized by CD8+ T‐cell infiltrations of the liver, subsequently regulating the recruitment of macrophages and activation of hepatic stellate cells.

Support or Funding Information

This work was supported by an American Diabetes Association Mentor‐Based Postdoctoral Fellowship (7‐10‐MI‐05), UNCF‐Merck Postdoctoral Science Research Fellowship, the Vanderbilt CTSA grant UL1 TR000445 from NCATS/NIH and K01HL121010 to AK. AHH is supported by a Merit Award from the Veterans Affairs (5I01BX002195), an Established Investigator Award from the American Heart Association (12EIA827), and an Innovation Award from the American Diabetes Association (1‐17‐IBS‐140). Confocal Microscopy was performed through the use of the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 and EY08126). Flow Cytometry experiments were performed in the VMC Flow Cytometry Shared Resource, which is supported by the Vanderbilt Digestive Disease Research Center (DK058404) from whom AHH received scholarship funds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SIRT3 Activation Inhibits Development of Diabetic Kidney Disease
Authors: Myakala Komuraiah , Wang Xiaoxin , Lewien Pierce , Wang Dong , Luo Yuhuan , Herman‐Edelstein Michal , Rosenberg Avi Z. , Levi Moshe
Published: June 2021

Diabetes continues to be the leading causes of kidney disease in the U.S. and around the world. Despite beneficial interventions implemented in patients with diabetes that mitigate some of its negative effects, kidney disease still progresses in most of these patients because the pathogenesis of diabetic kidney disease remains poorly understood. Recent evidence indicates that impairments in mitochondrial dynamics and function plays an important role in pathogenesis of diabetic kidney disease and brings promise to treatment strategies for improving mitochondrial function and the related pathways that may prevent or slow the progression of kidney disease.

We aimed to test the influence of one key mitochondrial protein deacetylase, Sirtuin 3 (SIRT3) as a potential target for therapy in diabetic kidney disease. In kidney biopsies from human patients with diabetic nephropathy, there are decreases in SIRT3 mRNA and protein abundance in glomeruli and tubules. To test the effect of SIRT3 on the prevention of diabetic kidney disease, we conducted a study comparing three groups of mice: A control group, a diabetic group (db/db), and a diabetic group treated with nicotinamide riboside (NR), a precursor for NAD+, which is a co‐substrate required for SIRT3 activity. Treatment of diabetic mice with NR resulted in a) increase in renal SIRT3 activity as determined by significant decreases in total acetylated proteins and acetylated SOD2, significant decreases in b) albuminuria, c) mesangial expansion and glomerular area, d) accumulation of collagen IV protein, e) loss of synaptopodin and f) accumulation of 4‐HNE. In conclusion, SIRT3 plays an important role in diabetic kidney disease and increasing the SIRT3 activity may prevent the progression of diabetic kidney disease.

Support or Funding Information

NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activator Protein 1 and Caspase 8 Mediate Palmitate‐Induced Cardiomyocyte Apoptosis
Authors: Oh Charles , Lee John , D'Souza Karen , Migrino Raymond , Thornburg Kent , Reaven Peter
Published: June 2021

Introduction

Lipoapoptosisof cardiomyocytes may be the basis for diabetic cardiomyopathy. Wepreviously demonstrated that p38α mitogen activated protein kinase (MAPK) mediatespalmitate (PA)‐induced cardiomyocyte apoptosis, and that specific p38α siRNAmarkedly and dose‐dependently attenuates apoptosis in cardiomyocytes treated with PA. We tested the hypothesis that p38α‐dependent pathway consists, in part, of c‐fos subunit of Activator Protein 1 (AP‐1) and caspase8, and that this p38α/AP‐1/caspase 8 cascade mediate PA‐induced cardiomyocyte apoptosis.

Methods

Human adult ventricular cardiomyocytes line (AC16 cells) were exposed to high physiological levels of PA. p38α‐dependent pathway was evaluated using p38α and c‐fos siRNA knockdown, and caspase 8 inhibitor.

Results

With PA treatment, dose‐dependent increases in c‐fos mRNA (1.83 fold and 3.13 fold control, at 150 μM and 300 μM respectively, n=6, p<0.01), in c‐fos protein (1.00 fold and 2.67 fold, at 150 μM and 300 μM respectively, n=3, p<0.05), and a much greater increase in phospho‐c‐fos (2.64 fold and 8.94 fold, at 150 μM and 300 μM respectively, n=, p<0.01) were measured. Transcription of caspase 8 also increased (1.40 fold and 1.80 fold, at 150 μM and 300 μM respectively, n=5, p<0.05) but procaspase 8 protein level did not.

p38α knockdown dose‐dependently blocked the PA‐induced increase in phospho‐c‐fos protein level(to 62%, 45%, and 30% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmol si‐p38α knockdown groups, n=5, p<0.01), but total c‐fos level did not change significantly. Interestingly, procaspase 8 level was also dose‐dependently reduced by p38α knockdown (75%, 65%, and 59% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmolsi‐p38α knockdown groups, n=5, p<0.05).

With c‐fos knockdown, PA‐induced apoptosis was reduced 39% (n=3, p<0.05) 24 h post‐transfection, and by 53% after 48 h post‐transfection (n=3, p<0.01). Caspase 8 inhibition for 24 h reduced apoptosis by 39% (n=3, p<0.01).

Conclusions

This study in cardiomyocytes demonstrated that PA increases expression and phosphorylation of c‐fos subunit of AP‐1, and caspase 8 transcription, and further that p38α knockdown attenuates PA‐induced increases in c‐fos and phospho‐c‐fos, and decreases caspase 8. Translational knockdown of c‐fos attenuated PA‐induced cardiomyocyte apoptosis, as did inhibition of caspase 8. Our results support the hypothesis that PA‐induced apoptosis is mediated by p38α, AP‐1/c‐fos, and caspase8.

Support or Funding Information

This work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&D5I01CX000598grant (to P.R.), and VA Merit grant BLRDI01BX007080(to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. The contents and the views do not represent the views of the Department of Veterans Affairs or the US government.
Targeting and tracing cardiac myocytes with Fgf1 expression in F1A‐CreERT2 transgenic mice
Authors: Chiu Ing‐Ming , Chung Yu‐Fen , Chen Mei‐Shu , Jiang Si‐Tse , Hsu Yi‐Chao
Published: June 2021

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and spliced to the first protein coding exon alternatively, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line Fgf1A promoter (F1A)‐driven inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A‐CreERT2 founder mice, similar to that of Fgf1A mRNA. The F1A‐CreERT2 mice were crossed with ROSA26R mice, and the F1 mice were analyzed. The punctate blue LacZ‐positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2‐mediated recombination in the tissues is monitored through punctate blue LacZ‐positive signals, indicating the in situ localization of F1A‐positive cells. Interestingly, these F1A‐positive cells with punctate blue signals were colocalized with cardiomyocytes expressing a‐actinin, a‐myosin heavy chain and cardiac troponin T, suggesting cardiomyocyte‐specific expression of Fgf1A. Our data suggested that the F1A‐CreERT2 mouse line could be used for time‐dependent and lineage tracing of Fgf1‐expressing cells in vivo.

Support or Funding Information

Ministry of Science and Technology, Taiwan;

Ministry of Health and Welfare, Taiwan;

National Health Research Institutes, Taiwan

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the role of pancreatic β‐cell CD36 in the development of Type 2 Diabetes
Authors: Kolar Elizabeth Anne , Gajrawala Eshaa , Deeney Jude , Hamilton James
Published: June 2021

CD36 is a transmembrane scavenger receptor for oxidized LDL (oxLDL). Expression of CD36 and subsequent uptake of oxLDL into macrophages is central to foam cell formation in atherosclerotic plaques. CD36 is also reported to play a pivotal role in glucotoxicity‐induced β‐cell dysfunction in Type 2 Diabetes (T2D). The aim of this study is to investigate mechanistic functions of CD36 that contribute to β‐cell dysfunction and the onset of T2D. For this study, rat INS‐1 pancreatic β‐cells were cultured in either low (5 mM) or high (11 mM) glucose conditions. Cells grown in high glucose exhibited a left shift in glucose stimulated insulin secretion (GSIS), showing that the cells are more sensitive to lower glucose levels. These cells also contained more lipid droplets, as shown by Nile Red staining. Using immunofluorescence and Western blotting, we demonstrated that CD36 expression increased with increasing glucose concentrations. When cells were incubated with DiI‐labeled oxLDL to investigate uptake, cells that were grown in high glucose exhibited increased labeling. Interestingly, oxLDL uptake by INS‐1 cells grown in 11 mM glucose can be suppressed by the addition of the ω‐3 fatty acid, docosahexaenoic acid (DHA). Studies investigating the effect of DHA and additional fatty acids on uptake of oxLDL by INS‐1 cells and effects on GSIS are ongoing. We propose that CD36 plays a role in how β‐cells respond to high glucose levels, similarly found in T2D. The increase in oxLDL uptake via CD36 in these cells may contribute to glucolipotoxic effects in β‐cells that results in aberrant insulin secretion in T2D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designing a Model Skin Cell Membrane to Investigate the Extent of Nanoparticle Absorption
Authors: Yoder Brandi , Sostarecz Audra Goach
Published: June 2021

The Langmuir Monolayer Technique is a useful method for modeling the epithelial cell membrane and for investigating the organization of membrane molecules. Liposomes with fluorescence analysis are useful in interpreting molecule interactions with the hydrophilic and hydrophobic regions of the membrane. Zinc oxide particles were introduced to monolayers of lipids found in the stratum corneum and also to liposomes of the same composition to investigate how these particles interact within the cell membrane. This process will allow us to ultimately determine if the nanoparticles in over the counter beauty products embed our skin. In our lab, the compression isotherms of four lipids, cholesterol, cholesterol sulfate, lignoceric acid, and N‐oleoyl‐d‐sphingosine, which are major components of the epithelial cell membrane have been established at body temperature conditions. Ceramide molecules have also been analyzed and incorporated to create a more realistic model system. Liposomes were used as a technique to study the bilayer due to the ability to analyze them via fluorescence spectroscopy by incorporation of a fluorescent ceramide. The ultimate goal is to determine whether or not the addition of the zinc oxide will increase the fluidity of our membrane system utilized for modeling epithelial 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.
Harnessing glycocalyx interactions to modulate differentiation and development
Authors: Huang Mia L. , Michalak Austen L. , Tota Ember M. , Smith Raymond Alexander , Trieger Greg W. , Godula Kamil
Published: June 2021

The cellular fate of embryonic stem cells and their subsequent development into differentiated cells are governed by precise growth factor signaling events at the stem cell surface. Such events are often orchestrated by heparan sulfate proteoglycans (HSPGs), which template growth factor binding to their corresponding receptors. Given the structural complexity of HSPGs, however, methods to manipulate them to study their function has been limited. Here, we report a suite of chemical strategies that exploit the function of HSPGs to control pluripotency or trigger the differentiation of mouse embryonic stem cells. To this end, we employ small molecules targeting the glycosaminoglycan (GAG) side chains of HSPGs to maintain pluripotency, and design GAG‐mimetic molecules that can recapitulate the functions of native GAGs.

Support or Funding Information

NIH Pathway to Independence Award (1K99HD09029201, to MLH)

NIH Director's New Innovator Award (1DP2HD08795401, to KG)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping Specific Cellular Sialoglycans Using Glycosyltransferases
Authors: Wu Zhengliang L. , Person Anthony D. , Burroughs Barbara , Singh Ravinder , Tatge Timothy , Manning Timothy , Wu Guoping , Sackstein Robert , Kalabokis Vassili
Published: June 2021

The spatial and temporal decoration of glycans with neuraminic acid (sialylation) on cell surface involves in many biological processes, ranging from providing a physical barrier, shielding galactose and mannose residues from respective receptors, to generating specific epitopes that guide cell adhesion events. For examples, sialylated Lewis X structure serves as a ligand for selectins and is essential for leukocyte homing process; sialylated T/Tn antigens are involved in oncogenic processes and cancer invasiveness; while α‐(2,3)‐linked sialic acids serve as receptors for avian influenza vial infection, α‐(2,6)‐linked sialic acids serve as receptors for human influenza viral infection. Mapping the distribution of these glycans on cell surface is essential for us to understand their biological roles. Here, using HELA, HUVEC and CHO cells as models, we describe techniques for imaging some specific sialo‐glycans using glycosyltransferases including ST6Gal1, ST3Gal1, ST6GalNAc1, FUT2, and FUT9, with respective clickable nucleotide sugar donors. Our data suggest that while small percentages of terminal lactosamine (precursors for Lewis X structure) and Tn antigen may not be sialylated, almost all N‐glycans and Core‐1 O‐glycans on HELA cells are sialylated. Our data also indicate that while N‐glycan and lactosamine are more enriched on cell edges, where they may participate in cell‐cell interactions, sialylated Core‐1 O‐glycan and Tn antigen are more evenly distributed on cell surface. Despite the notion that CHO cells show no evidence for ST6Gal1 gene expression, we found that CHO cells are covered with α‐(2,6)‐linked sialic acid, which is also confirmed by sialic acid probing on CHO expressed proteins.

Support or Funding Information

This work was supported by R&D Systems/Bio‐techne

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multi‐omics Studies Identify the Role of PPAR Pathway in Rheumatic Heart Disease Patients with Chronic Atrial Fibrillation
Authors: Li Ming‐Yang , Chen Huan‐Xin , Hou Hai‐Tao , Wang Jun , Yuan Chao , Yang Qin , He Guo‐Wei
Published: June 2021

Background

Atrial fibrillation (AF) is one of the most common arrhythmia encountered in the clinical practice. The estimated prevalence of AF in the general population is as high as 1–2%. In this study, we aimed to identify the molecular changes and mechanisms responsible for chronic atrial fibrillation in rheumatic heart disease (RHD).

Methods

Patients with rheumatic heart diseases (N=162) were enrolled in this study and classified into chronic AF group and sinus rhythm (SR) group based on their clinical manifestation. The atrial tissue (N=62) and plasma (N=100) samples were collected from the patients undergoing valve replacement surgery. Multi‐omics Studies were fulfilled with RNA Sequencing (RNA‐seq), isobaric tags for relative and absolute quantification (iTRAQ) and gas chromatography‐mass spectrometer (GC‐MS). ELISA was used to validate the proteins.

Results

The differential genes/proteins were identified in the tissue or in the plasma samples between RHD‐AF patients and RHD‐SR patients. Combined analysis of transcriptomics, proteomics, and metabolomics revealed that the PPAR signaling pathway plays an important role in AF. The expression of multiple proteins were associated with PPARα,β,γ in tissue samples. On the other hand, only PPARα downstream alternations were identified in the study of plasma samples (APO‐A1 and APO‐A2). These proteins may be potential biomarkers for the diagnosis and treatment of AF in the future.

Conclusions

We for the first time identified the differentially expressed genes/proteins and related metabolites from plasma and tissue in chronic AF patients. Our study suggests that AF may impair the energy metabolism in the heart by altering the PPAR pathway, thus presenting metabolic remodeling in the heart.

Support or Funding Information

Supported by grants from the National Natural Science Foundation of China (81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2014BHKY010, 2012BWKZ008, 2016BWKY007, 2016BWKZ003).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Novel Non‐Coding Products of the Pax‐5 Gene and Their Clinical Significance in Lymphoid Cancers
Authors: Hannay Brandon , Dumas Pascal , Veilleux Vanessa , LeBlanc Nicolas , Robichaud Gilles A.
Published: June 2021

Circular RNAs (circRNAs) are a newly identified class of RNA which are highly expressed and conserved in mammalian cells. With the intrinsic property of being resistant to exonucleolytic activity due to their circular configuration, circRNAs are remarkably stable compared to their linear RNA species counterpart. CircRNAs regulate many biological processes and their aberrant expression is often associated with disease progression. Recently, we have characterized the expression of circRNAs of the potent Pax‐5 oncogene in B cell cancers. In this present study, we set out to identify and establish the expression profiles of Pax‐5 circRNAs in B cell cancers and also, to evaluate the potential of these non‐coding products to act as disease biomarkers. Using RT‐PCR in B cell lines, Pax‐5 circRNAs were amplified with divergent primers. PCR products were then sequenced and aligned for identification and validation. Pax‐5 circRNAs were also evaluated by qPCR from peripheral blood mononuclear cells (PBMCs) from healthy donors and clinical samples. Four circRNA isoforms of the Pax‐5 gene were isolated, sequenced and identified from various B cell models. These latter Pax‐5 circRNA variants consisted of a circular single RNA strand characterized with either exons 2, 3, 4; exons 2, 3, 4, 5; exons 2, 3, 4, 5, 6; or exons 2, 3, 4, 5, 6, 7, 8 of the human Pax‐5 gene. Profiling of Pax‐5 circRNA expression levels in clinical samples reveal an overexpression in chronic lymphocytic leukemia in comparison to other B cell cancer lesions and healthy donors. Altogether, we describe 4 novel gene products (i.e. circRNAs) of the Pax‐5 oncogene in B cell cancers. Preliminary results also support a potential role for Pax‐5 circRNAs as prognostic or diagnostic biomarkers for specific B cell cancers. Further studies are ongoing to validate the correlation between Pax‐5 circRNA levels and disease progression. In addition, the mechanistic elucidation of Pax‐5 circRNAs in cancer processes will also potentially identify new avenues for B cell cancer therapeutic interventions.

Support or Funding Information

This work was supported by grants from the New Brunswick (NB) Innovation Foundation, the Canadian Breast Cancer Foundation, the Canadian Breast Cancer Society/QEII Foundation, the NB Health Research Foundation and by the Beatrice Hunter Cancer Research Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential response to apigenin in African American triple‐negative breast cancer in reducing TNF‐α mediated rise in CXCL1
Authors: Soliman Karam F. , Bauer David F. , Mazzio Elizabeth A.
Published: June 2021

Triple‐negative breast cancer (TNBC) is characterized by the absence of estrogen, progesterone and HER2 receptors. African Americans have higher death rates from TNBC attributable to inadequate preventative health care, late‐stage detection worsened by this being an inherent aggressive malignancy with limited treatment options with the exclusion of hormone receptor‐based chemotherapies. This health disparity may equally be a function of phenotype, where breast cancer can evade the immune system by sequestering tumor‐associated neutrophils and macrophages, in an environment rich in TNF‐α. In this study, we investigated the effects of apigenin, a natural compound from parsley to reduce TNF‐α mediated rise in chemokine (C‐X‐C motif) ligand 1 (CXCL1) in TNBC cell lines of ethnic origin: Caucasian (MDA‐MB‐231) and African American (MDA‐MB‐468). CXCL1 is a known key mediator in neutrophil recruitment and degranulation. The data obtained show that TNF‐α induced greater CXCL1 transcript and protein levels, which were attenuated by apigenin only in the MDA‐MB‐468 cell line. The implications of this warrant further investigation as to the capacity of natural or anti‐cancer agents to act differentially based on the ethnic phenotype of TNBC.

Support or Funding Information

Supported by the National Institutes of Health, National Institute on Minority Health and Health Disparities, RCMI grant (8G12MD007582‐28.) and COE grant (P20 MD006738).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relation of Lsd, Ecstasy and Alcohol Used by Undergraduate Students
Authors: Mendes Maria Clara Chaves , Lauar Gabriela Caetano Vilela , Miranda Renato Pricoli , Taricano Ingrid Dragan
Published: June 2021

The purpose of this investigation was to compare the individual and combined consumption of LSD – lysergic acid diethylamide, the psychedelic drug that causes visual hallucinations, a distorted sense of time and a feeling of euphoria, ecstasy and alcohol by undergraduation people in São Paulo, Brazil. Young people can mix hallucinogens with other drugs like ecstasy or alcohol to increase the effects. In fact, the average age which many people start using LSD is around 20, but the number of people who abuse LSD today is far less than in the 1960s and 1970s and one factor limiting higher LSD use is a lack of availability.

METHODOLOGY

The instrument used for data collection was Google FormsTM. The instant addition of responses to the data set makes this tool appealing for research purposes. Additionally, the respondents involved in the study has been regularly using Google FormsTM, and it was therefore considered the most appropriate method for data collection. The results of the survey was statistically analyzed. Within the various areas the group of students from medical, biology, nursing, engineering, architecture, psychology, and other undergraduate areas, were chosen because their experience using these tools.

RESULTS

Data obtained showed that in a sample of 907 students, 71 (7.82%) students have used LSD, and 42 (59.15%) of these also used ecstasy, but 40 (56.33%) in this group used alcohol with LSD or ecstasy in their experiences. The undergraduate areas that consumed the most, is health (medical students), 29 (40.84%) for LSD and 46 (64.78%) for ecstasy. The undergraduate areas that consumed less than others is biology, 4 (5,6%) for LSD and only one (1,4%) for ecstasy.

CONCLUSIONS

In fact, medical students have a high incidence of LSD and ecstasy use with or without alcohol. It's surprising because they supposedly know the damages of this practice, more than the students of other areas. This work was conducted in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration. CAAE(Br)67881517.1.0000.5511. All participants had full understanding and signed informed consent document.

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 Effects of Daily Docosahexaenoic Acid Administration on High Fat Chow‐Induced Enhanced Sensitivity to Dopaminergic Drugs
Authors: Hernandez‐Casner Caroline , Beltran Nina , Ramos Jeremiah , Serafine Katherine
Published: June 2021

Eating a diet high in fat can lead to several negative health consequences, including obesity, type 2 diabetes and dopamine system dysfunction. For example, rats eating high fat laboratory chow are more sensitive to the behavioral effects of drugs acting on dopamine systems, including direct‐(e.g., quinpirole) and indirect‐acting (e.g., cocaine) dopamine receptor agonists. Dietary supplementation with omega‐3 fatty acids, such as fish oil, can successfully prevent and treat this high fat chow‐induced enhanced sensitivity to dopaminergic drugs. Fish oil contains two omega‐3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), therefore the relative contribution of each fatty acid to the beneficial effects of fish oil are not known. To test the hypothesis that daily injections of DHA will prevent high fat diet‐induced enhanced sensitivity to the behavioral effects of cocaine (1.0–17.8 mg/kg), adolescent female rats ate standard laboratory chow (17% kcal from fat), or high fat chow (60% kcal from fat) in combination with daily injections of DHA or vehicle. Eating high fat chow enhanced sensitivity of female rats to cocaine‐induced locomotion as compared to rats eating standard chow alone. That is, the cocaine‐induced locomotion dose‐response curve was shifted upward in rats eating high fat chow, as compared to rats eating standard chow. Daily injections of DHA did not prevent this effect. Rats receiving daily injections of DHA (regardless of type of chow) gained significantly more weight than rats eating standard chow alone or high fat chow alone. While these results might suggest that EPA, rather than DHA, is the likely omega‐3 fatty acid driving the aforementioned effects of fish oil, only one dose of DHA has been examined thus far. Future experiments will focus on examining different doses of DHA alone and in combination with various doses of EPA to determine the relative contribution of each omega‐3 fatty acid in mediating the beneficial effects of fish oil. These data add to a growing literature regarding the health benefits of fish oil and omega‐3 fatty acids.

Support or Funding Information

Research reported in this abstract was supported by the National Institute of General Medical Sciences of the National Institute of Health under linked Award Numbers RL5GM118969, Tl4GM118971, and UL1GM118970.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacokinetics and Pharmacodynamics of Morphine and its Major Metabolites Following Intravenous Administration of Four Doses to Horses
Authors: Hamamoto Briana D. , Steffey Eugene P. , Knych Heather K.
Published: June 2021

Objective

To understand the pharmacokinetics of morphine and correlate the serum values of morphine and its metabolites to pharmacodynamic data.

Materials and Methods

Using a balanced randomized 5‐way crossover design; ten horses received an intravenous injection of saline and morphine at doses of 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, and 0.5 mg/kg. This was repeated until all horses received all treatments. A minimum washout period of 2 weeks was observed between treatments. Blood samples were collected at various time post drug administration, concentrations of morphine, M3G and M6G determined using liquid chromatography‐mass spectrometry and pharmacokinetic analysis performed. Horses were outfitted with step counters and Holter monitors. Gastrointestinal borborygmi, respiration, defecation, and overall demeanor were assessed. Concentrations of parent compound, M3G and M6G were correlated to pharmacodynamic data to better understand the effects of the active metabolites.

Results

Morphine is metabolized into two metabolites in the horse: morphine‐ 6 glucuronide (M6G) and morphine‐3 glucuronide (M3G). Both metabolites and parent compound were detected in the serum as soon as 5 minutes after administration. M3G was found in much higher concentrations than both M6G and parent compound. Concentrations of metabolites and parent compound were dose dependent with the highest concentrations seen in the 0.5 g/kg dose group. Plasma concentrations of morphine fit best in a three‐compartment model and were characterized by rapid distribution and slower elimination phases. Individual pharmacokinetic parameters for metabolites were obtained by noncompartmental analysis. Higher concentrations of M3G correlated with an increase in step count, increase in heart rate, and decrease in gastrointestinal borborygmi seen in the high dose group.

Conclusions and Potential Impact

This project delivers basic pharmacokinetic and pharmacodynamic knowledge of morphine administration to horses and provides valuable information regarding morphine metabolism in horses and the characteristics of these metabolites. The large amount of M3G may be contributing to the neuroexcitatory effects seen in horses after high doses of morphine. Further exploration of the individual effects of the metabolites would be helpful in potentially identifying a novel analgesic. This would be a large advance in veterinary medicine as the number of analgesics available to equine patients currently is limited an often come with severe side effects such as renal failure, gastrointestinal slowing, and increased CNS activity.

Support or Funding Information

California Horse Racing Board Pharmacology Research Funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression Analysis of Prostaglandin E and F Receptors in Rat Uterus, Mesenteric Artery and Uterine Artery for Development of a Surrogate Assay of Uterine Contractility
Authors: Roberts Melissa , Ferguson Greg , Karras James , van Duin Marcel , Lindstrom Beatriz
Published: June 2021

Preterm labor occurs in nearly 10% of U.S. pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Prostaglandins, specifically of the E and F series, play a key role in myometrial contractility and have been recognized as activators of parturition. As such, modulation of select prostaglandin signaling pathways provides a potential approach to pharmaceutical intervention of preterm labor. Prostaglandin E2 is the most abundantly produced prostanoid and functions through four subtypes of G protein coupled receptors (EP1, EP2, EP3, and EP4). It is generally accepted that EP1 and EP3 serve as pro‐contractility receptors while EP2 and EP4 are relaxatory receptors that promote myometrial quiescence. Prostaglandin F2α (PGF2α) functions through the FP receptor and plays an essential role in uterine contractility during preterm or term labor and delivery. The dynamicity and complexity of prostaglandin pharmacology is an important factor to consider during assay development for the testing of molecules which may not be selective to one receptor subtype.

Translational ex vivo assays that enhance throughput of drug candidate screening and decrease the use of experimental animals are increasingly desirable. The objective of this research was to determine the relevance of employing either mesenteric resistance artery or uterine artery segments as surrogate tissues to the uterus in the evaluation of prostaglandin‐induced smooth muscle contraction. The relative expression of EP1, EP2, EP3, EP4, and FP receptor mRNA was determined by RT‐qPCR and compared between the uterine body, uterine artery, and mesenteric artery in the rat. Additionally, the efficacy of PGF2α in eliciting contraction was measured by isometric wire myography and compared between the uterine and mesenteric artery.

The results of this study demonstrate that rat mesenteric artery may have utility as a model in the screening of prostaglandin receptor‐targeting molecules. Together, these findings will contribute to the identification of suitable and efficient ex vivo assays by which new pharmacological approaches for the treatment of preterm labor can be evaluated.

Support or Funding Information

Ferring Research Institute, San Diego, CA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative proteomics of Zebrafish liver in 5 POPs mixture exposure
Authors: Gao Yan , Kwon Oh Kwang , Na An Yae , Kim Ki‐Tae , Lee Sangkyu
Published: June 2021

Type 2 diabetes mellitus (T2DM) is a major public medical problem with ill‐defined pathophysiological mechanisms and multifactorial etiology. Increasing epidemiological studies have consistently suggested an association between persistent organic pollutant (POPs) and risk of developing T2DM. The POPs have also been reported to negatively affect reproductive, central nervous, or the immune system. In this study, the potential mechanism of POPs induced T2DM was investigated by quantitative proteomics. Five general POPs mixtures, p,p’‐DDT, heptachlor, β‐HCH, hexachlorobenzene and chlorodane were administered to zebrafish for 3 months. Proteomic analysis revealed upregulation of protein Pentaxin in zebrafish. Pentaxin is homologous to C‐reactive protein (CRP) in human, which is widely recognized as an independent risk factor for type 2 diabetes. This finding showed that POPs might be a diabetes inducer. Thus, this study increased the understanding of the relationship between POPs and T2DM in biological and proteomics aspects.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EVALUATION OF THE POTENCY AND SELECTIVITY OF THE NOVEL FAAH INHIBITOR BIA 10‐2474 IN COMPARISON WITH PF‐04457845 AND JNJ‐42165279
Authors: Bonifacio Maria Joao , Sousa Filipa , Aires Catia , Loureiro Ana I. , Fernandes‐Lopes Carlos , Palma Nuno , Moser Paul , Soares‐da‐Silva Patricio
Published: June 2021

In January 2016 one person died and others suffered adverse neurological effects during a Phase I clinical trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10‐2474. We have compared the potency and selectivity of BIA 10‐2474 with two other mechanism‐based FAAH inhibitors PF‐04457845 and JNJ‐42165279 which appeared safe in human testing.

In whole cells, BIA 10‐2474 inhibited FAAH with an IC50 of 74.4 and 18 nM after 1h and 24h incubation respectively. This compares with 26.2 and 41.5 nM for JNJ‐42165279 at 1 and 24h and 4.3 nm for PF‐04457845 at 1h. All three compounds were characterised as irreversible FAAH inhibitors. Interestingly, BIA 10‐2474 and JNJ‐42165279 were more potent FAAH inhibitors in whole cells as compared to a membrane preparation (4–6 fold more active) when tested after 1h incubation whereas PF‐04457845 was equipotent in the two models (4.3 and 2.0 nM respectively). Neither BIA 10‐2474 nor JNJ‐42165279 accumulated inside the cells.

Activity based protein profiling in vitro identified only ABHD6 as an off‐target for BIA 10‐2474 with no activity against other rat brain serine hydrolase enzymes up to 100 mM. In contrast PF‐04457845 and JNJ‐42165279 both interacted with ABHD11. BIA 10‐2474 had no significant activity against 220 other human off‐targets in binding and enzyme studies up to 50 μM. In contrast, PF‐04457845 showed significant activity (>50% inhibition) at 37 sites at 50 μM, including cannabinoid receptors, serotonin receptors, and several ion channels.

In vivo, oral administration of BIA 10‐2474 inhibited rat brain and liver FAAH with ED50s of 13.5 and 6.2 mg/kg PO respectively and increased brain levels of fatty acid amides over the same dose range. This compares with 4.3 and 2.0 mg/kg PO for PF‐04457845 and 12.5 and 3.7 mg/kg PO for JNJ‐42165279. Exposure levels of BIA 10‐2474 in plasma and brain were consistent with the potency identified in whole cells and there was no evidence for accumulation in the brain of either BIA 10‐2474 nor its main metabolites following repeated administration.

These data suggest that the in situ and in vivo potency and the selectivity of BIA 10‐2474 is comparable to other clinically tested FAAH inhibitors. These data also demonstrate that there is no unusual accumulation of BIA 10‐2474 or its metabolites in cells nor in the CNS. In conclusion, these results fail to identify an off‐target activity or a mechanism that could explain the clinical trial accident.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered dopamine transporter function and immunoregulation in PD macrophages
Authors: Mackie Phillip , Gopinath Adithya , Saadatpour Leila , Pino Jose , Khoshbouei Habibeh
Published: June 2021

Parkinson's Disease (PD), the second‐most common neurodegenerative disorder, has three key hallmarks: dysregulated dopamine (DA) signaling in part due to degeneration of Substantia Nigra neurons, intracellular aggregates of a‐synulcein, and neuroinflammation. However, the origin and interplay of these features remains unclear. DA substitution therapy, the standard pharmacological treatment, only alleviates symptoms with limited efficacy. Interestingly, plasma DA levels are altered in PD, and DA can regulate immune cell function in the periphery. Additionally, the dopamine transporter (DAT), a potent regulator of DA tone in the CNS, also exists on leukocytes. However, although it is characterized in the CNS and is a therapeutic target for some neurological disorders, its function in the periphery is largely unknown. Our study asks how DAT regulation of plasma DA modulates the peripheral immune system. To begin to address this hypothesis, we first used biochemical and microscopy approaches to examine DAT distribution and function in the healthy monocyte‐derived human macrophages (MDM). We found DAT constitutively exists in human MDM and is localized to the plasma membrane and in the intracellular space (n=10 biological replicates). We demonstrated the DAT molecules on the human MDM are functional as measured by uptake of IDT307, a fluorescent DAT substrate, and of 3H‐DA (Km=3.178). In addition we found DAT is subjected to canonical trafficking such as PKC‐dependent endocytosis as measured by Total Internal Reflection Florescence Microscopy (TIRF‐M). These data are consistent with the overarching hypothesis that immuno‐activation of human MDM regulates DAT activity and thus dopamine transmission. We discovered lipopolysaccharide (LPS)‐immuno‐activation of human macrophages recruits DAT to the membrane leading to increased surface DAT localization (n= 4 biological replicates P < 0.05). Contrary to our prediction, our preliminary data suggests a decrease in nomifensine‐sensitive, DAT‐mediated substrate uptake. Furthermore, we have also found that basal DAT‐dependent uptake in PD macrophages is elevated (p<0.0001, n=5 biological replicates), and our preliminary data suggests reduced sensitivity to LPS‐induced trafficking. Using simultaneous whole cell patch clamp electrophysiology and amperometry, we are currently testing the hypothesis that LPS‐induced TLR4 activation of macrophages stimulates DAT trafficking to the membrane and stabilizes the transporter in an efflux promoting conformation. We then plan to test the hypothesis that PD macrophages are immunocompromised, exhibiting an altered DAT response to LPS‐stimulation.

Support or Funding Information

R21NS103108 R21DA043895

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single Amino Acid Mutations in the Na+/Taurocholate Cotransporting Polypeptide (NTCP) Result in Substrate‐Dependent Effects
Authors: Ruggiero Melissa , Malhotra Shipra , Karanicolas John , Swint‐Kruse Liskin , Hagenbuch Bruno
Published: June 2021

Nonsynonymous polymorphisms can significantly impact the function of a protein. However, so far, the outcome of such mutations at non‐conserved positions are difficult to predict. Previous work showed that some non‐conserved positions have an unexpected outcome when they are mutated: if multiple amino acids are substituted, the results show progressive alterations like a “rheostat”, rather than an “all‐or‐nothing” effect often seen for mutations at conserved positions (“toggle”). To begin to define rules for predicting mutation outcomes at rheostat positions, we used the Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a model transmembrane transporter. NTCP is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. NTCP is important for the enterohepatic circulation of bile acids and also mediates the transport of steroid hormones like estrone‐3‐sulfate (E3S) or drugs like rosuvastatin. We initially focused on the S267F polymorphism that decreased taurocholate transport, stimulated rosuvastatin uptake, and showed no effect on E3S transport. We replaced the serine with all other nineteen amino acids by site directed mutagenesis, transfected the cDNAs into human embryonic kidney cells (HEK293), and determined the uptake of tritiated substrates. In addition, we identified the conserved G102 and the non‐conserved Y146 positions using multiple sequences alignments and homology modeling based on the structures of two bacterial homologues. Both positions fall in mobile regions of NTCP. Interestingly, mutations at S267 resulted in a clear rheostat behavior for taurocholate uptake, with S267F showing only 15% of the function of wild‐type NTCP. Similarly, rheostat behavior was observed for E3S and rosuvastatin, but several mutants transported these substrates 2‐ to 10‐fold higher than wild‐type NTCP, demonstrating that the different amino acid replacements resulted in substrate‐dependent effects. As expected, the non‐conserved Y146 demonstrated rheostat behavior for all three substrates. However, the conserved G102 turned out to be a rheostat position for taurocholate transport but a toggle for the uptake of E3S and rosuvastatin. In summary, we have determined that rheostat positions exist in human NTCP. Furthermore, the functional consequences of single amino acid mutations at the three positions studied so far, varied depending on the substrate tested. Therefore, functional predictions of amino acid mutations seem to be more challenging and will require further experiments at additional positions to potentially define a unifying set of rules.

Support or Funding Information

NIH R01 GM077336, P30 GM118247, W.M. Keck Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endoglin targeting inhibits tumor angiogenesis in non‐small cell lung cancer
Authors: Chen Chiao‐Yun , Sheu Ming‐Jyh
Published: June 2021

Endoglin, a transforming growth factor‐β co‐receptor, is highly expressed on angiogenic endothelial cells in many different solid tumors, including lung cancer. In patient samples, increased endoglin expression after the anti‐VEGF treatment was found. In contrast, in vitro TRC105, an endoglin‐neutralizing antibody, increased VEGF signaling in endothelial cells. This suggest that co‐administration with both endoglin antagonist and anti‐VEGF treatment may increase and sustain anti‐angiogenesis status. Subsequently, targeting endoglin is currently undertaken in clinical trials for anti‐angiogenic therapy. We aimed to explore how aspidin‐BB affected the endoglin expression and then effects on angiogenesis in lung cancer cells. Aspidin BB, a phloroglucinol derivative from Dryopteris fragrans (L.) Schott has been reported to demonstrate anticancer activity. In the present study, we investigated how aspidin‐BB affected the endoglin expression and then effects on angiogenesis. The binding mode of aspidin‐BB within the active site of endoglin was analyzed using DS 4.5. Our results showed that aspidin‐BB has strong affinity to endoglin. The results gave us a hint that aspidin‐BB could bind with endoglin and affected the BMP‐9, the main endoglin ligand, induced Alk1/Smad1 and/or Alk5/Smad2 phosphorylation in human umbilical vein endothelial cells (HUVECs). Further study will be focused on co‐administration with both endoglin antagonist and anti‐VEGF treatment, and determine its possibility to increase and sustain anti‐angiogenesis status.

Support or Funding Information

This work was supported by Grants from Ministry of Science and Technology (MOST 103‐2320‐B‐039‐026; MOST104‐2320‐B‐039‐007) and China Medical University (CMU105‐ASIA‐19;CMU106‐ASIA‐14).

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