bluuebunny/crossref_metadata_2025 huggingface dataset:
Atrazine Inhibition of Murine Electron Transport Chain: A CURE Biochemistry Laboratory
Authors: Martin Debra J.
Published: June 2021

For the majority of students entering Biochemistry laboratory, basic biochemical skills are a new concept for them. Previous courses, such as lower division Introductory Biology and Genetics may have introduced them to some of the techniques; but overall, the concept of combining the techniques to actually apply it to research is lacking. Additionally, we want students to learn research skills, like interpreting evidence, reasoning, thinking critically, and conveying ideas and to be able to write like a scientist. Using a research based inquiry laboratory, students explore how a common herbicide might inhibit the electron transport chain (ETC). Additionally, they investigate how the herbicide could affect ETC when animals are exposed. By the end of the semester long course, the students submit a final paper in the format for submission to the Proceedings of the National Academy of Science. Since the implementation of this laboratory research project, pre/post Biochemistry knowledge assessment exam scores have increased 30%.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leigh Syndrome Cell Model Development
Authors: Alverado Joshua , DeCuzzi Nicholaus , Hsueh Jessica , Madira Sarah , Lanning Nathan
Published: June 2021

Leigh Syndrome (LS) is a neurological disease that stems from mutations in the subunits of mitochondrial respiratory chain subunits. These mutations decrease electron transport chain (ETC) deficiency, leading to decreased cellular ATP levels and increased reactive oxygen species (ROS) levels. There is currently no therapeutic approach to combat this degenerative disorder, and patients typically die by 8–10 years of age. There are also very few models with which to study this disease. We have generated LS cell models using both a chemical and genetic approach. Chemically, we inhibit ETC activity with low levels of rotenone, an ETC poison, in order to suppress ETC‐dependent ATP generation to levels seen in LS patients. Genetically, we use siRNA to knock down the ETC subunits that are mutationally inactivated in LS patients, also disrupting ETC function. Through these approaches, we have constructed LS cell models that can now be used to investigate potential therapeutics.

Support or Funding Information

NIH 1R15GM123382, CSUPERB New Investigator Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Systems Approach Toward Elucidating The Molecular Basis Of Tropane Alkaloid Metabolism
Authors: D'Auria John C.
Published: June 2021

Tropane and granatane alkaloids are key mediators of interspecies plant communication and valued by society for their pharmacological properties. Beyond their biological significance, the biocatalytic steps needed to generate tropane and granatane chemical scaffolds is not completely understood. This lack of knowledge of biosynthetic steps as well as kinetic and thermodynamic properties diminishes the scientific community's understanding of the chemistry of life. Tropane and granatane alkaloid metabolism consists of three modules: a first ring closure that forms a reactive N‐heterocyclic intermediate, a second ring closure that generates the tropane chemical scaffold and lastly modifications of the core chemical scaffold. The overarching question guiding this research is, how are the critical ring forming enzymes leading to these bicyclic compounds functioning at the molecular level? While tropane alkaloid metabolism has historically been studied in the Solanaceae, recent evidence indicates that non‐solanaceous species have independently evolved tropane metabolism using alternative biosynthetic machinery. Moreover, granatane alkaloids are structurally similar to tropane alkaloids and are hypothesized to use biocatalytic steps similar to those used to produce tropane alkaloids. Collectively, the tropane and granatane alkaloids constitute a comparative system for studying the mechanistic basis for the emergence and diversification of specialized metabolic enzymes. An integrated approach using biochemical, biophysical and molecular genetic currently being used to discover the molecular determinants of the bicyclic ring in tropane and granatane biosynthesis. Results from both the early and the terminal steps in the respective tropane and granatane pathways have begun to fill‐in gaps in our knowledge of their respective biosynthetic pathways as well as discover novel enzymes and divergent evolutionary mechanisms. For example, current data supports the hypothesis that a Type III Polyketide synthase is responsible for the extension off the first ring and ultimate formation of the second ring in the bicyclic tropane or granatane nucleus. We have identified Type III PKS enzymes and the genes encoding them from members of the Erythroxylaceae, Solanaceae, and Lythraceae. These enzymes all produce their respective predicted non‐cylclized polyketide enzyme product while sharing closer identity with PKS enzymes from within their own lineages.

Support or Funding Information

This research is funded via the National Science Foundation of the United States of America award ID. 1714236

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Daurichromenic Acid Synthase from Rhododendron dauricum
Authors: Taura Futoshi , Iijima Miu , Kurosaki Fumiya
Published: June 2021

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti‐HIV meroterpenoid isolated from Rhododendron dauricum (Ericaceae). We identified a novel cDNA encoding DCA synthase by transcriptome‐based screening from young leaves of R. dauricum. The gene coded for a 533‐amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino‐terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine‐112 and cysteine‐175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris, exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa, whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin.

Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells. With respect to this unique extracellular biosynthesis and accumulation of DCA, we speculated the possibility that DCA would serve as a chemical defense component outside of the scale cells, the outermost layer of plants. In addition, we demonstrated that DCA and grifolic acid are phytotoxic meroterpenoids that induce cell death in the cell cultures of their producer plant R. dauricum, and thus have to be sequestered in the apoplast to avoid self‐poisoning.

Support or Funding Information

This work was supported by JSPS/MEXT KAKENHI (nos. 15K07994 and 17H05436) to FT, and a Grant‐in‐Aid for JSPS Fellows (no. 17J10178) to MI.

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 Glutathione Maintenance in Protection against Advanced Glycation End Product Induced Neurite Degeneration in SH‐SY5Y Cells
Authors: Stochelski Mateusz Andrzej , Wilmanski Tomasz Maciej , Burgess John R.
Published: June 2021

Diabetes mellitus is one of the most common chronic diseases and peripheral neuropathy (PN) affects at least 50% of diabetic patients. Medications available for patients ameliorate symptoms (pain) but do not protect against cellular damage, and come with severe side effects leading to discontinued use. In our lab we use differentiated SH‐SY5Y cells treated with advanced glycation end products (AGE) as a model to mimic diabetic conditions and to study the mechanisms of oxidative stress (OS) mediated cell damage and antioxidant protection. Nacetylcysteine (NAC), a common supplement, was previously shown in our lab to fully protect against AGE‐induced damage. Here we report that NAC confers protection by maintaining intracellular glutathione (GSH), suggesting that GSH plays a critical role in neurite protection. Our lab has also shown that 3H‐1,2‐dithiole‐3‐thione (D3T), a cruciferous vegetable constituent and potent inducer of nuclear factor (erythroid‐derived 2)‐ like (Nrf2), can significantly increase cellular GSH concentrations and protect against H2O2–induced cell death. Paradoxically, D3T conferred no protection against AGE‐induced cell death or neurite degeneration. Furthermore, D3T increased reactive oxygen species (ROS) generation, depleted GSH, and increased glucose‐6‐phosphate dehydrogenase (G6PD) expression, suggesting its role as an NADPH generating agent may promote OS rather than provide protection. Inhibition of NADPH generation via G6PD, the protein product of a Nrf2‐responsive gene, with dehydroepiandrosterone was found to protect against AGE‐induced ROS generation, loss of viability, and neurite degeneration. These results suggest that using a strategy to protect against oxidative stress by upregulation of the endogenous antioxidant defense system via Nrf2 may backfire and promote further damage if the products of upregulation contribute to ROS generation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Citrus polymethoxyflavones (PMFs) inhibit 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced mouse skin JB6 P+ cell transformation through the activation of Nrf2 pathway
Authors: Su Zheng‐Yuan , Pan Guan‐Wu , Tsai Chung‐Liang , Wu Hao‐Xuan , Wei Guor‐Jien
Published: June 2021

Oxidative stress is one of the important factors to induce carcinogenesis, including skin cancers. Many bioactivities of polymethoxyflavones (PMFs), such as tangeretin and nobiletin, in citrus peels have been studied. In this study, the cancer chemopreventive potential potential and mechanism of tangeretin and nobiletin metabolites with low cytotoxicity were investigated. The results showed that PMF‐2, a tangertin derivate, at 50 μM showed the strongest activity to trigger Nrf2 pathway in human hepatoma HepG2‐C8 cells that were stably transfected with Nrf2‐antioxidant response element (ARE)‐luciferase plasmids. PMF‐2 also significantly suppressed 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced anchorage independent growth of mouse skin JB6 P+ cells. Additionally, PMF‐2 increased mRNA and protein expression of Nrf2 and Nrf2 downstream genes such as heme oxygenase‐1 (HO‐1), NAD(P)H dehydrogenase quinone 1 (NQO1), and UDP glucuronosyltransferase 1A (UGT1A). Furthermore, PMF‐2 might regulate the expression of Nrf2 and Nrf2 downstream genes through regulating DNA methylation and Histone modification, which was elucidated by the determination of DNMTs, HDACs, and KDMs protein expressions, in vitro DNA methylation, methylation‐specific PCR (MSP) of Nrf2 promoter. The relationship between Nrf2 downstream genes and histone H3 acetylation (H3ac) was also analyzed by chromatin immunoprecipitation (ChIP)‐qPCR assay. Therefore, PMF‐2 has skin cancer chemopreventive potential through activating Nrf2‐related pathway that might be regulated by epigenetics modification.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of sex hormones alters indices of prefrontal cortex and hippocampal insulin signaling and increases lipid content in a region‐specific manner independent of cardiac pressure overload in female aortic‐banded Yucatan miniature swine
Authors: Hayward Grant C. , Yang Alex J. , Leblanc Paul J. , Hiemstra Jessica A. , Edwards Jenna C. , Emter Craig A. , Olver T Dylan , MacPherson Rebecca E.
Published: June 2021

Estimates indicate ~50% of patients with heart failure with preserved ejection fraction (HFpEF) experience dementia. Interestingly, postmenopausal women represent the largest cohort of patients with HFpEF. Impaired insulin signaling in the brain has been implicated in dementia, and may be related to regional changes in the neuronal lipid composition. How such processes are effected by the loss of female sex hormones remains to be elucidated. Using a translationally relevant large animal model of pressure overload‐induced heart failure, we tested the hypotheses that the loss of female sex hormones alters proteins involved in brain insulin signaling, and that such changes are associated with an altered lipid profile. Twenty‐eight female Yucatan miniature swine were divided into four groups (n=7): control (CON), aortic banding (AB; pressure overload model of heart failure), ovariectomy (OVX), and AB with OVX (AB‐OVX). Pigs underwent OVX and AB at 7 and 8 months of age, respectively, and prefrontal cortex and hippocampus tissues were collected at 14 months. Estrogen receptor‐α (p<0.05) content was reduced with OVX in both cortical and hippocampal brain regions. In the cortex, OVX reduced total Akt (p<0.05) and synaptophysin (p<0.10), and in the hippocampus OVX reduced phospho‐Akt Ser473 (p<0.05) and phospho‐ERK (p<0.05). Neuronal lipid composition in the prefrontal cortex and hippocampus revealed increases in total lipids (p<0.10), saturated (p<0.10), and monounsaturated fatty acids (p<0.05) with OVX. The hippocampus had increased total (p<0.10), monounsaturated (p<0.05), and polyunsaturated fats (p<0.10). No changes were observed in OVX for polyunsaturated fats. In conclusion, the loss of female sex hormones alters Akt and MAPK/ERK protein content/pathway activation predominantly in the hippocampus, independent of cardiac pressure overload. Impaired indices of hippocampal insulin signaling were associated with a local accumulation of saturated and monounsaturated fats. Overall, the loss of female sex hormones may contribute to the development of dementia in a translational swine model of pressure overload HF with potential relevance to human HFpEF through metabolic pathways influencing cognition.

Support or Funding Information

NIH R01 HL112998, PI: CAE

American Heart Association #16POST2776005, PI: TDO

Alzheimer Society of Brant, Haldimand, Norfolk, Hamilton, and Halton, PI: REKM

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cross‐talk Between the Gut and the Brain in Rats Subjected to LPS‐Induced Inflammation: Role of α‐Synuclein
Authors: Doursout Marie‐Francoise , Gaskey Gregory , Peng Zhanglong , Bick Diane L. , Smith Keri , Devalottai Ria , Kowada Minoru , Schiess Mya
Published: June 2021

It is now well established that the involvement of the gastrointestinal tract is of great interest as a contributing factor to the development and progression of neurodegenerative diseases. The dysregulation of the Gut‐Brain‐Axis (GBA) may be associated with gastrointestinal manifestations preceding disorders of cognitive and/or memory and learning process, supporting the hypothesis that the pathological process is spread from the gut to the brain (Mayer et al. 2014). The extensive involvement of the gut in neurodegenerative diseases such as Parkinson's disease, even in its early stages, has led to the evaluation of enteric α‐synuclein as a possible biomarker (Forsyth et al. 2011). Therefore, the overall goal of the proposed study is to assess the time course of the compromised intestinal barrier capable of influencing brain function in rats challenged with LPS. Specifically, we hypothesize that alterations of the intestinal barrier (or leaky guts) enhances inflammation as well as the production of a unique protein α‐synuclein which has characteristic of degenerative diseases. To induce inflammation, Lipopolysaccharide (LPS) was administered intravenously (IV) in rats at a dose of 20 mg/kg (Doursout et al. 2013). A single dose LPS was elected based on previously reported results from a mouse model (Qin et al. 2007). Animals were divided into 2 groups (saline and LPS). Animals were divided into 2 sub‐groups (short term; 1 week and long term 4 weeks). Following sacrifice at each time point, tissues (gut and brain e.g. hippocampus and olfactory bulb) were harvested. We measured edema formation in the gut in rats subjected to LPS as compared to saline by wet/dry ratio. Alpha‐synuclein was determined using ELISA (R&D Systems) in small and large intestines as well as in the brain at each time point. Data are expressed as Mean ± SEM. Our data show that LPS induced edema in the gut, suggesting inflammation. Our data demonstrate that increases in α‐synuclein protein concentrations were time‐dependent in the hippocampus and in the olfactory bulb. We postulate that LPS induce an increase in α‐synuclein concentrations in the gut and spread to the brain at a later time point.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of adiponectin ameliorates non‐alcoholic fatty liver disease via inhibition of FoxO1 in type I diabetic rats
Authors: Xie Xiang , Xia Zhengyuan
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is a common liver disease which is previously associated with Type 2 Diabetes Mellitus (T2DM). Recent research has indicated Type 1 Diabetes Mellitus (T1DM) is also involved in the development of non‐alcoholic fatty liver disease, whereas the underlying mechanisms are not utterly understood. Forkhead box O1 (FoxO1) and adiponectin (APN) have been proposed to play an important role in the processes in NAFLD in T1DM. We herein investigated the effects of FoxO1 and APN on the NAFLD and the underlying mechanism in T1DM. Serum AST and ALT was determined by commercially available kits. Blood glucose levels were measured by One Touch Ultra Glucose Meter. Relevant protein expression was tested by Western blot analysis. We found that serum AST and ALT were both increased in T1DM rats, which was ameliorated by application of APN or block of FoxO1. Moreover, APN and FoxO1 inhibitor both decreased the expression of FoxO1 which was significantly increased in diabetic rats. However, the inhibition of FoxO1 didn't alter the expression of APN and its receptors. We also found that Akt1 expression was significantly declined in diabetic rat which was restored by APN. Together, these results suggest that APN ameliorates NAFLD via inhibition of FoxO1 through Akt1/FoxO1 signaling pathway. The present study demonstrated the effort of T1DM on NAFLD and presented a novel role of APN in the underlying mechanism.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sustained crosstalk between TLR2 and IL‐1R1 exacerbates neurodegeneration in Parkinson's Disease
Authors: Johnson Marguerite Elizabeth , Kortagere Sandhya
Published: June 2021

Current evidence suggests the causal role of inflammation in the pathogenesis of Parkinson's Disease (PD). PD is a neurodegenerative disorder that predominantly affects dopaminergic neurons within the substantia nigra (SNc) region of the brain and is typically accompanied by the accumulation of alpha‐synuclein containing fibrils called Lewy Bodies (LB). Studies have demonstrated that alpha‐synuclein fibrils function as damage‐associated molecular patterns (DAMPs) to induce an inflammatory response via Toll‐like Receptor 2 (TLR2) activation on both neurons and microglia. The role of TLR2‐mediated inflammation in neurodegeneration is further supported by increased microglial activation, pro‐inflammatory cytokines (e.g. TNF‐a and IL‐1B), and TLR2 protein expression in LBs. Two recent reports have shown that IL‐1R1 antagonism attenuates inflammation and behavioral deficits in PD animal models. Despite these studies, the mechanism by which both TLR2 and IL‐1R1 contribute to neurodegeneration in PD remains elusive. Recently, our lab has demonstrated that activation of TLR2 by PAM3CSK4 induces an increase in IL‐1R1 expression in SH‐SY5Y cells. Further, activation of TLR2 or IL‐1R1 leads to an increase in phosphorylation of p38‐MAPK and ERK1/2 and an increase in cytokine levels. These results suggest a crosstalk between TLR2 and IL‐1R1 may be essential to produce degeneration of the dopaminergic neurons in the substantia nigra. Sustained crosstalk between TLR2 and IL‐1R1 could lead to exacerbation of this proinflammatory response in PD.

Support or Funding Information

Drexel 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.
Molecular GPS: Receptor and G‐protein dynamics that drive selectivity in GPCRs
Authors: Sandhu Manbir , Dysthe Matthew , Sivaramakrishnan Sivaraj , Vaidehi Nagarajan
Published: June 2021

Upon binding to agonists, G‐protein coupled receptors (GPCRs) couple selectively to specific G‐proteins (such as Gs, Gi, and Gq) or β‐arrestins to activate specific signaling pathways. Some GPCR agonists exhibit differential responses to signaling pathways, a phenomenon called “functional selectivity”. The mechanism by which the GPCR‐agonist pairing leads to G‐protein selectivity is relatively obscure. Structure based design of G‐protein selective ligands remains a challenge due to the paucity of information on the structural elements of the ligand‐GPCR‐G‐protein complexes that lead to G‐protein selectivity. We have used a combination of a multi‐resolution Molecular Dynamics (MD) method for enhanced conformational sampling of protein complexes, called the Generalized Newton‐Euler Inverse Mass Operator (GNEIMO) method and a genetically encoded FRET biosensor technique called Systematic Protein Affinity Strength Modulation (SPASM), to measure G‐protein specificity in live cells. The combination of GNEIMO and SPASM form a powerful, complementary and scalable toolset for studying the structural basis of G‐protein selectivity in several GPCR‐G‐protein pairings. SPASM sensors measure the interaction (through FRET) of two proteins tethered by a flexible α‐helical ER/K_linker; this tool permits measurement of non‐cognate GPCR‐G‐protein interactions which cannot be measured by traditional biochemical assays used for cell signaling. This allows for an unprecedented comparison of relative intrinsic affinities of different G‐proteins to a specific GPCR. In this study, we have generated the conformational ensemble for 7 class A GPCRs bound to a full agonist and a C‐terminal peptide from Gαs‐, Gαi‐, and Gαq‐proteins. We predicted the putative hotspot residues on the G‐protein that leads to selectivity and tested these mutations using SPASM for switching specificity between Gs, Gi and Gq receptors. We observe Gs peptide binding to a different location in the intracellular surface of GPCRs compared to Gi and Gq peptides. The homologous, anionic residues, E355 (Gq) and D350 (Gi), orient the G protein C‐terminus away from the Gs‐specific cavity in the GPCR interface. Gi binding is further distinguished from Gq at the conserved position of G352 (Gi) and N357 (Gq). The smaller G352 residue functions as a size‐filter for the smaller cavity of Gi‐signaling GPCRs. This work provides insight to molecular mechanisms governing G‐protein selectivity, and is imperative to the development of functionally selective ligands.

Support or Funding Information

NIH R01 GM117923‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxycholesterol protects against LXR‐mediated steatosis through Epstein‐Barr virus‐induced GPCR 2 (EBI2) in human and mouse hepatocytes
Authors: Huang Jin , Kang Saeromi , Park Soo‐Jin , Im Dong‐Soon
Published: June 2021

Background

Oxycholesterols are metabolites of cholesterol and several of them can act on the G protein‐coupled receptor, Epstein‐Barr virus‐induced G‐protein coupled receptor 2 (EBI2). Pivotal functions of oxycholesterols in immune regulation have been proven. However, the function of oxycholesterols in hepatocytes has not been fully investigated.

METHODS

An in vitro model of liver X receptor (LXR)‐mediated hepatocellular steatosis was used to assess the role of oxycholesterols and EBI2 in hepatocytes.

RESULTS

T0901317, a specific LXR activator, induced lipid accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by 7α,25‐dihydroxycholesterol (DHC), the most potent agonist of GPR183. The protective effects of DHC were inhibited by NIBR189, a specific EBI2 antagonist. T0901317 treatment induced expression of SREBP‐1c and ChREBP, key transcription factors for lipid synthesis. DHC inhibited the induction of SREBP‐1c and ChREBP proteins in a EBI2‐dependent manner. Using specific inhibitors of cellular signaling components, DHC‐induced inhibition of lipid accumulation was found to be mediated through Gi/o proteins, p38 MAPKs, PI3K, and AMPK. Furthermore, the protective effect of DHC was confirmed in HepG2 cells and mouse primary hepatocytes.

CONCLUSION

Therefore, the present data suggest that DHC‐EBI2 signaling in hepatocytes may protect against lipid accumulation in the liver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Targeting and Inactivation of Glutaminase and CDK4/6 in Mantle Cell Lymphoma
Authors: Gibbs Bettine E. , Masamha Chioniso P.
Published: June 2021

Mantle Cell Lymphoma (MCL) is an aggressive B‐cell non‐Hodgkin's lymphoma (NHL) subtype that accounts for 7% of all NHL cases. Upon relapse, standard salvage regimens are ineffective and the disease is considered clinically incurable. Hence, novel therapeutic strategies that effectively target deregulated pathways that contribute to MCL pathogenesis are warranted. The hallmark of MCL is a chromosomal translocation t (11:14) event that results in the constitutive expression of the G1‐phase cell cycle regulatory oncogene, cyclin D1 (CCND1), resulting in uncontrolled cell proliferation. Cyclin D1 binds to and activates cyclin dependent kinases 4 and 6 (CDK4/6) and the complex phosphorylates the tumor suppressor gene product retinoblastoma (RB), a negative regulator of the cell cycle. MCL patients whose tumors express cyclin D1 have reduced survival. Uncontrolled cell proliferation results in metabolic alterations of tumor cells to support the energy and biosynthetic needs of unregulated cell growth. Adaptations by malignant cells involve changes in the expression of genes involved in metabolism. This results in both increased glucose and glutamine uptake and metabolism in tumor cells. Recent studies in metabolically reprogrammed tumor cells have shown increased levels of glutaminase, the enzyme involved in the first steps of glutamine metabolism. We hypothesize that molecular targeting of proliferation and glutamine metabolism will induce synergistic tumor cell death. In vitro studies with a MCL cell line were completed using Palbociclib, a potent and selective inhibitor of CDK4/6 which was recently approved by the FDA for the treatment of HR+/HER2− metastatic breast cancer. MTT assays were also carried out using BPTES which inhibits enzymatic activity by binding to the glutaminase tetramer, stabilizing it in an inactive conformation. Cell viability assays show that treatment of MCL cells with Palbociclib resulted in a dose dependent reduction in cell viability. Treatment with 5μM BPTES alone resulted in a slight reduction in cell viability. However, treatment with 5μM BPTES sensitizes cells to treatment with different concentrations of Palbociclib. Studies are ongoing using different concentrations of BPTES with a series of concentrations of Palbociclib to determine the synergistic effects of the combination treatment. As studies continue, quantification of synergy will be relayed using the Chou‐Talalay method with computerized simulation. Preliminary cell cycle assays using flow cytometry show that treatment of cells with Palbociclib induce G1‐phase cell cycle arrest. Establishing a treatment regimen that achieves a synergistic therapeutic effect may allow for integration of a novel molecular targeted approach to treating MCL.

Support or Funding Information

Butler University Holcomb research award, AFPE Gateway to Research award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term Somatic Hypoalgesia as a Marker of Pathological Conditions in Gastrointestinal Tract
Authors: Filaretova Ludmila , Punin Yuriy , Sudalina Maria , Yarushkina Natalia
Published: June 2021

Pain is traditionally considered as an evolutionarily developed defensive response to noxious stimulus. Short‐term pain suppression in response to stressful factors (stress‐induced analgesia) is an important adaptive reaction that is required for survival if further injury or death were threatened. In contrast, long‐term pain suppression may be considered as a sign of pain abnormality induced by disturbance of endogenous pain mechanisms under circumstances of pathology. Indomethacin (IM) is commonly used in clinic; however, it has a side ulcerogenic effect on the gastrointestinal tract. Previously we have shown that the formation IM‐induced injury in gastrointestinal tract is accompanied by a decrease in somatic pain sensitivity lasting, a least, for 72 h after IM injection (long‐term somatic hypoalgesia). Here we tested hypothesis that suppression of pathological process in gastrointestinal tract may prevent somatic hypoalgesia and normalize pain regulation in conscious rats. For this we studied effects of “stress preconditioning” on the gastrointestinal tract and somatic pain sensitivity under circumstances of ulcerogenic action of IM (35 mg/kg, sc). Preliminary (24 h) fasted rats were subjected one of four exposures (“stress preconditioning”) before IM administration: a) cold restraint (30 min, t=10 degree C, 1 h before IM); b) CRF (1.25 mkg/kg, ip, 30 min before IM) administration; c) capsaicin (0.5/kg, sc, 1 h before IM) administration, d) forced treadmill running (30 min, 1 h before IM). Pain sensitivity was assessed by tail flick latencies (tail flick test) or number of pain responses induced by acetic acid (“writhing test”). IM administration caused the formation of gastric erosion (2–4 h after injection) and small intestine injury (24, 48, 72 h after injection) that was accompanied by an increase in tail flick latencies (long‐term somatic hypoalgesia). Somatic hypoalgesia 48 h after IM injection was also supported by a decrease in number of pain responses induced by acetic acid. IM‐induced pathological process in the gastrointestinal tract was followed by changes in plasma corticosterone levels, cardiovascular (heart rate, blood pressure) and somatic (weights of thymus and adrenals, body weight) parameters. Each preconditioning exposure attenuated IM‐induced gastric mucosa injury and prevented somatic hypoalgesia. The results obtained suggest that long‐term somatic hypoalgesia may be one of characteristics of pathological process in the gastrointestinal tract induced by ulcerogenic action of IM.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) №14‐15‐00790.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circadian Disruption Accelerates Atherosclerosis in ApolipoproteinE‐deficient Mice
Authors: Pendergast Julie S. , Chalfant Jeffrey M. , Howatt Deborah A. , McGann Eric G.
Published: June 2021

The circadian system is a network of molecular clocks located throughout the body. These clocks coordinate daily rhythms of behavior and physiology with environmental cycles. Shift work, which chronically disrupts circadian rhythms, increases the risk of developing cardiovascular diseases. The mechanisms by which this happens, however, are largely unknown. Our long‐term goal is to determine how disruption of circadian rhythms causes cardiovascular diseases. Atherosclerosis is the build‐up of plaques in arteries and can lead to myocardial infarction and stroke. In this study, we sought to investigate whether circadian disruption accelerates atherosclerosis in mice. Wild‐type mice do not develop atherosclerotic lesions. Therefore, we studied C57BL/6J ApolipoproteinE‐deficient (ApoE−/−) mice because they spontaneously develop atherosclerotic lesions. We first characterized circadian rhythms in ApoE−/− mice. We found that circadian behavioral rhythms, including free‐running periods of activity in constant darkness and constant light, phase angles of entrainment, and phase shifts to light pulses, were similar between wild‐type and ApoE−/− mice. These data show that ApoE−/− mice had no deficits in their circadian behavior rhythms or light responsiveness. Next, we determined if molecular circadian rhythms in tissues were altered in ApoE−/− mice. We analyzed the expression of the circadian timekeeping protein, PERIOD2, in central and peripheral tissues using a luciferase reporter. We found that PERIOD2::LUCIFERASE rhythms in tissues were similar in wild‐type and ApoE−/− mice. We next sought to determine the effect of circadian disruption on atherosclerosis in ApoE−/− mice. Housing mice in constant light profoundly disrupted locomotor activity rhythms in ApoE−/− mice. We also found that atherosclerotic lesion areas were increased in ApoE−/− mice housed in constant light for 3 months compared to those in control 12h light‐12h dark. Together, these data demonstrate that ApoE−/− mice have normal circadian rhythms and chronic circadian disruption accelerates atherosclerosis in ApoE−/− mice.

Support or Funding Information

This study was funded by National Institutes of Health grant P20GM103527, the Gertude F. Ribble Trust, and the University of Kentucky.

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 Impact of Primary Myoblast Transplantation on Functional Vasodilation Following Arteriogenesis in Mice with Diet‐Induced Obesity
Authors: Tran Christopher Minh , Hamzeinejad Vahid , Cardinal Trevor Ryan
Published: June 2021

Peripheral arterial occlusive disease (PAOD) is characterized by reduced blood flow to the lower limbs, causing claudication and tissue loss. Proximal collateral arteries are natural bypasses to atherosclerotic occlusions in major arteries. A variety of cell‐based therapies have been tested clinically to enhance collateral arteriogenesis. Unfortunately, their efficacy has been poor, despite promising results from early clinical and preclinical studies, possibly because most preclinical studies are performed with mice that lack co‐morbidities present in most patients that are known to impair arteriogenesis. Therefore, additional cell‐types must be explored in animal models that more closely recapitulate the clinical phenotype to determine if pro‐regenerative therapies are a viable treatment for PAOD. Myogenic cells are an attractive candidate, given their co‐localization with the skeletal muscle vasculature niche and their ability to enhance angiogenesis. Therefore, we tested the hypothesis that primary myoblasts would rescue functional vasodilation in the superficial gracilis collateral following arteriogenesis in mice with diet‐induced obesity (DIO).

In these studies, C57Bl/6 mice were fed a high‐fat diet from 6‐weeks of age; experiments were performed at about 4‐months of age. First, we performed a pilot study to evaluate functional vasodilation (vasodilation in response to electrically‐stimulated gracilis muscle contraction) in unoperated animals. As expected, vasodilation was impaired in the right (42±4 vs. 58±4 μm) and left (40±5 vs. 52±5 μm), as compared to lean controls.

Having established impaired vasodilation in mice with DIO, we ligated the femoral artery in mice to model obesity and the chronic ischemia aspect of PAOD and measured functional vasodilation at day‐7 following surgery. Pilot data suggests that functional vasodilation is reduced in mice with DIO at day‐7 following surgery (resting: 80±10 μm, dilated: 82±8 μm) and that cell transplantation appears to enhance arteriogenesis and improve functional vasodilation (resting: 93±8 μm, dilated: 108±8 μm). Additional experiments are necessary to confirm these findings, but the data suggest that myoblasts strengthen the arteriogenic response. Future studies may involve additional time points to investigate the time course and duration of myoblast‐rescued arteriogenesis and functional vasodilation following arterial occlusion.

Support or Funding Information

Research was funded by the California Institute of Regenerative Medicine.

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 effect of docosahexaenoic acid on macrophage polarization
Authors: Ding Shih‐Torng , Peng Yu‐Ju , Chen Yu‐Shan
Published: June 2021

Macrophages are one of the most important immune cells in the innate immune system. The native monocytes (M0) differentiate into different forms of activated macrophage depending on the cytokines in the microenvironment. The activated macrophages include classic/M1 macrophages and alternative/M2 or regulatory macrophages (Mreg). Docosahexaenoic acid (DHA), a long chain n‐3 polyunsaturated fatty acid is a common dietary supplement which against numerous diseases. In vitro study, we pre‐treated native THP‐1 cells with DHA and induced polarization by PMA. In THP‐1‐derived macrophages, pre‐treatment of DHA reduced the expression of M1 and increased M2/Mreg markers. In vivo study, oral administration of DHA decreased M1 and increased M2‐like macrophages in mice. Moreover, DHA decreased M1‐stimulating cytokines (IL‐12 and IFN‐γ) and increased M2/Mreg‐stimulating cytokines (IL‐4 and IL‐10) in blood. In conclusion, our results showed that DHA inhibited M1 and induced M2 macrophage polarization in THP‐1 cells and mice.

Support or Funding Information

The researcht was supported by Ministry of Science and Technology, 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.
Human Placental Stem Cell Therapy in Stroke: Endothelial/Smooth Muscle Mechanisms Underlying Protection
Authors: Barzegar Mansoureh , Minagar Alireza , Wang Yuping , Yun Winny Jungmi , Boyer Christen , Cananzi Sergio Giuseppe , Alexander Steven Jonathan
Published: June 2021

Following thrombolytic therapy in stroke, ischemia/reperfusion (I/R) mediated inflammation impairs maintenance of blood brain barrier (BBB), enhance expression of endothelial and leukocyte adhesion molecules and perturbs normal blood flow regulation. Currently, benefits of stem cell therapy (SCT) in stroke are thought to reflect secretion of protective and trophic factors which limit acute brain I/R injury. We studied human placenta mesenchymal stem cells (hPMSC) as a potent, high‐yield and inexpensive tool in SCT for human stroke therapy. We found that acute intravenous hPMSC administration significantly reduced infarct size (p<0.05) at 24h in our acute (1h) model of middle cerebral artery occlusion (MCAO) in rats. Our hypothesis was that acute benefits of hPMSCs in stroke might reflect suppression of post‐I/R immune, inflammatory and BBB disturbances which limit the onset and progression of stroke injury following restoration of blood flow. We first examined hPMSC support of BBB via mobilization of junctional proteins in human brain endothelial cells (hCMEC‐D3) in conjunction with FITC‐Dextran and FITC‐biotin‐gelatin avidin permeability assays plus western blotting and immunolocalization of junctional proteins. We also assessed the ability of hPMSC to suppress inflammation driven hCMEC‐D3 binding of macrophages (RAW246.7). Lastly we studied the influence of hPMSC on contractility of human brain vascular smooth muscle cells (hBVSMC) in collagen gel contraction assays.

hPMSC significantly (p<0.001) enhanced barrier integrity at 24h in oxygen/glucose deprived/reperfused (OGDR) brain endothelial monolayers (in both FITC‐dextran and FITC‐biotin assays. We found that hCMEC‐D3 which were contact‐‐independently co‐cultured with hPMSCs expressed significantly higher levels of VE‐cadherin and α‐catenin under OGDR conditions (p<0.001); surprisingly, we did not see significant increases in occludin or claudin‐5 expression. Additionally, the spatial localization of VE‐cadherin and α‐catenin at hCMEC‐D3 junctions was apparently increased under OGDR, but not control conditions after hPMSC treatment. We also saw that hPMSC significantly (p<0.0001) reduced binding of macrophages to activated brain endothelial monolayers. Lastly, we found that hBVSMC contraction was significantly increased by hPMSC (p=0.0001), an effect which was reversed by Angiotensin II Type 1 receptor inhibition (Losartan, 10 uM) (p=0.004), but not by thromboxane A2 or endothelin‐1 blockade.

We conclude that hPMSC release stable factors which beneficially and acutely influence endothelial barrier integrity, interactions with immune cells and vasoregulation to improve recovery in stroke.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lymphatic Wall Remodeling with Systemic and Tissue‐Associated Inflammation in Obese Zucker Rats
Authors: Trujillo Andrea N. , Breslin Jerome W.
Published: June 2021

Lymphatic vessels have an intimate relationship with adipose tissue, and a growing number of reports tie dysfunction of lymphatics to obesity. A large gap currently exists in the knowledge of the pathophysiological mechanisms involved in obesity‐induced lymphatic dysfunction. The goal of this study was to investigate the extent to which obesity gives rise to inflammation and remodeling of the wall of rat mesenteric collecting lymphatic vessels. ZUCKER‐Leprafa (obese) and ZUCKER‐Leprafa/+ (lean) male and/or female rats, in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education, were used for this study. Age groups included 8, 12 and 26–32 wks. Collecting lymphatic vessels were isolated from the mesentery and were mounted onto resistance‐matched glass micropipettes to determine passive wall mechanics. These experiments were performed in a 37°C albumin physiological salt solution (APSS without Ca2+) bath with luminal pressure stepped from 1–5 cm H2O. In addition, mesenteric arcades with adipose tissue and collecting lymphatics were dissected, fixed in 4% paraformaldehyde, processed and embedded in paraffin for sectioning and immunostaining with antibodies to rat‐specific CD68 (macrophage marker) and caveolin‐1 (adipocyte marker) for the quantification of crown‐like structures (CLSs). Blood (serum) and peri‐lymphatic adipose tissue (PLAT) were also collected and analyzed with the currently available panel of 67 inflammatory biomarkers through the Rat Biomarker Testing Service by RayBiotech. The results show that lymphatic wall thickness is significantly greater in obese Zucker rats compared to age‐matched counterparts. Wall:lumin ratio (X100) and medial cross‐sectional area were also significantly greater in obese versus lean male rats at all ages. Many CLSs were observed surrounding mesenteric collecting lymphatics isolated from obese male 12 and 26–32 wk rats. Additionally, CLSs significantly increased with age. In an analyses of serum biomarkers obtained from 12‐wk old lean and obese, male and female rats, we observed that many of the markers in PLAT (IL‐6, IL‐13, adiponectin) and in serum (GM‐CSF, IL‐4, Gas1) were differentially regulated by obesity and sex. The data support the concept that obesity causes an inflammatory environment in the mesentery that is associated with lymphatic vessel wall remodeling and changes to passive wall mechanics. The data also show significant differences in the profiles of inflammatory mediators increased by obesity in male and female Zucker rats, highlighting the importance of studying both sexes.

Support or Funding Information

Supported by NIH L40HL097863

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 coronary vascular function in a mouse model of Hypertrophic Cardiomyopathy
Authors: Muller‐Delp Judy Marie , Maraj Joshua J. , Huke Sabine
Published: June 2021

Mice with the troponin T (TnT)‐I79N mutation, which is associated with hypertrophic cardiomyopathy in humans, develop arrhythmias during stress despite the absence of an overt anatomical substrate. The arrhythmias are linked to the occurrence of energy deprived regions in the ventricular myocardium, but the underlying mechanism is not completely understood. Preliminary data indicate that vascular dysfunction that results in ischemia is at least in part responsible for the regional energy deprivation. In this study we are investigated whether cardiac microvascular function is altered in TnT‐I79N mice.

We isolated resistance arterioles from the hearts of troponinTnT‐I79N mice and non‐transgenic mice, and evaluated vasodilatory responses to endothelium‐dependent (flow) and endothelium‐independent (DeaNONOate) stimuli. Contractile responses to pressure (myogenic responsiveness) and endothelin were also assessed. Flow‐induced vasodilation was significantly increased in coronary arterioles from TnT‐I79N mice (maximal dilation 81±9%) as compared to coronary arterioles from non‐transgenic mice (maximal dilation 58±9%). Endothelium‐independent responses to DeaNONOate were not altered in coronary arterioles from TnT‐I79N mice. Contractile responses to endothelin were significantly reduced in arterioles from TnT‐I79N mice (44±6% in TnT‐I79N arterioles vs. 64±5% in non‐transgenic arterioles; P<0.01). Myogenic responses to increasing transmural pressure were not altered in arterioles from TnT‐I79N mice.

Altogether, these data indicate heightened responsiveness of the endothelium of coronary resistance arterioles in the hearts of TnT‐I79N mice. Since the mutant protein is exclusively expressed in the cardiomyocytes, this indicates an indirect effect of the cardiomyocytes on the vasculature.

Support or Funding Information

NIH R01 to Sabine Huke

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