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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/28245898/glutamate-impairs-mitochondria-aerobic-respiration-capacity-and-enhances-glycolysis-in-cultured-rat-astrocytes
#2
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
#3
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/28163917/mitochondrial-mutations-and-metabolic-adaptation-in-pancreatic-cancer
#4
Rae-Anne Hardie, Ellen van Dam, Mark Cowley, Ting-Li Han, Seher Balaban, Marina Pajic, Mark Pinese, Mary Iconomou, Robert F Shearer, Jessie McKenna, David Miller, Nicola Waddell, John V Pearson, Sean M Grimmond, Leonid Sazanov, Andrew V Biankin, Silas Villas-Boas, Andrew J Hoy, Nigel Turner, Darren N Saunders
BACKGROUND: Pancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited...
2017: Cancer & Metabolism
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
#5
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
#6
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/27872984/cinnamic-acid-derivatives-enhance-the-efficacy-of-transarterial-embolization-in-a-rat-model-of-hepatocellular-carcinoma
#7
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/27816874/prevailing-vitamin-d-status-influences-mitochondrial-and-glycolytic-bioenergetics-in-peripheral-blood-mononuclear-cells-obtained-from-adults
#8
Emily K Calton, Kevin N Keane, Mario J Soares, Jordan Rowlands, Philip Newsholme
BACKGROUND: Circulating peripheral blood mononuclear cells (PBMCs) are exposed to metabolic and immunological stimuli that influence their functionality. We hypothesized that prevailing vitamin D status [25(OH)D] would modulate the bioenergetic profile of PBMCs derived from humans. MATERIALS AND METHODS: 38 participants (16 males, 22 females) ranging in body fat from 14-51% were studied. PBMCs were isolated from whole blood, counted and freshly seeded for bioenergetic analysis using the Seahorse XF(e)96 flux analyser...
December 2016: Redox Biology
https://www.readbyqxmd.com/read/27604740/advanced-and-high-throughput-method-for-mitochondrial-bioenergetics-evaluation-in-neurotrauma
#9
Jignesh D Pandya, Patrick G Sullivan, Lai Yee Leung, Frank C Tortella, Deborah A Shear, Ying Deng-Bryant
Mitochondrial dysfunction is one of the key posttraumatic neuropathological events observed in various experimental models of traumatic brain injury (TBI). The extent of mitochondrial dysfunction has been associated with the severity and time course of secondary injury following brain trauma. Critically, several mitochondrial targeting preclinical drugs used in experimental TBI models have shown improved mitochondrial bioenergetics, together with cortical tissue sparing and cognitive behavioral outcome. Mitochondria, being a central regulator of cellular metabolic pathways and energy producer of cells, are of a great interest for researchers aiming to adopt cutting-edge methodology for mitochondrial bioenergetics assessment...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27494181/identification-of-kca3-1-channel-as-a-novel-regulator-of-oxidative-phosphorylation-in-a-subset-of-pancreatic-carcinoma-cell-lines
#10
Ilya Kovalenko, Andrea Glasauer, Laura Schöckel, Daniel R P Sauter, Alexander Ehrmann, Florian Sohler, Andrea Hägebarth, Ivana Novak, Sven Christian
Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries and overall 5-year survival rates of less than 5%. The most frequent mutations in PDAC are gain-of-function mutations in KRAS as well as loss-of-function mutations in p53. Both mutations have severe impacts on the metabolism of tumor cells. Many of these metabolic changes are mediated by transporters or channels that regulate the exchange of metabolites and ions between the intracellular compartment and the tumor microenvironment...
2016: PloS One
https://www.readbyqxmd.com/read/27392747/bioenergetic-profiling-of-trypanosoma-cruzi-life-stages-using-seahorse-extracellular-flux-technology
#11
Sheena Shah-Simpson, Camila F A Pereira, Peter C Dumoulin, Kacey L Caradonna, Barbara A Burleigh
Energy metabolism is an attractive target for the development of new therapeutics against protozoan pathogens, including Trypanosoma cruzi, the causative agent of human Chagas disease. Despite emerging evidence that mitochondrial electron transport is essential for the growth of intracellular T. cruzi amastigotes in mammalian cells, fundamental knowledge of mitochondrial energy metabolism in this parasite life stage remains incomplete. The Clark-type electrode, which measures the rate of oxygen consumption, has served as the traditional tool to study mitochondrial energetics and has contributed to our understanding of it in T...
August 2016: Molecular and Biochemical Parasitology
https://www.readbyqxmd.com/read/27271893/mitochondrial-stress-tests-using-seahorse-respirometry-on-intact-dictyostelium-discoideum-cells
#12
Sui Lay, Oana Sanislav, Sarah J Annesley, Paul R Fisher
Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27231905/role-of-mitochondrial-dna-copy-number-alteration-in-human-renal-cell-carcinoma
#13
Chen-Sung Lin, Hui-Ting Lee, Ming-Huei Lee, Siao-Cian Pan, Chen-Yeh Ke, Allen Wen-Hsiang Chiu, Yau-Huei Wei
We investigated the role of mitochondrial DNA (mtDNA) copy number alteration in human renal cell carcinoma (RCC). The mtDNA copy numbers of paired cancer and non-cancer parts from five resected RCC kidneys after radical nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively...
May 25, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27127201/mitochondrial-reactive-oxygen-species-mediate-lysophosphatidylcholine-induced-endothelial-cell-activation
#14
Xinyuan Li, Pu Fang, Yafeng Li, Yin-Ming Kuo, Andrew J Andrews, Gayani Nanayakkara, Candice Johnson, Hangfei Fu, Huimin Shan, Fuyong Du, Nicholas E Hoffman, Daohai Yu, Satoru Eguchi, Muniswamy Madesh, Walter J Koch, Jianxin Sun, Xiaohua Jiang, Hong Wang, Xiaofeng Yang
OBJECTIVE: Hyperlipidemia-induced endothelial cell (EC) activation is considered as an initial event responsible for monocyte recruitment in atherogenesis. However, it remains poorly defined what is the mechanism underlying hyperlipidemia-induced EC activation. Here, we tested a novel hypothesis that mitochondrial reactive oxygen species (mtROS) serve as signaling mediators for EC activation in early atherosclerosis. APPROACH AND RESULTS: Metabolomics and transcriptomics analyses revealed that several lysophosphatidylcholine (LPC) species, such as 16:0, 18:0, and 18:1, and their processing enzymes, including Pla2g7 and Pla2g4c, were significantly induced in the aortas of apolipoprotein E knockout mice during early atherosclerosis...
June 2016: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/27111768/triclosan-is-a-mitochondrial-uncoupler-in-live-zebrafish
#15
Juyoung Shim, Lisa M Weatherly, Richard H Luc, Maxwell T Dorman, Andy Neilson, Ryan Ng, Carol H Kim, Paul J Millard, Julie A Gosse
Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here, we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24-hour post-fertilization (hpf) zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XF(e) 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well...
December 2016: Journal of Applied Toxicology: JAT
https://www.readbyqxmd.com/read/27038461/measuring-bioenergetics-in-t-cells-using-a-seahorse-extracellular-flux-analyzer
#16
Gerritje J W van der Windt, Chih-Hao Chang, Erika L Pearce
This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways...
April 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/26803635/analysis-of-tlr-induced-metabolic-changes-in-dendritic-cells-using-the-seahorse-xf-e-96-extracellular-flux-analyzer
#17
Leonard R Pelgrom, Alwin J van der Ham, Bart Everts
Engagement of Toll-like receptors (TLRs) on dendritic cells (DCs) triggers the expression of a large set of genes involved in DC activation and maturation, which allow them to act efficiently as antigen-presenting cells. Recently, it has become clear that TLR signalling in DCs also results in dramatic metabolic changes that are integral to their changed biology. Here, we describe a detailed protocol on how DC metabolism can be studied after TLR stimulation using the 96-well format Extracellular Flux (XF(e)96) Analyzer from Seahorse Bioscience, a machine that allows one to simultaneously assess rates of oxidative phosphorylation and glycolysis in real-time, in live cells and in a high-throughput manner...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/26523474/seahorse-xfe-24-extracellular-flux-analyzer-based-analysis-of-cellular-respiration-in-caenorhabditis-elegans
#18
Anthony L Luz, Latasha L Smith, John P Rooney, Joel N Meyer
Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XF(e) 24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans...
November 2, 2015: Current Protocols in Toxicology
https://www.readbyqxmd.com/read/26236724/hypoxia-induces-a-metabolic-shift-and-enhances-the-stemness-and-expansion-of-cochlear-spiral-ganglion-stem-progenitor-cells
#19
Hsin-Chien Chen, Jen-Tin Lee, Cheng-Ping Shih, Ting-Ting Chao, Huey-Kang Sytwu, Shiue-Li Li, Mei-Cho Fang, Hang-Kang Chen, Yi-Chun Lin, Chao-Yin Kuo, Chih-Hung Wang
Previously, we demonstrated that hypoxia (1% O2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer...
2015: BioMed Research International
https://www.readbyqxmd.com/read/26204657/bioenergetic-characterization-of-h9c2-cells-using-the-extracellular-flux-analyzer
#20
Ileana Scurtu, A Sturza, Ioanna Zinuca Pavel, Roxana Popescu, Andreea Privistirescu, Oana Maria Duicu, Danina Mirela Muntean
UNLABELLED: The aim of the present work was to standardize the working methodology for assessing the bioenergetic profile of H9c2 cardiomyoblasts cells, with reference to the optimization of cell seeding number and the establishment of favorable concentrations for the classic modulators of mitochondrial respiratory function, in particular the one of a classical uncoupler, FCCP. MATERIAL AND METHODS: The extracellular flux analyzer (XF, Seahorse Bioscience) is a novel high-throughput instrument able to monitor the metabolism of living cells by simultaneously measuring mitochondrial respiration and glycolysis...
April 2015: Revista Medico-chirurgicală̆ a Societă̆ţ̜ii de Medici ş̧i Naturaliş̧ti Din Iaş̧i
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