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https://www.readbyqxmd.com/read/28276032/identification-of-disease-causing-mutations-by-functional-complementation-of-patient-derived-fibroblast-cell-lines
#1
Laura S Kremer, Holger Prokisch
Diagnosis of mitochondrial disorders is still hampered by their phenotypic and genotypic heterogeneity. In many cases, exome sequencing, the state-of-the-art method for genetically diagnosing mitochondrial disease patients, does not allow direct identification of the disease-associated gene but rather results in a list of variants in candidate genes. Here, we present a method to validate the disease-causing variant based on functional complementation assays. First, cell lines expressing a wild-type cDNA of the candidate genes are generated by lentiviral infection of patient-derived fibroblasts...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28276021/assessing-mitochondrial-bioenergetics-in-isolated-mitochondria-from-various-mouse-tissues-using-seahorse-xf96-analyzer
#2
Arcangela Iuso, Birgit Repp, Caroline Biagosch, Caterina Terrile, Holger Prokisch
Working with isolated mitochondria is the gold standard approach to investigate the function of the electron transport chain in tissues, free from the influence of other cellular factors. In this chapter, we outline a detailed protocol to measure the rate of oxygen consumption (OCR) with the high-throughput analyzer Seahorse XF96. More importantly, this protocol wants to provide practical tips for handling many different samples at once, and take a real advantage of using a high-throughput system. As a proof of concept, we have isolated mitochondria from brain, heart, liver, muscle, kidney, and lung of a wild-type mouse, and measured basal respiration (State II), ADP-stimulated respiration (State III), non-ADP-stimulated respiration (State IVo), and FCCP-stimulated respiration (State IIIu) using respiratory substrates specific to the respiratory chain complex I (RCCI) and complex II (RCCII)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28245898/glutamate-impairs-mitochondria-aerobic-respiration-capacity-and-enhances-glycolysis-in-cultured-rat-astrocytes
#3
Xu Yan, Zhong Fang Shi, Li Xin Xu, Jia Xin Li, Min Wu, Xiao Xuan Wang, Mei Jia, Li Ping Dong, Shao Hua Yang, Fang Yuan
OBJECTIVE: To study the effect of glutamate on metabolism, shifts in glycolysis and lactate release in rat astrocytes. METHODS: After 10 days, secondary cultured astrocytes were treated with 1 mmol/L glutamate for 1 h, and the oxygen consumption rates (OCR) and extra cellular acidification rate (ECAR) was analyzed using a Seahorse XF 24 Extracellular Flux Analyzer. Cell viability was then evaluated by MTT assay. Moreover, changes in extracellular lactate concentration induced by glutamate were tested with a lactate detection kit...
January 2017: Biomedical and Environmental Sciences: BES
https://www.readbyqxmd.com/read/28237843/characterization-of-energy-and-neurotransmitter-metabolism-in-cortical-glutamatergic-neurons-derived-from-human-induced-pluripotent-stem-cells-a-novel-approach-to-study-metabolism-in-human-neurons
#4
Blanca I Aldana, Yu Zhang, Maria Fog Lihme, Lasse K Bak, Jørgen E Nielsen, Bjørn Holst, Poul Hyttel, Kristine K Freude, Helle S Waagepetersen
Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography...
February 24, 2017: Neurochemistry International
https://www.readbyqxmd.com/read/28197868/plasma-levels-of-immune-factors-and-sex-steroids-in-the-male-seahorse-hippocampus-erectus-during-a-breeding-cycle
#5
Tingting Lin, Xin Liu, Dongxue Xiao, Dong Zhang
To better understand the endocrine- and immune-response pattern during reproduction in a fish species having parental care behaviors and also to accumulate the endocrine- and immune-related data for future explanations of the low reproductive efficiency in seahorse species, the variations of immune factors and sex steroids in the plasma of the male lined seahorse Hippocampus erectus at different breeding stages, i.e., pre-pregnancy, pregnancy (early, middle, and late periods), and post-pregnancy, were investigated in the present study...
February 14, 2017: Fish Physiology and Biochemistry
https://www.readbyqxmd.com/read/28179998/phlpp-regulates-hexokinase-2-dependent-glucose-metabolism-in-colon-cancer-cells
#6
Xiaopeng Xiong, Yang-An Wen, Mihail I Mitov, Mary C Oaks, Shigeki Miyamoto, Tianyan Gao
Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect...
2017: Cell Death Discovery
https://www.readbyqxmd.com/read/28110213/cannabidiol-attenuates-ogd-r-induced-damage-by-enhancing-mitochondrial-bioenergetics-and-modulating-glucose-metabolism-via-pentose-phosphate-pathway-in-hippocampal-neurons
#7
Shanshan Sun, Fangyuan Hu, Jihong Wu, Shenghai Zhang
Deficient bioenergetics and diminished redox conservation have been implicated in the development of cerebral ischemia/reperfusion injury. In this study, the mechanisms underlying the neuroprotective effects of cannabidiol (CBD), a nonpsychotropic compound derived from Cannabis sativa with FDA-approved antiepilepsy properties, were studied in vitro using an oxygen-glucose-deprivation/reperfusion (OGD/R) model in a mouse hippocampal neuronal cell line. CBD supplementation during reperfusion rescued OGD/R-induced cell death, attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers...
April 2017: Redox Biology
https://www.readbyqxmd.com/read/28047565/su-g-tep3-10-radiation-induces-prompt-live-cell-metabolic-fluxes
#8
D Campos, W Peeters, K Nickel, B Burkel, J Bussink, R Kimple, A van der Kogel, K Eliceiri, M Kissick
PURPOSE: To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. METHODS: Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH...
June 2016: Medical Physics
https://www.readbyqxmd.com/read/28006775/fgf-2-transcriptionally-down-regulates-the-expression-of-bnip3l-via-pi3k-akt-foxo3a-signaling-and-inhibits-necrosis-and-mitochondrial-dysfunction-induced-by-high-concentrations-of-hydrogen-peroxide-in-h9c2-cells
#9
Qian Chen, Xiaosong Chen, Conghui Han, Ying Wang, Tao Huang, Yingdong Du, Zhen Dong
BACKGROUND/AIMS: Cardiovascular disease is a growing major global public health problem. Necrosis is one of the main forms of cardiomyocyte death in heart disease. Oxidative stress is regarded as one of the key regulators of cardiac necrosis, which eventually leads to cardiovascular disease. Many pharmacological and in vitro studies have suggested that FGF-2 can act directly on cardiomyocytes to maintain the integrity and function of the myocardium and prevent damage during oxidative stress...
2016: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/27997810/glut-1-dependent-glycolysis-is-increased-during-aging-related-lung-fibrosis-and-phloretin-inhibits-lung-fibrosis
#10
Soo Jung Cho, Jong-Seok Moon, Chang-Min Lee, Augustine M K Choi, Heather W Stout-Delgado
RATIONAL: Aging is associated with metabolic diseases such as type 2 diabetes mellitus, cardiovascular disease, cancer and neurodegeneration. Aging contributes to common processes including metabolic dysfunction, DNA damage and reactive oxygen species generation. While glycolysis has been linked to cell growth and proliferation, the mechanisms by which the activation of glycolysis by aging regulates fibrogenesis in lung remain unclear. OBJECTIVES: To determine if glucose transport 1 (GLUT1)-induced glycolysis regulates age-dependent fibrogenesis of the lung...
December 20, 2016: American Journal of Respiratory Cell and Molecular Biology
https://www.readbyqxmd.com/read/27889640/inhibition-of-autophagy-with-bafilomycin-and-chloroquine-decreases-mitochondrial-quality-and-bioenergetic-function-in-primary-neurons
#11
Matthew Redmann, Gloria A Benavides, Taylor F Berryhill, Willayat Y Wani, Xiaosen Ouyang, Michelle S Johnson, Saranya Ravi, Stephen Barnes, Victor M Darley-Usmar, Jianhua Zhang
Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics...
April 2017: Redox Biology
https://www.readbyqxmd.com/read/27872984/cinnamic-acid-derivatives-enhance-the-efficacy-of-transarterial-embolization-in-a-rat-model-of-hepatocellular-carcinoma
#12
Luke R Wilkins, David L Brautigan, Hanping Wu, Hooman Yarmohammadi, Ewa Kubicka, Vlad Serbulea, Norbert Leitinger, Wendy Liu, John R Haaga
INTRODUCTION: We hypothesize that the combination of transarterial embolization (TAE) plus inhibition of lactate export will limit anaerobic metabolism and reduce tumor survival compared to TAE alone. The purpose of this study was to test this hypothesis in a rat model of hepatocellular carcinoma (HCC). METHODS: Rat N1-S1 hepatoma cells were assayed in vitro using the Seahorse XF analyzer to measure extracellular acidification (lactate excretion) comparing effects of the addition of caffeic acid (CA) or ferulic acid (FA) or UK-5099 with control...
March 2017: Cardiovascular and Interventional Radiology
https://www.readbyqxmd.com/read/27791989/stimulation-of-the-toll-like-receptor-3-promotes-metabolic-reprogramming-in-head-and-neck-carcinoma-cells
#13
Mathieu Veyrat, Sylvère Durand, Marion Classe, Tanja Matijevic Glavan, Natalie Oker, Nikiforos-Ioannis Kapetanakis, Xiaojun Jiang, Aurore Gelin, Philippe Herman, Odile Casiraghi, David Zagzag, David Enot, Pierre Busson, Benjamin Vérillaud
In this study, a possible link between the innate immune recognition receptor TLR3 and metabolic reprogramming in Head and Neck carcinoma (HNC) cells was investigated. The effects of TLR3 stimulation/knock-down were assessed under several culture conditions in 4 HNC cell-lines by cell growth assays, targeted metabolomics, and glycolysis assays based on time-resolved analysis of proton release (Seahorse analyzer). The stimulation of TLR3 by its synthetic agonist Poly(A:U) resulted in a faster growth of HNC cells under low foetal calf serum conditions...
October 25, 2016: Oncotarget
https://www.readbyqxmd.com/read/27784195/mitochondrial-spare-respiratory-capacity-is-negatively-correlated-with-nuclear-reprogramming-efficiency
#14
Yan Zhou, Rasha Abdelkadhem Al-Saaidi, Paula Fernandez-Guerra, Kristine K Freude, Rikke Katrine Jentoft Olsen, Uffe Birk Jensen, Niels Gregersen, Poul Hyttel, Lars Bolund, Lars Aagaard, Peter Bross, Yonglun Luo
Nuclear reprogramming efficiency has been shown to be highly variable among different types of somatic cells and different individuals, yet the underlying mechanism remains largely unknown. Several studies have shown that reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) requires remodeling of mitochondria and a metabolic shift from an oxidative state to a more glycolytic state. In this study, we evaluated the nuclear reprogramming efficiency in relation to mitochondrial bioenergetic parameters of fibroblasts from seven different human individuals...
February 1, 2017: Stem Cells and Development
https://www.readbyqxmd.com/read/27764131/generating-rho-0-cells-using-mesenchymal-stem-cell-lines
#15
Mercedes Fernández-Moreno, Tamara Hermida-Gómez, M Esther Gallardo, Andrea Dalmao-Fernández, Ignacio Rego-Pérez, Rafael Garesse, Francisco J Blanco
INTRODUCTION: The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. METHODOLOGY: Two immortalized hMSC lines (3a6 and KP) were used; 143B...
2016: PloS One
https://www.readbyqxmd.com/read/27741233/extracellular-redox-regulation-of-intracellular-reactive-oxygen-generation-mitochondrial-function-and-lipid-turnover-in-cultured-human-adipocytes
#16
Albert R Jones Iv, Tova Meshulam, Marcus F Oliveira, Nathan Burritt, Barbara E Corkey
BACKGROUND: Many tissues play an important role in metabolic homeostasis and the development of diabetes and obesity. We hypothesized that the circulating redox metabolome is a master metabolic regulatory system that impacts all organs and modulates reactive oxygen species (ROS) production, lipid peroxidation, energy production and changes in lipid turnover in many cells including adipocytes. METHODS: Differentiated human preadipocytes were exposed to the redox couples, lactate (L) and pyruvate (P), β-hydroxybutyrate (βOHB) and acetoacetate (Acoc), and the thiol-disulfides cysteine/ cystine (Cys/CySS) and GSH/GSSG for 1...
2016: PloS One
https://www.readbyqxmd.com/read/27718558/a-novel-methodology-for-bioenergetic-analysis-of-plasmodium-falciparum-reveals-a-glucose-regulated-metabolic-shift-and-enables-mode-of-action-analyses-of-mitochondrial-inhibitors
#17
Tomoyo Sakata-Kato, Dyann F Wirth
Given that resistance to all drugs in clinical use has arisen, discovery of new antimalarial drug targets is eagerly anticipated. The Plasmodium mitochondrion has been considered a promising drug target largely based on its significant divergence from the host organelle as well as its involvement in ATP production and pyrimidine biosynthesis. However, the functions of Plasmodium mitochondrial protein complexes and associated metabolic pathways are not fully characterized. Here, we report the development of novel and robust bioenergetic assay protocols for Plasmodium falciparum asexual parasites utilizing a Seahorse Bioscience XFe24 Extracellular Flux Analyzer...
December 9, 2016: ACS Infectious Diseases
https://www.readbyqxmd.com/read/27710853/hyperglycemia-induced-damage-to-mitochondrial-respiration-in-renal-mesangial-and-tubular-cells-implications-for-diabetic-nephropathy
#18
Anna Czajka, Afshan N Malik
Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer...
September 17, 2016: Redox Biology
https://www.readbyqxmd.com/read/27702678/parental-care-improves-immunity-in-the-seahorse-hippocampus-erectus
#19
Tingting Lin, Dong Zhang, Xin Liu, Dongxue Xiao
In the present study, the sexual dimorphism in immune response in the seahorse Hippocampus erectus in which males compete for mates and invest heavily in parental care was assessed. Variability in immunocompetence in virginal seahorses with differing levels of sexual maturity (i.e., immaturity, early maturity and maturity) and with different mating statuses (i.e., virginal, experienced mating failure and experienced mating success) were analyzed by evaluating immune parameters in the plasma. Additionally, ultrastructural characteristics of the inner epithelium of the brood pouch were compared between males that had experienced mating failure and those that had succeeded...
November 2016: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/27698021/macrophages-promote-oxidative-metabolism-to-drive-nitric-oxide-generation-in-response-to-trypanosoma-cruzi
#20
Sue-Jie Koo, Imran H Chowdhury, Bartosz Szczesny, Xianxiu Wan, Nisha J Garg
Trypanosoma cruzi is the causative agent of chronic chagasic cardiomyopathy. Why macrophages (mφs), the early responders to infection, fail to achieve parasite clearance is not known. Mouse (RAW 264.7) and human (THP-1 and primary) mφs were infected for 3 h and 18 h with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (nonpathogenic), which represent mφ stimulation and infection states, respectively. Mφs incubated with lipopolysaccharide and gamma interferon (LPS/IFN-γ) and with interleukin-4 (IL-4) were used as controls...
December 2016: Infection and Immunity
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