bluuebunny/crossref_metadata_2025_split huggingface dataset:
Methylmalonic Acid Compromises Energy Metabolism and Decreases the Expression of Neuronal Differentiation Genes in SH‐SY5Y Human Neuroblastoma Cells
Authors: da Costa Renata Torres , dos Santos Marcella Bacelar , de Almeida Raquel Pascott , Ribeiro César Augusto João 
Published: April 2020

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L‐methylmalonyl‐CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and symptoms during metabolic decompensation commonly appear in newborns. Considering that the mechanisms underlying neurological alterations of this disorder are not fully understood, this study aimed to investigate the toxic effects of MMA in a neuroblastoma cell line presenting neuronal features. Undifferentiated and retinoic acid (RA)‐differentiated human neuroblastoma SH‐SY5Y cells were exposed to MMA (1–10mM) for 24 or 48 hours and cell viability, glucose consumption and mitochondrial metabolic function were analyzed. Additionally, the expression of some molecular markers of neuronal differentiation was also quantified after co‐exposition with 10 μM RA and 1 mM of MMA for 3 and 7 days.MMA significantly reduced cell viability after 48‐hour period exposition in undifferentiated and RA differentiated SH‐SY5Y cells. It was also observed an increase in glucose consumption in cells exposed to MMA. Regarding oxygen consumption, there was an increase in respiratory parameters in RA differentiated SH‐SY5Y cells when compared to undifferentiated cells. MMA significantly reduced basal, ATP‐linked and maximal respiration, as well as the reserve respiratory capacity of cells after 48‐hour exposition in both undifferentiated and RA differentiated SH‐SY5Y cells. Regarding the effects of MMA in the neuronal differentiation process, it was observed that co‐exposition to RA and MMA significantly decreased the expression of enolase 2 (ENO2) and synaptophysin (SYP) genes after 7 days of exposition.In summary, MMA cell viability and oxygen consumption in SH‐SY5Y neuronal cells, suggesting that this organic acid compromises cell respiration, at resting and reserve capacity states, which might influence the cell capacity to meet energetic demands. In addition, glucose uptake was increased after exposition to MMA, corroborating that aerobic metabolism is compromised. The expression of mature neuronal genes was also reduced, suggesting that MMA possibly compromises neuronal differentiation, which can be involved in symptoms presented by affected patients during brain development. Finally, our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.Support or Funding InformationThis study was supported by grants from FAPESP (2015/25541‐0), CAPES and UFABC.
Developing of a Muscle Atrophy Model
Authors: Garcia-Pelagio Karla , Martinez-Mendez Sheiren , Hinojosa-Rodriguez Cindy , Jimenez-Estrada Ismael , Segura-Alegria Bertha 
Published: April 2020

Muscle atrophy is one of the most important and frequent problems observed in patients in Intensive Care Units following a surgery, immobilization, extended periods of bed rest as well as in sports medicine. All of these involves loss of muscle mass and decreased muscle strength which increases the risk for future health problems. Immobilization changes the function as well as structure of the peripheral neuromuscular system, and alters both protein degradation and synthesis. Therefore, effective therapeutic strategies to treat muscle wasting induced by immobilization are urgently required but first is necessary to validate and standardize the immobilization technique. In this work we develop a reproducible well‐characterized injury model using a modified Thomas‐ splint validated with mechanical force measurements, histology (H&E and NADH), morphology and ultrasound imaging. Forty young Wistar male rats were divided into two groups: 1) Control‐ no immobilized extremities (CON; n=10) and 2) Unilateral right hindlimb immobilization at 3, 7 or 14 days (Im 3, Im 7, Im 14; n=10 for each group). Results showed a significant increment in body weight for Im 7 and Im 14 compared to CON. Experiments performed in tibialis anterior (TA) of Im muscle showed morphological changes in muscle weight and muscle diameter compared to CON confirmed by Ultrasound imaging. These changes were accompanied with a decrement in peak isometric tetanic force (Po) of immobilized muscles of 20%, 41% and 28% at 3, 7 and 14 days post injury, respectively. Fatigue time for all Im muscles was higher than CON ones (Im 3= 23%, Im 7=33% and Im 14=42%). Metabolic changes using NADH technique denoted an early increase of oxidative fibers and a decrement of glycolytic fibers at 7 days post‐injury compared to CON. In here, we developed, standardized and characterized an immobilization technique that can be used as a first approach to use effective therapeutic strategies to avoid muscle atrophy.Support or Funding InformationSupported partially by UNAM ‐PAPIIT RA207818 and CONACyT AI‐S‐17636 to KPGP.
OCT3 gated intracellular β1AR activation enhances excitation‐contraction coupling in heart
Authors: Wang Ying , Zhao Meimi , Shi Qian , Xu Bing , Xiang Yang Kevin 
Published: April 2020

Rationaleβ1AR‐adrenergic receptor (β1AR) plays a central role in sympathetic regulation of cardiac excitation‐contraction (EC) coupling. Stimulation of β1AR by catecholamines enhances cardiomyocyte contraction and Ca2+transient via the cAMP‐protein kinase A (PKA) pathway. Organic cation transporter 3 (OCT3) plays a role in cleaning up the extracellular catecholamines and transporting a portion of catecholamine into myocardium. Recent progress found that β1AR is also present at intracellular membranes. However, how the intracellular β1AR was activated, and the role of OCT3 in regulating intracellular β1AR signaling and EC‐coupling remains unknown.ObjectiveTo characterize the presence and activation of intracellular β1AR‐adrenergic receptors and their impact on cardiac EC‐couplingMethods and ResultsWe used a genetic deletion of OCT3 to inhibit the entrance of catecholamine into the cells. We used isolated myocytes to measure β1AR‐induced contractility and Ca2+transients. We used PKA activity reporters to detect local activation via intracellular β1AR. We found that catecholamine‐induced inotropic effects were diminished in OCT3‐knock out hearts. OCT3 deficiency significantly attenuated norepinephrine (NE) induced PKA activities at the sarcoplasmic reticulum, myocyte contractility, and Ca2+transient. Additional data show β1AR associates with sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) at the sarcoplasmic reticulum (SR). Inhibition of OCT3 blocked the transportation of NE, and attenuated β1AR‐induced PKA activity at the SR and EC‐coupling.ConclusionOur finding demonstrates a previously unrecognized intracellular β1AR‐mediated local PKA activity at SR that is gated by OCT3. Upon catecholamine stimulation, this SR‐localized β1AR signal plays a critical role in promoting E‐C coupling and cardiac contractile function. Thus, internal β1AR signaling may presents a novel therapeutic target in cardiac diseases treatment.Support or Funding InformationNIH HL113413VABX009200
The Multi‐functional Enzyme CHIP Regulates Microtubule Dynamics
Authors: Altinok Selin , McDonough Holly , Schisler Jonathan C. 
Published: April 2020

The
Carboxy‐terminus of
Hsc70
Interacting
Protein (CHIP) controls several aspects of protein quality control machinery through its ability to function as a co‐chaperone, autonomous chaperone, and ubiquitin ligase. CHIP is ubiquitously expressed in all tissues, but interestingly, CHIP expression is higher in certain tissues such as heart, muscle and brain, suggesting an increased dependence on CHIP function in these organ systems. This concept is supported by studies that show how altered CHIP function associates with several ailments such as cardiac diseases, neurological disorders, and cancer. Microtubules (MTs) are a part of the cellular cytoskeleton that provides shape and structure to cells. In addition to these roles, MTs play a crucial role in cell division, migration, intracellular organization and trafficking. Dynamic assembly and disassembly of MTs upon various stimuli is important for the proper functioning of differentiated cells and is a process orchestrated by multiple proteins. Dysregulation in the MT reorganization machinery is implicated in a similar spectrum of diseases associated with the loss of CHIP function, however, there has yet to be a direct connection between CHIP and MT dynamics. We performed a proteomics screen on wild type and CHIP−/− mouse embryonic fibroblasts and identified differential regulation of proteins that regulate several aspects of MT function, such as STMN1 and PDLIM. Given the importance of CHIP in protein homeostasis, we hypothesized that CHIP is necessary for proper MT dynamics in response to cellular stimulations. We found that CHIP is required for the phosphorylation of stathmin, a regulator of MT assembly, and for the tubulin polymerization in C2C12 myoblast cells following insulin and glucose stimulation. This stimulation triggers an important MT‐dependent process to mediate glucose uptake. Furthermore, ex vivo studies using muscle fibers isolated from CHIP−/− mice failed to form MT lattices after glucose stimulation, consistent with a new role for CHIP in regulating MT in muscle. Altered MT dynamics, as well as CHIP loss of function, are also implicated in neurodegenerative diseases, particularly those associated with proteinopathies. To determine if CHIP contributes to MT regulation in neurons, we used an immunoprecipitation‐mass spectrometry approach and identified changes in interactions between CHIP and MT proteins during proteotoxic stress. Future experiments will focus on validating the interaction data and further dissecting the specific functions of CHIP that contribute to the regulation of MT dynamics using biochemical approaches and imaging. In conclusion, for the first‐time, our data couple CHIP to MT dynamics, and suggest that CHIP may contribute to cell protection by regulating the proteins that are vital to MT dynamics.Support or Funding InformationFunding was provided by a grant to J.C.S. from the National Institute of Aging (R01‐GM061728).
Evaluation of Glutaredoxin 1 as a Novel Marker of Renal Damage after Ischemia‐Reperfusion Injury in Mice
Authors: Godoy Jose R. , O’Sullivan Meghan , Bethel-Lebel Herdest , Moncada Marcela H. , Illanes Oscar , Gellert Manuella , Lillig Christopher Horst 
Published: April 2020

Renal ischemia‐reperfusion injury (IRI) is a significant cause of acute renal failure and delayed graft function in clinical patients. Tissue damage in renal IRI is caused in part by the activation of the immune response and the generation of reactive oxygen species (ROS), both taking place mainly during the reperfusion phase, once the blood supply to the tissue is restored. Glutaredoxin 1 (Grx1), a cytosolic protein that reduces protein disulfides as well as glutathionylated proteins, plays a protective role in ROS‐mediated damage to the cell. In a previous work, we detected increased levels of Grx1 in the urine of mice 24h after renal IRI
1. Recently, Grx1 was shown to be elevated in the serum of patients with chronic kidney disease
2. To determine the usefulness of Grx1 as a possible biomarker of ischemic renal damage we evaluated serum, urinary, as well as intra‐renal levels of Grx1 in a mouse model of renal IRI. Different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) were compared in order to follow Grx1 fluctuation levels in the kidney, serum, and urine. Serum Grx1 levels, analyzed by ELISA, were correlated with several renal function parameters such as serum creatinine, proteinuria, glomerular diameter, and tubular thickness. After IRI, Grx1 serum levels increased much earlier than creatinine (4h versus 24h). Western blot analyses showed a decrease of Grx1 in the kidney of mice with surgically induced IRI, compared to the sham group. Additionally, immunohistochemistry revealed an increased apical localization of Grx1 in cells of the distal convoluted tubules of the mice exhibiting IRI. Urinary levels of Grx1, even if increased in the first hours after renal IRI, were not significantly altered. These findings provide initial supporting evidence for the use of a redox protein such as Grx1 as a novel biomarker for assessing renal damage under experimental conditions. Further studies are necessary to validate these results.Support or Funding InformationThis work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine.
Investigation of Curcumin and its Analogs as Adenosine Receptor Agonists for Treating Chronic Pain
Authors: Hamilton Luke J. , Pattabiraman Mahesh , Chandra Surabhi 
Published: April 2020

Chronic pain debilitates millions of people throughout the world. From the mid‐1990s until the present, the principle pharmacologic treatment for chronic pain has been long‐term administration of opioid medications. However, between 1999 and 2007, the number of deaths related to opioid drug overdose rose throughout the world and more than tripled in the USA, causing concern. In response, studies have been initiated to discover new analgesics that carry a lower risk of addiction and overdose‐related death. All four adenosine receptor (AR) subtypes (A1AR, A2AAR, A2BAR, A3AR) have been investigated as targets for analgesic drugs, but concerns about cardiovascular side effects are impeding translation to the clinic. Drugs that are selective for A3AR offer a solution, because they inhibit pain without cardiovascular effects. Our aim is to clarify the structural characteristics of molecules that make them more selective for A3AR over other ARs. To generate ARs for testing, three human embryonic kidney cell lines are being grown, expressing A2A, A2B, and A3ARs respectively. We have selected curcumin as our test molecule because it has documented analgesic properties and because its binding affinity for ARs has not previously been described. We use photochemistry to alter curcumin, changing it from trans‐trans to cis‐trans. Both forms of curcumin are then evaluated for their binding affinity (Ki) for each of the AR subtypes using fluorescent ligand competitive binding assays. Results thus far show that isomerized curcumin binds to ARs more strongly than unmodified curcumin. Further binding assays will reveal whether isomerized curcumin is selective for A3AR over other ARs. Further testing with cAMP ELISAs will reveal whether binding of isomerized curcumin is associated with receptor activation. Activation of A3AR will result in depressed cAMP levels because A3AR is coupled to Gαi. Activation of A2AAR and A2BAR will result in elevated cAMP levels, because both A2 ARs are coupled to Gαs. In summary, the structure activity relationships discovered by this study will help future pharmacologists to design molecules that target A3ARs for the alleviation of chronic pain.Support or Funding InformationWe thank the Great Plains IDeA CTR program (NIGMS # 1U54GM115458‐01) and the American Chemical Society Petroleum Research Fund (# 54862‐UR4) for their funding support.
Functional Implications of Tyrosine Hydroxylase Immunoreactivity in Rat Brain
Authors: Ayles Brittney , Rahmani Sana , Kiriakedis Satomi , Li Hui-Yun 
Published: April 2020

Hypothalamo‐pituitary‐adrenal axis is involved in the stress regulation. Understanding the cellular mechanisms involved in this system is critical in studying stress response. Tyrosine hydroxylase (TH), the rate‐limiting enzyme of catecholamine synthesis, has been suggested to play a pivotal role in regulating the neuronal functions under stress. Previous findings showed the brainstem ascending TH‐immunoreactive fibers participate in the cellular activation of paraventricular nucleus of the hypothalamus in response to neurogenic stress. Current study aimed to study the role of TH in regulating brain areas which are involved in the neural circuits in stress response. Immunocytochemical staining of TH confirmed the TH‐immunoreactive positive neurons are found in the locus coeruleus, ventral lateral medulla, nucleus of the solitary tract and zona incerta; wihile TH‐ir fibers are widely found in the forebrain region including the striatum, paraventricular nucleus of hypothalamus, and amygdala. These are areas rich in neurons bearing dopamine receptor, corticotropin‐releasing factor and oxytocin. Using double immunohistochemical staining, we are able to demonstrate the functional significance of tyrosine hydroxylase inputs to these different populations of neurons.Support or Funding InformationWe are grateful to Oregon Tech for supporting undergraduate research as well as Dr. Paul Sawchenko for his generosity in donating rat brain tissues.
Airway Relaxation Mechanisms and Structural Basis of Osthole to Improve Lung Function in Asthma
Authors: Tu Yaping , Wang Sheng , Xie Yan , Huo Yan-Wu , Li Yan , Abel Peter W. , Jiang Haihong , Zou Xiaohan , Jiao Hai-Zhan , Kuang Xiaolin , Wolff Dennis W. , Huang You-Guo , Casale Thomas B. , Wei TaoTao , Cao Zhengyu 
Published: April 2020

Background and objectiveOveruse of β2‐adrenoceptor agonist bronchodilators causes receptor desensitization, resulting in decreased efficacy and an increased risk of death in asthma patients. This is a critical unmet problem in asthma management, and novel drugs are urgently needed. Here, we report that osthole, a coumarin derived from a traditional Chinese medicine, induces complete relaxation of preconstricted airways in mouse precision‐cut lung slices, irrespective of β2‐adrenoceptor desensitization.MethodsMouse precision‐cut lung slices as an in vitro model were used to analyze airway relaxation. The cAMP level in human airway smooth muscle (ASM) or HEK293 cells was determined by phosphorylation of vasodilator‐stimulated phosphoprotein using Western blot or cAMP dependent reporter luciferase assay. The secretion of prostaglandin E2 (PGE2) in ASM cells or lung slices was measured by ELISA assay. The effect of osthole on airway hyperresponsiveness (AHR) was measured in both house dust mite (HDM)‐sensitized mice and naïve regulator of G‐protein signaling 2 (RGS2) KO mice. The binding sites of osthole with PDE4D5 catalytic domain were analyzed by crystallization and X‐ray diffraction.ResultsOsthole administered in vivo ameliorates AHR, a hallmark of asthma, in murine asthma models. Mechanistic studies revealed that osthole inhibits phosphodiesterase 4D (PDE4D) activity to amplify endogenous PGE2 signaling in ASM cells, which eventually triggers cAMP/protein kinase A‐dependent relaxation of airways. The crystal structure of the PDE4D complexed with osthole reveals that osthole binds to the catalytic site to prevent cAMP binding and hydrolysis. Together, our studies elucidate a specific molecular target and mechanism by which osthole induces airway relaxation.ConclusionIdentification of osthole binding sites on PDE4D will guide further development of novel therapeutics for β2‐adrenoceptor agonist‐resistant asthma.Support or Funding InformationThis work was supported in part by the National Institutes of Health (R01HL116849) and Nebraska State LB595 Research Program, USA; the National Laboratory of Biomacromolecules and the Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences (CAS); the CAS/SAFEA International Partnership Program for Creative Research Teams; the National Key R&D Program of China (2017YFA0205501); the National Natural Science Foundation of China (21777192 and 31500623)
Role of WNK1/OSR1 in maintaining endothelial tight junction integrity
Authors: Jaykumar Ankita , Plumber Sakina , Stippec Steve , Earnest Svetlana , Cobb Melanie 
Published: April 2020

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.Support or Funding InformationAmerican Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb