bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reprogramming Cancer Cell Metabolism through the Combinatorial Action of PK11195 and 5‐Azacytidine
Authors: Duran Daniel , Steinsaltz Matthew , Anstett Sarah , Custance Michaeal , O'Donnell Robert 
Published: June 2021

Deregulated cellular energetics is a widely observed phenotype across numerous cancer cell lines, making it a logical target for cancer therapies. This aberrant metabolism commonly manifests as the “Warburg Effect,” wherein fractional pyruvate oxidation results from a tumor cell's over‐reliance on glycolysis. One of the simplest molecular explanations for this effect is the loss of a multisubunit mitochondrial pyruvate carrier (MPC) that shuttles pyruvate into the mitochondria. The MPC‐1 and MPC‐2 genes code for distinct subunits of this carrier protein and are commonly silenced in cancer cell genomes. In this investigation, we use a DNA methyltransferase inhibitor, 5‐Azacytidine, in combination with a constitutive androstane receptor (CAR) inhibitor, PK‐11195, to re‐express the coregulated MPC subunit genes. Through reverse transcription PCR, we demonstrate that the combinatorial action of these two drugs in the HTB‐4 cell line results in a 6.36 and 3.54 fold increase for the MPC‐1 and MPC‐2 genes, respectively. Additionally, in the MDA‐MB‐435 cell line, the MPC‐1 and MPC‐2 genes were upregulated by a factor of 3.54 and 3.18, respectively. Preliminary western blot analysis on whole cell lysates reveals no distinction between the levels of the 12 kDa MPC‐1 subunit from treated and untreated samples. Currently, experiments are underway to test whether the treatment results in a more oxidative phenotype that is indicative of pyruvate oxidation within the mitochondria. Finally, PK‐11195 also binds a peripheral benzodiazepine receptor (PBR) on the surface of the outer mitochondrial membrane, subsequently limiting the rate of glycolysis through inhibition of hexokinase. Cytotoxicity assays reveal that while PK‐11195 introduces a glycolytic block on both cell lines, supplementation with fatty acids rescues them from the glycolytic stress. Collectively, our work demonstrates the significance of both restoring the mitochondrial pyruvate carrier, and working around other metabolic pathways, as a means of repressing the Warburg effect.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNMT3A mutations in AML patients cause loss and gain of function and differential regulation by protein partners
Authors: Sandoval jonathan Enrique 
Published: June 2021

Eukaryotic DNA methylation, an integral component of the epigenetic machinery, prevents genomic instability by regulating the expression of oncogenes and tumor suppressor genes. The importance of dysregulated DNA methylation in diverse blood cancers including Acute Myeloid Leukemia (AML) pathogenesis is highlighted by the strong correlation between mutations in the de novo DNA methyltransferase gene, DNMT3A, and adult patients with poor prognoses. We show that clinically observed DNMT3A mutations result in dramatic changes in enzyme activity, including mutations that lead to 6‐fold hypermethylation and 3‐fold hypomethylation on the p15 human promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN‐AML) constitutes 40–50% of all AML cases, is the most epigenetically diverse AML subtype and has pronounced changes in DNA methylation in non‐CpG regions. We identified a subset of mutations in DNMT3A that lead to a 2–8 fold enhancement in the enzyme's ability to perform non‐CpG methylation. Many of the mutations studied map to regions on the protein that are well known to interact with partner proteins, which themselves contribute to AML, such as the DNA base excision repair (BER) enzyme Thymine DNA glycosylase (TDG). Here we show that DNMT3A mutations cause significant and diverse changes in the ability of these regulatory partner proteins to affect DNMT3A function. Our results present a link between DNMT3A mutations and the disruption of the epigenetic landscape in AML.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ACE and AGTR1 are differentially methylated in a hypertensive versus normotensive cohort of Kenyans
Authors: Shelton Dimond A. , Rice Nancy A. 
Published: June 2021

Cardiovascular diseases (CVD) are emerging as the primary cause of global mortality and are disproportionately affecting low and middle‐income countries (LMICs). The leading risk factor for CVD is hypertension (HTN), a multifactorial disease with no single genetic cause. Epigenetic control, i.e. methylation, of blood pressure regulatory genes likely contributes to the etiology of this disease. From a rural population of Kenyans previously identified to have a high incidence of HTN, we investigated the cytosine‐phosphate‐guanine dinucleotide (CpG) methylation pattern of two promoter regions that regulate renin‐angiotensin aldosterone system (RAAS) gene expression. Specifically, angiotensin II receptor type 1 (AGTR1) and angiotensin‐converting enzyme (ACE) genes, that function in maintaining salt and water homeostasis, were analyzed. The promoter regions of AGTR‐1 and ACE contain 11 and 19 CpG islands, respectively, making them a prime target for methylation driven regulation. Briefly, genomic DNA was isolated from buccal cells isolated from either hypertensive or normotensive individuals using standard alkaline lysis. DNA was quantified, and subsequently unmethylated cytosines were converted to uracils by demethylation using bisulfite treatment. Gene‐specific CpG islands were amplified by PCR and sequenced. From preliminary results, ACE promoter methylation is increased by 10 % (50.4 ± 3 vs 60.1 ± 4; p <0.05) and AGTR1 promoter methylation is decreased by 8 % (61.0 ± 8.2 vs 53.8 ± 4.0; p, 0.05) in hypertensive individuals versus normotensive, respectively. Specifically, the 18th CpG dinucleotide in ACE was unmethylated in all samples isolated from normotensive or prehypertensive individuals, yet was methylated in the majority of samples isolated from participants with elevated blood pressure. These results may suggest a role for this dinucleotide in regulating ACE gene expression in endothelial dysfunction.Support or Funding InformationThis work was funded by NIH‐NCRR P20 RR16481 (NAR) as well as support from the Kentucky Academy of Sciences (NAR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DNA Methylation of Multiple Genes involved in Bladder Cancer among Saudi Population
Authors: Siddiqui Amna , Yaqinuddin Ahmed , Alshehri Faizah A. 
Published: June 2021

ObjectiveTo identify bladder cancer specific methylated DNA sequences for Saudi population in order to detect and predict bladder cancer progression.MethodsIn this study, we analyzed DNA methylation levels of 94 tumor suppressor genes loci in 24 bladder tissues (19 bladder cancer samples and 5 control samples taken from histologically normal bladders). DNA Methylation analysis was done using Human Tumor Suppressor Genes EpiTect Methyl II Complete PCR Array from Qiagen TM.ResultsWe identified significant difference in DNA hypermethylation levels at APC, BRCA1, CDH1, CDH13, CDKN2A, DAPK1, ESR1, FHIT, MGMT, RASSF1, SOCS1, TIMP3, TP73, VHL, WIF1, E2F1, ERBB2, H1C1, OPCML, SFN, SFRP1, SFRP2, SPARC, and TERT between controls and cancerous samples. It was also observed that CADM1, DKK3 and SCGB3A1 were deferentially methylated in non‐muscle invasive versus muscle invasive bladder cancer samples. Additionally, DNA hypermethylation of ESR1 was notified as the novel tumor suppressor gene specific for Saudi population in bladder cancer.ConclusionOur findings suggest that these aberrant DNA methylation patterns in bladder cancer are disease and population specific and have a potential to develop as distinct DNA methylation based bio markers in future.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‐drug gene expression overlaps reveal potential targets for pharmaco‐epigenetic interventions in drug addiction
Authors: Padilla Adriana Mercedes , Ghezzi Alfredo Mercedes 
Published: June 2021

Sustained exposure to drugs leads to homeostatic adaptations in the brain brought on by responsive changes in gene regulation mechanics at synapses which modify neuronal circuitry. This genetic plasticity is involved in the persistence of symptoms of drug addiction even after extended periods of abstinence and give rise to behavioral phenotypes such as tolerance and dependence. After exposure to sedatives such as alcohol we have observed epigenetic modulations induced by ethanol exposure that activate the expression of genes producing an excitative state which opposes the drug's inhibitory effects. Congruently, addiction to opiate drugs such as heroin and fentanyl, or the effects of the noncompetitive NMDA antagonist ketamine, have also been found to be mediated by an epigenetic machinery. Although genes and possible mechanisms involved in the development of drug addiction have been uncovered, the use of an epigenetic reversible system as a potential pharmacologic treatment is still unknown. Here we use a combination of behavioral assays with the drosophila model system and genome‐wide analyses of gene expression to investigate the common molecular responses to the different drugs. We exposed groups of wild‐type flies to single sedating doses of vaporized ethanol, morphine or ketamine, and perform genome‐wide analyses of gene expression using RNA‐seq. Results from RNA‐sequencing experiments allow for cross‐analyses of the gene expression profiles of each addiction‐inducing drug. Identifying and mapping genetic overlaps specify regions in the genome that are potentially regulated during the adaptations underlying drug addiction. Commonalities in drug‐responsive genetic expression propose novel targets for pharmaco‐epigenetic interventions at molecularly modulated sites. Findings might further elucidate the intricacies that are at work within the flow of genetic plasticity indispensable for the development of addiction to drugs.Support or Funding InformationDepartment of Biology and NeuroID Program, University of Puerto Rico ‐ Rio Piedras, San Juan, PR 00931This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring Cytotoxic Effects In Non‐Cancerous Cells via Docetaxel
Authors: Couvertier Iyonna , Dhimolea Eugen , Mitsiades Constantine 
Published: June 2021

Therapeutic chemotherapy drugs are known to induce cytotoxicity in both non‐cancerous cells and in malignant cells. However, it is still unknown if the chemotherapy drug Docetaxel induces cytotoxic events in non‐cancerous cells. Our approach to measuring cytotoxic events was H&E staining (hematoxylin and eosin) followed by immunohistochemistry (IHC) with Estrogen Receptor α (ERα), Androgen Receptor α (ARα) or Ki‐67 antibodies to determine if the cytotoxicity occurred in cancerous or non‐cancerous cells. In the MCF7 breast cancer cell line, chemotherapy drug Docetaxel induced cytotoxic effects in 44.20% of malignant breast cancer cells and 34.86% in non‐malignant cells as classified according to their response to the ERα antibody with a standard deviation of 26.89%. Patient derived prostate cancer cells grown in mice generated from the MDA‐MB‐231 cell line were also treated with Docetaxel. An average of 28.3% of prostate cancer cells exhibited ARα monoclonal antibody sensitivity, and 28.3% of prostate cancer cells displayed cytotoxic effects after administration of Docetaxel with a standard deviation of 34.3%. In conclusion, Docetaxel was shown to induce cytotoxic effects in both in prostate cancer cells and non‐malignant cells however it induced a greater toxic effect in nonmalignant breast cancer 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.
Differential Gene Expression Analysis of Brain Tissue RNA From Mucolipidosis IV Knockout Mice
Authors: Chacon Jonathan , Rosas Lauren , Cuajungco Math P. 
Published: June 2021

Mucolipidosis IV (MLIV) is an autosomal recessive disorder that is characterized by progressive neuronal and retinal degeneration. MLIV is caused by loss of function mutations in the human Mucolipin‐1 (MCOLN1) gene, whose protein product, Transient Receptor Potential Mucolipin‐1 (TRPML1), serves as a lysosomal non‐selective cation channel. We have previously reported abnormal zinc levels in MLIV patient fibroblasts, human MCOLN1‐knockdown cells, and Mcoln1‐knockout (KO) mouse brain tissues. Collectively, these observations suggest that the loss of cellular zinc homeostasis may play an underlying role in MLIV pathogenesis. To investigate whether these irregularities are reflected in the gene expression patterns of zinc buffering and transport proteins, we performed RNA sequencing (RNA‐seq) to analyze the brain transcriptome of three Mcoln1 KO mice and three wild‐type (WT) littermate controls. Gene ontology data revealed significant differential gene expression in the category of “Zinc Ion Transmembrane Transporter Activity.” Gene cluster analyses of zinc transport and zinc buffering genes revealed a distinct reduction in brain transcript levels of solute carrier 30a3 (Slc30a3; also known as the efflux zinc transporter, ZnT3) and transmembrane (Tmem)‐163 genes in KO mice, both of which facilitate zinc transport within neuronal synaptic vesicles. Comparison of RNA‐seq data between individual assembly and combined assembly of KO and WT sequencing reads showed distinct transcript‐level variability that could be explained by inter‐subject differences. Validation using real‐time quantitative polymerase chain reaction (qPCR) confirmed this effect. Nevertheless, both RNA‐seq and qPCR results consistently showed a decrease in ZnT3 transcript levels in KO mice compared to WT littermate control mice. These data imply that abnormal ZnT3 expression could be a mitigating factor for the observed zinc dyshomeostasis in MLIV cells. Overall, our findings suggest that the loss of human TRPML1 protein function perturbs the expression of specific genes necessary for intracellular zinc homeostasis. Future studies will investigate whether the reduction of ZnT3 transcript levels also reflect decreased protein levels in brain tissues of MLIV KO versus control mice.Support or Funding InformationThis work was funded by NIH R15 NS101594 and NIH R25 MD010397.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Membrane‐anchored E‐cadherin/AGO2 complex promote non‐canonical miRNA biogenesis of miR‐451a
Authors: Li Jie‐Ning , Kuo Yao‐Lung , Ku Wen‐Hui , Lu Yu‐Jhan , Wang Ming‐Yang , Chen Pai‐Sheng 
Published: June 2021

Micro‐RNAs are small non‐coding RNAs which regulate target mRNAs expression. Canonical miRNA biogenesis requires two‐step processing conducted by Drosha/DGCR8 and Dicer/AGO2 complexes in the nucleus and cytoplasm, respectively. Aberrant miRNA biogenesis have been reported in malignant human cancers. E‐cadherin is a junctional protein which localizes on plasma membrane to maintain cell‐cell adhesion. Downregulation of E‐cadherin is widely observed in cancers and correlates with epithelial–mesenchymal transition (EMT), it maintains cell‐cell contact and epithelial‐like phenotype to suppress migratory and invasive abilities of cancer cells. Although abnormal regulation of miRNA biogenesis has been reported in cancer, the integrated spatial and temporal regulatory pathways remains unclear. Here, we study the physical interaction between E‐cadherin and AGO2, and the effects on miRNA biogenesis. We found that E‐cadherin enhance the accumulation of AGO2 protein on cell membrane. Membrane‐anchored E‐cadherin/AGO2 complex promotes AGO2‐mediated specific miRNA, miR‐451a, biogenesis in a Dicer‐independent manner. Furthermore, the induced miR‐451 targeted EPH receptor A6 (EphA6) 3′UTR and downregulated EphA6, a metastasis‐promoting gene family member in breast cancer. Our findings demonstrate the spatial and temporal regulation of an AGO2‐dependent miRNA biogenesis pathway modulated by E‐cadherin, which provides new insight into the reciprocal interaction between EMT and miRNA biogenesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delivery of DNA encoding RNA Therapeutics to Alter the Expression of Oncogenic Transcripts in Glioblastoma
Authors: Gallagher Kevin R. , Falotico Sarah C. , Sivetz Nicole , Muralidharan Koushik , Khan Arbaz , Hicks Martin J. 
Published: June 2021

Glioblastoma multiforme (GBM) is the most common central nervous system (CNS) malignancy. The median survival is only 14 months. It is often characterized by the activation of one or more tyrosine kinase receptors, particularly epidermal growth factor receptor (EGFR). This receptor is dysregulated in about 60% of GBM tumors. EGFR amplification, over‐expression and constitutive activation leads to uncontrolled growth and proliferation of GBM. Although a great deal is known about the aberrant biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. Although anti‐sense RNAs and small interfering RNAs can be used to target and silence gene expression, exogenously expressed RNAs are susceptible to extracellular and intracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we take advantage of a natural noncoding RNA gene architecture and the miRNA expression pathway along with an anti‐sense targeted approach to alter EGFR expression. In addition, we make use of a polycistronic delivery system to express RNAs targeting splicing and alternative poly‐A signal/G‐rich elements of the EGFR transcript. DNA delivery vectors were transfected into human GBM cell lines. Results show that our vectors were expressed at high levels with subsequent reduction in full‐length EGFR mRNA expression and concomitant activation of alternative therapeutic isoforms. Current strategies include using the polycistronic delivery mechanism to target additional oncogenic transcripts and adapting to a mouse model of GBM.Support or Funding InformationBristol‐Myers Squibb, Johnson & Johnson, and the Independent College Fund of New JerseyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A1543 is a Potential Bridge Between Cellular Redox State and RNA Processing
Authors: Thomas Patrick , Cameron Jeffrey C. 
Published: June 2021

Synechococcus Sp. PCC 7002 is a rapidly growing, genetically tractable, photosynthetic microbe of industrial interest. Understanding how gene expression and carbon fixation are regulated in PCC 7002 is essential for its advancement as an industrial microbe. Towards that aim, we are investigating a cyanobacterial homolog to E. coli RNase II/R, A1543, that we hypothesize regulates gene expression and other cellular processes in response to changes in cellular redox state.A1543 is an RNA binding protein with three RNA binding domains ‐ two OB folds and an S1 domain. Multiple alignment of PCC 7002 A1543 to RNase II/R homologues in other cyanobacteria show conservation across the genre of two cysteines. Threading of the A1543 sequence onto the E. coli RNase II crystal structure shows that these two cysteines sit in L12 and b2 of the first OB fold, within 4 angstroms of each other, and thus could potentially interact via a disulfide bond.To characterize the role of A1543 in vivo we constructed a segregated A1543 knockout line, DA1543, which shows, as previously reported in the literature, defects in both pigmentation and growth. Additionally, we found that our DA1543 line shows resistance to the carbonic anhydrase acetazolamide compared to wild‐type. This phenotype had been characterized before in a related species, Synechocystis sp. 6803. We have found concentrations of acetazolamide that is lethal and inhibitory to both wildtype and DA1543 cells in several different growth conditions. Interestingly, at 3% CO2, acetazolamide has a greater effect on growth than in air for both cell lines.A1543 is a potential link between the cellular redox state and RNA processing in cyanobacterial cells via changes of RNA binding behavior through oxidation or reduction of a disulfide bridge in a conserved OB‐fold. Understanding if this type of regulation is occurring and how it affects cyanobacterial gene expression and carbon fixation will enable further development of Synechococcus Sp. PCC 7002 as an industrial research organism.Support or Funding InformationPatrick Thomas is supported by the NIH T32 Biophysics training grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Molecular Determinants that Mediate Protein/RNA Interactions at the Synapse
Authors: Mirkovic Jovan , Hossain Md. Faruk , Stolfi Alberto , Wang Ruizhi , Stitzel Henry , Christiaen Lionel , Ruggiu Matteo 
Published: June 2021

The NOVA family of RNA‐binding proteins consists of neuron‐specific splicing regulators that are targeted in an autoimmune neurodegenerative motor disorder. They are sequence‐specific splicing factors, with a modular structure consisting of three KH domains that mediate protein‐RNA interactions.One of the targets of NOVA is a neuron‐specific isoform of the ubiquitously expressed gene AGRIN. This splice variant, termed Z+ AGRIN, is secreted by neurons and is found at the synaptic cleft at the neuromuscular junction (NMJ), a synapse between a nerve cell and a muscle cell, and the best‐studied model synapse. By inducing the clustering of acetylcholine receptors (AChR) on the postsynaptic membrane, Z+ AGRIN promotes the formation, maintenance, and development of the NMJ. The Z exons are also of particular interest as they are potential mutation sites in congenital neuromuscular diseases.The study of NOVA function in vertebrates is complicated by the presence of two NOVA genes termed NOVA1 and NOVA2. The tunicate Ciona robusta is a marine invertebrate that is the closest living relative to vertebrates. Interestingly, Ciona has only one Nova gene. We used the NOVA‐AGRIN paradigm to study the evolution of nerve cell specific protein‐RNA interactions. We cloned and characterized Nova proteins from Ciona and generated Agrin minigenes that recapitulate the action of Nova in a tissue culture cell system. Here, we identify the specific Agrin pre‐mRNA nucleotide sequences that mediate Nova‐dependent splicing of Z+ agrin in tunicates.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.