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Seahorse analyzer

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
Tomoyo Sakata-Kato, Dyann F Wirth
Given that resistance to all drugs in clinical use has arisen, discovery of new anti-malarial 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...
October 9, 2016: ACS Infectious Diseases
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
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...
October 1, 2016: Fish & Shellfish Immunology
Sue-Jie Koo, Imran Chowdhury, Bartosz Szczesny, Xianxiu Wan, Nisha J Garg
BACKGROUND: 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. METHODS: Mouse (RAW 264.7) and human (THP-1 and primary) mϕs were infected with T. cruzi TcI isolates, SylvioX10/4 (SYL, virulent) and TCC (non-pathogenic), for 3 h and 18 h that represent mϕ stimulation and infection states, respectively. Mϕs incubated with LPS/IFN-γ and IL-4 were used as controls...
October 3, 2016: Infection and Immunity
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
N C N Perera, G I Godahewa, Jehee Lee
Copper-zinc-superoxide dismutase (CuZnSOD) from Hippocampus abdominalis (HaCuZnSOD) is a metalloenzyme which belongs to the ubiquitous family of SODs. Here, we determined the characteristic structural features of HaCuZnSOD, analyzed its evolutionary relationships, and identified its potential immune responses and biological functions in relation to antioxidant defense mechanisms in the seahorse. The gene had a 5' untranslated region (UTR) of 67 bp, a coding sequence of 465 bp and a 3' UTR of 313 bp. The putative peptide consists of 154 amino acids...
October 2016: Fish & Shellfish Immunology
Michaela C Hohnholt, Vibe H Andersen, Lasse K Bak, Helle S Waagepetersen
Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A...
August 22, 2016: Neurochemical Research
David Liuwantara, Yi Vee Chew, Emmanuel J Favaloro, Joanne M Hawkes, Heather L Burns, Philip J O'Connell, Wayne J Hawthorne
INTRODUCTION: The instant blood-mediated inflammatory reaction (IBMIR) causes major loss of islets after transplantation and consequently represents the initial barrier to survival of porcine neonatal islet cell clusters (NICC) after xenotransplantation. METHODS: This study used novel assays designed to characterize the various immunologic components responsible for xenogeneic IBMIR to identify initiators and investigate processes of IBMIR-associated coagulation, complement activation and neutrophil infiltration...
June 2016: Transplantation Direct
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
Sonali Nashine, Marilyn Chwa, Mina Kazemian, Kunal Thaker, Stephanie Lu, Anthony Nesburn, Baruch D Kuppermann, M Cristina Kenney
PURPOSE: Variations in mitochondrial DNA (mtDNA) and abnormalities in the complement pathways have been implicated in the pathogenesis of age-related macular degeneration (AMD). This study was designed to determine the effects of mtDNA from AMD subjects on the complement pathway. METHODS: Transmitochondrial cybrids were prepared by fusing platelets from AMD and age-matched Normal subjects with Rho0 (lacking mtDNA) human ARPE-19 cells. Quantitative PCR and Western blotting were performed to examine gene and protein expression profiles, respectively, of complement markers in these cybrids...
2016: PloS One
Steven Mink, Subir K Roy Chowdhury, Jose Gotes, Zhao-Qin Cheng, Krika Kasian, Paul Fernyhough
BACKGROUND: The development of lactic acidemia (LA) in septic shock (SS) is associated with an ominous prognosis. We previously showed that the mechanism of LA in SS may relate to impaired hepatic uptake of lactate, but the mechanism was not clear. Uptake of lactate by the liver occurs by a membrane-associated, pH-dependent, antiport system known as the monocarboxylate transporter. In the hepatocyte, lactate can then be metabolized by oxidative phosphorylation or converted to glucose in the cytosol...
December 2016: Intensive Care Medicine Experimental
M Kubota, Y B Shui, M Liu, F Bai, A J Huang, N Ma, D C Beebe, C J Siegfried
PURPOSE: The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells. METHODS: Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro...
August 2016: Free Radical Biology & Medicine
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
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...
2016: International Journal of Molecular Sciences
Riccardo Natoli, Matt Rutar, Yen-Zhen Lu, Joshua A Chu-Tan, Yuwei Chen, Kartik Saxena, Michele Madigan, Krisztina Valter, Jan M Provis
PURPOSE: Light is a requirement for the function of photoreceptors in visual processing. However, prolonged light exposure can be toxic to photoreceptors, leading to increased reactive oxygen species (ROS), lipid peroxidation, and photoreceptor cell death. We used the 661W mouse cone photoreceptor-like cell line to study the effects of pyruvate in protecting these cells from light-induced toxicity. METHODS: 661W cells were exposed to 15,000 lux continuous bright light for 5 hours and incubated in Dulbecco's modified eagle medium (DMEM) with various concentrations of pyruvate...
May 23, 2016: Current Eye Research
Gavin Pharaoh, Daniel Pulliam, Shauna Hill, Kavithalakshmi Sataranatarajan, Holly Van Remmen
Mice deficient in the electron transport chain (ETC) complex IV assembly protein SURF1 have reduced assembly and activity of cytochrome c oxidase that is associated with an upregulation of components of the mitochondrial unfolded protein response (UPR(MT)) and increased mitochondrial number. We hypothesized that the upregulation of proteins associated with the UPR(MT) in response to reduced cytochrome c oxidase activity in Surf1(-/-) mice might contribute to increased stress resistance. To test this hypothesis we asked whether primary cultures of fibroblasts from Surf1(-/-) mice exhibit enhanced resistance to stressors compared to wild-type fibroblasts...
August 2016: Redox Biology
James Hamilton, Jessica J Pellman, Tatiana Brustovetsky, Robert A Harris, Nickolay Brustovetsky
Alterations in oxidative metabolism and defects in mitochondrial Ca(2+) handling have been implicated in the pathology of HD, but existing data are contradictory. We investigated the effect of human mHtt fragments on oxidative metabolism and Ca(2+) handling in isolated brain mitochondria and cultured striatal neurons from the R6/2 mouse model of HD. Nonsynaptic and synaptic mitochondria isolated from the brains of R6/2 mice had similar respiratory rates and Ca(2+) uptake capacity compared to mitochondria from WT mice...
April 30, 2016: Human Molecular Genetics
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
Yi Yang, Lei Zhang, David R Lynch, Thomas Lukas, Kreshnik Ahmeti, Patrick M A Sleiman, Eanna Ryan, Kimberly A Schadt, Jordan H Newman, Han-Xiang Deng, Nailah Siddique, Teepu Siddique
OBJECTIVE: To identify the genetic defect for adult-onset primary lateral sclerosis (PLS) in a family with 5 patients. METHODS: Whole-exome sequencing was performed to identify the shared genetic variants in 3 affected members in a PLS family with 5 affected individuals. Sanger sequencing was used for validation of the variants and for cosegregation analysis. Mitochondrial activity for both patients and unaffected siblings was measured using a SeaHorse metabolic analyzer...
April 2016: Neurology. Genetics
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