bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examination of downstream targets of the FKH‐8 transcription factor in dopaminergic neurons
Authors: Anthony Austin , Nelms Brian 
Published: June 2021

The transcription factor forkhead‐8 (FKH‐8) is enriched in dopamine (DA) neurons and is required for DA neuron function. Disruption of the fkh‐8 gene mimics the swimming‐induced paralysis (SWIP) phenotype characteristic of loss of the dopamine transporter (DAT). DAT is responsible for the reuptake of excess dopamine from the synaptic cleft after its pre‐synaptic release, and loss of DAT function results in excess dopamine in the synaptic cleft and spillover into extra‐synaptic areas. This spillover of dopamine results in the hyperdopaminergic state seen via the SWIP phenotype.However, neither the expression nor function of DAT‐1 are altered in fkh‐8 mutants compared to wildtype worms, and FKH‐8 does not seem to impact genes in the dopamine synthesis pathway. While FKH‐8 has been shown to regulate important DA neuron functions through phenotypic assays, downstream genes have yet to be identified. Because FKH‐8 acts independently of the well‐studied DAT‐1 transporter, it is likely that its targets will include a number of novel genes whose impact on dopamine signaling have yet to be revealed.Cell‐specific RNA sequencing comparing gene expression in dopamine neurons isolated from wildtype vs. mutant organisms will identify what genes are important in dopamine signaling in the model organism Caenorhabditis elegans. More specifically, it will reveal targets and gene expression patterns that are altered by loss of FKH‐8. in dopamine neurons.We anticipate that using this unbiased approach to identify novel genes critical for these functional outcomes in C. elegans will provide fundamental new biological insights as well as potential novel molecular targets for therapeutic intervention.Support or Funding InformationNSF CREST BioSS Center Grant HRD15‐47757This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Role of Cyclin Dependent Kinase 5 (Cdk5) in Insulin Related Neurodegenerative Disease
Authors: Tran Brandon , Aguanno Ann 
Published: June 2021

Cyclin Dependent Kinase 5 (Cdk5) is a member of the cyclin dependent kinase family of proteins that play key roles in the regulation of the cell cycle. The activity of these enzymes is dependent upon association with a cyclin activating partner. Unlike other members of this family, however, Cdk5 appears to play no role in cell cycle regulation. Cdk5 is ubiquitously expressed in all cells. However maximum kinase activity is found in post‐mitotic neurons and glial cells, where Cdk5's cyclin activating partners exhibit high expression. Known substrates of Cdk5 include proteins that modulate cell morphology and motility. Our lab has shown that Cdk5 plays an essential role in the developing nervous system primarily through studies of chemical inhibition of Cdk5 in a neuronal model. Specifically, we employed the well‐known PC12 cell line which can be induced to transdifferentiate into neuronal‐like cells. Inhibition of Cdk5 during this process reduced the neuronal characteristics these cells typically exhibit, mimicking neuronal degradation or degeneration. Multiple published studies support Cdk5's role in the developing mammalian nervous system. Recent investigations have shown an increased correlation between the likelihood of developing neurodegenerative diseases and insulin resistance associated with Type 2 diabetes. Insulin resistance results in the over‐secretion of insulin (hyperinsulinemia) into the blood stream and causes a down regulation of insulin receptors in peripheral tissue. Although acute hyperinsulinemia in the central nervous system (CNS) may have cognitive enhancing effects, chronic hyperinsulinemia appears to be neurotoxic. We are exploring this potential relationship between insulin levels and the neuron by exploiting our neuronal model cell line, PC12. Using this model, we examine the role of normal and aberrant Cdk5 activity in the response of differentiated neuronal‐like PC12 cells to varying concentrations of insulin in culture. Our aim is to test the reports of deregulation of brain insulin signaling as an early marker in AD. Here we show our preliminary results of the transdifferentiation of PC12 cells while Cdk5 activity is manipulated under hyperinsulinemic conditions. Our findings underscore the value of this model as a means to examine the potential role of Cdk5 as a link between AD and Type 2 diabetes. Given the increased Type 2 Diabetes diagnosis and the aging of American society implications for this study are self‐evident.Support or Funding InformationThe Rose M. Badgely Charitable Trust, Joseph Alexander Foundation, Sister Colette Mahoney Science Research Grant and the Marymount Manhattan College Faculty Award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measuring T‐cell avidity and enrichment using an acoustic force‐based technology
Authors: Scheper Wouter , Merino‐Rodriguez Elena , Braster Rens , Sitters Gerrit , Oswald Felix , Schumacher Ton , Candelli Andrea 
Published: June 2021

The key driver for effective immunotherapies is the overall strength binding of the immune cell and the target cell(e.g. tumor cells). The overall strength is known as ‘avidity’, a parameter reflecting interaction efficiency. One of the main obstacles has been the generation of immunotherapies promoting effective and long lasting immune responses, due to the lack of tools measuring this parameter. Here, we have made used of acoustic forces as a novel method to isolate immune cells based on their avidity to specific targets, such as (tumor) cells. The force that cell target need to experience to be separated is called ‘rupture force’. In this study, we were able to identify the rupture force of tumor specific and non specific T cells and separate these different populations applying controllable acoustic forces. T cells engineered with a melanoma antigen recognizing T cell receptor needed 6 times more relative force than the control, nonspecific T cells, to be separate from their melanoma cells. These findings indicate that the specific melanoma T cells bind with higher efficiency and higher avidity than the nonspecific ones, and that they can be separated with this method. Furthermore, 1.4 to 3.6‐fold enrichment of high avidity T cells from a mixed specific:nonspecific (1:10) T cell population was obtained after their isolation using acoustic forces and confirmed using FACS measurements. Therefore, we demonstrate here that acoustic forces can be used as a tool to measure avidity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancing the anti‐tumor efficacy of chimeric antigen receptor‐expressing T cells with naturally occurring plant stilbenes
Authors: Soles Andrea , Richardson Elle , Barber Amorette 
Published: June 2021

Despite advances in treatment options, cancer remains the second leading cause of death in the United States. Many therapies use immune cells, specifically T cells, for cancer therapy. To enhance tumor recognition by T cells, chimeric antigen receptors consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Our lab has genetically engineered a chimeric PD1 (chPD1) receptor and has shown that chPD1‐expressing T cells kill tumor cells, secrete inflammatory cytokines, reduce tumor burden, and increase survival in multiple tumor types. A current focus of cancer research is the development of combination therapies that enhance anti‐tumor efficacy without increasing harmful side effects. Recently, a family of naturally occurring plant compounds called stilbenes have been shown to enhance T cell function. Therefore, we tested if stilbenes enhanced chPD1 T cell function in breast cancer. Addition of resveratrol, a stilbene found in berries, grapes, peanuts, and other plants, decreased proliferation, enhanced tumor cell killing, and increased secretion of proinflammatory cytokines (IFNγ, GM‐CSF, TNFα, IL‐17, IL‐2, and IL‐21) from chPD1 T cells. Presence of resveratrol also increased skewing of T cell differentiation to a central memory phenotype through reduction of AKT and mTORc1 signaling, reduced expression of T‐bet and BLIMP‐1 transcription factors, and increased expression of Bcl‐6 and Eomes transcription factors. Hence, combination of resveratrol and chimeric antigen receptor‐expressing T cells may enhance anti‐tumor immune responses through inhibition of the AKT and mTORc1 pathways and skewing to a long‐lived central memory phenotype.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Higher‐Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling
Authors: Fu Tianmin , Pan Liqiang , Zhao linlin , Wu Hao , Chou James 
Published: June 2021

Receptor clustering on cell membrane plays important roles in the signaling of many immunoreceptors and this mechanism has previously been attributed to the extracellular and/or the intracellular interactions. Here, we report an unexpected finding that for death receptor 5 (DR5), a Type‐1 receptor in the tumor necrosis factor receptor (TNFR) superfamily, the single‐pass transmembrane helix (TMH) alone in the receptor directly assembles a defined higher‐order structure to drive signaling, and that this higher‐order structure is in turn inhibited by the unliganded extracellular domain. Nuclear magnetic resonance structure of the DR5 TMH in bicelles shows distinct faces of the TMH that mediate trimeric and dimeric interactions, allowing formation of dimer‐trimer interaction networks on membrane. Single TMH mutations that disrupt either trimerization or dimerization, as well as double mutations that disrupt both all abolish ligand‐induced receptor activation. Surprisingly, proteolytic removal of the DR5 extracellular domain, which releases the TMH from physical restraints of the pre‐ligand receptor association, can fully activate downstream signaling in the absence of ligand binding. A dimer‐disruptive, but not trimer‐disruptive mutant, decreases DR5 pre‐ligand association, demonstrating dimerization of DR5 before ligand engagement. Collectively, our data suggest a novel model of receptor signaling in which TMH clustering alone can drive DR5 signal transduction, and binding of ligands or agonistic antibodies at the extracellular domain helps to overcome the pre‐ligand autoinhibition.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyphenon E Alters Protein Levels in PC‐3 Cells
Authors: Couming Chris , Carastro L. Michael 
Published: June 2021

Prostate cancer (PCa) is the most commonly diagnosed cancer in American men. Polyphenon E (Poly E) is a caffeine‐free standardized green tea extract reported to have chemopreventative activity. However, the mechanisms through which Poly E exhibits chemopreventative activity have not yet been fully elucidated. Our working hypothesis was that Poly E alters expression of specific genes to inhibit PCa cell proliferation. We previously conducted DNA microarray and qRT‐PCR analyses of RNA samples isolated from PCa cells (PC‐3) treated with varying levels of Poly E. The qRT‐PCR analysis assessed CASP‐8 and ATR expression changed not more than 2‐fold under Poly E treatments. The aim of the current study was to determine protein levels by Western blotting. PC‐3 cells were treated with Poly E (0 mg/L, 100mg/L, 200mg/L, and 300mg/L) for 24 hours. Protein was extracted and Western blot analysis was performed for ATR and CASP‐8 proteins. Results from Western Blot analysis for ATR indicated increased protein expression (1.43‐fold) at the 200mg/L Poly‐E treatment level compared to the untreated control, but were essentially unchanged at the 100mg/L and 300mg/L Poly E treatments (0.99‐fold and 0.91‐fold, respectively). These data were consistent with observed qRT‐PCR data. Western blot analysis for CASP‐8 indicated decreased protein expression levels of 0.36‐fold, 0.40‐fold and 0.38‐fold at the 100, 200, and 300 mg/L Poly E treatment levels, respectively. This result exhibited a trend similar to our CASP‐8 qRT‐PCR, but exhibited greater degree of decreases.Support or Funding InformationThis work was supported by a University of Tampa David Delo Research Professor Grant (to L.M.C).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence that endurance exercise improves cardiac function in POLG mice
Authors: Hill Blake , Fu Vivian , Ugras George , Prolla Tomas 
Published: June 2021

Aging is caused by a variety of factors, but a common thread is impaired function in the organelle central to cellular metabolism and energy production: the mitochondrion. Each cell contains ~500–1000 mitochondria (depending on tissue) and each mitochondrion contains its own DNA (~2–10 copies). The mitochondrial DNA (mtDNA) accumulate mutations with age in several species including humans and it has long been suggested that mitochondria are central to human aging. Highlighting the crucial role of mitochondria to aging, a genetic lesion that increases mtDNA damage causes premature aging. This lesion resulted in a residue substitution (D257A) in the highly conserved exonuclease domain of the sole mammalian mtDNA polymerase, POLG, impairing its proofreading ability. Homozygous knock‐in mice with the POLG‐D257A substitution (POLG mice) show a profound aging syndrome with reduced life span and premature aging including weight loss, decreased subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, and cardiac dysfunction. Recently endurance exercise was shown to reverse this aging syndrome. Here the exercise protocol was repeated and followed by measurements of blood and cardiac function. Aging in POLG mice was associated with reduced levels of hemoglobin and white blood cells. Endurance exercise failed to prevent age‐related changes. By contrast, many cardiac parameters as measured by high‐resolution echocardiography showed statistically significant differences between young and old sedentary POLG mice. Statistically significant differences were also found between old POLG mice that were either sedentary or exercised. The results indicate that exercise improves some measures of cardiac dysfunction in POLG mice and suggest that interventions may exist to overcome the deleterious effects of mtDNA damage.Support or Funding InformationThis study was funded by a grant from Breakout Labs to CyteGen Corp.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of SNF1 Gene on Glycolytic Flux Addressed to Aerobic Fermentation (Crabtree Effect) of Saccharomyces cerevisiae
Authors: Carrillo Andres , Dufoo‐Hurtado Miguel David , Madrigal‐Perez Luis Alberto 
Published: June 2021

The alcoholic fermentation is a biotechnological process, which has been employed for more than one hundred years. Despite the ample researches carry out about this, there are specific aspects that are still not fully understood, an example of this is the Crabtree effect. The Crabtree effect is a metabolic phenomenon that occurs when the concentration of fermentable sugars in the medium is higher than 0.015% (w/v) and is characterized by the increase of glycolytic flux, an augment in the production of metabolites of fermentation and respiration repression. In spite of the wide quantity of information on the Crabtree effect is still not clear the molecular basis by which originates. It has been proposed that sucrose non‐fermenting protein‐1 (Snf1p) plays a key role in the unleashing of the Crabtree effect. Since in high concentrations of glucose, Snf1p is found dephosphorylated; while in low concentrations of glucose the protein Snf1p is phosphorylated and participate in the de‐repression of genes repressed by glucose. However, it has been suggested that both Snf1p phosphorylated and dephosphorylated have an essential participation in the metabolic transition of fermentative metabolism to the respiratory metabolism. This study aimed to evaluate the influence of the deletion of SNF1 gene in the glycolytic flux addressed to the Crabtree effect of S. cerevisiae. For this purpose, we use the Saccharomyces cerevisiae genetic background BY4742 and its mutant in the gene SNF1 (snf1Δ). Here, we provide evidence that snf1Δ mutants grown in YPD medium supplemented with 10% glucose did not present any effect upon glycolytic flux. In this regard, NADH/NAD+ ratio, extracellular acidification rate and transcription levels of genes HXK2 and PFK1 did not show a significative difference between strains snf1Δ and wild‐type. Surprisingly, the strain with the deletion in the gen SNF1 exhibits an increased oxygen consumption in comparison with wild‐type. The results obtained in this study suggest by first time that even when Snf1p is found dephosphorylated in high concentrations of glucose could be necessary for carrying out the repression of the respiration.Support or Funding InformationPRODEP partially funded this work (project number: ITESCH‐EXB‐002).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Importance of Hydrophobic Bulk at the Rotor‐Stator Interface of E. coli ATP Synthase
Authors: Dodd Michaela , Pressley Benjamin , Steed Ryan 
Published: June 2021

As the primary producer of adenosine triphosphate (ATP), the chemical energy currency of cells, F1Fo ATP synthase is essential for life. Using the H+ electrochemical gradient, the membrane‐embedded Fo complex generates torque on the cytoplasmic F1 complex, which synthesizes ATP from ADP and phosphate. While much is known about the mechanism of ATP synthesis, aspects of Fo functionality, including the roles of interactions between subunit a (stator) and the c10 ring (rotor), remain unresolved. In E. coli, Phe54 and Ile55 on subunit c have been found in a previous study to be important for H+ movement through Fo during ATP‐driven H+ pumping. We show here that cF54C and cI55C mutations cannot pump or passively translocate protons through Fo. ATP‐driven H+ pumping activity was partially restored by systematically adding steric bulk to Cys54 through chemical modifications with methanethiosulfonate derivatives of increasing size; however, similar restoration did not occur for Cys55. Additionally, we used site‐directed mutagenesis to replace Phe54 or Ile55 with hydrophobic amino acids of various size, all of which supported H+ pumping activity. While the chemical modification of Cys54 suggested that hydrophobic bulk at this position is important for H+ pumping, the chemical properties required at these positions remain unclear.Support or Funding InformationSupported by the North Carolina GlaxoSmithKline Foundation and the University of North Carolina Asheville Undergraduate Research ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of the SNF1 Deletion in the Glycolytic Pathway of Saccharomyces cerevisiae Grown at 1% Glucose
Authors: Martinez‐Ortiz Cecilia , Dufoo‐Hurtado Miguel David , Madrigal‐Perez Luis Alberto 
Published: June 2021

The alcoholic fermentation is an amphibolic process that allows the formation of ATP at substrate‐level, keeps active the glycolysis through re‐oxidation of NADH, and generate metabolites of biotechnological interest. Saccharomyces cerevisiae is a positive Crabtree, which performs the alcoholic fermentation in oxygen presence, increasing the glycolytic flux accompanied by repression of respiration. However, the molecular mechanism of the Crabtree effect is still not clear. The sucrose non‐fermenting protein‐1 (Snf1p) is a cellular energy sensor that modulates the switch between respiration and alcoholic fermentation. Therefore, Snf1p could be a key piece in the molecular puzzle of the alcoholic fermentation related to the Crabtree effect. For this reason, this work aimed to determine whether the deletion in SNF1 gene affects the alcoholic fermentation at 1% glucose. For this purpose, the BY4742 genetic background was used and its mutant in the SNF1 gene. In this way, it was found: 1) that the absence of SNF1 gene affects growth. 2) A decrease in extracellular acidification rate in SNF1 mutant. 3) NADH/NAD ratio does not have any change in the strains. However, the amount NAD(P)H showed an increase in SNF1 mutant. 4) Transcription levels of HXK2 gene displayed an increase in SNF1 mutant; while transcription levels of PFK1 gene decrease. 5) The basal respiration and the maximum respiratory capacity diminished in SNF1 mutant. All data suggest that deletion in SNF1 gene favors the formation of the anabolic species NADPH, probably by a transcriptional regulation of glycolysis, not observing an effect in the alcoholic fermentation.Support or Funding InformationPRODEP partially funded this work (project number: ITESCH‐EXB‐002).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The A2B Adenosine Receptor Antagonist Alloxazine Exacerbates NASH and Disrupts Cellular Folate Metabolism in Methionine and Choline Deficient Mice
Authors: Eudy Brandon James , Silva Robin da 
Published: June 2021

Nonalcoholic fatty liver disease is characterized by hepatic lipid accumulation of greater than 5% and is a rising healthcare problem in the United States. Approximately 25–30% of the US population may have a fatty liver and 10–15% of these patients will progress to more severe disease known as nonalcoholic steatohepatitis (NASH). There are no approved treatments for NASH and the factors that result in the transition from fatty liver to NASH are not well defined. Folate and one‐carbon metabolism are implicated in the pathogenesis of NASH and are important pathways for the biosynthesis of many important molecules like creatine, phosphatidylcholine, purines, and thymidine. Additionally, the purine adenosine modulates immune cells by signaling to cell surface receptors on macrophages in the liver. In the present study, mice were fed a methionine and choline deficient diet to induce NASH lesions and treated with a putative A2B adenosine receptor antagonist, alloxazine, to probe the role of this adenosine receptor in NASH. MCD diet feeding reduced purine and thymidine nucleotides in the liver; potentially limiting the availability of crucial molecules for regeneration of liver tissue. MCD mice showed reduced activating transcription factor 4 (ATF4) expression, which has been shown to regulate mitochondrial folate metabolism and formate production which is required for purine biosynthesis. Alloxazine treatment exacerbated hepatic lipid accumulation and caused severe lobular inflammation in MCD mice. Alloxazine also disrupted hepatic folate metabolism and reduced the concentration of formate without altering concentrations of S‐adenosylmethionine and S‐adenosylhomocysteine. These data suggest that in addition to its function as an A2B adenosine receptor antagonist, alloxazine also impairs cellular folate metabolism. These data provide a novel link between folate and lipid metabolism, and inflammation in NASH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.