bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effects of Ezh2 Mutation on Kras‐Driven Adenocarcinoma
Authors: Mandias Hannah Caroline Rattu , Mao Suifang , Xue Bin , Fuziwara Cesar Seigi , He Lin 
Published: April 2020

Lung cancer is the deadliest form of cancer worldwide. Non‐small cell lung cancer (NSCLC) is the most common type of lung cancer, constituting an estimated 84% of diagnoses. Conducting research regarding the metastasis of lung adenocarcinomas is especially pertinent, as the 5‐year survival rate for localized NSCLC is an estimated 50% versus 6% in metastatic lung cancer. Here we investigate the activation effects of Polycomb Repressive Complex 2 (PRC2), an epigenetic regulator commonly found to be dysregulated in human lung cancer, as a driver of metastasis. We employed genetically engineered mouse models harboring activating point mutations in Kras and inactivating mutations in the p53 pathway (KP model) which develop lung adenocarcinomas but exhibit low penetrance and long latency for metastasis. As a resolution, our lab has generated a KPE mouse model which carries a mutation that induces Ezh2 activation, a core component of PRC2. We hypothesize that KPE is an effective and robust metastatic model which displays a shorter latency period than previously established lung adenocarcinoma models. In this project we used a histological approach to characterize morphological properties of primary tumors as well as distinct lymph node metastases in KPE mouse models. By visualizing cellular phenotypes through Hematoxylin & Eosin staining, we intended to quantify tumor area and composition of lymphatic micrometastases. The results illustrated that mice with the Ezh2 mutation in addition to the Kras and p53 mutations experienced a shorter latency period as well as a higher occurrence of lymph node metastases as opposed to the KP model.Support or Funding InformationThis project is supported in part by National Science Foundation Grant 1758007.
Regulation of DNA methyltransferase 3a Expression by Prostaglandin E2 in Human Fibroblasts
Authors: Seutter Sara , Mercando Jason , Sen Neil , Penev Boris , Auker Bennet , Shamseddin Seyed M. , Green Kevan , Borghaei Ruth 
Published: April 2020

IntroductionPeriodontitis is common chronic inflammatory condition that not only results in loss of teeth, but contributes to a variety of systemic conditions, including cancer. Previously we showed that exposure of human gingival fibroblasts (HGF) derived from patients with periodontitis to PGE2 decreases expression of DNMT1, DNMT3a and TET1, but increases global levels of methylated and hydroxymethylated DNA, acting predominantly through the EP4 receptor. The current study compares the effects of PGE2 on expression of these genes in non‐inflamed fibroblasts and aims to further elucidate the signaling pathways involved.MethodsReal time PCR and ELISA are used to measure expression of DNMT1, DNMT3a, TET1 and EP receptors 1–4 in untreated HGF and IMR90 lung fibroblasts and those treated with PGE2. The effects of actinomycin D, as well as EP receptor agonists and antagonists and signaling pathway activators and inhibitors on mRNA expression of these genes will also be assessed by qPCR.ResultsResults so far show that PGE2 decreases expression of DNMT1 in IMR90 cells to an extent similar to that observed in HGF, but levels of DNMT3a are increased rather than decreased, and levels of TET1 are unaffected in IMR90 cells. Although all four EP receptors are expressed in both HGF and IMR90, the relative levels of EP4 expression are lower.ConclusionThe ability of this inflammatory mediator to influence DNA methylation is consistent with the association of chronic inflammation with increased risk of cancer. However, the fact that the effects are cell and context dependent indicate that more information about the specific signaling pathways involved in regulating expression of these important enzymes is needed. Further studies will be aimed at elucidating the roles of the PGE2 receptors and their signaling pathways in mediating the cell‐specific effects.
Essential Amino Acid Supplement lowers Intrahepatic Lipid in Individuals with Alcohol Use Disorder
Authors: Coker Melynda S. , Ladd Kaylee , Kim Josh , Murphy Carl J. , DeCort Ryan , Newcomer Bradley R. , Wolfe Robert R. , Coker Robert H. 
Published: April 2020

Excess alcohol consumption has been ranked as a top risk factor for death and disability. Supervised treatment for alcohol use disorder (AUD) is unlikely for most individuals; fatty liver will likely develop and the risk of liver disease increases. Essential amino acid supplementation (EAAS) has been demonstrated to reduce intrahepatic lipid (IHL) in pre‐clinical and clinical trials for nonalcoholic fatty liver disease. Therefore, the purpose of our study was to evaluate the influence of EAAS on IHL, body composition, and blood lipids in individuals with mild to moderate AUD. Following consent, determination of eligibility and medical screening, 18 young to middle‐aged participants were randomly assigned to a low dose (LD: 8 grams of EAAS BID) or high dose (HD: 13 grams of EAAS BID) group and consumed the supplement for 4 weeks. Pre‐ and post‐EAAS administration, IHL was determined using magnetic resonance imaging/spectroscopy, body composition was analyzed using dual energy x‐ray absorptiometry, and blood parameters were measured by LabCorp. T‐tests were used for statistical analysis and considered significant at P<0.05. While there was no change in IHL (Δ−0.1±0.0 %) in the LD group, there was a reduction in IHL (Δ−0.2±0.0 % water peak) in the HD group (p=0.02). Fat mass, lean tissue mass, bone mineral content, and blood lipids were not altered. Post‐EAAS phosphatidylethanol was unchanged and remained elevated in LD @ 422±224 ng/ml and HD @ 429±196 ng/ml, indicating chronic and excessive alcohol consumption. Based on these results, we suggest that 13 grams of EAAS may lower IHL in individuals with mild to moderate AUD and potentially provide additional opportunities for effective long‐term treatment.Support or Funding InformationResearch reported in this publication was supported by Essential Blends, LLC and by an Institutional Development Award (IDeA) (P20GM103395) and the Biomedical Learning and Student Training Program (UL1GM118991, TL4GM118992, or RL5GM118990) from the National Institute of General Medical Sciences under the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Elucidation of Terpenoid Biosynthesis in Sclerotinia
Authors: Lemire Colin Andrew , Mafu Sibongile 
Published: April 2020

Terpenes are a large class of specialized metabolites found in plant and fungus species that have been widely studied for their medicinal and cytotoxic properties. The plant pathogenic fungus Sclerotinia homoeocarpa was previously shown to produce eleven nortetralabdane compounds that exhibit strong antiplasmodial and growth inhibitive properties. Our objective is to elucidate the biosynthesis of these unique terpene compounds in S. homoeocarpa. Genome mining identified two terpene cyclases as well as multiple clustered cytochromes P450 (CYPs). Combinatorial expression in Escherichia coli and GC‐MS analysis of extracts revealed that one cyclase, ShTPS1, is a bifunctional diterpene synthase that produces the 20‐carbon compound pimara‐8(14),15‐diene. This diterpene likely acts as a scaffold to be modified by downstream CYPs into nortetralabdanes. The second cyclase, ShTPS2, produces several 25‐carbon sesterterpenes, one of the least well‐characterized subsets of terpenes and a frontier for pharmaceutical research. The discovery of ShTPS2 is uniquely interesting due to its promiscuity and the rarity of characterized bifunctional sesterterpene synthases ‐ terpene synthases with a chimeric prenyltransferase domain. Homology modeling of ShTPS2 identified targets for site‐directed mutagenesis in the prenyltransferase and terpene cyclase domains. Mutations in these areas reveal both the functional plasticity of the enzyme and that the promiscuity may result from cationic intermediates of a single terpene cyclase pathway. This research contributes to understanding the diversity of terpene biosynthesis in fungi and allows for the biosynthesis of compounds of interest that are naturally produced at low levels, enabling future work to investigate their function.Support or Funding InformationThis work was supported by the Massachusetts Agricultural Experiment Station as well as the University of Massachusetts Amherst Commonwealth Honors College and Department of Biochemistry and Molecular Biology.
Transfer RNA Abundance Alters Modification Levels in Response to Peroxide Stress in the Radiation Resistant Fungus Cryptococcus neoformans
Authors: Kelley Melissa S. , Yoshida George M. , Myers Ryan A. , Jora Manasses , Limbach Patrick A. 
Published: April 2020

Cryptococcus neoformans is a radiation resistant fungus that has been found growing at the site of the nuclear reactor meltdown in Chernobyl, Ukraine. The Defense Threat Reduction Agency (DTRA) funds research on this organism to gain fundamental knowledge on radiation survival. Many DTRA projects cause soldiers to be exposed to radiation, therefore, gaining this information may have future therapeutic implications. Radiation causes cellular damage through the radiolysis of water into reactive oxygen species (ROS) – such as hydrogen peroxide. Cells have mechanisms such as antioxidants to regulate ROS levels. When ROS is too high, nucleic acids may be damaged. Transfer RNA (tRNA) – the molecule that carries the amino acid to the translational machinery – directly affect protein production. Hence, the integrity of tRNA is critical to antioxidant defenses and general protein synthesis. Chemical modifications to tRNA are abundant and crucial in all domains of life. Peroxide is known to cause changes in tRNA modification 5‐methylcytidine (m5C) levels in the model fungus S. cerevisiae. This work sets out to evaluate the effects of peroxide exposure on tRNA modification and tRNA transcript abundance in C. neoformans. Using liquid chromatography coupled with mass spectrometry (LC‐MS), modifications were relatively quantified and found to respond differently than in S. cerevisiae. Moreover, RNA sequencing information indicated that there are changes in tRNA abundance in response to peroxide. Since the modifying enzyme transcripts were not altered, the tRNA transcript abundance was reflected in the detected modification levels. Therefore, C. neoformans exhibits an increase in certain tRNA transcripts which then causes an increase in those tRNAs’ modifications. In conclusion, C. neoformans responds to peroxide stress via regulation of the tRNA pool – consequently altering the modifications within it.Support or Funding InformationThis work was funded by the Defense Threat Reduction Agency (DTRA) ‐ a branch of the US Department of Defense (DoD). Further support has been provided by the University of Cincinnati.
Cytochrome P450‐Derived Epoxy Lipids of N‐3 PUFAs Protect the Heart From Ischemia‐Reperfusion Injury by Regulating Mitochondrial Sirtuin 3
Authors: Darwesh Ahmed M. , Altamimi Tariq R. , Jamieson K. Lockhart , Bassiouni Wesam , Zhang Hao , Oudit Gavin Y. , Lopaschuk Gary D. , Seubert John M. 
Published: April 2020

ObjectiveCytochrome P450 epoxygenases can metabolize dietary n‐3 polyunsaturated fatty acids (n‐3 PUFAs) to epoxy lipids, which have known cardioprotective effects; however, it remains unclear how this cardioprotection occurs. Our recent data demonstrate that cytochrome P450 epoxygenase‐derived metabolites of the n‐3 PUFA docosahexaenoic acid (DHA), 19,20‐epoxydocosapentaenoic acid (19,20‐EDP), exhibit protective effects toward cardiac mitochondria. Maintaining mitochondrial quality is essential to limit myocardial damage following ischemia‐reperfusion (IR) injury. The mitochondrial deacetylase sirtuin 3 (SIRT3) plays a pivotal role in the maintenance of mitochondrial function and cardiac energy metabolism. We hypothesize that the cardioprotective mechanism(s) of 19,20‐EDP against IR injury involve(s) stimulating mitochondrial SIRT3.MethodsLangendorff and isolated working heart perfusions were performed in male and female C57BL/6 mice to assess changes in cardiac function and energy metabolism in response to IR injury. Hearts were perfused with either vehicle, 19,20‐EDP (1 μM) and/or the SIRT3 inhibitor nicotinamide (30 μM) for a 20 min baseline perfusion, following which hearts were subjected to 30 min of global no flow ischemia and 40 min of reperfusion. Glucose and palmitate oxidation rates were measured to detect alterations in cardiac energy metabolism. Mitochondrial fractions were assessed for changes in SIRT3 activity and total lysine acetylation levels. Mitochondrial SIRT3 downstream targets were assessed in human ventricular tissues obtained from individuals with ischemic heart disease (IHD) included in the Human Explanted Heart Program and compared to non‐failing control hearts (NFC) collected from unused transplant donors through the Human Organ Procurement and Exchange Program at the University of Alberta. In addition, mitochondrial respiration was assessed in cardiac fibers isolated from the human tissues.ResultsHearts perfused with 19,20‐EDP demonstrated improved post‐ischemic functional recovery, glucose oxidation and cardiac efficiency compared to control groups. The cardioprotective effects were associated with enhanced mitochondrial SIRT3 activity and reduced global protein acetylation. We observed increased acetylation of the mitochondrial antioxidant superoxide dismutase 2 in tissues obtained from IHD human myocardium suggesting decreased antioxidant capacity. Treatment with 19,20‐EDP also markedly improved mitochondrial respiration in permeabilized human fibers obtained from IHD hearts. Importantly, the cardioprotective effects of 19,20‐EDP was abolished by the SIRT3 inhibitor nicotinamide.ConclusionWe demonstrate that 19,20‐EDP‐mediated cardioprotective mechanisms against IR injury involve preservation of mitochondrial SIRT3 activity, which results in improved cardiac efficiency.
Metabolic and Contractile Pathophysiology Following Volumetric Muscle Loss Injury
Authors: Basten Alec M. , Raymond-Pope Christiana J. , Dalske Kyle A. , Call Jarrod A. , Greising Sarah M. 
Published: April 2020

Volumetric muscle loss (VML) injury, due to abrupt and significant loss of tissue, exceeds the endogenous regenerative ability of the skeletal muscle, leading to long‐term functional impairment and disability. There is a well‐established physiologic relationship between skeletal muscle mass and metabolism, but the implications of VML injury on this relationship are widely unexplored. Recent investigations have identified oxidative and mitochondrial dysfunction associated with VML injury; however, the local metabolic myofiber response is not completely understood. We hypothesized that significant metabolic and contractile limitations would be observed in the remaining myofibers following VML injury, suggestive of motor unit reorganization. To evaluate the metabolic properties following VML, adult C57BL/6 male mice (n=16) underwent a sham procedure or multi‐muscle full thickness VML injury to the gastrocnemius, soleus, and plantaris muscles (21.1±0.7mg). Two months post‐VML, terminal histologic evaluation of capillarity, mitochondrial content, fibrotic deposition, and myosin heavy chain distribution was completed. Additionally, maximal contractile function and force‐frequency response of the posterior muscle compartment were evaluated. Maximum passive torque at 20 degrees dorsiflexion was significantly greater in the VML group (5.5±0.22 mN·m) compared to sham (2.3±0.13 mN·m). Maximum isometric torque was significantly less in VML (334±28 mN·m) compared to sham (557±66 mN·m). VML injury resulted in slower torque production and elevated twitch:tetani ratio, perhaps indicating a slower muscle fiber phenotype in the tissue remaining. Collectively, this work suggests that motor unit reorganization may alter muscle fiber phenotype following VML injury, likely limiting long‐term muscle functionality.Support or Funding InformationSupported by W81XWH‐18‐1‐0710 & T32AR050938.
The Impact of High Dietary Sodium on Cerebral Blood Flow Regulation
Authors: Migdal Kamila U. , Robinson Austin T. , Watso Joseph C. , Babcock Matthew C. , Serrador Jorge R. , Farquhar William B. 
Published: April 2020

High salt consumption is linked to increased stroke risk. In rodents, high dietary sodium has been shown to impair cerebral blood flow regulation. In humans, cerebrovascular reactivity to carbon dioxide (CO2) is a validated method for assessing the cerebrovasculature where a reduction in cerebrovascular reactivity is associated with cerebrovascular disease. Additionally, the ability for the brain to maintain cerebral blood flow despite changes in arterial blood pressure is termed cerebral autoregulation (CA). Dysfunctional CA is also predictive of strokes and all‐cause mortality. However, the effects of a high dietary sodium consumption on cerebrovascular reactivity and dynamic CA in healthy, young adults is unknown.PurposeWe tested the hypothesis that ten days of high dietary sodium feeding would impair cerebral blood flow regulation in healthy young adults.MethodsEight adults (6M/2F; age: 29 ± 11yrs; BMI: 26±3 kg/m2, Mean‐±SD) participated in a controlled feeding study that consisted of 10 days of either low‐ (LS: 1 g sodium/day), medium‐ (MS: 2.3 g sodium/day), or high‐sodium (HS: 7 g sodium/day) diets, in randomized order separated by at least a month. Urinary sodium excretion was determined to ensure compliance on diets. Transcranial Doppler of the right middle cerebral artery (MCA) velocity was assessed while participants lay supine. On the tenth day of the diet, cerebrovascular reactivity to high CO2 (via 8% CO2; hypercapnia) and low CO2 (via hyperventilation; hypocapnia) were assessed. Dynamic CA between spontaneous changes in mean arterial pressure to MCA velocity using a transfer function analysis was also measured. Data were analyzed using one‐way repeated measures ANOVA. Tukey post hoc comparisons were used when appropriate.ResultsUrinary sodium excretion (LS= 64±56, MS= 82±19, HS=250±213 mmol/24h, P=0.04) increased in a step‐wise manner. Brachial blood pressures were not different (systolic blood pressure= P=0.45; diastolic blood pressure: P=0.17). Cerebrovascular reactivity to hypercapnia (LS= 2.4±0.6, MS= 1.7±0.5, HS=2.5±0.7 %/mmHg, P=0.09) and to hypocapnia (LS= 1.8±0.6, MS= 1.9±0.6, HS=2.0±0.7 %/mmHg, P=0.72) were not different. Dynamic CA was also not different in the very low frequency (phase: LS= 71±23, MS= 72±38, HS=80±39°, P=0.69) and low frequency band (phase: LS= 42±14, MS= 45±11, HS=47±14°, P=0.62).ConclusionDespite the diets, these preliminary data suggest that high dietary sodium may not impair cerebral blood flow regulation in healthy young adults.Support or Funding InformationAcknowledgementsNIH Grant 1RO1HL128388
The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Monocytes
Authors: Peng Qianman , Shen Jianzhong 
Published: April 2020

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor induces up‐regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, it has been shown by others that monocyte TF is more important than endothelial TF in terms of provoking pro‐thrombotic state. Thus, we aimed to study whether human monocytes express P2Y2 receptor and its role in control of TF expression. RT‐PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP‐1 cells. Consistent with this, stimulation of the THP‐1 cells by ATP or UTP at micromolar concentration dramatically increased TF protein expression measured by Western blotting, which was abolished by AR‐C118925XX, a selective P2Y2 receptor antagonist, suggesting a key role for P2Y2 receptor. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA, which was maximal at 2h and preceded with an increased TF pre‐mRNA, indicating enhanced TF gene transcription. However, we also observed a significant increase in TF mRNA stability after UTP and ATP treatment. Furthermore, activation of the monocyte P2Y2 receptor by a low dose of UTP or ATP (1μM) significantly activated the MAPK pathways including ERK1/2, JNK, and p38, along with their downstream transcription factors including c‐Jun, c‐Fos and ATF‐2, whereas blocking the ERK1/2, JNK or p38 pathways respectively, all significantly suppressed P2Y2 receptor‐mediated TF expression. These findings demonstrate for the first time that the P2Y2 receptor mediates TF expression in human monocytes through new mechanisms involving ERK1/2, JNK and p38, and that both transcriptional and posttranscriptional mechanisms contribute to a pro‐thrombotic status in monocytes through the P2Y2‐TF axis, highlighting monocyte P2Y2 receptor may be a new drug target for the prevention and/or treatment of relevant thrombotic diseases.Support or Funding InformationSupported by NIH funding 1R01HL125279‐01A1 (JS)
Measuring the Kinetics of G‐protein‐coupled Receptor Signaling and Biased Agonism.
Authors: Hughes Thomas Edward , Tewson Paul , Hoare Sam 
Published: April 2020

Quantifying the time course of G‐protein‐coupled receptor signaling can reveal biological properties of GPCR function such as receptor desensitization, pathway selectivity, and spatiotemporally compartmentalized signaling. Applying signaling kinetics to modern receptor research and drug discovery requires assay modalities that can monitor signaling responses over time, and data analysis methods to extract the kinetic drug parameters. Here we describe a suite of fluorescent biosensors for monitoring multiple pathways of GPCR signaling (Gs, Gi, Gq and arrestin). These biosensors are genetically‐encoded and amenable to kinetic analysis, with continuous “real time” responses detected on plate readers or imaging systems. A kinetic data analysis framework is applied to the time course data using familiar equations on commonly‐used curve‐fitting software (e.g. GraphPad Prism). The analysis yields the initial rate of signaling (kTau), a biologically‐meaningful parameter of ligand efficacy at generating the signal. Biased agonism assessment is simplified using this approach, and demonstrated using arrestin recruitment and second messenger signaling via the AT1 angiotensin and GLP‐1 receptors. Bias is quantified as the ratio of the initial rates of signaling reported by each assay. This platform provides a straightforward method that investigators can use to quantify GPCR receptor pharmacology and biased signaling with the application of kinetic analysis to understanding receptor function and to the development of new therapeutics.Support or Funding InformationThis work was supported by grants from the National Institutes of HealthR44 GM125390‐02 and R44 DA050357‐01