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'''Bioenergetics Vienna''', 1st Bioenergetics DE-CH-AT Meeting +
'''Brain bioenergetics – From behavior to pathology, Lausanne, CH''' +
'''COST MitoEAGLE WG and MC Meeting, 2019, Belgrade, Serbia.''' +
'''Demo O2k-Course at MiPsummer 2009.''' Baton Rouge, USA; 2009 June 21. +
'''ESCI meeting, Genoa, IT''' +
'''FASEB, West Palm Beach, FL, US''' +
'''FAT4BRAIN 1st Online ESR Workshop, 2020''' +
'''FAT4BRAIN 2nd Online ESR Workshop, 2021''' +
'''FAT4BRAIN 3rd Online ESR Workshop, 2022''' +
'''FAT4BRAIN 4th Online ESR Workshop, 2022''' +
'''FAT4BRAIN ESR Workshop, 2023''' +
'''FAT4BRAIN Final rview meeting, Virtual, 2023''' +
'''FAT4BRAIN Kick-off meeting, Riga, Latvia, 2019''' +
'''FAT4BRAIN Midterm Review meeting, Virtual, 2021''' +
'''MiP2023, Obergurgl, Austria, 2023.''' +
'''Mitochondrial Medicine 2017, Washington DC, USA.''' +
'''Oroboros Distributor Meeting'''. Virtual. +
'''Oroboros Installation and startup support session'''. +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. +
'''Research to Practice 2016, Singapore, SG''' +
'''SFRR-E 2016, Budapest, HU; [http://sfrr-e-2016.hu/ SFRR-E 2016].''' +
''By author request, this abstract is not made available online.'' +
''No abstract available'' +
10th European Algae Industry Summit, Reykjavik, Iceland, 2020 +
10th Int. CeBiTec Research Conference, Bielefeld, Germany, 2021 +
10th Italian Meeting on Mitochondrial Diseases , Virtual, 2020 +
11th Annual Congress of Cardiology, Suzhou, China, 2019 +
13th Targeting Mitochondria Congress, Berlin, 2022 +
13th congress of EBSA, Vienna, Austria, 2021 +
13th ÖGMBT Annual Meeting, Virtual, 2021 +
14th congress of EBSA, Stockholm, Sweden, 2023 +
17th Chinese Biophysics Congress, Tianjin , China, 2019 +
18th Annual Meeting of the Japan Mitochondrial Association, Kurume, 2018 +
19th Chinese Biophysics congress, Anhui Province, China, 2021 +
2019 World conference on Movement and Cognition, Tel-Aviv, Israel, 2019 +
2020 World conference on Movement and Cognition, Paris, France, 2020 +
20th Biennial Meeting of SFRR International, Virtual, 2021 +
20th International Botanical Congress (IBC), Madrid, ES, 2024 +
22nd GFB conference, Bedoin, France, 2023 +
24th Kalorimetrietage, Braunschweig, Germany, 2021. +
25th Krakow Conference on Endothelium, Krakow, Poland. +
28th ECSS Congress, Paris, France, 2023 +
28th Congress of the Polish Physiological Society, Virtual, 2021 +
2nd Annual Mitochondria-Targeted Drug Development, Boston MA, US, 2022. +
2nd International Munich ROS Meeting, Munich, Germany, 2018 +
2nd Mitochondria Conference, Lisbon, Portugal, 2023. +
32nd APS Annual Convention, Chicago, USA, 2020 +
37th Annual Meeting of the ISHR-ES, Porto, Portugal, 2023 +
3rd edition - Metabolism and Cancer Meeting, Marseille, France, 2019 +
55th ESCI meeting, Virtual, 2021 +
56th ESCI meeting, Bari, Italy, 2022 +
57th ESCI meeting, Prague, Czech Republic, 2023 +
6th Research Day, Innsbruck, Austria, 2023 +
7th European Phycological Congress, Zagreb, Croatia, 2019 +
9th Annual Conference of the SMRM, Hyderabad, India. +
9th ÖGMBT Annual Meeting & 8th Life Science Meeting, Innsbruck, Austria +
A brief accout of '''mitochondrial mitophagy'''. +
A brief accout of the '''sirtuin family'''. +
'''Authors:''' [[Axelrod Christopher L]] … '''Authors:''' [[Axelrod Christopher L]], [[Kirwan John P]] Obesity mediates the onset of lipid-induced insulin resistance, increasing the risk of type 2 diabetes. The inability of mitochondria to maintain core functions such as ATP synthesis, redox homeostasis, organelle quality control, and/or preservation of inheritance is proposed to link obesity-related insulin resistance to the onset and progression of type 2 diabetes, yet evidence remains elusive. To parse out the contributions of obesity versus peripheral insulin resistance, healthy weight adults were infused with an intralipid solution followed by evaluation of skeletal muscle mitochondrial function. The lipid infusion reduced insulin sensitivity and dampened mitochondrial membrane potential while increasing markers of mitochondrial fission and increasing the presence of autophagic vesicles, consistent with activation of the quality control machinery. Despite this, respiratory capacity and mitochondrial content were unaltered. From these studies, we concluded that activation of mitochondrial fission and quality control were early events in the onset of insulin resistance to defend cellular energy homeostasis. Subsequently, we conducted a cross-sectional analysis of individuals across the insulin sensitivity spectrum. We observed that markers of fission and quality control were markedly altered in patients with obesity and type 2 diabetes relative to obesity alone and healthy weight despite no apparent differences in respiratory capacity. Mitochondrial volume was incrementally lower in patients with obesity and type 2 diabetes relative to healthy weight. Collectively, we conclude that preservation of bioenergetic function in patients with obesity and type 2 diabetes is achieved by chronic activation of the quality control machinery which occurs at the expense of mitochondrial volume.y which occurs at the expense of mitochondrial volume. +
'''Authors:''' [[Barkova Daria]] … '''Authors:''' [[Barkova Daria]], [[Ukropec Jozef]], [[Nemec Michal]], [[Slobodova L]], [[Schoen M]], [[Tirpakova V]], [[Krumpolec P]], [[Sumbalova Zuzana]], [[Vician M]], [[Sedliak M]], [[Ukropcova Barbara]] '''Introduction:''' Regular exercise supports healthy ageing and reduces risk of elderly chronic diseases. Respirometry is an important tool in understanding the physiological adaptations in response to physical activity at cellular level. Previously, we showed that 3-month exercise training increases muscle metabolism in the elderly. Present study is aimed to assess the effects of long-term training on muscle oxidative capacity in the subset of individuals continuing regular training for 5 years. '''Methods:''' Volunteers (n=60, 66.9±1.2 years, 27.1±3.9 kg/m2) were recruited for 3-month intervention study: 36 of them underwent aerobic-strength training, 24 volunteers were active controls. A volunteer subpopulation continued aerobic-strength training for next 5 years (n=15), and is compared to non-exercising controls (n=15). Body composition, glucose tolerance, insulin sensitivity and other metabolic parameters were assessed. Samples of m. vastus lateralis obtained by biopsy were used for measurement of muscle mitochondria oxygen consumption by O2k high-resolution respirometry, applying RP1 SUIT protocol. '''Results and discussion:''' Three-month exercise training enhanced muscle mitochondrial respiration rate in the elderly undergoing exercise training compared to controls. So far, two individuals completed follow up phenotyping after 5 years training. A slight deterioration in anthropometric (increased BMI by ~ 8 % and visceral fat content by ~ 36%) and metabolic parameters was observed, together with a reduction in muscle mitochondrial respiration (by ~ 15 %). Short-term training improved the whole-body and muscle metabolism in the elderly. Obtaining data from exercising and non-exercising cohorts (currently ongoing) will allow us to assess the impact of a long-term intervention.ongoing) will allow us to assess the impact of a long-term intervention. +
'''Authors:''' [[Brunetta Henver Simionato]] … '''Authors:''' [[Brunetta Henver Simionato]], [[Palermo Ruiz Gabriel]], [[Ludwig Raissa]], [[Ruberti Olivia]], [[Bechara Luiz]], [[Consonni Silvio]], [[Rodrigues Bruno]], [[Ferreira Julio Cesar B]], [[Mori Marcelo AS]] The negative effects of high-fat high-sucrose (HFHS) diet consumption on heart function are exacerbated in mice lacking DICER in adipocytes (AdicerKO). These findings suggest a protective role of adipocyte-derived microRNAs on heart physiology. Exercise training is known to have a protective role in cardiometabolic diseases. However, it is not known whether chronic aerobic training is able to rescue heart dysfunction in HFHS-fed AdicerKO mice. Here, we fed AdicerKO mice with a HFHS diet for 12 weeks, after confirming the deleterious effects of the diet on these mice, we submitted them to moderate aerobic training for 8 weeks, 5 days/week for 60 minutes each section while keeping them on HFHS-diet. Chronic aerobic training restored end-systolic volume and stroke volume in the hearts of HFHS-fed AdicerKO mice without changing ejection fraction. In addition, aerobic exercise increased left ventricle diameter in both, systolic and diastolic, phases. Notably, HFHS-fed AdicerKO-trained mice presented lower heart rate with no differences in systolic blood pressure compared to HFHS-fed AdicerKO sedentary mice. Mechanistically, chronic exercise training lowered mitochondrial H2O2 emission and oxidative stress alongside greater lipid- and succinate-supported mitochondrial respiration. Importantly, these effects were not followed by changes in triacylglycerol content within the left ventricle or fibrosis. In summary, chronic aerobic training is capable to rescue heart function of HFHS-fed AdicerKO mice in association with improvements in mitochondrial bioenergetics and redox balance.ssociation with improvements in mitochondrial bioenergetics and redox balance. +
'''Authors:''' [[Cardoso Luiza HD]] … '''Authors:''' [[Cardoso Luiza HD]], [[Donnelly Chris]], [[Komlodi Timea]], [[Doerrier Carolina]], [[Gnaiger Erich]] '''Introduction:''' Multiple mt-matrix dehydrogenases reduce NAD+ to NADH+H+, which is oxidized by CI (N-junction). Convergent electron flow through several mt-Complexes (CI, CII, CETF, etc) reduces electron transfer system (ETS)-reactive ubiquinone (UQ) to ubiquinol (UQH2), which is oxidized by CIII (Q-junction). The aim of our study was to analyze the relationships between the N- and Q-redox states and electron transfer rates. '''Methods:''' Respiration and N- or Q-redox fractions were measured simultaneously with the Oroboros NextGen-O2k. Multiple protocols were used with sequential titrations of substrates, inhibitors, and uncouplers [1, 2]: N-pool with pyruvate&glutamate&malate, mouse liver mitochondria; Q-pool with succinate&rotenone, octanoylcarnitine&malate or palmitoylcarnitine&malate, permeabilized HEK 293T. After substrates, ADP, CCCP and antimycin A were titrated. '''Results and discussion:''' Varying energy supply upstream of the Q-junction by using combinations of substrates and ETS-inhibitors in the noncoupled state, the Q-pool became reduced in direct proportion to respiration. In contrast, varying downstream energy demand in the absence of ADP (LEAK), by ADP activation (OXPHOS), and by uncoupler titrations (ET capacity), the N- and Q-pools were reduced in indirect proportion to respiration. The opposite correlations between redox state and respiratory rate were explained by the contrasting effects of varying electron push from different fuel substrates of the ETS or electron pull modulated by coupling and corresponding energy demand. Special emphasis on the interaction between fatty acid oxidation, CI, and CII – all involving separate electron entries into the Q-junction [3] – is particularly relevant in the context of obesity and bioenergetics studies.# Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. https://doi.org/10.26124/bec:2021-0003# Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. https://doi.org/10.26124/bec:2020-0002 # Gnaiger E (2023) Complex II ambiguities ― FADH2 in the electron transfer system. https://doi.org/10.26124/mitofit:2023-0003.v4 E (2023) Complex II ambiguities ― FADH2 in the electron transfer system. https://doi.org/10.26124/mitofit:2023-0003.v4 +
'''Authors:''' [[Chicco Adam J]] … '''Authors:''' [[Chicco Adam J]], [[Le Catherine H]], [[Mulligan Christopher M]], [[Whitcomb Luke A]], [[Evans Amanda E]], [[Routh Melissa A]], [[Sparanga Genevieve C]] Cardiolipin (CL) is a tetra-acyl mitochondrial phospholipid that supports the optimal function of several mitochondrial membrane proteins and processes. In the healthy mammalian heart, the majority of CL species contain four linoleate acyl chains (L4CL). A marked depletion of cardiac L4CL is paralleled by an increase in CL species containing docosahexaenoic acid (DHA) in hyperphagic obese (''Lepob''; OB) mice despite no change in dietary fat composition [1], but the mechanisms and functional relevance of these changes are unclear. We hypothesized that this shift in CL composition results from increased activity of delta-6 desaturase (D6D), the rate limiting enzyme in the biosynthesis of DHA and conversion of linoleate into highly unsaturated ω-6 fatty acids, by altering the distribution of fatty acids available for CL remodeling. To test this, we administered the selective D6D inhibitor SC-26196 (100 mg/kg/d in chow) to 4-5 month-old OB or lean (C57Bl/6) mice for 4 weeks. As hypothesized, D6D inhibition reversed obesity-related changes in cardiac CL composition, restoring L4CL and DHA-enriched CL species to within 5 % of levels in lean mice, which paralleled reciprocal shifts in the linoleate and DHA levels of total myocardial phospholipids. Obesity-related decreases in cardiac mitochondrial respiratory control by ADP (with NS pathway substrates) and greater mitochondrial H2O2 release during both LEAK and OXPHOS states were also abolished by D6D inhibition. These results corroborate accumulating evidence that cardiac CL composition is strongly influenced by the membrane fatty acids available for CL remodeling [2-3], and may impact the bioenergetic efficiency of mitochondrial respiration. # Han X, Yang J, Yang K, ZhongdancZ, Abendschein DR, Gross RW (2007) Alterations in Myocardial Cardiolipin Content and Composition Occur at the Very Earliest Stages of Diabetes: A Shotgun Lipidomics Study Biochemistry https://doi.org/10.1021/bi7004015 # Le CH et al. (2014) Delta-6-desaturase links PUFA metabolism with phospholipid remodeling and disease progression in heart failure. https://doi.org/10.1161/CIRCHEARTFAILURE.113.000744 # Oemer G, Edenhofer ML, Wohlfarter Y, Lackner K, Leman G, Koch J, Cardoso LHD, Lindner HH, Gnaiger E, Dubrac S, Zschocke J, Keller MA (2021) Fatty acyl availability modulates cardiolipin composition and alters mitochondrial function in HeLa cells. https://doi.org/10.1016/j.jlr.2021.100111 and alters mitochondrial function in HeLa cells. https://doi.org/10.1016/j.jlr.2021.100111 +
'''Authors:''' [[Giordano Luca]] … '''Authors:''' [[Giordano Luca]], [[Nolte A]], [[Wittig Ilka]], [[Pak Oleg]], [[Knoepp F]], [[Ramser K]], [[Wahl J]], [[Cabrera A]], [[Huettemann Maik]], [[Grossman Lawrence]], [[Pecina Petr]], [[Ghofrani HA]], [[Seeger W]], [[Weissmann Norbert]], [[Giehl K]], [[Sommer Natascha]] '''Introduction:''' Hypoxia in the lung alveoli triggers the contraction of the small precapillary pulmonary arteries, i.e., hypoxic pulmonary vasoconstriction (HPV), avoiding life-threatening hypoxemia. Pulmonary arterial smooth muscle cells (PASMCs) are involved in HPV, with the mitochondrial cytochrome c oxidase (COX) subunit 4 isoform 2 (Cox4i2) playing an essential role in the acute oxygen sensing1. Nonetheless, the molecular mechanism by which Cox4i2 sensitizes the whole COX remains unclear. '''Methods:''' We analysed superoxide production by MitoSOX, oxygen consumption by high-resolution respirometry, redox changes of the electron transport system (ETS) by RAMAN spectroscopy, and supercomplex formation by blue native gel analysis of PASMCs isolated from wild type (WT) and Cox4i2 knockout mice (Cox4i2 KO) exposed to normoxia or hypoxia. To figure out the role of Cox4i2-specific cysteine residues we generated mouse epithelial (CMT167) cells overexpressing either Cox4i1, or WT Cox4i2, or Cox4i2 mutants (C41S, C55A, C109S), and we tested their superoxide production and oxygen affinity. '''Results:''' Respiration, abundance, and COX assembly were similar in WT and Cox4i2 KO PASMCs. On the contrary, hypoxia-induced production of superoxide and the reduction of ETS components (NADH, ubiquinol, cytochrome c) was prevented in Cox4i2 KO PASMCs. CMT167 cells expressing either Cox4i1, or Cox4i2 mutants lacked hypoxia-induced superoxide production, which was detected only in cells expressing WT Cox4i2. Overexpression of Cox4i1, or Cox4i2, or Cox4i2 mutants did not affect oxygen affinity. Our findings suggests that Cox4i2 does not alter superoxide production by rearrangement of supercomplexes, but by the reduction of the ETS, likely mediated by the cysteine residues.Sommer N, Hüttemann M, et al. Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing. Circ Res. 2017;121(4):424-38.r Acute Pulmonary Oxygen Sensing. Circ Res. 2017;121(4):424-38. +
'''Authors:''' [[Guerrier Lisa]] … '''Authors:''' [[Guerrier Lisa]], [[Malpuech-Brugere C]], [[Bacoeur-Ouzillou O]], [[Cassagnes L]], [[Pezet D]], [[Gagniere J]], [[Richard R]], [[Touron Julianne]] '''Introduction:''' Adipose tissue (AT), as an endocrine organ, plays an important role in health and diseases, but unlike skeletal muscle, its energy metabolism have been under investigated due to technical limitations. Nevertheless, according to recent studies, mitochondria could play a predominant role in AT disorders and their activity could depend on adiposity level (1-3). This study aims to evaluate mitochondrial activity and metabolism of human visceral and subcutaneous white AT and their relationship with body mass index (BMI) and composition. '''Methods:''' Sixty-two patients undergoing digestive surgery, without chemotherapy, nor parietal infection, have been included in the study with BMI ranging from 15.4 to 51.9 kg·m-2. Their body composition was assessed by computed tomographic (CT) image at third lumbar vertebra (L3). Mitochondrial function was measured in situ in digitonin-permeabilized adipocytes using high resolution respirometry and a substrate/inhibitor titration approach (Figure) (4,5). Protein accumulation of mitochondrial and lipid metabolism key elements was evaluated by Western-blot. '''Results and discussion:''' Results showed a negative correlation between maximal mitochondrial respiration and BMI (p<0.05) as well as with AT surface, regardless of the anatomical location, though, OXPHOS respiration was significantly higher in visceral (2.22±0.15 pmol·sec-1·mg-1) than in the subcutaneous AT (1.79±0.17 pmol·sec-1·mg-1). Thus, mitochondrial function can be studied with small amount of AT despite its low mitochondrial density and can be discriminated according to AT depot and BMI. Further analyses are required to know whether the observed differences are quantitative and/or qualitative, as well as to identify the mechanisms involved # Ling Y et al. (2019) Persistent low body weight in humans is associated with higher mitochondrial activity in white adipose tissue. https://doi.org/10.1093/ajcn/nqz144# Fischer B, Schöttl T, Schempp C, Fromme T, Hauner H, Klingenspor M, Skurk T (2015) Inverse relationship between body mass index and mitochondrial oxidative phosphorylation capacity in human subcutaneous adipocytes. https://doi.org/10.1152/ajpendo.00524.2014# Wessels B, Honecker J, Schöttl T, Stecher L, Klingenspor M, Hauner H, Skurk T (2019) Adipose Mitochondrial Respiratory Capacity in Obesity is Impaired Independently of Glycemic Status of Tissue Donors. https://doi.org/10.1002/oby.22435# Kraunsøe R, Boushel R, Neigaard Hansen C, Schjerling P, Qvortrup K, Støckel M, Mikines KJ, Dela F (2010) Mitochondrial respiration in subcutaneous and visceral adipose tissue from patients with morbid obesity. https://doi.org/10.1113/jphysiol.2009.184754# Sahl RE, Frederikke Høy Helms E, Schmücker M, Flensted-Jensen M, Ingersen A, Morville T, Dela F, Wulff Helge J, Larsen S (2021) Reliability and variation in mitochondrial respiration in human adipose tissue. https://doi.org/10.1080/21623945.2021.1991617en S (2021) Reliability and variation in mitochondrial respiration in human adipose tissue. https://doi.org/10.1080/21623945.2021.1991617 +
'''Authors:''' [[Holloway Graham P]] … '''Authors:''' [[Holloway Graham P]], [[Petrick Heather L]], [[van Loon LJC]] Mitochondria play a key role in metabolic homeostasis, with impaired mitochondrial biology directly linked with numerous pathological conditions, including skeletal muscle atrophy, insulin resistance and heart dysfunction. Our team has focused on identifying nutritional approaches that preserve mitochondrial bioenergetics as a preventative medicine approach. In particular, we have studied dietary nitrate, which can be consumed through foods such as beets and green leafy vegetables or supplementation, as this compound appears to positively affect mitochondrial bioenergetics in diverse tissues. Additionally, we have shown that dietary nitrate can prevent high-fat diet-induced cardiac dysfunction, whole-body insulin resistance, dyslipidemia, and hepatic dysfunction. Moreover, we have recently uncovered that nitrate prevents skeletal muscle disuse-mediated reductions in mitochondrial protein synthesis rates (FSR), mitochondrial protein content, respiration and prevented the normal increase mitochondrial reactive oxygen species (ROS) emission during limb immobilization. While these physiological outcomes are likely in part linked to the serial reduction of nitrate to systemic nitric oxide (NO)-mediated vasodilation, we have also utilized fecal microbial transplantation from nitrate-fed donors to prevent HFD-induced cardiac dysfunction in the absence of increasing serum nitrate or reducing blood pressure. Given these systemic, reproducible, and consistent effects, nitrate appears to represent a viable therapeutic approach to improve mitochondrial bioenergetics to combat compromised cardiometabolic health in diverse situations.promised cardiometabolic health in diverse situations. +
'''Authors:''' [[Jasinska Joanna]] … '''Authors:''' [[Jasinska Joanna]], [[Bednarczyk Piotr]], [[Kalenik B]], [[Kulawiak Bogusz]], [[Wrzosek A]], [[Szewczyk Adam]] Recent studies point out that mitochondria are not only a source of ATP in the cell, but more and more data indicate their role related to Ca2+ buffering, production of reactive oxygen species (ROS) and activation of intracellular signaling pathways of necrosis and apoptosis. Recent studies clearly indicate that mitochondrial potassium channels (mitoK) present in the inner mitochondrial membrane play an important protective role in the ischemia-reperfusion processes of myocardial cell damage. These results were obtained using low molecular weight chemicals. Due to the lack of selective modulators of potassium channels, we opted for an alternative approach to modulate the activity of mitoK channels by changing the redox state of the respiratory chain, which we have demonstrated in previous studies. Some respiratory chain proteins are thought to absorb infrared (IR) light. Cytochrome c oxidase (COX) may be important in these mechanisms because it has four metal redox centers: binuclear CuA, CuB, heme a, and heme a3. All these metal centers are able to absorb light waves in the IR region. Data obtained in our laboratory indicate that COX may be functionally linked to mitochondrial high-conductance Ca2+-activated potassium channels (mitoBKCa) in the U87 cell line1. Using the patch-clamp technique with the illumination system, we exposed the mitoBKCa channel. We observed that in the presence of ferricyanide, channel activity was inhibited and that mitoBK channel activity could be restored by 820 nm illumination, suggesting that COX is involved in the modulation of mitoBK channel activity.# Szewczyk A and Bednarczyk P (2018) Modulation of the Mitochondrial Potassium Channel Activity by Infrared Light. https://doi.org/10.1016/j.bpj.2017.11.288ed Light. https://doi.org/10.1016/j.bpj.2017.11.288 +
'''Authors:''' [[Karabatsiakis Alexander]] … '''Authors:''' [[Karabatsiakis Alexander]], [[Manrique Juan-Salinas]], [[Stoll Thomas]], [[Hennessy Thomas]], [[Hill Michelle M]], [[Dietrich Detlef E]] Major depressive disorder (MDD) is characterized by impairments in mental and physical performance. Despite intensified hypothesis-driven research, applicable biomarkers for MDD are missing. Research showed that MD is associated with impaired mitochondrial bioenergetic functioning in peripheral blood mononuclear cells (PBMC). However, deeper biomolecular insights into bioenergetic and associated biochemical changes in blood underlying the pathophysiology of MDD are necessary to identify new biomarker candidates. Here, the biochemistry of PBMC-surrounding blood was analyzed using a hypothesis-free biomarker identification approach combining metabolite and lipid fingerprinting. Biochemical fingerprints of serum were compared between female individuals (N = 44) with and without MDD. Serum extracts were separated by liquid chromatography and detected with time-of-flight mass spectrometry. The data was analyzed by multiple group comparisons and correlations, as well as two multivariate classification procedures. Next, our previously identified alterations in mitochondrial bioenergetics in PBMC were co-considered as an outcome for our biomarker identification approach. Consequently, the most promising compound was tested for correlation with mitochondrial respiration. Nine biomarker candidates discriminated between MDD and non-MDD with high predictive accuracy (90.9 %). The detected compounds are involved in lipid and amino acid-metabolism. ''9,10-dihydroxy-octadenedioic acid'' was revealed as a robust biomarker candidate with a predictive accuracy of 81.8 % and significant mean positive correlation with parameters of mitochondrial respiration (r = 0.31-0.48, p<0.01). Our fingerprinting results highlight novel biomarker candidates and associated pathways for MDD research. The unraveled biochemical pathways indicate a modulated association of MDD with inflammation, oxidative stress, and mitochondrial bioenergetics. The biomarker candidates have to be replicated in independent cohorts of all ages & sexes.be replicated in independent cohorts of all ages & sexes. +
'''Authors:''' [[Kopecky Jan]] … '''Authors:''' [[Kopecky Jan]], [[Zouhar Petr]], [[Janovska Petra]], [[Bardova K]], [[Otahal J]], [[Vrbacky Marek]], [[Mracek Tomas]], [[Adamcova K]], [[Lenkova L]], [[Funda J]], [[Cajka T]], [[Drahota Zdenek]], [[Stanic S]], [[Rustan Arild C]], [[Horakova Olga]], [[Houstek Josef]], [[Rosmeissl M]] Heat production is essential for maintaining a constant body temperature, and is an important component of energy balance. Well-described mechanisms involved in heat generation include shivering of muscle and non-shivering thermogenesis (NST) in brown adipose tissue (BAT). Thermogenesis in BAT, which is dependent on the presence of the mitochondrial protein UCP1, is the focus of interest for its potential use in the treatment of obesity. Other mechanisms of NST and their significance are relatively poorly understood. We have shown [1] that obesity-resistant A/J mice acclimated to cold failed to increase adrenergically stimulated NST in BAT and activated NST in skeletal muscle instead. Heat generation in muscle involved increased calcium ion cycling in the endoplasmic reticulum associated with higher mitochondrial oxidative activity. The involvement of different thermogenic mechanisms could be related to the different susceptibility to obesity. The resistance of A/J mice to obesity may result, at least in part, from their ability to activate NST in muscle. Such mechanism may provide a more promising way to treat obesity than potential therapies based on increasing thermogenesis in BAT, as the capacity of skeletal muscle of adult human to burn fat energy stores is several fold greater than in BAT. Thus, only a relatively small increase in thermogenesis in muscle could significantly reduce adipose tissue deposition. How to achieve such an increase is a challenge for further research. # Janovska P et al., 2023, Mol Metab. https://doi.org/10.1016/j.molmet.2023.101683Metab. https://doi.org/10.1016/j.molmet.2023.101683 +
'''Authors:''' [[Mahapatra Gargi]] … '''Authors:''' [[Mahapatra Gargi]], [[Gao Zhengrong]], [[Bateman James R III]], [[Lockhart Samuel Neal]], [[Bergstrom Jaclyn]], [[Craft Suzanne]], [[Molina Anthony JA]] Impaired glucose tolerance (IGT), including prediabetes and diabetes, increases risk of developing age related disorders, including Alzheimer’s disease (AD). We analyzed mitochondrial bioenergetics in platelets collected from 208 adults, 55 years and older, with or without insulin sensitivities (112 normoglycemics (NG), 96 IGTs). Platelets from IGT participants exhibited unique bioenergetic profiles exemplified by higher mitochondrial respiration than NG. IGT platelets exhibited higher glucose-dependent maximal (Max) and spare respiratory (SRC) capacities compared to NG, and higher fatty acid oxidation-dependent maximal coupled (MaxOXPHOS) and uncoupled (MaxETS) respiration compared to NG. Correlating bioenergetics from all 208 participants combined with glucose measures (OGTT_120, OGTT_AUC, and HbA1c) revealed significant positive associations. Most associations were unaltered with age, sex, and BMI adjustments. Further separating NG and IGT participants and correlating platelet respiration with glucose measures revealed distinct trends in NG versus IGT group. In NG, previously observed associations remained intact, and new significant positive associations emerged between platelet bioenergetics and HbA1c. Associations in IGT group were overall negative. Identifying systemic mitochondrial mechanisms that associate with glucose intolerance in older adults will help in monitoring pathological progression of AD in relation to comorbidities such as insulin sensitivity, and supports the development of minimally invasive biomarkers of AD.# Mahapatra G, Gao, Bateman JR III, Lockhart SN, Bergstrom J, DeWitt AR, Piloso JE, Kramer PA, Gonzalez-Armenta JL, Amick A, Casanova R, Craft S, Molina AJA (2022) Blood-Based Bioenergetic Profiling Reveals Differences in Mitochondrial Function Associated with Cognitive Performance and Alzheimer’s Disease Alzheimer's & Dementia 2022. https://doi.org/10.1002/alz.12731 & Dementia 2022. https://doi.org/10.1002/alz.12731 +
'''Authors:''' [[Nagwani Amit K]] … '''Authors:''' [[Nagwani Amit K]], [[Kaczmarek L]], [[Kmita H]] '''Introduction:''' Tardigrades are considered as one of the toughest animals on Earth due to their remarkable ability to withstand extreme condition. An example of these conditions is hypomagnetic field (HMF, static magnetic field with an intensity <5 μT), which is known to influence the metabolic processes including mitochondria functioning. However, very few studies considering HMF impact were performed for organisms able to survive under extreme conditions and considered as suitable for outer space colonization. Therefore, we decided to check the impact of HMF on the tardigrade ''Paramacrobiotus experimentalis'' focusing on mitochondria functionality reflected by the mitochondrial inner membrane potential (ΔΨ) having regard to age and sex. '''Methods:''' Females and males from 3 different age classes (i.e., 30-60, 150-180 and >300 days) were extracted from laboratory culture and divided into experimental and control groups exposed to HMF and standard magnetic field (SMF), respectively, for three different durations i.e., 7 days, 15 days and 30 days. The HMF treatment was performed in a special anti-magnetic chamber whereas SMF treatment was performed in a climate chamber. TMRM staining of intact animals was used to estimate ΔΨ. '''Results and discussion:''' The calculated FITMRM index indicated HMF-related changes in Δψ dependent on age and sex. Accordingly, HMF effect was most pronounced for the oldest animals and males appeared to be more sensitive to HMF than females that correlated with the survival rate. The results provide an insight into mechanisms of HMF effect that could be useful for organization of space travels and living outside the Earth. # Mo W, Liu Y, He R. (2014) Hypomagnetic field, an ignorable environmental factor in space? https://doi.org/10.1007/s11427-014-4662-x# Binhi VN, Prato FS (2017) Biological effects of the hypomagnetic field: An analytical review of experiments and theories. https://doi.org/10.1371/journal.pone.0179340# Conley CC (1970) A Review of the biological effects of very low magnetic fields. NASA. Technical Note; TN D-5902: 1–27. https://ntrs.nasa.gov/citations/19700024915# Erdmann W, Idzikowski B, Kowalski W, Kosicki J, Kaczmarek Ł (2021) Tolerance of two anhydrobiotic tardigrades Echiniscus testudo and Milnesium inceptum to hypomagnetic conditions. https://doi.org/10.7717/peerj.10630scus testudo and Milnesium inceptum to hypomagnetic conditions. https://doi.org/10.7717/peerj.10630 +
'''Authors:''' [[Owesny Patricia]] … '''Authors:''' [[Owesny Patricia]], [[Hegemann N]], [[Kuebler WM]], [[Ost Mario]], [[Grune T]], [[Ott C]] Cardiac aging is a multifactorial process, which is associated with increased oxidative stress, cell death and mitochondrial abnormalities. These factors can lead to an overall impairment of cardiac function and substrate utilization [1,2]. With the increased prevalence of obesity and related comorbidities, especially coronary heart disease, it was proposed that obesity could present a condition of premature heart aging [3]. Therefore, our aim is to compare the impact of obesity and aging on heart function, as well as the cardiac energy metabolism, focusing on mitochondria. Our experimental design of diet-induced obesity contains three different age groups (22, 76 and 106 weeks), where male C57BL/6J mice receive either a High fat/High-carb or a Standard diet for 8 weeks. After dietary intervention, mice underwent echocardiographic or metabolic treadmill analysis. Heart tissue was used for the Oroboros O2k measurement of mitochondrial bioenergetics. In further studies of cardiac energy metabolism Western blot and qPCR in heart tissue and isolated cardiomyocytes were performed. Echocardiography revealed a decline in cardiac output in mice 76 and 106 weeks of age with a further decrease by High fat/High-carb diet. Interestingly, these effects were more pronounced in 76 weeks group. In the same group we investigated indications of an impaired mitochondrial energy metabolism, specifically associated with cardiomyocytes. Although, loss of cardiac function with age has been previously described, we demonstrate here a key role for mitochondrial energy metabolism in this loss of function. # Houtkooper R H, Argmann C, Houten S M, Cantó C, Jeninga E H, Andreux P A, Thomas C, Doenlen R, Schoonjans K, Auwerx J (2011), The metabolic footprint of aging in mice. https://doi.org/10.1038/srep00134. # Lazzeroni D, Villatore A, Souryal G, Pili G, Peretto G (2022), The Aging Heart: A Molecular and Clinical Challenge. https://doi.org/10.3390/ijms232416033.# Ren J, Dong F, Cai G-J, Zhao P, Nunn J M, Wold L E, Pei J (2010), Interaction between age and obesity on cardiomyocyte contractile function: role of leptin and stress signaling. https://doi.org/10.1371/journal.pone.0010085.n and stress signaling. https://doi.org/10.1371/journal.pone.0010085. +
'''Authors:''' [[Petrick Heather L]] … '''Authors:''' [[Petrick Heather L]], [[Aussieker T]], [[Fuchs CJ]], [[Hermans WJ]], [[Betz MW]], [[Pinckaers PJM]], [[Snijders T]], [[van Loon LJC]], [[Holloway Graham P]] '''Introduction:''' Mitochondrial ADP sensitivity represents an important control point in oxidative phosphorylation. The sensitivity of mitochondria to ADP is lower in high-lipid environments, in aging males, and in young females compared to young males. However, the interaction between sex, age, and body composition (fat mass) in the regulation of mitochondrial ADP sensitivity remains unknown. '''Methods:''' Vastus lateralis muscle biopsies were obtained from healthy, recreationally active, young males (n=21, 24±4 y, 22.7±2.2 kg/m2 BMI), young females (n=20, 21±2 y, 21.7±2.2 kg/m2), older males (n=13, 76±5 y, 25.8±2.5 kg/m2), and older females (n=6, 70±6 y, 23.4±3.0 kg/m2). Permeabilized fibers were prepared to measure mitochondrial ADP sensitivity. Whole-body DEXA scans were performed. Data (mean±SD) were analyzed using two-way ANOVAs. '''Results and discussion:''' Body fat percentage was higher in females and older individuals (main effects). While maximal mitochondrial respiration did not differ between groups, mitochondrial ADP sensitivity was affected by sex and age. Specifically, in younger individuals mitochondrial ADP sensitivity was lower in females compared with males (~15 % higher apparent ADP Km, p=0.02). Older males also showed ~15% lower mitochondrial ADP sensitivity compared with young males (p=0.04). In contrast to young individuals, mitochondrial ADP sensitivity was numerically greater (~15 %) in older females when compared with older males (p=0.14) and younger females (p=0.12). However, there were no correlations between body fat percentage and mitochondrial apparent ADP Km in any group. We speculate that sex-based differences in mitochondrial ADP sensitivity are impacted by estrogen as opposed to body composition, as this response is lost with aging. ADP sensitivity are impacted by estrogen as opposed to body composition, as this response is lost with aging. +
'''Authors:''' [[Phang Howard J]] … '''Authors:''' [[Phang Howard J]], [[Gerwick W]], [[Molina Anthony JA]] Mitochondrial bioenergetic decline is a well known biological hallmark of aging, suggesting that mitochondria-targeting therapeutics have great potential in treating age-related diseases and conditions [1]. Despite this, their efficacy within the context of human aging remains largely unknown. We sought to develop a phenotypic screening platform to identify agents that directly modulate mitochondrial function in human cells. Marine natural products (MNP) represent a large, under-explored chemical space with immense therapeutic potential [2]. We screened a MNP library of 125 pure compounds at 10, 1, and 0.1 µg/mL incubated for 24 hours with with primary human dermal fibroblasts (pHDF) as summarized in Figure 1. We leveraged the San Diego Nathan Shock Center which houses 50+ pHDF lines derived from healthy donors across a spectrum of adult age. Cultured pHDF retain age-related phenotypes including mitochondrial bioenergetic decline, which presents a robust opportunity to identify bioenergetic effects within the context of human aging [3]. Thus, we used pHDF from a donor representative of an “older” phenotype (74 years of age) to ensure aging relevance. We identified numerous compounds that modulate mitochondrial function in a dose-dependent manner. Our primary outcomes were change in basal or maximal respiration using high throughput respirometry (Agilent Seahorse XFe96). This screening platform successfully identified compounds with stimulatory as well as inhibitory effects on respiratory capacity. Future steps include further validation of hit compounds using high-resolution respirometry on the Oroboros O2k. These studies will elucidate mechanistic effects on the electron transfer system as well as effects on cells of different donor ages. # Murphy MP, Hartley RC (2018) Mitochondria as a therapeutic target for common pathologies. https://doi.org/10.1038/nrd.2018.174.# Liang X, Luo D, Luesch H (2018) Advances in exploring the therapeutic potential of marine natural products. https://doi.org/10.1016/j.phrs.2019.104373. # Auburger G, Klinkenberg M, Drost J, Marcus K, Morales-Gordo B, Kunz WS, Brandt U, Broccoli V, Reichmann H, Gispert S, Jendrach M (2012). Primary Skin Fibroblasts as a Model of Parkinson's Disease. https://doi.org/10.1007/s12035-012-8245-1. Parkinson's Disease. https://doi.org/10.1007/s12035-012-8245-1. +
'''Authors:''' [[Piel Sarah]] … '''Authors:''' [[Piel Sarah]], [[cManus Meagan J]], [[Heye K]], [[Beaulieu F]], [[Fazeliniae H]], [[Janowska Joanna I]], [[McTurk B]], [[Starr Jonathan]], [[Gaudio H]], [[Patel N]], [[Hefti MM]], [[Smalley M]], [[Hook JF]], [[Kohli NV]], [[Bruton J]], [[Hallowell T]], [[Delso N]], [[Roberts A]], [[Lin Y]], [[Ehinger Johannes K]], [[Karlsson Michael]], [[Berg RA]], [[Morgan RW]], [[Kilbaugh Todd J]] '''Introduction:''' Despite advancements in cardiopulmonary resuscitation (CPR), secondary neurological injury remains the key determinant of successful recovery from cardiac arrest (CA) [1-3]. Currently, there are no established clinical therapies that preserve neurological function [4]. We previously found that acute decline in mitochondrial health up to 24 hours post-CA correlated with poor neurological outcome [5-6]. Here, we tested the potential of dimethyl fumarate (DMF), a derivative of the TCA-cycle intermediate fumaric acid shown to enhance mitochondrial bioenergetics [7], to improve mitochondrial injury in brain and heart following successful resuscitation after CA. '''Methods:''' Female piglets representing toddler age underwent asphyxia, followed by ventricular fibrillation, cardiopulmonary resuscitation and defibrillation until return of spontaneous circulation. Subsequently, animals received daily treatment with DMF or vehicle. Sham animals underwent identical anesthesia protocols and instrumentation without CA. After 4 days, animals (n=5 of each group) were euthanized, tissues were harvested and their mitochondrial function, quantity and proteomic profile was analyzed. '''Results and discussion:''' Mitochondrial content and function, as measured by citrate synthase activity and high-resolution respirometry, was reduced at 4 days following CA. In contrast, myocardial mitochondria demonstrated a complete restoration of mitochondrial content and function despite persistent changes in mitochondrial ultrastructure. DMF treatment prevented 25 % of the long-term proteomic changes in the brain, including proteins related to mitochondrial bioenergetics and oxidative stress. In addition, myocardial mitochondrial morphology was normalized by DMF. In this model of CA, mitochondria sustained persistent damage in an organ-specific manner. DMF partially prevents these long-term mitochondrial changes in myocardium and brain.# Berg RA et al: Incidence and Outcomes of Cardiopulmonary Resuscitation in PICUs. Crit Care Med 2016; 44(4):798-808# Slomine BS, Silverstein FS, Christensen JR, et al: Neurobehavioural outcomes in children after In-Hospital cardiac arrest. Resuscitation 2018; 124:80-89# Laver S, Farrow C, Turner D, et al: Mode of death after admission to an intensive care unit following cardiac arrest. Intensive Care Med 2004; 30(11):2126-2128# Neumar RW et al: Post-Cardiac Arrest Syndrome. Circulation 2008; 118(23):2452-2483# Lautz AJ, Morgan RW, Karlsson M, et al: Hemodynamic-Directed Cardiopulmonary Resuscitation Improves Neurologic Outcomes and Mitochondrial Function in the Heart and Brain. Critical care medicine 2019; 47(3):e241-e249# Kilbaugh TJ, Sutton RM, Karlsson M, et al: Persistently Altered Brain Mitochondrial Bioenergetics After Apparently Successful Resuscitation From Cardiac Arrest. Journal of the American Heart Association 2015; 4(9):e002232# Hayashi G, Jasoliya M, Sahdeo S, et al: Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans. Human molecular genetics 2017; 26(15):2864-2873ice and humans. Human molecular genetics 2017; 26(15):2864-2873 +
'''Authors:''' [[Pytlak Karolina]] … '''Authors:''' [[Pytlak Karolina]], [[Maliszewska – Olejniczak K]], [[Sek Aleksandra]], [[Szewczyk Adam]], [[Bednarczyk Piotr]], [[Kulawiak Bogusz]] Human bronchial epithelial (HBE) cells form an external barrier in the airways and are constantly exposed to factors such as urban dust. Recently, the large conductance calcium-activated potassium (mitoBKCa) channel has been identified in the inner mitochondrial membrane of HBE cells. The pore-forming subunit of the channel is encoded by the ''KCNMA1'' gene, which also encodes plasma membrane BKCa channels. Mitochondrial potassium channels regulate mitochondrial membrane potential, oxygen consumption, mitochondrial volume and reactive oxygen species synthesis. Activation of mitoBKCa induces cytoprotection of cardiac and brain tissue. In our project, we applied CRISPR/Cas9 technique to disrupt ''KCNMA1'' gene in the HBE cell line (16HBE14o- cells). The newly formed line showed no mitoBKCa channel activity. We also noticed changes related to the deregulation of the cell cycle. The loss of mitoBKCa significantly affected mitochondrial function. We observed a decrease in the rate of mitochondrial respiration. Furthermore, we analyzed the organization of respiratory chain complexes using Blue Native electrophoresis. In addition, analysis of the expression of selected genes encoding mitochondrial proteins showed changes in cells with disrupted ''KCNMA1'' gene. Nevertheless, a thorough understanding of the observed mitochondrial dysfunction requires further study.We conclude that the presence of the mitoBKCa channel in HBE cells is essential for the preservation of mitochondrial function and is important for the proper function of these cells as part of the human airways. for the preservation of mitochondrial function and is important for the proper function of these cells as part of the human airways. +
'''Authors:''' [[Sadler Daniel]] … '''Authors:''' [[Sadler Daniel]], [[Treas L]], [[Ross T]], [[Sikes JD]], [[Britton SL]], [[Koch LG]], [[Borsheim Elisabet]], [[Porter Craig]] '''Introduction:''' Low cardiorespiratory fitness (CRF) is associated with a greater risk for metabolic disease. The potential for early life exercise training to overcome metabolic perturbations imparted by low intrinsic CRF remains unknown. We tested the hypothesis that early life exercise training would overcome whole-body and tissue metabolic defects imparted by low CRF.'''Methods:''' At 26 days of age, rat low-capacity runners (LCR, ''n''=20) and high-capacity runners (HCR, ''n''=20) generated by artificial selection were assigned to either sedentary control (CTRL, n=10) or voluntary wheel running (VWR, ''n''=10) for 6 weeks. Post-intervention, whole-body metabolic phenotyping was performed, and the respiratory function of isolated skeletal muscle and liver mitochondria assayed. Quantitative proteomics were performed on tissue samples.'''Results and discussion:''' HCR-VWR performed 1.8-fold greater volume of wheel running than LCR-VWR (P<0.001). In LCR, VWR reduced body fat (''P''<0.001), increased total daily energy expenditure (+16 %, ''P''=0.030), and enhanced glucose tolerance (''P''=0.040). Muscle mitochondrial respiratory function was unaffected by VWR in both strains, although VWR increased muscle mitochondrial protein content (both ''P''<0.05). VWR enhanced the respiratory capacity of HCR hepatic mitochondria (+23 %, ''P''=0.040). Proteomic analyses revealed lower capacity for fatty acid oxidation in muscle and liver of LCR-CTRL versus HCR-CTRL, which was not rescued by VWR. VWR reduced hepatic pyruvate kinase abundance in both strains (both ''P''<0.013), indicating VWR may shift fuel preferences of hepatic mitochondria. These results reveal early life exercise training partially overcomes the metabolic phenotype imparted by low intrinsic CRF, although proteomic adaptations to early exercise training remain influenced by intrinsic CRF. to early exercise training remain influenced by intrinsic CRF. +
'''Authors:''' [[Saleem Ranim]] … '''Authors:''' [[Saleem Ranim]], [[Scott Graham R]] '''Introduction:''' High-altitude environments are characterized by cold temperatures and low O2 levels (hypoxia). Small mammals at high altitude thus face the metabolic challenge of maintaining thermogenesis to cope with cold in a hypoxic environment that can constrain aerobic ATP supply. Circulatory O2 delivery by the heart is essential for supporting tissue O2 demands, but it is unclear whether evolved or plastic changes in cardiac mitochondria help overcome constraints on thermogenesis in high-altitude environments. '''Method:''' We examined this issue in deer mice (Peromyscus maniculatus). Mice from populations native to high altitude and low altitude were born and raised in captivity, and adults were acclimated to warm (25 °C) normoxia or cold (5 °C) hypoxia (~12 kPa O2 for 5-6 weeks) in a full-factorial design. Mitochondrial function was studied by high-resolution respirometry and fluorometry in permeabilized tissue from left ventricles and was complemented by assays of several metabolic and antioxidant enzymes. '''Results and discussion:''' Mitochondrial capacities for oxidative phosphorylation and electron transport were similar between populations and were unaffected by acclimation to cold hypoxia, as were activities of citrate synthase and cytochrome oxidase. However, exposure to cold hypoxia increased activities of lactate dehydrogenase, which were also greater in highlanders than in lowlanders, likely to augment capacities for lactate oxidation. Furthermore, mitochondrial emission of reactive oxygen species was lower in highlanders than in lowlanders across environments, associated with lower levels of lipid peroxidation and protein carbonyls. Therefore, phenotypic plasticity and evolved changes in cardiac mitochondria help deer mice cope with metabolic challenges at high altitude. evolved changes in cardiac mitochondria help deer mice cope with metabolic challenges at high altitude. +
'''Authors:''' [[Saucedo-Rodriguez Maria Jose]] … '''Authors:''' [[Saucedo-Rodriguez Maria Jose]], [[Pecina Petr]], [[Cunatova Kristyna]], [[Vrbacky Marek]], [[Cajka T]], [[Mracek Tomas]], [[Pecinova Alena]] '''Introduction:''' Succinate dehydrogenase (SDH) connects the TCA cycle by oxidizing succinate to fumarate and the respiratory chain by transferring electrons to ubiquinone. Mutations in SDH subunits have been associated with tumorigenesis as well as mitochondrial diseases. In this project, we focused on the flavoprotein subunit A of SDH (SDHA) which is primarily associated with inherited mitochondrial disease [1] and investigated the consequences of this subunit loss in HEK cells (SDHA KO). '''Methods:''' We performed structural and functional characterizations of the SDHA KO model involving protein electrophoresis to study OXPHOS complexes and subcomplexes, label-free quantification of protein levels, measurement of cellular respiration using high-resolution respirometry and determination of NAD+/NADH levels. '''Results and discussion:''' Together with SDHA, other SDH subunits were downregulated as well, leading to the absence of assembled SDH. Moreover, a secondary downregulation of the majority of complex I and IV subunits was observed. The cellular respiratory capacity was severely decreased in the model, with SDH-dependent respiration completely abolished and complex I-dependent respiration attenuated reflecting the downregulation of respiratory chain complexes in general. Finally, the NAD+/NADH ratio was increased in SDHA KO compared to the controls, indicating complex rearrangement of the TCA. The SDHA KO cells thus represent a suitable model to study metabolic rewiring and the effect of pathogenic SDHA mutations. # Rustin, P., Munnich, A., & Rötig, A. (2002). Succinate dehydrogenase and human diseases: new insights into a well-known enzyme. https://doi.org/10.1038/sj.ejhg.5200793 d human diseases: new insights into a well-known enzyme. https://doi.org/10.1038/sj.ejhg.5200793 +
'''Authors:''' [[Schoenfeld Peter|Schönfeld P]], … '''Authors:''' [[Schoenfeld Peter|Schönfeld P]], [[Reiser G]] Distinct hypothalamic neurons sense blood levels of fatty acids (FA) and, thereby regulate caloric intake. Astrocytes have some capacity of β-oxidation. But, there are ongoing discussions on this question: Do neurons generally burn FA for energy generation?Respiration and membrane potential of mitochondria of rat brain (RBM) and, for comparison, of liver (RLM) were measured without and with octanoate (l-octanoylcarnitine). In addition, H2O2 generation was measured with Amplex Red.In line with previous studies, we found no evidence for a noteworthy β-oxidation of FA by RBM. This fits with theoretical considerations (1) and values obtained for capacities of enzymes of β-oxidation (2). But, these results contradict those of a previous study (3), reporting that RBM incubated with mixtures of FA (carnitine derivatives) plus other substrates (e.g. succinate) show substantial β-oxidation. What could be possible reasons for disregarding FA as energy substrates by neurons? These are mainly: (a) Harmful activities of non-esterified long-chain FA on mitochondria. (b) Burning of FA costs more oxygen than glucose burning with respect to the energy yield. (c) FA oxidation by mitochondria is associated with more sites of superoxide generation. (d) Neurons are equipped with poor antioxidative capacity. In conclusion, burning of FA would expose neurons to intolerably high oxidative stress.# Speijer D (2011] Oxygen radicals shaping evolution: Why fatty acid catabolism leads to peroxisomes while neurons do without it. https://doi.org/10.1002/bies.201000097# Yang SY, He XY, Schultz H (1987) Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase. https://doi.org/10.1016/S0021-9258(18)45161-7# Panov A, Orynbayeva Z, Vavilin V, Lyakhovich V (2014) Fatty acids in energy metabolism of the central nervous system. https://doi.org/10.1155/2014/472459tabolism of the central nervous system. https://doi.org/10.1155/2014/472459 +
'''Authors:''' [[Sobotka Lubos]] … '''Authors:''' [[Sobotka Lubos]], [[Sobotka Ondrej]] Obesity is associated with insulin resistance, which is the cause of subsequent metabolic complications, including increased morbidity. Despite several decades of efforts to prevent the growth of obesity, its incidence continues to increase. We do not even know what ratio of nutrients is optimal for preventing obesity and insulin resistance, and the optimal ratio of carbohydrates to lipids has not been proven. Some studies, including calorimetric measurements performed at our workplace, have shown that the oxidation of individual substrates does not correspond to their ratio in the given diet. However, this apparent paradox makes sense because food intake in humans is intermittent and usually does not occur during increased or even maximal physical activity. Energy and metabolic substrates are stored in the body during intake and are subsequently mobilized during periods of starvation and physical activity. As a result, the human body is never in true energy balance; storage and subsequent mobilization of energy is necessary for a functioning organism. In addition, carbohydrates, fats and proteins are not only a source of energy, but also important substances with many functions [1]. After ingestion of a mixed meal, carbohydrates (especially glucose) are used for both oxidation and non-oxidative pathways (antioxidant, anaplerotic, cataplerotic processes). Only a relatively small fraction of glucose is a source for new lipid synthesis. Ingested fats are preferentially stored in adipose tissue and does not influence carbohydrate oxidation. The lack of glucose can explain more insulin resistance in whole organism than Randle cycle measured in vitro conditions [2].# Sobotka L, Sobotka O. The predominant role of glucose as a building block and precursor of reducing equivalents. https://doi.org/10.1097/mco.0000000000000786 # Sobotka O, et al. Should Carbohydrate Intake Be More Liberal during Oral and Enteral Nutrition in Type 2 Diabetic Patients? https://doi.org/10.3390/nu15020439in Type 2 Diabetic Patients? https://doi.org/10.3390/nu15020439 +
'''Authors:''' [[Stanic Sara]] … '''Authors:''' [[Stanic Sara]], [[Janovska Petra]], [[Zouhar Petr]], [[Bardova K]], [[Otahal J]], [[Vrbacky Marek]], [[Mracek Tomas]], [[Adamcova K]], [[Lenkova L]], [[Funda J]], [[Cajka T]], [[Drahota Zdenek]], [[Rustan Arlid C]], [[Horakova Olga]], [[Houstek Josef]], [[Rosmeissl M]], [[Kopecky Jan]] '''Introduction:''' Non-shivering thermogenesis (NST) is an energy-dissipating process that occurs in brown adipose tissue (BAT) and is activated by the adrenergic system. Earlier studies found that cold induces adrenergically activated NST in obesity-prone C57BL/6 (B6) mice, but not in obesity-resistant A/J mice. To investigate this difference, we studied the effect of cold acclimation on muscle NST. '''Methods:''' Palmitoyl carnitine oxidation and cytochrome c oxidase (COX) activity (TMPD+ascorbate and KCN) was measured in muscle homogenates of A/J and B6 mice acclimated to 30 °C or to 6 °C using Oroboros Oxygraph. In parallel, amount of mitochondrial supercomplexes was assessed by Blue native electrophoresis. '''Results and discussion:''' As expected, muscle of A/J mice exhibited higher amount of Scaf1 dependent supercomplex III2IV than muscle of B6 mice, and this amount was further increased by cold acclimation. Both palmitoyl carnitine oxidation and COX activity were induced by cold in A/J but not in B6 mice. The higher oxidation capacity of muscle of cold acclimated A/J mice, possibly connected with supercomplex composition, may indicate that muscle represents the site of alternative NST instead of BAT in these mice. The distinct mechanism of NST could correspond to obesity resistance of this strain. # Janovska P. et al (2023) Impairment of adrenergically-regulated thermogenesis in brown fat of obesity-resistant mice is compensated by non-shivering thermogenesis in skeletal muscle. https://doi.org/10.1016/j.molmet.2023.101683rmogenesis in skeletal muscle. https://doi.org/10.1016/j.molmet.2023.101683 +
'''Authors:''' [[Stankova Pavla]] … '''Authors:''' [[Stankova Pavla]], [[Peterova E]], [[Dusek J]], [[Elkalaf Moustafa]], [[Cervinkova Zuzana]], [[Kucera Otto]] '''Introduction:''' In our previous study in a murine model of nonalcoholic steatohepatitis (NASH), we found reduced succinate-activated hepatic mitochondrial respiration and accumulation of succinate, a proinflammatory, profibrogenic, and oncogenic metabolite [1]. According to preliminary studies, telmisartan, an angiotensin II type 1 receptor blocker, positively affects insulin resistance and liver steatosis. This project aimed to investigate the effect of telmisartan on NASH in mice. '''Methods:''' The NASH was induced in male mice fed a western-style diet (WD) for 36 weeks. During the last 6 weeks of the experiments, mice were administered daily telmisartan (oral gavage, 5 mg/kg b.w./day). Liver and epididymal fat histological changes were evaluated (Hematoxylin-eosin, Sirius red). Body parameters, plasma liver profile (VetScan), hepatic triglycerides, cholesterol, and the expression of selected proteins (WB/ELISA) and genes (qRT-PCR) were assessed. Mitochondrial respiration of liver homogenates was measured by high-resolution respirometry (OROBOROS Oxygraph-2k). Using Reporter Gene assay, telmisartan's activation of nuclear receptors was evaluated on HepG2 cells. '''Results and discussion:''' Administration of telmisartan to mice fed a WD reduced absolute and relative liver weight and visceral adipose tissue weight, activities of ALT and AST, liver steatosis, and inflammation grade. These effects were accompanied by a significant increase in succinate-activated respiration in the ET state and the activity of succinate dehydrogenase. We confirmed that telmisartan is a PPAR-γ partial agonist and described the activating effect of telmisartan on the CAR receptor for the first time. Telmisartan appears to be a promising safety drug for treating NASH that affects metabolism at multiple levels. # Staňková P, Kučera O, Peterová E, Elkalaf M, Rychtrmoc D, Melek J, Podhola M, Zubáňová V, Červinková Z (2021) Western Diet Decreases the Liver Mitochondrial Oxidative Flux of Succinate: Insight from a Murine NAFLD Model. https://doi.org/10.3390/ijms22136908ght from a Murine NAFLD Model. https://doi.org/10.3390/ijms22136908 +
'''Authors:''' [[Stiles Linsey]] … '''Authors:''' [[Stiles Linsey]], [[Fernandez-del-Rio L]], [[Beninca C]], [[Acin-Perez R]], [[Shirihai Orian]] Impaired mitochondrial function has been shown to play a key role in diseases of metabolism and aging. Respirometry is the gold standard measurement of mitochondrial function, as it is an integrated metabolic readout of the final step of the electron transport chain (ETC). However, analysis of mitochondrial respiratory function in tissue requires processing and measurement of freshly isolated mitochondria. This requirement makes respirometry impracticable for standard clinical practice, clinical studies, retrospective studies, and higher throughput respirometry. We have validated a methodology to measure maximal mitochondrial oxygen consumption rates through Complex I, II, and IV of the ETC in previously frozen biological samples using Agilent XF Analyzers. Additionally, Complex V (CV) ATP hydrolytic activity can be measured with the pH channel. These measurements of Complex I-V activities are specific as demonstrated by inhibition with ETC inhibitors. Additionally, these approaches can be applied to tissue homogenates, which simplifies the sample preparation and reduces the required starting material compared with isolating mitochondria. We find that primary changes in the maximal respiratory capacity, detected in fresh tissue, are preserved in frozen samples. These techniques to measure mitochondrial maximal respiratory function and CV hydrolytic activity in frozen samples makes clinical mitochondrial assessment more feasible and adds a complementary approach to investigate the role of mitochondrial function in disease onset and progression.tochondrial function in disease onset and progression. +
'''Authors:''' [[Timon-Gomez Alba]] … '''Authors:''' [[Timon-Gomez Alba]], [[Cardoso Luiza HD]], [[Doerrier Carolina]], [[Garcia-Souza Luiz F]], [[Gnaiger Erich]] '''Introduction:''' Mitochondrial dysfunction in muscle tissue is associated with obesity (mitObesity) and its comorbidities. Many drugs and nutraceuticals used to treat these conditions target mitochondria. Early diagnosis of mitObesity is crucial for understanding the link between obesity, mitochondrial dysfunction, and its associated chronic comorbidities. Respirometry of mitochondrial preparations can assess electron transfer pathways and coupling in oxidative metabolism with high diagnostic resolution [1]. '''Methods:''' We developed a standardized protocol for functional diagnosis of mitochondrial defects using high-resolution respirometry [2]. This substrate-uncoupler-inhibitor titration (SUIT) protocol analyzes fatty acid oxidation (FAO) by adding 0.1 mM malate and octanoylcarnitine, with consideration of malate-linked anaplerosis to avoid overestimation of FAO [3-4]. The protocol is extended to stimulate the NADH-linked pathway by adding pyruvate and glutamate. Then succinate and glycerophosphate are titrated to investigate convergent CoQ-reducing pathways. A stepwise titration of uncoupler CCCP allows quantification of the electron transfer capacity. Residual oxygen consumption is assessed after inhibition by rotenone and antimycin A. '''Results and discussion:''' To quantify FAO, malate was needed to avoid inhibition by accumulating acetyl-CoA. However, in the presence of mitochondrial malic enzyme, 2 mM malate stimulated respiration through the NADH-linked pathway in liver and brain mitochondria. Anaplerotic activity above endogenous respiration was minimized at a low (0.1 mM) malate concentration and subtracted from respiration obtained after addition of octanoylcarnitine. This SUIT reference protocol can be used as a general diagnostic tool for bioenergetic profiling in various sample preparations from different cell types, tissues, and organisms. # Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. https://doi.org/10.26124/bec:2020-0002# Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. https://doi.org/10.1007/978-1-4939-7831-1_3 # Ojuka E, Andrew B, Bezuidenhout N, George S, Maarman G, Madlala HP, Mendham A, Osiki PO (2016) Measurement of β-oxidation capacity of biological samples by respirometry: a review of principles and substrates. https://doi.org/10.1152/ajpendo.00475.2015# Bernstine EG, Koh C, Lovelace CC (1979) Regulation of mitochondrial malic enzyme synthesis in mouse brain. https://www.doi.org/10.1073/pnas.76.12.6539synthesis in mouse brain. https://www.doi.org/10.1073/pnas.76.12.6539 +
'''Authors:''' [[Torres-Quesada Omar]] … '''Authors:''' [[Torres-Quesada Omar]], [[Strich Sophie]], [[Feichtner Andreas]], [[Schwaighofer Selina]], [[Doerrier Carolina]], [[Schmitt Sabine]], [[Gnaiger Erich]], [[Stefan Eduard]] Protein kinases play an important role in numerous signaling pathways regulating cell proliferation, cell cycle, and metabolism. Deregulation of kinase functions have been connected to various human diseases, such as cancer [1]. In recent years, kinase inhibitors have gained recognition by aiming to block single or multiple oncogenic kinase pathways [2]. In these lines, blockade of kinase activities has been shown to converge on the central energetic organelle, the mitochondria [3, 4]. Furthermore, colon cancer cells rely on mitochondrial OXPHOS as major source of energy, contradicting the Warburg effect [5]. To increase the understanding of small molecule-based kinase blockers and their cell-type-specific adverse effects, we set out to record the impact of kinase drugs on mitochondrial respiration using High-resolution FluoRespirometry in several colon cancer cell models. We observed that the impact of kinase inhibitors depends on the mutational background of the tested cancer cell lines as well as on cell culture medium formulations [6]. First, we detected off-target effects of sunitinib, an FDA-approved multikinase blocker, only in a more physiological cell culture medium as compared with classical formulations. Second, mitochondrial profiling of the glycolytic kinase inhibitor PFK158 revealed off-target mitochondrial dysfunction. Third, we were able to show that inhibition of kinase signaling is connected to mitochondrial reactive oxygen species (ROS), which can be influenced by protein kinase modulators. In summary, cell-based mitochondrial bioenergetic profiles have the power to identify off-target effects of kinase inhibitors and allow a detailed mechanistic insight on drug-induced perturbations in cancer cell metabolism.# Cohen, P, Cross, D, Jänne, PA (2021). Kinase drug discovery 20 years after imatinib: progress and future directions. Nat Rev Drug Discov. 20(7):551-569. https://doi.org/10.1038/s41573-021-00195-4 # Zhang J, Yang PL, Gray NS (2009). Targeting cancer with small molecule kinase inhibitors. https://doi.org/10.1038/nrc2559 # Wallace, DC (2012). Mitochondria and Cancer Nat. Rev. Cancer, 12, 685–698. https://doi.org/10.1038/nrc3365 # Torres-Quesada O, Strich S, Stefan E (2022). Kinase perturbations redirect mitochondrial function in cancer. Bioenerg. Commun. 2022,13. https://doi.org/10.26124/bec:2022-0013# Sun, X., Zhan, L., Chen, Y. et al. (2018) Increased mtDNA copy number promotes cancer progression by enhancing mitochondrial oxidative phosphorylation in microsatellite-stable colorectal cancer. Sig Transduct Target Ther 3, 8. https://doi.org/10.1038/s41392-018-0011-z # Torres-Quesada, O, Doerrier, C, Strich, S, Gnaiger, E, Stefan, E (2022). Physiological Cell Culture Media Tune Mitochondrial Bioenergetics and Drug Sensitivity in Cancer Cell Models. Cancers, 14, 3917. https://doi.org/10.3390/cancers14163917ancers, 14, 3917. https://doi.org/10.3390/cancers14163917 +
'''Authors:''' [[Ullrich Volker]] … '''Authors:''' [[Ullrich Volker]], [[Heidler Juliana]], [[Schildknecht S]], [[Daiber Andreas]], [[Frensch M]], [[Wittig Ilka]], [[Bruene B]] '''Introduction:''' Micelles containing cardiolipin (CL) and phosphatidylcholine in presence of cytochrome ''c'' (Cytc) and H2O2 were reported to catalyze peroxidationes of typical peroxidase substrates but also of CL itself (Kagan et. al, Biochem. 45,4998, 2006). This can be explained by complex formation of CL with Cytc under removal of the Met80 sixth ligand of the heme. '''Methods:''' O2 consumption was measured polarographically (Orobos Instruments) and diene formation spectrally at 237 nm. '''Results and Discussion:''' Cytc addition to CL micelles caused a burst of O2 uuptake that could be repeated until CL or O2 were depleted. About 4.5 mol of O2/mol CL were taken up forming products with mainly 2,4,6 or 8 additional O-atoms. Diene formation initially followed the same kinetics but stopped or was reversed before O2 uptake was completed. In presence of 2 M KCl Cytc acted catalytically with slower kinetics in three phases and showed oxidative modifications of the protein. The required peroxide tone originated from autoxidized CL and was upregulated during progress of the reaction. Significance for the process of opening of the permeability pore is suggested. autoxidized CL and was upregulated during progress of the reaction. Significance for the process of opening of the permeability pore is suggested. +
'''Authors:''' [[Valencia Ana P]] … '''Authors:''' [[Valencia Ana P]], [[Melhorn Susan J]], [[Schur Ellen A]], [[Marcinek David J]] '''Introduction:''' Weight loss (WL) promotes counterregulatory mechanisms that may involve mitochondrial (MITO) function to limit cardiometabolic benefit. This study compared T-cell MITO function in states of obesity (OB), active weight loss (OB-WL), weight loss plateau (OB-PL), regain (OB-RG), and healthy weight (HWC). '''Methods:''' Participants with obesity (61.5%female, 39.5±10.8 yr, BMI 36.7±6.4) underwent a 24-week WL intervention and transmitted their daily weight for 18 months. T-cells (CD3+) were isolated from blood samples obtained at baseline, 6-month, or 12-months. We measured MITO respiratory capacity (MITO-RC) (XFe Analyzer) and sensitivity of membrane depolarization with ADP (IC50) (O2K Fluorespirometer). '''Results:''' Compared to HWC, MITO-RC was lower in OB T-cells (4.1±1.7 vs. 3.3±1.0 pmol O2/106 cells, p<0.05), and even lower in OB-PL (3.0±0.7, p<0.05) and OB-RG (2.7±0.3, p<0.05). Maximal membrane potential was also lower in the OB group and remained low in all phases of WL. IC50 did not differ in T-cells between HWC and OB but was lower in OB-WL and OB-PL (156±15 vs. 7±1 & 22±5, p<0.05). '''Conclusions:''' T-cell MITO respiratory capacity is reduced in obesity and further aggravated in response to WL, particularly following a plateau. However, WL improved ADP sensitivity, suggesting a potential counterregulatory mechanism to meet energy demand. Findings suggest that the MITO function of T-cells is not restored by WL to resemble HWC and is rather altered in a way that could potentially limit cardiometabolic benefit of WL. L to resemble HWC and is rather altered in a way that could potentially limit cardiometabolic benefit of WL. +
'''Authors:''' [[Vujacic-Mirski Ksenija]] … '''Authors:''' [[Vujacic-Mirski Ksenija]], [[Sudowe S]] The impairment of mitochondrial respiration, observed in neurodegenerative and cardiovascular disease, diabetes, cancer and migraine headaches, has emerged as a biomarker of mitochondrial dysfunctions [1]. Newer research are also trying to link conditions such as chronic fatigue, depression and other behaviour/mood disorders with mitochondrial malfunctioning [2]. In our study, we examined 88 (relatively) healthy volunteers, ages from 23 to 68, from which 36 individuals were taking some sort of medication (such as for asthma, high blood pressure, mood disorders), but they considered themselves fit and healthy. Volunteers were ask to follow their normal routines day prior the test. The blood was drawn 3 h before PBMCs isolation, followed by immediate Seahorse XF Cell Mito Stress Test (Agilent) on SeahorseXF96e instrument (Agilent). Our analysis consisted of carefully examining parameters of mitochondrial respiration: basal respiration, ATP-linked respiration, reserve capacity, maximal respiration, proton leak, non-mitochondrial respiration as well as bioenergetics health index (BHI) [3]. We observed difference between people who took some sort of medication for chronic but manageable comorbidities and completely healthy individuals. There was significant difference between BHI, reserve capacity, coupling efficiency and proton leak. We also observed that people who had regular sport activities (in the healthy group without any medication) seem to have lower proton leak. This difference was not significant but points out to the lifestyle impact to mitochondria [4]. # Petrus, A T et al. (2019) Assessment of platelet respiration as emerging biomarker of disease. https://doi.org/10.33549/physiolres.934032# Zvěřová M et al. (2019) Disturbances of mitochondrial parameters to distinguish patients with depressive episode of bipolar disorder and major depressive disorder. https://doi.org/10.2147/NDT.S188964# Chacko, Balu K et al. (2014) The Bioenergetic Health Index: a new concept in mitochondrial translational research. https://doi.org/10.1042/CS20140101 # Janssen, Joëlle J E et al. (2022) Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females. https://doi.org/10.1152/ajpendo.00365.2021w-fit females. https://doi.org/10.1152/ajpendo.00365.2021 +
'''Authors:''' [[Whitcomb Luke A]] … '''Authors:''' [[Whitcomb Luke A]], [[Li Puma Lance C]], [[Zilhaver PT]], [[Izon CS]], [[Chicco Adam J]] Myocardial ischemia causes pathological increases in cardiomyocyte mitochondrial calcium (Ca++), which trigger a series of events that contribute to cell death and myocardial necrosis. Previous studies in our lab and others indicate that metabolites of phosholipid-derived arachidonic acid (AA), an omega-6 polyunsaturated fatty acid (PUFA), contribute to mitochondrial permeability transition pore (mPTP) opening in response to Ca++ overload, leading to mitochondrial swelling, rupture, and release of reactive oxygen species (ROS) [1,2]. We hypothesized that age-related increases in these parameters result in part from greater mitochondrial production of AA from its abundant membrane PUFA precursor linoleic acid (LA) in response to Ca++ overload. To test this hypothesis, we evaluated effects of 50-400 µM Ca++ on O2 consumption, ROS release and mPTP opening in cardiac mitochondria isolated from young (3 mo) and aged (24 mo) BALB/c mice in the presence or absence of an inhibitor of delta-6 desaturase (D6D), the rate-limiting enzyme in AA biosynthesis from LA. Results demonstrate that cardiac mitochondria from old mice release more ROS during oxidative phosphorylation and undergo more mPTP opening in response to Ca++ overload than mitochondria from young mice. D6D inhibition significantly attenuates these responses in both young and old mitochondria, but had greater impacts on old, largely abolishing the effect of aging on both ROS release and mPTP opening. Similar attenuation of mPTP opening was seen following inhibition of lipoxygenase enzymes (Baicalein), consistent with the hypothesized links between mitochondrial AA synthesis, eicosanoid production and mPTP in regulating responses of cardiac mitochondria to Ca++ overload. # Moon SH, Jenkins CM, Liu X, Guan S, Mancuso DJ, Gross RW (2012) Activation of mitochondrial calcium-independent phospholipase A2 gamma by divalent cations mediating arachidonate release and production of downstream eicosanoids. https://10.1074/jbc.M111.336776 # Moon SH, Jenkins CM, Kiebish MA, Sims HF, Mancuso DJ, Gross RW (2012) Genetic Ablation of Calcium-independent Phospholipase A2γ (iPLA2γ) attenuates calcium-induced opening of the mitochondrial permeability transition pore and resultant cytochrome ''c''. https://doi.org/10.1074/jbc.M112.373654 uced opening of the mitochondrial permeability transition pore and resultant cytochrome ''c''. https://doi.org/10.1074/jbc.M112.373654 +
'''Authors:''' [[Wohlfarter Yvonne]] … '''Authors:''' [[Wohlfarter Yvonne]], [[Eidelpes R]], [[Yu RD]], [[Sailer S]], [[Koch Jakob]], [[Karall Daniela]], [[Scholl‑Buergi S]], [[Amberger A]], [[Hillen HS]], [[Zschocke J]], [[Keller Markus A]] '''Introduction:''' Human 17β-Hydroxysteroid dehydrogenase 10 (HSD10) is a crucial enzyme located in mitochondria that participates in isoleucine catabolism and is part of the mitochondrial RNase P complex [1,2]. Mutations in the ''HSD10B17'' gene have been linked to HSD10 disease, which causes progressive cardiomyopathy and cognitive function loss [3]. Recently, HSD10 has been reported to possess a phospholipase C-like activity towards cardiolipins, which are essential mitochondrial membrane lipids involved in various processes such as super-complex assembly, cristae formation, and apoptotic signaling cascades [4]. The transacylase tafazzin is remodeling cardiolipin side chains, and its deficiency leads to high levels of monolyso-cardiolipins and abnormal cardiolipin patterns [5]. '''Methods:''' To explore the role of HSD10 in cardiolipin homeostasis, we carried out a comprehensive analysis of cardiolipin profiles in different cellular contexts by means of LC-MS/MS [6]: We investigated the impact of HSD10 knockdown in wild-type cells, in a tafazzin-deficient background, and in fibroblasts derived from HSD10-deficient patients. Additionally, by supplementation with fatty acids such as linoleic acid and palmitic acid we simulated different lipid environments. '''Results and Discussion:''' We found no evidence for the enzyme function of HSD10 to be involved in cardiolipin homeostasis in all conditions examined [6]. Thus, its previously reported cardiolipin cleaving function is likely to be regarded as an ''in vitro'' artefact. However, the HSD10's structural importance in the mitochondrial RNase P complex underscores its essential role in cellular function [7]. We show that the enzyme has evolved with significant evolutionary constraints to maintain this structure, possibly at the expense of achieving a high degree of substrate specificity and reaction rates [6].# Zschocke J, Ruiter JPN, Brand J, et al (2000) Progressive Infantile Neurodegeneration Caused by 2-Methyl-3-Hydroxybutyryl-CoA Dehydrogenase Deficiency: A Novel Inborn Error of Branched-Chain Fatty Acid and Isoleucine Metabolism. https://doi.org/10.1203/00006450-200012000-00025# Bhatta A, Dienemann C, Cramer P, Hillen HS. (2021) Structural basis of RNA processing by human mitochondrial RNase P. https://doi.org/10.1038/s41594-021-00637-y# Zschocke J. (2012) HSD10 disease: clinical consequences of mutations in the HSD17B10 gene. https://doi.org/10.1007/s10545-011-9415-4# Boynton TO, Shimkets LJ. (2015) Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases. https://doi.org/10.1101/gad.268482.115# Oemer G, Koch J, Wohlfarter Y, Lackner K, Gebert REM, Geley S, et al. (2022) The lipid environment modulates cardiolipin and phospholipid constitution in wild type and tafazzin-deficient cells. https://doi.org/10.1002/jimd.12433# Wohlfarter Y, Eidelpes R, Yu RD, Sailer S, Koch J, Karall D, et al. (2022) Lost in promiscuity? An evolutionary and biochemical evaluation of HSD10 function in cardiolipin metabolism. https://doi.org/10.1007/s00018-022-04682-8# Zschocke J, Byers PH, Wilkie AOM. (2023) Mendelian inheritance revisited: dominance and recessiveness in medical genetics. https://doi.org/10.1038/s41576-023-00574-0 n medical genetics. https://doi.org/10.1038/s41576-023-00574-0 +
'''Authors:''' [[Yardeni Tal]] … '''Authors:''' [[Yardeni Tal]] Both mitochondrial DNA (mtDNA) lineages and the gut microbiota have been correlated with altered risk for a variety of human diseases including obesity. However, the mechanisms by which mtDNA variation and the gut microbiota modulate disease risk remains unknown. Our hypothesis is that both the gut microbiota and the immune system are modulated by the mitochondrial genome, in part through mitochondrial reactive oxygen species (mtROS) production, forming a critical link between the gut microbiota and disease initiation and progression.Our studies showed significant differences in gut microbiota in our conplastic mice which differ in their mtDNA lineages. Further, the transfer of the gut microbiota from a host of one mitochondrial genotype to a host of different mitochondrial genotypes shifted the gut microbiota composition toward that of the recipient animal. Moreover, we showed that host mtROS levels modulated the composition of the gut microbiota. Those conplastic mice also exhibit markedly different capacities to sustain melanoma tumor growth. Relative to control mtDNA (mtDNAB6) mice, the mice harboring NZB mtDNAs (mtDNANZB) have strong anti-tumor immune response while those with129 mtDNA (mtDNA129) are the opposite. Reduction of mtROS by expression of mitochondrial catalase (mtCAT)Tg only in the hematopoietic cells changed the gut microbiota and obviated the anti-tumor effects on the mtDNANZB and mtDNAB6 mice. These observations suggest that disease severity (melanoma), and gut microbiota are regulated by the mtDNA's regulation of mtROS production in host immune cells, pointing to new potential pathways for understanding diseases etiology.lation of mtROS production in host immune cells, pointing to new potential pathways for understanding diseases etiology. +
'''Authors:''' [[Zweck Elric]] … '''Authors:''' [[Zweck Elric]], [[Piel Sarah]], [[Chadt A]], [[Al-Hasani H]], [[Kelm M]], [[Szendroedi Julia]], [[Roden Michael]], [[Granata Cesare]] '''Introduction:''' Ketone bodies (KB) are important substrates for the heart, particularly during heart failure [1], kidney [2], brain, skeletal muscle, and other organs [3]. Despite their significant role in health and disease [4], very limited research is available investigating KB-linked ATP production in mammalian tissues [5]; moreover, no optimized protocols exist to assess the interplay of key enzymes involved in ketolysis and their respective contribution to OXPHOS capacity. '''Methods:''' β-hydroxybutyrate (HBA)- and acetoacetate (ACA)-linked mitochondrial respiration was assessed in the heart left ventricle (LV), kidney, liver, brain, and soleus of ~18-24-week-old C57BL/6J female mice (n=6-8). A novel protocol combining KB-linked and complex I (CI)+CII-linked mitochondrial respiration was also devised. '''Results and discussion:''' The Km for HBA was similar (~1 mM) in all tested organs. However, maximal HBA-linked respiration was different between organs (p<0.001), i.e., greater in the LV and liver (~32 pmol O2·s-1·mg-1), and lowest in the brain (5.2 pmol O2·s-1·mg-1). This protocol allows to determine β-hydroxybutyrate dehydrogenase activity in the liver. The Km for ACA and maximal ACA-linked respiration were greater in the kidney compared to the other tested organs (all p<0.050). Our novel KB+CI+CII combined respiration protocol indicated that the KB contribution to maximal respiration is 2- to 4-fold greater in the kidney (37.4 %) compared to all other organs (all p<0.050), confirming the kidney’s reliance on KB metabolism [2]. Taken together, our novel protocols demonstrate an organ-specific response of mitochondrial respiration to different KBs. # Aubert, G., et al., The failing heart relies on ketone bodies as a fuel. Circulation, 2016. 133(8): p. 698-705. https://doi.org/10.1161/CIRCULATIONAHA.115.017355# Forbes, J.M. and D.R. Thorburn, Mitochondrial dysfunction in diabetic kidney disease. Nature Reviews Nephrology, 2018. 14(5): p. 291-312. https://doi.org/10.1038/nrneph.2018.9# Robinson, A.M. and D.H. Williamson, Physiological roles of ketone bodies as substrates and signals in mammalian tissues. Physiological reviews, 1980. 60(1): p. 143-187. https://doi.org/10.1152/physrev.1980.60.1.143# Puchalska, P. and P.A. Crawford, Metabolic and signaling roles of ketone bodies in health and disease. Annual review of nutrition, 2021. 41: p. 49-77. https://doi.org/10.1146/annurev-nutr-111120-111518# Petrick, H.L., et al., In vitro ketone‐supported mitochondrial respiration is minimal when other substrates are readily available in cardiac and skeletal muscle. The Journal of Physiology, 2020. 598(21): p. 4869-4885. https://doi.org/10.1113/JP280032en other substrates are readily available in cardiac and skeletal muscle. The Journal of Physiology, 2020. 598(21): p. 4869-4885. https://doi.org/10.1113/JP280032 +
'''Authors:'''[[Kolonics Attila]] … '''Authors:'''[[Kolonics Attila]], [[Kawamura T]], [[Szipoecs R]], [[Radak Z]] Aging leads to a loss of muscle mass and a decline in skeletal muscle function (1) leading to imbalance between glucose and lipid metabolism (2). Low exercise capacity is highly correlated with skeletal muscle dysfunction and metabolic disorders (3). Age-associated factors intrinsic to the muscle, including defects in NAD+ synthesis (4), reduced mitochondrial copy number (5), and epigenomic changes affecting the expression of metabolic genes (6) reported. We aimed to characterize mitochondrial fitness of liver in an inborn low- versus high-capacity runners (LCR/HCR) aged female rats to study the spread of metabolic dysfunction. LCR/HCR rats (44th generation, 24 months old) used were artificially selected from genetically heterogeneous N:NIH stock (7). NAD(P)H lifetime imaging (FLIM) characterized liver metabolism in frozen tissues; basal and succinate induced ROS production was evaluated by Amplex Red in the presence of horseradish peroxidase, ΔΨmt by TMRE in intact liver mitochondria. HCR group was less vulnerable to metabolic disorder comparing to LCR group proofed by decreased body mass and increased VO2max. It was further supported by mitochondrial analysis of intact liver mitochondria. Basal ROS production showed no difference between LCR and HCR groups although succinate induced ROS production was higher in LCR group at identical ΔΨmt. NAD(P)H FLIM uncovered subtle alterations: LCR groups had significantly less free NADH comparing to HCR groups (Fig.1). In conclusion, epigenetic changes induced decline of metabolism correlated with deterioration of liver mitochondrial fitness. Succinate induced ROS-production at same membrane potential negatively correlated with free NADH-level. # Frontera WR, Hughes VA, Lutz KJ, Evans WJ (1985) A cross-sectional study of muscle strength and mass in 45- to 78-yr-old men and women. J Appl Physiol. 1991 Aug;71(2):644-50 http://doi.org/10.1152/jappl.1991.71.2.644 # Gheller BJ, Riddle ES, Lem MR, Thalacker-Mercer AE (2016) Understanding Age-Related Changes in Skeletal Muscle Metabolism: Differences Between Females and Males. Annu Rev Nutr. 2016 Jul 17;36:129-56 http://doi.org/10.1146/annurev-nutr-071715-050901# Biolo G, Cederholm T, Muscaritoli M (2014) Muscle contractile and metabolic dysfunction is a common feature of sarcopenia of aging and chronic diseases: from sarcopenic obesity to cachexia. Clin Nutr. 2014 Oct;33(5):737-48 http://doi.org/10.1016/j.clnu.2014.03.007# Yoshino J, Mills KF, Yoon MJ, Imai S (2011) Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab. 2011 Oct 5;14(4):528-36 http://doi.org/10.1016/j.cmet.2011.08.014# Barazzoni R, Short KR, Nair KS (2000) Effects of aging on mitochondrial DNA copy number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart. J Biol Chem. 2000 Feb4; 275(5): 3343-7 http://doi.org/10.1074/jbc.275.5.3343# Jiang MH, Fei J, Lan MS, Lu ZP, Liu M, Fan WW, Gao X, Lu DR (2008) Hypermethylation of hepatic Gck promoter in ageing rats contributes to diabetogenic potential. Diabetologia. 2008 Aug;51(8):1525-33 http://doi.org/10.1007/s00125-008-1034-8# Koch LG, Britton SL (2007) Artificial selection for intrinsic aerobic endurance running capacity in rats. Physiol Genomics. 2001 Feb 7;5(1):45-52 http://doi.org/10.1152/physiolgenomics.2001.5.1.45 running capacity in rats. Physiol Genomics. 2001 Feb 7;5(1):45-52 http://doi.org/10.1152/physiolgenomics.2001.5.1.45 +
'''Authours:''' [[Kayastha Pushpalata]] … '''Authours:''' [[Kayastha Pushpalata]], [[Wieczorkiewicz Filip]], [[Kaczmarek Lukasz]], [[Poprawa Izabela]] Tardigrada (water bears) are well known for their ability to undergo cryptobiosis and survival in extreme conditions. The best-known type of cryptobiosis for their survival is anhydrobiosis i.e. response to lack of water. In this state tardigrades are able to tolerate high pressure, very high and low temperatures, space vacuum, and high levels of UV, and ionizing radiation. These results in various ultrastructural changes in tardigrades, including in mitochondria. We analyzed the effect of different temperatures (20 °C, 35 °C, 37 °C, 40 °C and 42 °C) on the ultrastructure of mitochondria in the tardigrade ''Paramacrobiotus experimentalis'' Kaczmarek et al. 2020. Analyzes were conducted in active specimens, specimens in anhydrobiosis (tun), and rehydrated specimens. The analysis will provide knowledge about changes in the ultrastructure of tardigrades caused by different temperatures. Our results will also determine whether anhydrobiosis protects against temperature-induced ultrastructural changes.s against temperature-induced ultrastructural changes. +
'''BACKGROUND & AIMS:''' Liver ischemi … '''BACKGROUND & AIMS:''' Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage.'''METHODS:''' Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 ''in vivo''.'''RESULTS:''' PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes ''in vitro''. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism.'''CONCLUSIONS:''' Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.ducing or preventing I/R-induced liver injury. +
'''BACKGROUND AND AIMS:'''We investigated … '''BACKGROUND AND AIMS:'''We investigated whether in patients with liver cirrhosis reduced muscle strength is related to dysfunction of muscle mitochondria.'''METHODS:''' The mitochondrial respiratory capacity of the tibial anterior muscle was evaluated in seven patients and eight healthy control subjects by 31P nuclear magnetic resonance spectroscopy (31PMRS) to express ATP turnover in vivo and by respirometry of permeabilized fibres from the same muscle to express the in vitro capacity for oxygen consumption.'''RESULTS:''' Maximal voluntary contraction force for plantar extension was low in the patients (46% of the control value; P < 0.05), but neither the capacity for mitochondrial ATP synthesis, V(max-ATP) (0.38 ± 0.26 vs. 0.50 ± 0.07 mM s(-1) ; P = 0.13) nor the in vitro VO(2max) (0.52 ± 0.21 vs. 0.48 ± 0.21 μmol O2 (min g wet wt.)(-1) P = 0.25) were lowered correspondingly. Also, the activity of citrate synthesis and the respiratory chain complexes II and IV were similar in patients and controls. However during the contractions, the contribution to initial anaerobic ATP production from glycolysis relative to that from PCr was reduced in the patients (0.73 ± 0.22 vs. 0.99 ± 0.09; P < 0.01).'''CONCLUSIONS:''' The results demonstrate that the markedly lower capacity for force generation in patients with liver cirrhosis is unrelated to their capacity for muscle ATP turnover, but the attenuated initial acceleration of anaerobic glycolysis suggests that these patients could be affected by a central limitation to force generation.ted by a central limitation to force generation. +
'''BACKGROUND:''' Mild cold exposure and o … '''BACKGROUND:''' Mild cold exposure and overfeeding are known to elevate energy expenditure in mammals, including humans. This process is called adaptive thermogenesis. In small animals, adaptive thermogenesis is mainly caused by mitochondrial uncoupling in brown adipose tissue and regulated via the sympathetic nervous system. In humans, skeletal muscle is a candidate tissue, known to account for a large part of the epinephrine-induced increase in energy expenditure. However, mitochondrial uncoupling in skeletal muscle has not extensively been studied in relation to adaptive thermogenesis in humans. Therefore we hypothesized that cold-induced adaptive thermogenesis in humans is accompanied by an increase in mitochondrial uncoupling in skeletal muscle.'''METHODOLOGY/PRINCIPAL FINDINGS:''' The metabolic response to mild cold exposure in 11 lean, male subjects was measured in a respiration chamber at baseline and mild cold exposure. Skeletal muscle mitochondrial uncoupling (state 4) was measured in muscle biopsies taken at the end of the respiration chamber stays. Mild cold exposure caused a significant increase in 24h energy expenditure of 2.8% (0.32 MJ/day, range of -0.21 to 1.66 MJ/day, ''p''<0.05). The individual increases in energy expenditure correlated to state 4 respiration (''p''<0.02, ''R''(2) = 0.50).'''CONCLUSIONS/SIGNIFICANCE:''' This study for the first time shows that in humans, skeletal muscle has the intrinsic capacity for cold induced adaptive thermogenesis via mitochondrial uncoupling under physiological conditions. This opens possibilities for mitochondrial uncoupling as an alternative therapeutic target in the treatment of obesity. therapeutic target in the treatment of obesity. +
'''BEC tutorial-Living Communications. Fro … '''BEC tutorial-Living Communications. From Peter Mitchell’s protonmotive force to protonmotive pressure: elements of the science of bioenergetics. Preceding the '''[[MiPNet27.04 IOC155 Schroecken AT |Oroboros O2k-Workshop on high-resolution respirometry]]'''. Schroecken, Austria; 2022.[[File:Gnaiger 2020 BEC MitoPathways.jpg|left|100px|link=Gnaiger_2020_BEC_MitoPathways|Gnaiger 2020 BEC MitoPathways]]The [[mitochondrial membrane potential]] is an element of the science of bioenergetics, linked to the control of respiratory flux and related mitochondrial functions. A [https://pubmed.ncbi.nlm.nih.gov/?term=mitochondrial+membrane+potential PubMed search] on ‘mitochondrial membrane potential’ yields 40 000 results and 3452 for 2021 (search 2022-09-20), with a linear increase during the past 20 years. [[Gnaiger_2020_BEC_MitoPathways#Chapter_8._Protonmotive_pressure_and_respiratory_control |Chapter 8]] on ‘Protonmotive pressure and respiratory control’ of [[Mitochondrial Pathways]] (Gnaiger 2020) introduces a novel perspective on Peter Mitchell’s protonmotive force, which incorporates the mitochondrial membrane potential. If you find the reading is tough, you are not alone. Join this BEC tutorial-Living Communications for a fundamental introduction into the relevant concepts of physical chemistry, which differ from [[Force#Thermodynamic_ignorance |misleading chapters in bioenergetics textbooks]]. A retreat with plenty of informal discussions and group interactions takes you on a journey to visit chemical potential differences versus potential gradients, Gibbs [[energy]] versus Gibbs [[force]], quantities of capacity versus intensity, protonmotive force and [[motive unit]]s, [[flow]]s and [[force]]s, and finally protonmotive [[pressure]]. This will introduce students (and teachers) to a new understanding of mitochondrial membrane potential and the protonmotive force, connecting the ideal gas equation, osmotic pressure, the [[Boltzmann constant]] and [[gas constant]] with [[Fick 1855 Pogg Ann |Fick’s]] and [[Einstein 1905 Ann Physik 549 |Einstein’s diffusion equation]]. If theory gets dry and grey, join for a swim in lake Körbersee, for a Walk&Talk in the colorful alpine environment of the Schröcken-Tannberg region, and a visit to the [https://www.alpmuseum.at/ Alpmuseum ufm Tannberg].s://www.alpmuseum.at/ Alpmuseum ufm Tannberg]. +
'''BEC tutorial-Living Communications. Mit … '''BEC tutorial-Living Communications. Mitochondrial membrane potential and Peter Mitchell’s protonmotive force: elements of the science of bioenergetics. Preceding the [[EMC2022 Prague CZ |EMC 2022 49th European Muscle Conference]], Prague, Czech Republic.[[File:Gnaiger 2020 BEC MitoPathways.jpg|left|100px|link=Gnaiger_2020_BEC_MitoPathways|Gnaiger 2020 BEC MitoPathways]]The [[mitochondrial membrane potential]] is an element of the science of bioenergetics, linked to the control of respiratory flux and related mitochondrial functions. A [https://pubmed.ncbi.nlm.nih.gov/?term=mitochondrial+membrane+potential PubMed search] on ‘mitochondrial membrane potential’ yields 40 000 results and 3452 for 2021 (search 2022-09-20), with a linear increase during the past 20 years. [[Gnaiger_2020_BEC_MitoPathways#Chapter_8._Protonmotive_pressure_and_respiratory_control |Chapter 8]] on ‘Protonmotive pressure and respiratory control’ of [[Mitochondrial Pathways]] (Gnaiger 2020) introduces a novel perspective on Peter Mitchell’s protonmotive force, which incorporates the mitochondrial membrane potential. If you find the reading is tough, you are not alone. Join this BEC tutorial-Living Communications for an introduction into the relevant concepts of physical chemistry, which differ from [[Force#Thermodynamic_ignorance |misleading chapters in bioenergetics textbooks]] on potential gradients, Gibbs ''[[energy]]'', protonmotive [[flow]] and [[force]], and finally protonmotive [[pressure]]. This will introduce students (and teachers) to a new understanding of mitochondrial membrane potential and the protonmotive force, connecting the ideal gas equation, osmotic pressure, the [[Boltzmann constant]] and [[gas constant]] with [[Fick 1855 Pogg Ann |Fick’s]] and [[Einstein 1905 Ann Physik 549 |Einstein’s diffusion equation]]. If theory gets tough, join for a [[MiPNet27.05 BEC tutorial-Living Communications pmF |follow-up retreat]].C tutorial-Living Communications pmF |follow-up retreat]]. +
'''BEC tutorial-Living Communications. Mit … '''BEC tutorial-Living Communications. Mitochondrial membrane potential and Peter Mitchell’s protonmotive force: elements of the science of bioenergetics. [[File:Gnaiger 2020 BEC MitoPathways.jpg|left|100px|link=Gnaiger_2020_BEC_MitoPathways|Gnaiger 2020 BEC MitoPathways]]The [[mitochondrial membrane potential]] is an element of the science of bioenergetics, linked to the control of respiratory flux and related mitochondrial functions. A PubMed search on ‘mitochondrial membrane potential’ yields nearly 40 000 results and 3442 for 2021 (search 2022-07-04), with a linear increase during the past 20 years. [[Gnaiger_2020_BEC_MitoPathways#Chapter_8._Protonmotive_pressure_and_respiratory_control |Chapter 8]] on ‘Protonmotive pressure and respiratory control’ of [[Mitochondrial Pathways]] (Gnaiger 2020) introduces a novel perspective on Peter Mitchell’s protonmotive force, which incorporates the mitochondrial membrane potential. If you find the reading is tough, you are not alone. Join this BEC tutorial-Living Communications for an introduction into the relevant concepts of physical chemistry, which differ from [[Force#Thermodynamic_ignorance |misleading chapters in bioenergetics textbooks]] on potential gradients, Gibbs ''[[energy]]'', protonmotive [[flow]] and [[force]], and finally protonmotive [[pressure]]. This will introduce students (and teachers) to a new understanding of mitochondrial membrane potential and the protonmotive force, connecting the ideal gas equation, osmotic pressure, the [[Boltzmann constant]] and [[gas constant]] with [[Fick 1855 Pogg Ann |Fick’s]] and [[Einstein 1905 Ann Physik 549 |Einstein’s diffusion equation]]. If theory gets tough, join for a [[MiPNet27.05 BEC tutorial-Living Communications pmF |follow-up retreat]].C tutorial-Living Communications pmF |follow-up retreat]]. +
'''Background ''' Mitochondrial dysfuncti … '''Background ''' Mitochondrial dysfunction is one of the most characteristic properties of the cancer cell. However, it is not known whether oxidative energy metabolism has already become altered in conditions of atrophic gastritis, a precancerous state of gastric disease. The purpose of our study was to comparatively characterize oxidative phosphorylation (OXPHOS) in the atrophic and nonatrophic gastric corpus mucosa.'''Methods''' Mucosal biopsies were taken from 12 patients with corpus dominant atrophic gastritis and from 12 patients with nonatrophic mucosa (controls). One part of the tissue samples was permeabilized with saponin for analysis of the function of the respiratory chain using high-resolution respirometry, and another part was used for histopathological examination. The serum level of pepsinogen I (S-PGI) was determined with a specific enzyme immunoassay (EIA).'''Results''' Compared to the control group, the maximal capacity of OXPHOS in the atrophy group was almost twofold lower, the respiratory chain complex I-dependent respiration, normalized to complex II-dependent respiration, was reduced, and respiratory control by ADP in the presence of succinate was increased in the atrophic corpus mucosa. In the whole cohort of the patients studied, serum S-PGI level correlated positively with complex I-dependent respiration or complex Idependent to complex II-dependent respiration ratio.'''Conclusions''' Corpus dominant atrophic gastritis is characterized by decreased respiratory capacity and relative deficiency of the respiratory complex I of mitochondria in the mucosa, the latter defect probably limiting mitochondrial ATP production and energetic support of the secretory function of the zymogenic mucosal cells.y function of the zymogenic mucosal cells. +
'''Background''' Mitochondrial dysfunction … '''Background''' Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined.'''Methodology/Principal Findings''' Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2.'''Conclusions/Significance''' We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease.hondrial integrity in Parkinson's disease. +
'''Background''': "Open peer review" (OPR) … '''Background''': "Open peer review" (OPR), despite being a major pillar of Open Science, has neither a standardized definition nor an agreed schema of its features and implementations. The literature reflects this, with numerous overlapping and contradictory definitions. While for some the term refers to peer review where the identities of both author and reviewer are disclosed to each other, for others it signifies systems where reviewer reports are published alongside articles. For others it signifies both of these conditions, and for yet others it describes systems where not only "invited experts" are able to comment. For still others, it includes a variety of combinations of these and other novel methods. '''Methods''': Recognising the absence of a consensus view on what open peer review is, this article undertakes a systematic review of definitions of "open peer review" or "open review", to create a corpus of 122 definitions. These definitions are systematically analysed to build a coherent typology of the various innovations in peer review signified by the term, and hence provide the precise technical definition currently lacking. '''Results''': This quantifiable data yields rich information on the range and extent of differing definitions over time and by broad subject area. Quantifying definitions in this way allows us to accurately portray exactly how ambiguously the phrase "open peer review" has been used thus far, for the literature offers 22 distinct configurations of seven traits, effectively meaning that there are 22 different definitions of OPR in the literature reviewed. '''Conclusions''': I propose a pragmatic definition of open peer review as an umbrella term for a number of overlapping ways that peer review models can be adapted in line with the aims of Open Science, including making reviewer and author identities open, publishing review reports and enabling greater participation in the peer review process. participation in the peer review process. +
'''Background''': Atherosclerosis is one o … '''Background''': Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues.'''Methods''': We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC).'''Results''': The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC.'''Conclusions''': Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease.'''General significance''': Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis.lin resistance-associated atherosclerosis. +
'''Background''': It has been hypothesized … '''Background''': It has been hypothesized that reduced axonal transport contributes to the degeneration of neuronal processes in Parkinson's disease (PD). Mitochondria supply the adenosine triphosphate (ATP) needed to support axonal transport and contribute to many other cellular functions essential for the survival of neuronal cells. Furthermore, mitochondria in PD tissues are metabolically and functionally compromised. To address this hypothesis, we measured the velocity of mitochondrial movement in human transmitochondrial cybrid "cytoplasmic hybrid" neuronal cells bearing mitochondrial DNA from patients with sporadic PD and disease-free age-matched volunteer controls (CNT). The absorption of low level, near-infrared laser light by components of the mitochondrial electron transport chain (mtETC) enhancesmitochondrial metabolism, stimulates oxidative phosphorylation and improves redox capacity. PD and CNT cybrid neuronal cells were exposed to near-infrared laser light to determine if the velocity of mitochondrial movement can be restored by low level light therapy (LLLT). Axonal transport of labeled mitochondria was documented by time lapse microscopy in dopaminergic PD and CNT cybrid neuronal cells before and after illumination with an 810 nm diode laser (50 mW/cm2) for 40 s. Oxygen utilization and assembly of mtETS complexes were also determined.'''Results''': The velocity of mitochondrial movement in PD cybrid neuronal cells (0.175 +/- 0.005 SEM) was significantly reduced (p < 0.02) compared to mitochondrial movement in disease free CNT cybrid neuronal cells (0.232 +/- 0.017 SEM). For two hours after LLLT, the average velocity of mitochondrial movement in PD cybrid neurites was significantly (p < 0.003) increased (to 0.224 +/- 0.02 SEM) and restored to levels comparable to CNT. Mitochondrial movement in CNT cybrid neurites was unaltered by LLLT (0.232 +/- 0.017 SEM). Assembly of complexes in the mtETC was reduced and oxygen utilization was altered in PD cybrid neuronal cells. PD cybrid neuronal cell lines with the most dysfunctional mtETC assembly and oxygen utilization profiles were least responsive to LLLT.'''Conclusion''': The results from this study support our proposal that axonal transport is reduced in sporadic PD and that a single, brief treatment with near-infrared light can restore axonal transport to control levels. These results are the first demonstration that LLLT can increase axonal transport in model human dopaminergic neuronal cells and they suggest that LLLT could be developed as a novel treatmentto improve neuronal function in patients with PD. treatment to improve neuronal function in patients with PD. +
'''Background''': Reactive oxygen species … '''Background''': Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H2DCF) but had no direct impact on the H2O2 production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).'''Methods''': H2O2 generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.'''Results''': We show that diazoxide reduces ROS production by mitochondrial Complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Qo site of Complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of Complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na+ or K+ excluding a role for putative mitochondrial KATP-channels.'''General significance''': A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of Complex II promotes transient generation of signaling ROS at Complex III (during preconditioning) or attenuates the production of deleterious ROS at Complex I (during ischemia and reperfusion).x III (during preconditioning) or attenuates the production of deleterious ROS at Complex I (during ischemia and reperfusion). +
'''Background''': Restriction of intracell … '''Background''': Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role ''in vivo'' is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (''Oncorhynchus mykiss''), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.'''Results''': Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria.'''Conclusions''': The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in lowperformance hearts.ion restrictions in lowperformance hearts. +
'''Background''': Tumor cells are characte … '''Background''': Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.'''Methodology/Principal Findings''': Various cellular and biochemical parameters were evaluated in lung cancer H460 cells treated with the histone deacetylase inhibitors (HDACis), sodium butyrate (NaB) and trichostatin A (TSA). NaB and TSA reduced glycolytic flux, assayed by lactate release by H460 cells in a concentration dependent manner. NaB inhibited the expression of glucose transporter type 1 (GLUT 1), but substantially increased mitochondria bound hexokinase (HK) activity. NaB induced increase in HK activity was associated to isoform HK I and was accompanied by 1.5 fold increase in HK I mRNA expression and cognate protein biosynthesis. Lactate dehydrogenase (LDH) and pyruvate kinase (PYK) activities were unchanged by HDACis suggesting that the increase in the HK activity was not coupled to glycolytic flux. High resolution respirometry of H460 cells revealed NaB-dependent increased rates of oxygen consumption coupled to ATP synthesis. Metabolomic analysis showed thatNaB altered the glycolytic metabolite profile of intact H460 cells. Concomitantly we detected an activation of the pentose phosphate pathway (PPP). The high O2 consumption in NaB-treated cells was shown to be unrelated to mitochondrial biogenesis since citrate synthase (CS) activity and the amount of mitochondrial DNA remained unchanged.'''Conclusion''': NaB and TSA induced an increase in mitochondrial function and oxidative metabolism in H460 lung tumor cells concomitant with a less proliferative cellular phenotype.h a less proliferative cellular phenotype. +
'''Background'''—Opening of the mitochondr … '''Background'''—Opening of the mitochondrial permeability transition pore (mPTP) is a crucial event in lethal reperfusion injury. Phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK3β) has been involved in cardioprotection. We investigated whether phosphorylated GSK3β may protect the heart via the inhibition of mPTP opening during postconditioning.'''Methods and Results'''—Wild-type and transgenic GSK3β-S9A mice (the cardiac GSK3β activity of which cannot be inactivated) underwent 60 minutes of ischemia and 24 hours of reperfusion. At reperfusion, wild-type and GSK3β-S9A mice received no intervention (control), postconditioning (3 cycles of 1 minute ischemia and 1 minute of reperfusion), the mPTP inhibitor cyclosporine A (CsA; 10 mg/kg IV), or the GSK3β inhibitor SB216763 (SB21; 70 µg/kg IV). Infarct size was assessed by triphenyltetrazolium chloride staining. The resistance of the mPTP to opening after Ca2+ loading was assessed by spectrofluorometry on mitochondria isolated from the area at risk. In wild-type mice, infarct size was significantly reduced by postconditioning, CsA, and SB21, averaging 39±2%, 35±5%, and 37±4%, respectively, versus 58±5% of the area at risk in control mice (P<0.05). In GSK3β-S9A mice, only CsA, but not postconditioning or SB21, reduced infarct size. Postconditioning, CsA, and SB21 all improved the resistance of the mPTP in wild-type mice, but only CsA did so in GSK3β-S9A mice.'''Conclusion'''—These results suggest that S9-phosphorylation of GSK3β is required for postconditioning and likely acts by inhibiting the opening of the mitochondrial permeability transition pore.pening of the mitochondrial permeability transition pore. +
'''Background:''' Appropriate control of m … '''Background:''' Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that coordinatemitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death.'''Results:''' We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one ofthe yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1.'''Conclusions:''' We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast. with nutritional status in budding yeast. +
'''Background:''' Determination of mitocho … '''Background:''' Determination of mitochondrial membrane potential ((m) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of ΔΨm induced by inhibitors of oxidative phosphorylation characterizerespective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis.'''Methods:''' ΔΨm in BSC-40 and HeLa G cell lines was determined by flow cytometry and spectrofluorometry. Its changes induced by specific mitochondrial inhibitors were evaluated using 3,3 ΔΨ-dihexyloxacarbocyanine iodide(DiOC6(3)), tetramethylrhodamine ethyl ester, and Mito-Tracker Red. Mitochondrial function was further characterized by oxygen consumption.'''Results:''' Inhibition of respiration by antimycin A or uncouplingof mitochondria by FCCP decreased ΔΨm in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of ΔΨmin HeLa G cells and an increase of ΔΨm in BSC-40 cells. Statistically significant differences in ΔΨm between the two cell lines were found with both flow cytometry and spectrofluorometry. Respirometry showed higher basaland FCCP-stimulated respiration in BSC-40 cells.'''Conclusion:''' Changes of ΔΨm and oxygen consumption showed that BSC-40 cells are more sensitive than HeLa G cells to inhibitors of mitochondrial function, suggesting that BSC-40 cells are more dependent than HeLa G cells onaerobic ATP production. Determination of ΔΨm changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry.ivity than the ones by spectrofluorometry. +
'''Background:''' Sepsis is a severe infla … '''Background:''' Sepsis is a severe inflammatory disorder with a high mortality in intensive care units mostly due to multiorgan failure. Mitochondrial dysfunction is regarded as a key factor involved in the pathogenesis of septic disorders, leading to a decline in energy supply. The aim of the present study was to evaluate whether application of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) could improve mitochondrial function and thus might serve as a potential energy source under inflammatory conditions. '''Materials and Methods:''' As an experimental approach, starved human endothelial cells and monocytes were incubated with hexanoic acid, heptanoic acid, octanoic acid, or glucose and subsequently subjected to high-resolution respirometry to assess mitochondrial function under baseline conditions. In a second set of experiments, cells were pretreated with tumor necrosis factor-α to mimic inflammation and sepsis. Results: We demonstrated that addition of SCFAs and MCFAs increases mitochondrial respiratory capacity at baseline and inflammatory conditions in both cell types. None of the fatty acids induced changes in mitochondrial DNA content or the generation of proinflammatory cytokines, indicating a beneficial safety profile. '''Conclusion:''' We deduce that SCFAs and MCFAs are suitable and safe sources of energy under inflammatory conditions with the capability to partly restore mitochondrial respiration. partly restore mitochondrial respiration. +
'''Basal respiration''' is well defined in physiology. Terminology in mitochondrial physiology gains quality by reference to established concepts. +
'''Bioblast 2022: Inaugural Conference of ''Bioenergetics Communications''''' +
'''Bioblast''' was launched as a glossary … '''Bioblast''' was launched as a glossary and index for high-resolution respirometry (Oroboros Instruments: OroboPedia) and Mitochondrial Physiology (MitoPedia), to find topics quickly, as a dynamic tool for summarizing definitions of terms, symbols and abbreviations. However, it´s potential benefits as an innovative, self-developing database make the '''Bioblast Wiki''' much more than a service by Oroboros.i''' much more than a service by Oroboros. +
'''COST CA22169 METAHEART Kick-off meeting MC1, Brussels, Belguim, 2023-10-18''' +
'''Call for research - We all need oxygen … '''Call for research - We all need oxygen – “The oceans are gasping for air”''' 1, 2, 3“Human dominion over planet Earth is driving profound changes that may culminate in extinction.” 4 Whilst there is wider research into the oceanic impact of climate change including warming and acidification,5, 6 and on oxygen content of oceans, there is very little research into the specific impact of acidification and related carbon dioxide changes on marine photosynthetic oxygen production.This is an important field of research as it also involves consideration of the consequent effects of excess atmospheric carbon dioxide, including warming, on oceanic and atmospheric oxygen, oxygen exchange between them and possibilities of tipping points whereby photosynthetic marine organisms may rapidly die off, potentially leading to severe existential consequences for aerobic life forms.We bemoan the loss of polar bears and rare alpine plants along with changes to weather and food, but as societies and individuals we are reluctant to severely moderate the day-to-day fossil fuel energy consumption that underlies 21st century life. When faced by the choice of polar bears vs cars, heating, laptops and phones, the polar bears lose!Ocean acidification is a more empirically evidenced phenomenon than climate change, however, it is also less prominent in the public psyche even though it springs from the same increased atmospheric carbon dioxide levels. Were research to be commissioned, though, that provides clear evidence of risk to oceanic oxygen production and therefore atmospheric oxygen levels, the conclusions could be far-reaching, including identifying a potential tipping point that may result in human extinction.This stark prospect would, arguably, be easier to convey to, and fix in the wider public consciousness than the more diffuse issues around climate change. Humans are reminded with every breath they take that oxygen is essential to their health function and, ultimately, their survival and existence as a species.7 The importance of the prospect of oxygen depletion for future generations would be easily understood by all, and so promote greater public engagement and cohesive demand for a global response to try and find viable energy alternatives to fossil fuels.iable energy alternatives to fossil fuels. +
'''Chemical biology approaches to assessing and modulating mitochondria, Buckinghamshire, UK''' +
'''Contents''' * Introduction [http://www. … '''Contents'''* Introduction [http://www.oroboros.at/fileadmin/user_upload/Reprints/O-MiPNet-Publ/MitoPathways2_Introduction.pdf pdf]* MitoPathways to Complex I: [[MiPNet11.04]]* MitoPathways to Complex II: [[MiPNet11.09]]* MitoPathways to Complexes I&II: [[MiPNet12.12]]* MitoPathways compilation: [[MiPNet12.13]] * MitoPathways - respiratory states: [[MiPNet12.15]]* Cell respiration and phosphorylation control: [[MiPNet08.09]] * HRR and phosphorylation control: [[MiPNet10.04]]* FCRs in isolated mitochondria: [[MiPNet12.11]]* O2k manual titrations: [[MiPNet09.12]]* O2k-paradigm: [[MiPNet09.01]]* [[Gnaiger 2012 MitoPathways References|References]]* The Oroboros - Feeding on negative entropy [http://www.oroboros.at/fileadmin/user_upload/Reprints/O-MiPNet-Publ/MitoPathways2_Introduction.pdf pdf]Reprints/O-MiPNet-Publ/MitoPathways2_Introduction.pdf pdf] +
'''Context''': Previous studies on leg ske … '''Context''': Previous studies on leg skeletal musculature have demonstrated mitochondrial dysfunction associated with type 2 diabetes mellitus (T2DM), but it is not known whether mitochondrial dysfunction is present in the upper extremities.'''Objective''': The aim of the study was to compare mitochondrial respiration and markers of mitochondrial content in skeletal muscle of arm and leg in patients with T2DM and obese control subjects.'''Patients''': Ten patients with T2DM (age, 52.3 ± 2.7 yr; body mass index, 30.1 ± 1.2 kg/m2) (mean ± SE) were studied after a 2-wk washout period of oral antihyperglycemic agents. Ten control subjects (age, 54.3 ± 2.8 yr; body mass index, 30.4 ± 1.2 kg/m2) with normal fasting and 2-h oral glucosetolerance test blood glucose levels were also included. Main Outcome Measure:Wemeasured mitochondrial respiration in saponin-treated skinned musclefibers from biopsies of m. deltoideus and m. vastus lateralis using high-resolution respirometry.'''Results''': In the arm, mitochondrial respiration and citrate synthase activity did not differ between groups, but mitochondrial respiration per milligram of muscle was significantly higher in the leg muscle of the control subjects compared to T2DM. Fiber type compositions in arm and leg muscleswere not different between the T2DM and control group, and maximum rate of O2 consumption did not differ between the groups.'''Conclusion''': The results demonstrate that reduced mitochondrial function in T2DM is only present in the leg musculature. This novel finding suggests that mitochondrial dysfunction is not a primary defect affecting all skeletal muscle but could be related to a decreased response to locomotor muscle use in T2DM. (J Clin Endocrinol Metab 95: 857–863, 2010)J Clin Endocrinol Metab 95: 857–863, 2010) +
'''Do you ever dream about an equation?'' … '''Do you ever dream about an equation?''The Mitchell’s dream series by [[Odra Noel]] is a dream on equations and shows a dream on the equation that penetrates all of biology since Peter D Mitchell started publishing on the protonmotive force equation [1]. Can we imagine how many dreaming was required until the chemiosmotic hypothesis emerged on energy coupling by the protonmotive force of oxidative phosphorylation in the bioblasts, which comprise the mitochondria, chloroplasts, bacteria and archaea? Seeing Odra Noel’s pictures on Mitchell’s dream provides insights into the equations of biophysics and biochemistry: these equations do not just belong to our books. They do belong to our cells, our [[bioblasts]], to the living world. It is the mitochondria that help us to understand these equations, since the equations are in the mitochondria, they are the visible parts of the mitochondria and open insights into function beyond the visible form – this is mitochondrial physiology.e form – this is mitochondrial physiology. +
'''Doerrier C, Draxl A, Wiethuechter A, Ei … '''Doerrier C, Draxl A, Wiethuechter A, Eigentler A, Gnaiger E (2015) Mitochondrial respiration in permeabilized fibers versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03(04):1-9.''' In the present study we compared mitochondrial function of permeabilized fibers and homogenate of heart muscle of mice. In addition, respiration of trout heart homogenate preparations were compared with permeabilized fibers, and the PBI-Shredder was successfully tested with preparation of trout liver.:» Product: [[Oroboros O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]][OROBOROS O2k-Catalogue | O2k-Catalogue]] +
'''Doerrier C, Gnaiger E (2003-2016) High- … '''Doerrier C, Gnaiger E (2003-2016) High-resolution respirometry and coupling-control protocol with living cells: ROUTINE, LEAK, ET-pathway, ROX. Mitochondr Physiol Network 08.09(11):1-8.'''An experiment on respiration of [[living cells]] is reported from an O2k-Workshop on high-resolution respirometry. Leukemia cells were incubated at a density of 1 million cells/ml in 2 ml culture medium in two O2k-Chambers operated in parallel. Cellular ROUTINE respiration, ''J''R, resulted in volume-specific oxygen consumption of 20 pmol·s-1·ml-1. Oxygen concentration changed by merely 6.4 and 6.5 µM in the two O2k-Chambers over a period of 5 min (<1% air saturation per minute). Inhibition by oligomycin (''JL''), and rotenone (residual oxygen consumption, ''J''ROX; after uncoupling) reduced respiration to 5 and 1 pmol·s-1·ml-1, while inducing the noncoupled state by the uncoupler FCCP revealed the capacity of the Electron transfer-pathway (ET-pathway) at ''JE'' of 50 pmol·s-1·ml-1. The ROUTINE control ratio, ''R/E'', was 0.4 (uncoupling control ratio, UCR=''E/R''=2.5), and the LEAK control ratio, ''L/E'', was 0.1 (''E/L''=12.0). This indicates tight coupling of OXPHOS, and a large ET-pathway excess capacity over ROUTINE respiration. The net ROUTINE control ratio, net''R''=(''R-L'')/''E'' was 0.30, indicating that 30% of ET-pathway capacity was activated for ATP production.Automatic correction for instrumental background amounted to 13% for ROUTINE respiration, but to >50% and 180% for ''JL'' and ''J''ROX, respectively, illustrating the importance of real-time correction. The experiment illustrates the sensitivity and resproducibility of high-resolution respirometry with the OROBOROS O2k. Calibrations and routine corrections provide the basis of the high accuracy required for mitochondrial respiratory physiology. Real-time analyses were performed, combining high-resolution with instant diagnostic information. In this update graphs are presented illustrating some features of DatLab.:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]os O2k-Catalogue | O2k-Catalogue]] +
'''EBEC2024 Satellite Oroboros O2k-Workshop: Mito&Chlora High-Resolution Respirometry and PhotoBiology'''. Innsbruck, Austria (2024 Sep 02-04). +
'''ET-pathway states''' are defined in [[mitochondrial preparations]] complementary to [[coupling-control state]]s in mitochondrial physiology. +
'''Essentials:''' The role of protein C (P … '''Essentials:''' The role of protein C (PC) activation in experimental autoimmune encephalitis (EAE) is unknown. PC activation is required for mitochondrial function in the central nervous system. Impaired PC activation aggravates EAE, which can be compensated for by soluble thrombomodulin. Protection of myelin by activated PC or solulin is partially independent of immune-modulation.'''SUMMARY:''' Studies with human samples and in rodents established a function of coagulation proteases in neuro-inflammatory demyelinating diseases (e.g. in multiple sclerosis [MS] and experimental autoimmune encephalitis [EAE]). Surprisingly, approaches to increase activated protein C (aPC) plasma levels as well as antibody-mediated inhibition of PC/aPC ameliorated EAE in mice. Hence, the role of aPC generation in demyelinating diseases and potential mechanisms involved remain controversial. Furthermore, it is not known whether loss of aPC has pathological consequences at baseline (e.g. in the absence of disease). To explore the role of thrombomodulin (TM)-dependent aPC generation at baseline and in immunological and non-immunological demyelinating disease models. Myelination and reactive oxygen species (ROS) generation were evaluated in mice with genetically reduced TM-mediated protein C activation (TMPro/Pro) and in wild-type (WT) mice under control conditions or following induction of EAE. Non-immunological demyelination was analyzed in the cuprizone-diet model. Impaired TM-dependent aPC generation already disturbs myelination and mitochondrial function at baseline. This basal phenotype is linked with increased mitochondrial ROS and aggravates EAE. Reducing mitochondrial ROS (p66Shc deficiency), restoring aPC plasma levels or injecting soluble TM (solulin) ameliorates EAE in TMPro/Pro mice. Soluble TM additionally conveyed protection in WT-EAE mice. Furthermore, soluble TM dampened demyelination in the cuprizone-diet model, demonstrating that its myelin-protective effect is partially independent of an immune-driven process. These results uncover a novel physiological function of TM-dependent aPC generation within the CNS. Loss of TM-dependent aPC generation causes a neurological defect in healthy mice and aggravates EAE, which can be therapeutically corrected.© 2016 International Society on Thrombosis and Haemostasis.ically corrected. © 2016 International Society on Thrombosis and Haemostasis. +
'''Expert/inn/en-Workshop Medizintechnik, … '''Expert/inn/en-Workshop Medizintechnik, Innsbruck, AT.'''== Time and Location ==13:00 until 17:00 at Standortagentur Tirol, Ing.-Etzelstr. 17, Innsbruck == General information (German) ==Wie vielen bereits bekannt ist, hat sich die Standortagentur Tirol dazu entschlossen – gemeinsam mit IMP – das Projekt „Tirol 2025“ zu starten, um strategische Handlungsfelder für Tirol in ausgewählten Branchen zu definieren. Und in der Zwischenzeit hat sich diesbezüglich viel getan.'''WAS BISHER GESCHAH...'''In den letzten Monaten wurden Gespräche mit hochkarätigen internationalen Expertinnen und Experten aus Wirtschaft, Wissenschaft und Kultur geführt sowie zahlreiche Zukunftsthemen rund um die Tätigkeitsfelder der Standortagentur untersucht. Aus dem generierten Wissen konnten daraufhin konkrete Zukunftshypothesen entwickelt werden, die von knapp 450 Befragten bezüglich Eintrittswahrscheinlichkeit sowie Art der Auswirkung auf Tirol und seinen Branchen bewertet wurden. Darauf aufbauend konnten aus weiteren 40 Interviews mit Tiroler Branchenexpert/innen zukünftige Kernthemen für die Branchencluster der Standortagentur identifiziert werden. '''DER NÄCHSTE SCHRITT...'''Im nächsten Schritt geht es nun darum, gemeinsam mit 8 bis 12 Experten pro Themencluster Lösungsansätze zu den zentralen Zukunftsherausforderungen für Unternehmen, für die Standortagentur sowie für die Politik zu entwickeln und zu diskutieren. Im Bereich Medizintechnik werden folgende Fragestellungen behandelt:* Wie könnten neue Geschäftsmodelle helfen die Erfolgsgeschichte der Tiroler Medizintechnikunternehmen auszubauen? * Was sind spannende, digitale Lösungsansätze um die Wettbewerbsfähigkeit Tiroler Medizintechnikunternehmen erhöhen zu können? * Wie können Tiroler Medizintechnikunternehmen durch eine branchenübergreifende Vernetzung (IT, Gesundheit,..) innovative Angebote entwickeln? * Wie müsste eine wirksame Förderpolitik für Tiroler Medizintechnikunternehmen aussehen? * Welche Vermarktungsansätze könnten Tiroler Medizintechnikunternehmen im Wettbewerb massiv weiterhelfen? * Welche Ansätze helfen Tiroler Medizintechnikunternehmen deren Effektivität und Effizienz in der Entwicklung und Herstellung zu steigern? * Welche Ansätze könnten (kleineren) Tiroler Medizintechnikunternehmen helfen, mit der Flut an neuen Regularien umzugehen? Ihr Mitwirken in diesem Prozess ist uns ein zentrales Anliegen, da es nur mithilfe von hochkarätigem Expertenwissen gelingen kann, effektive strategische Schritte in die Zukunft zu setzen.egische Schritte in die Zukunft zu setzen. +
'''FAT4BRAIN Advanced Virtual O2k-Workshop IOC149 on Amplex UltraRed, Virtual Event, 2021''' +
'''FAT4BRAIN Advanced Virtual O2k-Workshop IOC150 on TMRM and Calcium Green, Virtual Event, 2021''' +
'''FAT4BRAIN O2k-Workshop IOC159 on HRR for the assessment of mitochondrial bioenergetics.''' Riga, LV, 2023 +
'''FAT4BRAIN O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2022 October 03-08). +
'''FAT4BRAIN Online Workshop: Brain energy metabolism in emotion and cognition, 2021''' +
'''FAT4BRAIN Online Workshop: Central regulatory mechanisms of energy metabolism, 2021''' +
'''FAT4BRAIN School IOC147 on mt-functionality assessment in CNS-related applications, Virtual Event, 2020''' +
'''FAT4BRAIN Symposium - Long COVID and acetylcarnitines: From preclinical models to clinical applications and translation potential, Jena, Germany, 2022''' +
'''FAT4BRAIN Symposium - Novel drug target and pathway identification, Riga, Latvia, 2023''' +
'''FAT4BRAIN Virtual O2k-Workshop IOC148 on HRR for the assessment of mitochondrial bioenergetics, Virtual Event, 2021''' +
'''FAT4BRAIN Workshop IOC 151 on mitochondrial function in CNS-related applications: from pre-clinical to clinical studies, Innsbruck AT, 2022''' +
'''FEBS Advanced course - Frontiers in Molecular Biochemistry of Mitochondria.''' Warsaw, Poland; 2006 June 09. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[OROBOROS O2k-Catalogue | O2k-Catalogue]] +
'''Fasching M, Fontana-Ayoub M, Gnaiger E … '''Fasching M, Fontana-Ayoub M, Gnaiger E (2018) Mitochondrial respiration medium - MiR06. Mitochondr Physiol Network 14.13(06):1-4.''' <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>Mitochondrial respiration medium MiR06 was developed for oxygraph incubations of mitochondrial preparations. MiR06 = MiR05 plus catalase. MiR06Cr = MiR06+creatine.:» Product: [[MiR05-Kit]]R05-Kit]] +
'''Fontana-Ayoub M, Fasching M, Gnaiger E … '''Fontana-Ayoub M, Fasching M, Gnaiger E (2016) Selected media and chemicals for respirometry with mitochondrial preparations. Mitochondr Physiol Network 03.02(18):1-10.'''Different media for tissue preparation and respiration are used in investigations of mitochondrial function. Initial decisions on the composition of media and chemicals are decisive for long-term studies and crucial for comparability of results. As a guideline, we summarize an update of our experience with media and chemicals for high-resolution respirometry with isolated mitochondria, permeabilized cells, muscle fibres and tissue homogenates. Whereas optimization is necessary for specific experimental protocols, standardization will improve the comparability of results obtained in different laboratories. Efforts towards standardization are important for the advancement of mitochondrial physiology.:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''Gnaiger E (2014) Service of the polarog … '''Gnaiger E (2014) Service of the polarographic oxygen sensor OroboPOS. Mitochondr Physiol Network 19.01(B01):19-24.''' '''This is an old version, which applies up to O2k-Series F and to DatLab 5.''': ''New version:'' '''[[MiPNet19.18B POS-service|»MiPNet19.18B POS-service]]'''[[MiPNet19.18B POS-service|»MiPNet19.18B POS-service]]''' +
'''Gnaiger E, Doeller JE, Kraus D, Shiva S … '''Gnaiger E, Doeller JE, Kraus D, Shiva S, Brookes PS, Darley-Usmar VM (2011) NO effect on mitochondrial oxygen kinetics at low oxygen. O2k workshop Report. Mitochondr Physiol Network 08.12(07).''' »[http://www.bioblast.at/index.php/File:MiPNet08.12_NO-O2kWorkshop.pdf Versions]A single pilot experiment was carried out during an O2k workshop on high-resolution respirometry (IOC22). Respiration of isolated rat liver mitochondria was inhibited by addition of NO, which increased the sensitivity to oxygen >25-fold when compared to the half-saturation oxygen pressure, p50, in the absence of NO. Oxygen kinetics followed a monophasic hyperbolic function up to 2.2 kPa with NO (p50=0.93 kPa), compared to the standard oxygen range to 1.1 kPa without NO (p50=0.035 kPa).[[Image:MiPNet08.12.jpg|400px|centre|thumb|Figure 1. Oxygen dependence of mt-respiration and competitive inhibition by NO. The full line shows oxygen kinetics at state 3 with pyruvate and malate in the absence of NO, measured in the physiological oxygen range (from Gnaiger et al. 1998a). Dotted lines show inhibition of respiration by the indicated NO concentrations, where measurements were performed with low-resoltion respirometry and are restricted to the high oxygen range (from Koivisto et al. 1977). Extrapolations into the physiological oxygen range (shaded region) suggest sigmoidal oxygen kinetics, which requires testing by direct measurements at low oxygen (modified after Gnaiger, Kuznetsov 2002).]][[Aguirre_2010_Biochim_Biophys_Acta| Reference: Biochim Biophys Acta 1797: 557-565 (2010)]]:» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''Gnaiger E, Lassnig B (1997) DatLab 2. Analysis of oxygen kinetics. Mitochondr Physiol Network 02.05.''' :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]] +
'''Gnaiger E, Reck M (1997) DatLab 2 Analysis. High resolution of data in the lab. Mitochondr Physiol Network 02.07: 1-72.''' :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue ]] +
'''Gnaiger E, Rieger G (1999) From step ti … '''Gnaiger E, Rieger G (1999) From step titration to ramp injection: Uncoupling by FCCP with TIP. Mitochondr Physiol Network 04.05.''':» Product: [[O2k-Catalogue: TIP2k|TIP2k]], [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]Fully supported by the O2k-Core and control of the TIP2k by the software DatLab: The TIP2k can be programmed for multiple titrations and continuous injections. As an alternative to traditional step titration, the TIP offers the new option of ramp injection, providing maximum resolution of the concentration dependence of oxygen flux. This is illustrated by the recording of cellular respiratory flux as a function of a continuous increase of uncoupler (FCCP) concentration.''Titration:''Programmable, automatic titration regimes, with titration volumes of 0.05 to 250 µl, variable titration intervals and duration of titration pulses.''Injection:''Steady-state injection: Operation at quasi steady-states by continuous injection of substrates at limiting rates of consumption, providing new flexibility in experimental design by combining the technical advantages of closed and open systems. Programmable injection flows: 0.01 to 25 µl.s-1.Ramp injection (MiPNet04.05, see above): Ramp increase of effector concentrations by "continuous titration".DatLab software for feedback control by the the TIP2k: for steady-state respirometry at selected oxygen levels and pH-stat applications.ed oxygen levels and pH-stat applications. +
'''Highlights''' Heterozygous mice (αMHC& … '''Highlights'''Heterozygous mice (αMHC403/+) expressing the human hypertrophic cardiomyopathy (HCM) disease causing mutation ''Arg403Gln'' exhibit cardinal features of HCM.This study investigated the role of L-type Ca2+ channel (ICa-L) in regulating mitochondrial function in ''Arg403Gln'' (αMHC403/+) mice.Activation of ICa-L in αMHC403/+ mice caused a significantly greater increase in mitochondrial membrane potential and metabolic activity when compared to wild-type mice.Increases in mitochondrial membrane potential and metabolic activity were attenuated with ICa-L antagonists and when F-actin or β-tubulin were depolymerized.ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.'''Summary'''Heterozygous mice (αMHC403/+) expressing the human disease-causing mutation ''Arg403Gln'' exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+ mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+ mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+ cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+. These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+ mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the ''Arg403Gln'' mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity. +
'''How mitochondria work''' 10 years afte … '''How mitochondria work'''10 years after setting the foundations of the [[Mitochondrial Physiology Society]] (MiP2003, Schröcken, Austria) our search continues as to what mitochondrial physiology is. Mitochondrial physiology is the study of “''how mitochondria work''”. Animal physiology is the study of “''how animals work''” - says the title of a textbook by Knut Schmidt-Nielsen. Comparative physiology derives its fascination from the diversity of form and function. Organismic variation is studied in diverse environments and in extremes of physiological performance, with explosive activities and high power output in short bursts or endurance over prolonged periods of time with high efficiency. Diversity is nature’s treasure and the subject of comparative physiology. The famous August Krogh principle – Krogh received the Nobel Prize in 1920 - is frequently cited [1,2]: “''For a large number of problems there will be some animal of choice or a few such animals on which it can be most conveniently studied.''” This principle was first formulated in 1975 by another Nobel laureate who received the Prize in 1953 for the metabolic cycle that carries his name, Sir Hans Krebs [3,4]. This direct link between one of the most famous mitochondrial biochemists and the August Krogh principle that “''epitomized the very essence of comparative physiology''” [2] immediately raises the question: Why was comparative mitochondrial physiology not established some 30 to 40 years ago?y not established some 30 to 40 years ago? +
'''Implementing DORA principles by publishing in Bioenergetics Communications - beyond counting papers''' - presentation by Erich Gnaiger, BEC Editor-in-chief +
'''Importance''': The American Heart Assoc … '''Importance''': The American Heart Association ideal cardiovascular health (CVH) score is associated with the risk of cardiovascular disease (CVD) and mortality. However, it is unclear whether the number of years spent in ideal CVH is associated with morbidity or with mortality.'''Objective:''' To evaluate whether living longer with a higher CVH score in midlife is associated with lower risk of hypertension, diabetes, chronic kidney disease, CVD and its subtypes (coronary heart disease, stroke, congestive heart failure, and peripheral artery disease), or all-cause mortality in later life.'''Design, Setting, and Participants''': This prospective cohort study used data from 1445 participants from 1991 to 2015 who participated in the community-based Framingham Heart Study Offspring investigation conducted in Massachusetts. The CVH scores of participants were assessed at examination cycles 5, 6, and 7 (1991-1995; 1995-1998; and 1998-2001, respectively). Individuals were excluded from analyses of the association between duration of CVH score and outcomes if they had the outcome of interest at the seventh examination. The median follow-up was approximately 16 years. Data were analyzed from April 2018 to October 2019. The CVH score categories were poor for scores 0 to 7, intermediate for scores 8 to 11, and ideal for scores 12 to 14. A composite score was derived based on smoking status, diet, physical activity, resting blood pressure levels, body mass index, fasting blood glucose levels, and total serum cholesterol levels.'''Main Outcomes and Measures''': Number of events and number at risk for each main outcome, including incident hypertension, diabetes, chronic kidney disease, CVD, and all-cause mortality, after the seventh examination.'''Results''': Of 1445 eligible participants, the mean (SD) age was 60 (9) years, and 751 (52 %) were women. Number of events/number at risk for each main outcome after the seventh examination were 348/795 for incident hypertension, 104/1304 for diabetes, 198/918 for chronic kidney disease, 210/1285 for CVD, and 300/1445 for all-cause mortality. At the seventh examination, participants mostly had poor (568 [39 %]) or intermediate (782 [54 %]) CVH scores. For each antecedent (before examination cycle 7) 5-year duration that participants had intermediate or ideal CVH, they were less likely to develop adverse outcomes (hazards ratios of 0.67 [95 % CI, 0.56-0.80] for incident hypertension, 0.73 [95 % CI, 0.57-0.93] for diabetes, 0.75 [95 % CI, 0.63-0.89] for chronic kidney disease, 0.73 [95 % CI, 0.63-0.85] for CVD, and 0.86 [95 % CI, 0.76-0.97] for all-cause mortality) relative to living the same amount of time in poor CVH (referent group). No effect modification was observed by age or by sex.'''Conclusions and Relevance''': These results suggest that more time spent in better CVH in midlife may have salutary cardiometabolic benefits and may be associated with lower mortality later in life.ciated with lower mortality later in life. +
'''International Course on High-Resolution … '''International Course on High-Resolution Respirometry - Satellite to 1st SMRM.''' Hyderabad, India; 2011 December 08:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''Introduction''' Low blood pressure, in … '''Introduction'''Low blood pressure, inadequate tissue oxygen delivery and mitochondrial dysfunction have all been implicated in the development of sepsis-induced organ failure. This study evaluated the effect on liver mitochondrial function of using norepinephrine to increase blood pressure in experimental sepsis.'''Methods'''Thirteen anaesthetized pigs received endotoxin (Escherichia coli lipopolysaccharide B0111:B4; 0.4 μg/kg per hour) and were subsequently randomly assigned to norepinephrine treatment or placebo for 10 hours. Norepinephrine dose was adjusted at 2-hour intervals to achieve 15 mmHg increases in mean arterial blood pressure up to 95 mmHg. Systemic (thermodilution) and hepatosplanchnic (ultrasound Doppler) blood flow were measured at each step. At the end of the experiment, hepatic mitochondrial oxygen consumption (high-resolution respirometry) and citrate synthase activity (spectrophotometry) were assessed.'''Results'''Mean arterial pressure (mmHg) increased only in norepinephrine-treated animals (from 73 [median; range 69 to 81] to 63 [60 to 68] in controls [''P'' = 0.09] and from 83 [69 to 93] to 96 [86 to 108] in norepinephrine-treated animals [''P'' = 0.019]). Cardiac index and systemic oxygen delivery (''D''O2) increased in both groups, but significantly more in the norepinephrine group (''P'' < 0.03 for both). Cardiac index (ml/min per·kg) increased from 99 (range: 72 to 112) to 117 (110 to 232) in controls (''P'' = 0.002), and from 107 (84 to 132) to 161 (147 to 340) in norepinephrine-treated animals (''P'' = 0.001). ''D''O2 (ml/min per·kg) increased from 13 (range: 11 to 15) to 16 (15 to 24) in controls (''P'' = 0.028), and from 16 (12 to 19) to 29 (25 to 52) in norepinephrine-treated animals (''P'' = 0.018). Systemic oxygen consumption (systemic VO2) increased in both groups (''P'' < 0.05), whereas hepatosplanchnic flows, ''D''O2 and ''V''O2 remained stable. The hepatic lactate extraction ratio decreased in both groups (''P'' = 0.05). Liver mitochondria Complex I-dependent and II-dependent respiratory control ratios were increased in the norepinephrine group (Complex I: 3.5 [range: 2.1 to 5.7] in controls versus 5.8 [4.8 to 6.4] in norepinephrine-treated animals [''P'' = 0.015]; Complex II: 3.1 [2.3 to 3.8] in controls versus 3.7 [3.3 to 4.6] in norepinephrine-treated animals [''P'' = 0.09]). No differences were observed in citrate synthase activity.'''Conclusion'''Norepinephrine treatment during endotoxaemia does not increase hepatosplanchnic flow, oxygen delivery or consumption, and does not improve the hepatic lactate extraction ratio. However, norepinephrine increases the liver mitochondria Complex I-dependent and II-dependent respiratory control ratios. This effect was probably mediated by a direct effect of norepinephrine on liver cells. direct effect of norepinephrine on liver cells. +
'''Introduction''' Mitochondrial disorders … '''Introduction'''Mitochondrial disorders are extremely heterogeneous and can involve single tissue, such as the optic nerve to widespread pathologies including muscle disorders, peripheral neuropathies, encephalopathy, cardiomyopathies or complex multisystem disorders. The age at onset ranges from neonatal to adult life. Mitochondrial dysfunction is a relatively common disorder but the clinical and genetic variability makes it difficult to diagnose. Our primary hypothesis is that disturbance in mitochondrial respiratory chain can be diagnosed with blood test and skin biopsy, by combining structural (Blue native page) and functional information, with high-resolution respirometry of the respiratory chain in blood cells. This rapid diagnostic method can be used to diagnose the flora of undiagnosed and unknown encephalopathy in children today. '''Methods'''Our aim is to 1. Establish reference material for mitochondrial normal function in children through high resolution respirometry by diagnosing thrombocytes and fibroblasts. We also want to establish reference material for structural information with Blue Native PAGE (BNP) in fibroblasts. 2. We want to use these methods in children with known mitochondrial disease to confirm that our methods are usefull.3. We want to compare our methods to known methods today for diagnoses of mitochondrial disease (muscle biopsy).4. We want to see the benefits of treatment by comparing results through BNP and respirometry before and after treatment. 5. We want to use these methods for diagnosis of unknown encephalopathy in children.'''Results'''We have started collecting reference material from children from 0-17 years old. We collect blood and skin biopsy from healthy children that are having a small operation at the University Hospital in Lund. Our aim is to collect reference material from 60 children in different age groups. We also collect blood and skin biopsy from 30 newborn babies from the umbilical cord.We have also done respirometry on children that have both suspected mitochondrial disease and children with known mitochondrial disease. The results are promising. We have also taken skin biopsy from these children but we do not know the outcome yet. We have also started using our methods to look at children with autism and other encephalopathy. '''Conclusion'''Mitochondrial dysfunction has been difficult to diagnose. Our methods give us the opportunity to diagnose mitochondrial dysfunction in unknown encephalopathy in children by a more rapid and simple way than before.y a more rapid and simple way than before. +
'''Introduction''' Mitochondrial dysfuncti … '''Introduction'''Mitochondrial dysfunction has been suggested as a contributing factor to the pathogenesis of sepsis-induced multiple organ failure. Also, restoration of mitochondrial function, known as mitochondrial biogenesis, has been implicated as a key factor for the recovery of organ function in patients with sepsis. Here we investigated temporal changes in platelet mitochondrial respiratory function in patients with sepsis during the first week after disease onset.'''Methods'''Platelets were isolated from blood samples taken from 18 patients with severe sepsis or septic shock within 48 hours of their admission to the intensive care unit. Subsequent samples were taken on day 3 to 4 and day 6 to 7. Eighteen healthy blood donors served as controls. Platelet mitochondrial function was analyzed by high-resolution respirometry. Endogenous respiration of viable, intact platelets suspended in their own plasma or PBS glucose was determined. Further, in order to investigate the role of different dehydrogenases and respiratory complexes as well as to evaluate maximal respiratory activity of the mitochondria, platelets were permeabilized and stimulated with complex-specific substrates and inhibitors.'''Results'''Platelets suspended in their own septic plasma exhibited increased basal non-phosphorylating respiration (state 4) compared to controls and to platelets suspended in PBS glucose. In parallel, there was a substantial increase in respiratory capacity of the Electron transfer-pathway from day 1 to 2 to day 6 to 7 as well as compared to controls in both intact and permeabilized platelets oxidizing Complex I and/or II-linked substrates. No inhibition of respiratory complexes was detected in septic patients compared to controls. Non-survivors, at 90 days, had a more elevated respiratory capacity at day 6 to 7 as compared to survivors. Cytochrome c increased over the time interval studied but no change in mitochondrial DNA was detected.'''Conclusions'''The results indicate the presence of a soluble plasma factor in the initial stage of sepsis inducing uncoupling of platelet mitochondria without inhibition of the Electron transfer-pathway. The mitochondrial uncoupling was paralleled by a gradual and substantial increase in respiratory capacity. This may reflect a compensatory response to severe sepsis or septic shock, that was most pronounced in non-survivors, likely correlating to the severity of the septic insult.ting to the severity of the septic insult. +
'''Introduction''': Alzheimer's disease (A … '''Introduction''': Alzheimer's disease (AD) is the most frequent neurodegenerative disease, characterized by progressive decline in variety of higher brain functions - memory, orientation, and thinking. According to increasing evidences, mitochondrial insufficiencies contribute to pathology of AD; changes were described in AD brains, blood cells and human fibroblasts.'''Objectives''': On molecular level, oxygen and glucose metabolism is altered and energy metabolism is impaired.Mitochondrial abnormalities and alterations in mitochondrial enzymes, especially Complex I and cytochrome ''c'' oxidase, were observed. However, the cause and important aspects of AD mechanism have not yet been sufficiently clarified.'''Aims''': The aim of our study was to find whether kinetics of oxygen consumption is modified in AD patients. Further, we afford to suggest parameters that could be suitable as AD markers.'''Methods''': AD patients and healthy control group were included in the study. Respiratory rate of mitochondria, as measure of total activity of the system of oxidative phosphorylation (OXPHOS), was measured in mitochondria using oxygraph with Clark-type electrodes. High-resolution respirometry was performed in intact as well as in permeabilized platelets.'''Results''': Our results indicate significantly lower respiratory rate in intact platelets as well as lower respiratory capacity of Electron transfer-pathway in patients with AD compared to controls.'''Conclusions''': We propose that decrease in oxygen consumption may participate in pathophysiology of AD, and respiratory rate in platelets could be AD marker.tory rate in platelets could be AD marker. +
'''Introduction:''' Hydrogen sulfide (H< … '''Introduction:''' Hydrogen sulfide (H2S) is a potent inhibitor of cytochrome c oxidase (COX) and, thus, of mitochondrial respiration [1]. Since H2S was reported to induce a suspended animation-like status characterized by reduced energy expenditure and hypothermia [2], we sought to determine the effect of hypothermia on mitochondrial respiratory capacity and H2S-related COX inhibition. We further studied the influence of variations in pH on both variables.'''Methods:''' All measurements were conducted in digitonin-permeabilised cultured peritoneal macrophages using high-resolution respirometry [3] (Oxygraph-2 k, Oroboros, Austria). Maximum mitochondrial respiration (1 to 2 Mio cells/ml respiration medium) was achieved in the uncoupled state by adding pyruvate, malate, glutamate and succinate as respiratory substrates. Then, in one of the two chambers of the oxygraph, mitochondrial respiration was inhibited stepwise by incremental concentrations of the H2S donor Na2S (1 to 64 μM). In the parallel chamber, the identical inhibitor titration sequence was preceded by the inhibition of the respiratory chain by rotenone and antimycin A followed by the selective stimulation of CIV after addition of ascorbate and TMPD. COX excess capacity (% of ET-pathway) was calculated based on the ratio of inhibition of mitochondrial respiration with full operating respiratory chain versus the CIV-stimulated condition. This experimental sequence was repeated at 37 °C and 25 °C with a medium pH of 7.1 and then at 37°C with a pH of 6.8 and 7.7.'''Results:''' CIV excess capacity (median (quartiles)) was significantly higher at 25 °C than at 37 °C (134 (113; 140) vs 61 (47; 79)), most likely due to the almost halved mitochondrial respiratory capacity at hypothermia (50 (37; 63) vs 95 (81; 103) pmol O2/s × Mio cells). Changing the medium pH from 6.8 to 7.7 significantly increased the COX excess capacity (91 (79; 103) vs 71 (64; 82) pmol O2/s × Mio cells), which again was related to the significantly lower mitochondrial respiratory capacity with more acidic conditions (80 (70; 89) vs 94 (85; 98)).'''Conclusions:''' Our results suggest that COX excess capacity is temperature as well as pH dependent in peritoneal macrophages. This effect may protect cells from H2S toxicity at low temperatures and high pH values. in peritoneal macrophages. This effect may protect cells from H2S toxicity at low temperatures and high pH values. +
'''Journal publication 2021-11-16 in [[Fischer 2021 Antioxidants |»Antioxidants«]]'' … '''Journal publication 2021-11-16 in [[Fischer 2021 Antioxidants |»Antioxidants«]]'''</big>[[File:Fischer_2021_MitoFit_Fe_liver - graphical abstract.png|right|500px|Graphical abstract]] Iron is an essential co-factor for many cellular metabolic processes, and mitochondria are main sites of utilization. Iron accumulation promotes production of reactive oxygen species (ROS) via the catalytic activity of iron species. Herein, we investigated the consequences of dietary and genetic iron overload on mitochondrial function. C57/BL6N wildtype and ''Hfe-/-'' mice, the latter a genetic hemochromatosis model, received either normal diet (ND) or high iron diet (HI) for two weeks. Liver mitochondrial respiration was measured using high-resolution respirometry along with analysis of expression of specific proteins and ROS production. HI promoted tissue iron accumulation and slightly affected mitochondrial function in wildtype mice. Hepatic mitochondrial function was impaired in ''Hfe-/-'' mice on ND and HI. Compared to wildtype mice, ''Hfe-/-'' mice on ND showed increased mitochondrial respiratory capacity. ''Hfe-/-'' mice on HI showed very high liver iron levels, decreased mitochondrial respiratory capacity and increased ROS production associated with reduced mitochondrial aconitase activity. Although ''Hfe-/-'' resulted in increased mitochondrial iron loading, the concentration of metabolically reactive cytoplasmic iron and mitochondrial density remained unchanged. Our data shows multiple effects of dietary and genetic iron loading on mitochondrial function and linked metabolic pathways, providing an explanation for fatigue in iron-overloaded hemochromatosis patients and suggests iron reduction therapy for improvement of mitochondrial function. chromatosis patients and suggests iron reduction therapy for improvement of mitochondrial function. +
'''Journal publication 2022-03-03 in [[Zdrazilova 2022 PLOS ONE |»'''PLOS ONE 17:e0264496'''«]]'' … '''Journal publication 2022-03-03 in [[Zdrazilova 2022 PLOS ONE |»'''PLOS ONE 17:e0264496'''«]]'''Version 1 ('''v1''') '''2021-09-21''' [https://www.mitofit.org/images/1/15/Zdrazilova_2021_MitoFit_ace-sce.pdf doi:10.26124/mitofit:2021-0007]Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology and toxicology. Fibroblasts are cultured in monolayers but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached and suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell.No differences were observed in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. Multiple steps of uncoupler titrations in the O2k allowed for evaluation of maximum electron transfer capacity, which was higher than respiration obtained in the XF24 due to a limitation to two uncoupler titrations.Quantitative evaluation of respiration measured in different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Consistent results obtained with different platforms provide a test for reproducibility and allow for building an extended respirometric database. extended respirometric database. +
'''Journal publication 2022-03-21 in [[Fischer 2022 Metabolites |»Metabolites«]]'' … '''Journal publication 2022-03-21 in [[Fischer 2022 Metabolites |»Metabolites«]]'''</big>Iron is an essential component for metabolic processes including oxygen transport within hemoglobin, tricarboxylic acid (TCA) cycle activity and mitochondrial energy transformation. Iron deficiency can thus lead to metabolic dysfunction and eventually result in iron deficiency anemia (IDA) which affects approximately 1.5 billion people worldwide. Using a rat model of IDA induced by phlebotomy, we studied the effects of IDA on mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) and liver. Furthermore, we evaluated whether mitochondrial function evaluated by high-resolution respirometry in PBMCs reflects corresponding alterations in the liver. Surprisingly, mitochondrial respiratory capacity was increased in PBMCs from rats with IDA compared to controls. In contrast, mitochondrial respiration remained unaffected in livers from IDA rats. Of note, citrate synthase activity indicated an increased mitochondrial density in PBMCs, whereas it remained unchanged in the liver, partly explaining the different responses of mitochondrial respiration in PBMCs and liver. Taken together, these results indicate that mitochondrial function determined in PBMCs cannot serve as a valid surrogate for respiration in the liver. Metabolic adaptions to iron deficiency resulted in different metabolic reprogramming in the blood cells and liver tissue. ng in the blood cells and liver tissue. +
'''KEY POINTS:''' Genetic mutations in car … '''KEY POINTS:'''Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterised by myocyte remodeling, disorganisation of cytoskeletal proteins and altered energy metabolism. The L-type Ca2+ channel is the main route for calcium influx and critical to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network. We find that L-type Ca2+ channel kinetics are altered in cTnI-G203S cardiac myocytes, and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI-G203S cardiac myocytes. These responses occur as a result of impaired communication between the L-type Ca2+ channel and cytoskeletal protein F-actin, involving decreased movement of actin-myosin, and block of mitochondrial VDAC, resulting in a 'hypermetabolic' mitochondrial state. We propose that L-type Ca2+ channel antagonists such as diltiazem may be effective in reducing the cardiomyopathy by normalising mitochondrial metabolic activity.'''ABSTRACT:'''Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy (HCM). HCM is associated with disorganisation of cytoskeletal proteins and altered energy metabolism. The L-type Ca2+ channel (ICa-L ) plays an important role in regulating mitochondrial function. This involves a functional communication between ICa-L and mitochondria via the cytoskeletal network. We investigate the role of ICa-L in regulating mitochondrial function in 25-30-week old cardiomyopathic mice expressing human disease causing mutation Gly203Ser in cTnI (cTnI-G203S). The inactivation rate of ICa-L is significantly faster in cTnI-G203S myocytes (cTnI-G203S: τ1 = 40.68 ± 3.22, n = 10 versus wt: τ1 = 59.05 ± 6.40, n = 6, P < 0.05). Activation of ICa-L caused a greater increase in mitochondrial membrane potential (Ψm , 29.19 ± 1.85%, n = 15 versus wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 versus wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred due to impaired communication between ICa-L and F-actin, involving lack of dynamic movement of actin-myosin, and block of mitochondrial VDAC. Similar responses were observed in pre-cardiomyopathic mice. ICa-L antagonists nisoldipine and diltiazem decreased Ψm to basal levels. We conclude that the Gly203Ser mutation leads to impaired functional communication between ICa-L and mitochondria resulting in a 'hypermetabolic' state. This may contribute to development of cTnI-G203S cardiomyopathy because the response is present in young pre-cardiomyopathic mice. ICa-L antagonists may be effective in reducing the cardiomyopathy by altering mitochondrial function. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.e is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. +
'''Klinische MitochondrienMedizin und Umwe … '''Klinische MitochondrienMedizin und Umweltmedizin 2015, Internationales Wissenschaftsforum der Universität, Heidelberg, DE.'''Im März 2015 startet in Heidelberg bereits vierte Auflage eines erfolgreichen Curriculums Klinische MitochondrienMedizin und Umweltmedizin. Die Veranstaltung ist als ärztliche Fortbildung mit Ärztekammer-, Zahnärzte- und Apothekerkammer-Anerkennung und als Wahlpflichtmodul des KWKM-Masterstudiengangs an der Europa-Universität Viadrina konzipiert. An sechs intensiven Wochenenden (freitags und samstags) werden in Vorträgen und Übungen:* Grundlagen der Mitochondrien-Medizin,* aktuelle Forschungsergebnisse,* Diagnosemethoden und* Therapieverfahren der mitochondrialen Medizinu.a. in Verbindung mit der Umweltmedizin, Umwelt-Zahnmedizin, Frauenheilkunde und Psychotherapie erläutert. Ergänzend zu dem theoretischen Teil werden Hospitanten-Tage im Centrum für Integrative Medizin in Speyer angeboten, welches auf dem Gebiet der Mitochondrien-Medizin spezialisiert ist.r Mitochondrien-Medizin spezialisiert ist. +
'''Klinische MitochondrienMedizin und Umwe … '''Klinische MitochondrienMedizin und Umweltmedizin 2016, Internationales Wissenschaftsforum der Universität, Heidelberg, DE.''' [[Media:MitochondrialMedicine_2016.pdf| »Flyer]] Im März 2016 startet in Heidelberg bereits fünfte Auflage eines erfolgreichen Curriculums '''Klinische MitochondrienMedizin und Umweltmedizin'''. Die Veranstaltung ist als ärztliche Fortbildung mit Ärztekammer-, Zahnärzte- und Apothekerkammer-Anerkennung und als Wahlpflichtmodul des KWKM-'''Masterstudiengangs an der Europa-Universität Viadrina''' konzipiert. An sechs intensiven Wochenenden (freitags und samstags) werden in Vorträgen und Übungen:* Grundlagen der Mitochondrien-Medizin* Aktuelle Forschungsergebnisse* Diagnosemethoden* Therapieverfahren der mitochondrialen Medizinu.a. in Verbindung mit der Umweltmedizin, Umwelt-Zahnmedizin, Frauenheilkunde undPsychotherapie erläutert. Ergänzend zu dem theoretischen Teil werden Hospitanten-Tage im BioMedical Center in Speyer angeboten, welches auf dem Gebiet der Mitochondrien-Medizin spezialisiert ist. Mehr Informationen finden Sie hier: http://www.mito-medizin.de/fortbildung/ '''Termine 2016:''' :* Kurs A: 04. - 05.03:* Kurs B: 15. - 16.04:* Kurs C: 20. - 21.05:* Kurs D: 17. - 18.06:* Kurs E: 09. - 10.09:* Kurs F: 11. - 12.11Kurs E: 09. - 10.09 :* Kurs F: 11. - 12.11 +
'''Kuznetsov AV, Gnaiger E. Laboratory pro … '''Kuznetsov AV, Gnaiger E. Laboratory protocol: Complex I (NADH:Ubiquinone Oxidoreductase, EC 1.6.5.3). Mitochondrial membrane enzyme. Mitochondr Physiol Network 08.15.'''Complex I (CI) is the segment of the electron transport system (integral enzyme of the inner mitochondrial membrane) responsible for electron transfer from NADH to ubiquinone. CI is sensitive to different pathologies, particularly to oxidative stress, which is involved in ischemia-reperfusion injury, anoxia/ reoxygenation, aging, etc (Kuznetsov et al 2004; Rouslin & Millard 1981; Rouslin & Ranganathan, 1983; Rouslin, 1983). For the assessment of CI activity, among the ubiquinone isoprenologs, it is most convenient to use ubiquinone-1 (CoQ1) as electron acceptor, because of its relative water solubility. Importantly, CoQ1 yields one of the lowest rotenone insensitive rates and a high enzymatic rate. It is, therefore, the best electron acceptors for the CI assay.:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]][[Oroboros O2k-Catalogue]] +
'''Kuznetsov AV, Gnaiger E. Laboratory pro … '''Kuznetsov AV, Gnaiger E. Laboratory protocol: Lactate dehydrogenase. Cytosolic marker enzyme. Mitochondr Physiol Network 08.18.''' Lactate dehydrogenase (EC 1.1.1.27) is an enzyme, which catalyzes the last step in glycolysis. LDH is a soluble enzyme and localized in the cytosol (cytoplasm). LDH, therefore, is used as a quantitative marker enzyme for the intact cell, its activity providing information on cellular glycolytic capacity (Renner et al, 2003). Measurement of LDH release (leakage) is an important and frequently applied test for cellular membrane permeabilization (rupture) and severe irreversible cell damage. LDH leakage normally correlates well with CK release and the trypan blue viability test.:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]][[Oroboros O2k-Catalogue]] +
'''Lassnig B, Gnaiger E. Laboratory protoc … '''Lassnig B, Gnaiger E. Laboratory protocol: Isolation of rat liver mitochondria. Mitochondr Physiol Network 08.13.''' <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue]]roboros O2k-Catalogue]] +
'''Lectures on High-Resolution Respirometr … '''Lectures on High-Resolution Respirometry and Oroboros O2k Demonstration at BTK 1994.''' Innsbruck, Tyrol, Austria; 1994 September.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''Long Night of Research 2016: MitoFit – Training for the powerhouses of your blood- and muscle cells. Innsbruck, AT.''' +
'''Long Night of Research 2018: The diagnostic bioenergetic report – a milestone on the way to mitochondrial fitness and physical well-being. Innsbruck, AT.''' +
'''Long Night of Research 2020: The diagnostic bioenergetic report – a milestone on the way to mitochondrial fitness and physical well-being. Virtual Event.''' +
'''Meeting of the European Society for Clinical Investigation, Paris, FR''' +
'''Metabolic Energetics in Ecological, Cellular and Biomedical Research.''' Aberystwyth Wales United Kingdom; 1993 March 01-03. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''MiPschool, Obergurgl, Austria, 2023: Mitochondrial structure and function, respiratory supercomplexes, and respiratory control''' +
'''MitoCom Lecture''' '''2011-Nov-10, 8:1 … '''MitoCom Lecture''''''2011-Nov-10, 8:15 - 09:45'''. Medical University Innsbruck, Anichstr. 25, Chirurgie (8-U1-517) Seminarraum 2Speaker: '''[[Jezek P|Prof. Dr. Petr Jezek, Prague]]'''Host: [[Gnaiger E|Erich Gnaiger, DSL, MitoCom Tyrol]]'''Abstract''': Three-dimensional (3D) super-resolution microscopy, using a biplane detection scheme, termed biplane photo-activated localization microscopy (Biplane FPALM), enables imaging of volumes as thick as whole cells and reveals otherwise unaccessible details of cellular organization [1]. Hence, we attempted to visualize mitochondrial reticulum via the matrix space loaded with mitochondria-addressed Eos, while transfecting cells by lentiviral expression. Our 3D images of single Eos molecules in the matrix space have proven the continuous character of mitochondrial reticulum tubules, i.e., an existence of a highly interconnected major mitochondrial reticulum in insulinoma Ins1E and oxidative-phosphorylation-dependent glutaminolytic hepatoma HepG2 cells [2] (Figure).Also, using Eos-conjugate of mitochondrial transcription factor-A (TFAM), we have imaged nucleoids of mitochondrial DNA (mtDNA) in which TFAM represents a major assessor protein. Using PA-CFP2-TFAM and mitochondria-addressed Eos, the first 3D two color super-resolution images were obtained for mitochondrial reticulum together with the distribution of mt nucleoids in it. In intact cells we have found mt nucleoids of a narrow size distribution. The Biplane FPALM technique has proven to be robust and reliable for imaging of mitochondrion and related substructures.Supported by grants P302/10/0346 (GACR); ME09029 (Czech Ministry of Education); IAA500110701, and M200110902 (Academy of Sciences).) and 1R01GM091791-02 (NIH). Disclosure statement: J.B. declares significant financial interest in Vutara, Inc.[1] Juette MF, Gould TJ, Lessard MD, Mlodzianoski MJ, Nagpure BS, Bennett BT, Hess ST, Bewersdorf J (2008) 3D sub-100 nm resolution by biplane fluorescence photoactivation localization microscopy. Nat. Methods 5: 527-529.[2] Mlodzianoski MJ, Schreiner JM, Callahan SP, Smolková K, Dlasková A, Šantorová J, Ježek P, Bewersdorf J (2011) Sample drift correction in 3D fluorescence photoactivation localization microscopy. Opt. Express. 19: 15009-15019.microscopy. Opt. Express. 19: 15009-15019. +
'''MitoFit Open Seminar on respiration, cryopreservation and viability test in human blood cells'''. Innsbruck, AT +
'''Mitochondrial Medicine 2016, Seattle, W … '''Mitochondrial Medicine 2016, Seattle, WA, USA.''' The [[United Mitochondrial Disease Foundation]] Symposium has been recognized by many researchers, scientists, and families as THE symposium for mitochondrial disease in the world. 10 years ago, the UMDF had only a handful of exhibitors and less than 200 scientific attendees. We now have more exhibitors than space at times and close to 600 attendees … representing almost every state in the United States and more than 15 different countries from around the world.different countries from around the world. +
'''Mitochondrial Physiology (MiP) ''contin … '''Mitochondrial Physiology (MiP) ''continues a tradition of rigorous mitochondrial bioenergetics'' '''([http://www.mitophysiology.org quoting the International MiPsociety]). The company [[Oroboros Instruments]] Corp. values this tradition as a basis of our continuous instrumental development, which is part of our concept of Corporate Social Responsibility. In this spirit and with emphasis on our Educational Responsibility, we initiated and support the ''[[MiP-Collection]]''.[[MiP-Collection]]''. +
'''Mitochondrial capacity''': [[OXPHOS]] … '''Mitochondrial capacity''': [[OXPHOS]] capacity is evaluated in isolated mitochondria (mt) and permeabilized cells with physiological substrate cocktails to reconstitute tricarboxylic acid cycle function. As a consequence, convergent electron flow from Complexes CI+II of the electron transfer-pathway ([[ET-pathway]]) to the [[Q-junction]] exerts an additive effect on flux [1].'''Oxygen kinetics of mt-respiration''': The apparent ''K''m,O2 or ''c''50 [µM] (''p''50 [kPa]) of mt-respiration increases linearly with respiratory capacity controlled by metabolic state, from 0.2 to 1.6 µM determined by [[high-resolution respirometry]]. O2 gradients are significant only in large cells including cardiomyocytes. The apparent ''p''50 increases 100-fold in permeabilized muscle fibers due to diffusion gradients [2].'''mt-function at ''V''O2max''': Aerobic capacity of the human leg muscle exceeds maximum O2 uptake of isolated mitochondria [3] and v. lateralis during ''V''O2max [4]. Therefore, oxygen supply limits aerobic performance, proportional to the apparent mt-excess capacity [5]. mt-respiration is more sensitive to average ''p''O2 in heterogenous tissues than under homogenous conditions in vitro. Tissue heterogeneity increases the kinetic dependence of flux on average intracellular ''p''O2. High mt-density reinforces the steepness of oxygen gradients and oxygen heterogeneity in the tissue, contributing to the O2 limitation in athletic vs sedentary individuals at ''V''O2max [6]. This provides a functional rationale for the observation that hypoxia does not specifically trigger mt-biogenesis [7].Contribution to K-Regio ''[[MitoCom_O2k-Fluorometer|MitoCom Tyrol]]''.[1] [[Gnaiger 2009 Int J Biochem Cell Biol|Gnaiger 2009]]; [[Lemieux_2011_Int J Biochem Cell Biol|Lemieux et al 2011 Int J Biochem Cell Biol]] [2] [[Gnaiger_2003_Adv Exp Med Biol|Gnaiger 2003]]; [[Scandurra_2010_Adv Exp Med Biol|Scandurra, Gnaiger 2010 Adv Exp Med Biol]]. [3] Rasmussen et al 2001 AJP.[4] [[Boushel_2011_Mitochondrion|Boushel et al 2011 Mitochondrion]].[5] [[Gnaiger_1998_J_Exp_Biol|Gnaiger et al 1998 JEB]].[6] Richardson et al; Haseler et al JAP.[7] [[Pesta_2011_AJP|Pesta et al 2011 AJP]]; [[Jacobs_2011_J_Appl_Physiol|Jacobs et al 2011 JAP]].Jacobs_2011_J_Appl_Physiol|Jacobs et al 2011 JAP]]. +
'''Note''': Subscript ‘§’ indicates throug … '''Note''': Subscript ‘§’ indicates throughout the text those parts, where ''potential differences'' provide a mathematically correct but physicochemically incomplete description and should be replaced by ''stoichiometric potential differences'' ([[Gnaiger 1993 Pure Appl Chem |Gnaiger 1993b]]). A unified concept on vectorial motive transformations and scalar chemical reactions will be derived elsewhere (Gnaiger, in prep.). Appreciation of the fundamental distinction between ''differences of potential'' versus ''differences of stoichiometric potential'' may be considered a key to critically evaluate the arguments presented in Section 3 on the protonmotive force. Since this discussion appears to be presently beyond the scope of a MitoEAGLE position statement, Section 3 is removed from the next version and [[Gnaiger 2019 MitoFit Preprint Arch |'''final manuscript''']]. This section should become a topic of discussion within [[WG1 MitoEAGLE protocols, terminology, documentation |Working Group 1]] of the MitoEAGLE consortium, following a primary peer-reviewed publication of the concept of stoichiometric potential differences.t of stoichiometric potential differences. +
'''O2k-International course on high-resolu … '''O2k-International course on high-resolution respirometry and MiPNet meeting.''' Schroecken, Voralberg, Austria; 2004 September 15-21.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry and MiPNet workshop.''' Schroecken, Voralberg, Austria; 2006 December 13 to 17. :>> O2k-Workshop: [[Oroboros Events]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry and ROS/NO detection.''' Schroecken, Voralberg, Austria; 2005 September 13-16. +
'''O2k-International course on high-resolution respirometry.''' 2007 August 24, 9:00 a.m. to 3:00 p.m. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' 2007 July 18-22. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' 2007 September 1 and 6. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Gainsville, USA; 2009 February 23-25. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria; 2004 December 9-13. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria; 2007 April 13 to 17. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria; 2007 December 12-16. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria; 2008 April 04-08. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria; 2009 December 11 to 16. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg, Austria;2009 April 18 to 22. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken, Voralberg,Austria; 2009 July 30 to August 04. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry.''' Schroecken,Voralberg, Austria; 2008 July 12-16. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry: O2k, TIP-2k and DatLab 4.''' Schroecken, Voralberg, Austria; 2006 August 18-22. +
'''O2k-International course on high-resolu … '''O2k-International course on high-resolution respirometry: Oroboros O2k, TIP-2k and DatLab 4.''' Schroecken, Voralberg, Austria; 2006 April 21-25.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-International course on high-resolution respirometry: Oxygraph-2k, TIP-2k and DatLab 4.''' Schroecken, Voralberg, Austria; 2005 April 08-10. +
'''O2k-MultiSensor Workshop.''' Schroecken, Voralberg, Austria;2010 April 12 to 16. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Protocol for Oxygen flux''' In a … '''O2k-Protocol for Oxygen flux''' In a closed oxygraph chamber, the oxygen concentration declines over time as a result of respiratory processes. The time derivative, therefore, is a negative number. Why is then the ‘rate of oxygen consumption’ not expressed as a negative value? Why is the term ‘oxygen flux’ used in this context of chemical reactions? The rationale is based on fundamental concepts of physical chemistry and non-equilibrium thermodynamics.[[Image:O2k-Protocols.jpg|right|150px|link=http://www.oroboros.at/?o2k-protocols|O2k-Protocols contents]][[Image:MiPNet10.05.jpg|centre|500px|thumb]]Respiratory oxygen flux: On-line display of oxygen concentration (blue) and oxygen flux (respiration, red). Endogenous respiration of endothelial cells leads to oxygen depletion, followed by reoxygenations (dotted arrows). Cell membrane permeabilization by digitonin causes a decline of respiration to the resting level (without adenylates in the medium, -ANP). ADP titration activates respiration about 2-fold above the endogenous level of oxygen consumption.Eye-fitted slopes of oxygen chart recorder traces belong to the past. With [[DatLab|DatLab]], trends are resolved. Accuracy is improved by standard numerical corrections. Graphs and protocols are stored and printed ready for publication.'''Reference'''[[Gnaiger_1993_Pure_Appl_Chem| Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65: 1983-2002.]]:>> O2k-Protocols:[[O2k-Protocols| Overall contents]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]]os O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Training course on high-resolution respirometry.''' Innsbruck, Tyrol, Austria; 2003 December 11-13. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop and training course on high-resolution respirometry.''' Schroecken, Vorarlberg, Austria; 2003 September 09-12. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on High-Resolution Respiro … '''O2k-Workshop on High-Resolution Respirometry - Introductory and Advanced.'''Schroecken, Voralberg, Austria; 2010 December 11 to 16.The past O2k-Workshop on HRR ('''IOC60''') was a success based on long-term experience combined with continuous improvements and innovations. With introductory and advanced groups working in parallel, the needs of the participants could be met more specifically compared to introductory and advanced workshops organized separately. The next O2k-Workshop, therefore, will again offer parallel introductory and advanced workpackages.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on High-Resolution Respiro … '''O2k-Workshop on High-Resolution Respirometry - O2k-Basic and TPP-Basic.''' Schröcken, Vorarlberg, Austria; 2011 April 26 to May 1.[[File:O2k-TIP2k.jpg|right|200px|caption]]:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on High-Resolution Respirometry.''' Barcelona, Catalonia, Spain; 2012 May 29 to 30 :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on High-Resolution Respiro … '''O2k-Workshop on High-Resolution Respirometry: O2k-Basic and TPP-Basic.''' Schröcken, Vorarlberg, Austria; 2011 10 - 15 December :>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on High-Resolution Respirometry: O2k-Basic.''' Schroecken, Vorarlberg, Austria 2012 December 05 to 10 :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on high-resolution respiro … '''O2k-Workshop on high-resolution respirometry and mitochondrial physiology.''' Seoul, Korea; 2007 February 4. Satellite to [[ASMRM]] 2007.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on high-resolution respirometry.''' Innsbruck, Tyrol, Austria; 2001 October 04-05. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on high-resolution respirometry.''' Innsbruck, Tyrol, Austria; 2002 June 12-15. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on high-resolution respirometry.''' Sidney, Australia; 2012 April 02 to 04. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k-Workshop on high-resolution respirometry.''' Voralberg, Austria;2010 April 07 to 12. +
'''O2k-Workshop on high-resolution respirometry: O2k-Basic and TPP-Basic.''' Schroecken, Voralberg,Austria; 2012 April 11 to 16. :» O2k-Workshop: [[OROBOROS_Events|Current dates]] :» Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''O2k: Mitochondrial physiology (MiP) workshop on high-resolution respirometry.''' Schroecken AT, 27-31 March 2003. :>> O2k-Workshop: [[Oroboros Events| Current dates]] :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''OBJECTIVE''': Growing evidence suggests … '''OBJECTIVE''': Growing evidence suggests that mitochondria function is impaired in sepsis. Here, we tested the hypothesis that lipopolysaccharide would induce mitochondrial Ca2+ overload and oxygen utilization abnormalities as consequences of sarcoplasmic reticulum Ca2+ handling derangements that are typically observed in sepsis. As lipopolysaccharide-induced sarcoplasmic reticulum dysfunction was mainly characterized by reduced sarcoplasmic reticulum Ca2+ uptake and Ca2+ leak, we tested whether dantrolene, a sarco(endo)plasmic reticulum calcium ATPase leak inhibitor, would prevent mitochondrial and cardiac contractile dysfunction.'''DESIGN''': Randomized controlled trial.'''SETTING''': Experimental laboratory.'''SUBJECTS''': Male Sprague Dawley rats.'''INTERVENTIONS''': Sepsis was induced by injection of endotoxin lipopolysaccharide (10 mg/kg/intravenously). Assessment of contractile function and Ca2+ handling was performed 4 hr after lipopolysaccharide. The relative contribution of the different Ca2+ transporters to relaxation in intact cardiomyocytes was studied during successive electrically evoked twitches and caffeine stimulation. Sarcoplasmic reticulum vesicles and mitochondria from ventricles of rats treated or not with lipopolysaccharide were prepared to evaluate Ca2+ uptake-release and oxygen fluxes, respectively. Effects of dantrolene (10 mg/kg) treatment in rats were evaluated in sarcoplasmic reticulum vesicles, mitochondria, and isolated hearts.'''MEASUREMENTS AND MAIN RESULTS''': Lipopolysaccharide challenge elicited cardiac contractile dysfunction that was accompanied by severe derangements in sarcoplasmic reticulum function, i.e., reduced Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Functional sarcoplasmic reticulum changes were associated with modification in the status of phospholamban phosphorylation whereas SERCA was unchanged. Rises in mitochondrial Ca2+ content observed in lipopolysaccharide-treated rats coincided with derangements in mitochondrial oxygen efficacy, i.e., reduced respiratory control ratio. Administration of dantrolene in lipopolysaccharide-treated rats prevented mitochondrial Ca2+ overload and mitochondrial oxygen utilization abnormalities. Moreover, dantrolene treatment in lipopolysaccharide rats improved heart mitochondrial redox state and myocardial dysfunction.'''CONCLUSION:''' These experiments suggest that sarcoplasmic reticulum Ca2+ handling dysfunction is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction.tion is an early event during endotoxemia that could be responsible for, or contribute to, mitochondrial Ca2+ overload, metabolic failure, and cardiac dysfunction. +
'''OBJECTIVE:''' Impairments in mitochondr … '''OBJECTIVE:''' Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome.'''RESEARCH DESIGN AND METHODS:''' Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS).'''RESULTS:''' Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a Complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a Complex I protein) were reduced by 29 and 36% (''P'' < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control.'''CONCLUSIONS:''' Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the [[respiratory chain]] was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.tic neurons under high glucose concentration. +
'''OBJECTIVE:''' Increasing evidence point … '''OBJECTIVE:''' Increasing evidence points to the role of mitochondrial dysfunction in the pathogenesis of sepsis. Previous data indicate that mitochondrial function is affected in monocytes from septic patients, but the underlying mechanisms and the impact of these changes on the patients' outcome are unknown. We aimed to determine the mechanisms involved in mitochondrial dysfunction in peripheral blood mononuclear cells from patients with septic shock.'''DESIGN:''' A cohort of patients with septic shock to study peripheral blood mononuclear cell mitochondrial respiration by high-resolution respirometry analyses and to compare with cells from control subjects.'''SETTING:''' Three intensive care units and an academic research laboratory.'''SUBJECTS:''' Twenty patients with septic shock and a control group composed of 18 postoperative patients without sepsis or shock.'''INTERVENTIONS:''' Ex vivo measurements of mitochondrial oxygen consumption were carried out in digitonin-permeabilized peripheral blood mononuclear cells from 20 patients with septic shock taken during the first 48 h after intensive care unit admission as well as in peripheral blood mononuclear cells from control subjects. Clinical parameters such as hospital outcome and sepsis severity were also analyzed and the relationship between these parameters and the oxygen consumption pattern was investigated.'''MEASUREMENTS AND MAIN RESULTS:''' We observed a significant reduction in the respiration specifically associated with adenosine-5'-triphosphate synthesis ([[State 3]]) compared with the control group (5.60 vs. 9.89 nmol O2/min/10(7) cells, respectively, ''P'' < .01). Reduction of State 3 respiration in patients with septic shock was seen with increased prevalence of organ failure (''r'' = -0.46, ''P'' = .005). Nonsurviving patients with septic shock presented significantly lower adenosine diphosphate-stimulated respiration when compared with the control group (4.56 vs. 10.27 nmol O2/min/10(7) cells, respectively; ''P'' = .004). Finally, the presence of the functional F1Fo adenosine-5'-triphosphate synthase complex (0.51 vs. 1.00 ng oligo/mL/10(6) cells, ''P'' = .02), but not the adenine nucleotide translocator, was significantly lower in patients with septic shock compared with control cells.'''CONCLUSION:''' Mitochondrial dysfunction is present in immune cells from patients with septic shock and is characterized as a reduced respiration associated to adenosine-5'-triphosphate synthesis. The molecular basis of this phenotype involve a reduction of F1Fo adenosine-5'-triphosphate synthase activity, which may contribute to the energetic failure found in sepsis.ute to the energetic failure found in sepsis. +
'''OBJECTIVE:''' To determine biological m … '''OBJECTIVE:''' To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506).'''METHODS:''' INS-1 cells and isolated rat islets were incubated with vehicle or FK506 and harvested at 24-hr intervals. Cells were assessed for viability, apoptosis, proliferation, cell insulin secretion, and content. Gene expression studies by microarray analysis, quantitative polymerase chain reaction, and motifADE analysis of the microarray data identified potential FK506-mediated pathways and regulatory motifs. Mitochondrial functions, including cell respiration, mitochondrial content, and bioenergetics were assessed.'''RESULTS:''' Cell replication, viability, insulin secretion, oxygen consumption, and mitochondrial content were decreased (''P''<0.05) 1.2-, 1.27-, 1.77-, 1.32-, and 1.43-fold, respectively, after 48-hr FK506 treatment. Differences increased with time. FK506 (50 ng/mL) and cyclosporine A (800 ng/mL) had comparable effects. FK506 significantly decreased mitochondrial content and mitochondrial bioenergetics and showed a trend toward decreased oxygen consumption in isolated islets. Cell apoptosis and proliferation, mitochondrial DNA copy number, and ATP:ADP ratios were not significantly affected. Pathway analysis of microarray data showed FK506 modification of pathways involving ATP metabolism, membrane trafficking, and cytoskeleton remodeling. PGC1-α mRNA was down-regulated by FK506. MotifADE identified nuclear factor of activated T-cells, an important mediator of β-cell survival and function, as a potential factor mediating both up- and down-regulation of gene expression.'''CONCLUSIONS:''' At pharmacologically relevant concentrations, FK506 decreases insulin secretion and reduces mitochondrial density and function without changing apoptosis rates, suggesting that posttransplantation diabetes induced by FK506 may be mediated by its effects on mitochondrial function.ted by its effects on mitochondrial function. +
'''Objective''': Impairments in mitochondr … '''Objective''': Impairments in mitochondrial physiology may play a role in diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function.'''Research design and methods''': Rates of oxygen consumption were measured in mitochondria from dorsal root ganglia (DRG) of 12- to- 22-week streptozotocin (STZ)-induced diabetic rats, diabetic rats treated with insulin, and age-matched controls. Activities and expression of components of mitochondrial complexes and reactive oxygen species (ROS) were analyzed.'''Results''': Rates of coupled respiration with pyruvate + malate (P + M) and with ascorbate + TMPD (Asc + TMPD) in DRG were unchanged after 12 weeks of diabetes. By 22 weeks of diabetes, respiration with P + M was significantly decreased by 31-44% and with Asc + TMPD by 29-39% compared with control. Attenuated mitochondrial respiratory activity of STZ-diabetic rats was significantly improved by insulin that did not correct other indices of diabetes. Activities of mitochondrial complexes I and IV and the Krebs cycle enzyme, citrate synthase, were decreased in mitochondria from DRG of 22-week STZ-diabetic rats compared with control. ROS levels in perikarya of DRG neurons were not altered by diabetes, but ROS generation from mitochondria treated with antimycin A was diminished compared with control. Reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins.'''Conclusions''': Mitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ROS.hout a significant rise in perikaryal ROS. +
'''Objective'''—The enzyme telomerase and … '''Objective'''—The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function.'''Methods and Results'''—Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide–induced damage. TERT increases overall respiratory chain activity, which is most pronounced at Complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H2O2-induced apoptosis. Lung fibroblasts from 6-month-old TERT-/- mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT ''in vivo''.'''Conclusion'''—Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress–induced damage.iratory chain activity and protecting against oxidative stress–induced damage. +
'''Oroboros O2k-Workshop on High-Resolution Respirometry. Obergurgl, Tyrol, Austria; 2010 October 01 to 06. Satellite to [[MiP2010]].''' :>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''Oroboros O2k-Workshop on high-resolutio … '''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2022 October 03-08). Following the [[MiPNet27.05_Schroecken_BEC_tutorial-Living_Communications_pmP|BEC tutorial on mitochondrial membrane potential and protonmotive pressure]] (2022 Sep 30 - Oct 03).[[MiPNet27.05_Schroecken_BEC_tutorial-Living_Communications_pmP|BEC tutorial on mitochondrial membrane potential and protonmotive pressure]] (2022 Sep 30 - Oct 03). +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2023 June 19-24). +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2023 October 02-07). +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2024 June 17-22). +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria (2024 Sep 30 - Oct 05). +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria; 2019 September. +
'''Oroboros O2k-Workshop on high-resolution respirometry'''. Schroecken, Austria; 2019. +
'''Oroboros Virtual O2k-Workshop on high-resolution respirometry and PhotoBiology'''. +
'''Oroboros Virtual O2k-Workshop on high-resolution respirometry and and measurement of the redox state of the Q-pool'''. +
'''Oroboros Virtual O2k-Workshops on high-resolution respirometry''' were offered during the COVID-19 lockdown and are discontinued. +
'''Pesta D, Gnaiger E (2015) Preparation o … '''Pesta D, Gnaiger E (2015) Preparation of permeabilized muscle fibers for diagnosis of mitochondrial respiratory function. Mitochondr Physiol Network 14.14(02):1-5.''' Application of [[permeabilized muscle fibers]] and [[high-resolution respirometry]] offer a sensitive diagnostic test of mitochondrial dysfunction in small [[biopsy]] specimens of human muscle. By using these techniques in conjunction with multiple [[substrate-uncoupler-inhibitor titration]] (SUIT) protocols, respirometric studies of human and animal tissue biopsies improve our fundamental understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial myopathies.[[Image:MiPNet14.14.jpg|right|200px|thumb]]:>> Product: [[O2k-Catalogue: O2k-MultiSensor]], [[O2k-Core]], [[Oroboros O2k-Catalogue]][[Oroboros O2k-Catalogue]] +
'''Protocol''': Couple palmitoylcarnitine (10µM) + malate (1mM) on isolated mitochondria and permeabilized fibers. In such case the buffer is always supplemented with 2mg/ml of BSA. +
'''Protocol''': I use either palmitoyl-L-carnitine plus malate (25 µM + 2.5 mM) or palmitoyl-CoA + L-carnitine + malate (25 µM + 2 mM + 2.5 mM) as substrates. Respiratory control index is usually around 5-6 for healthy controls. +
'''Protocol''': Palmitate, Stearate, Oleate and Linoleate in intact cells. We use various BSA-fatty acid combinations that result in free fatty acid levels that are in the 2 to 12 nM range. +
'''Protocol''': final concentration of dig … '''Protocol''': final concentration of digitonin in chamber is 10μg/mlcell number in chamber is 2 millions cell/ml, cell type PBMC, Malate (2mM), Palmitoyl-DLcarnitine-HCl (20μM), ADP (2.5mM), pyruvate (5mM), glutamate (10mM), succinate (10mM), rotenone (0.1μM), malonic acid(5mM), myxothiazol (0.5μM), antimycin A (2.5μM), TMPD (0.5mM), azide (100mM)cin A (2.5μM), TMPD (0.5mM), azide (100mM) +
'''Purpose of review''': Mitochondrial con … '''Purpose of review''': Mitochondrial content and function vary across species, tissue types, and lifespan. Alterations in skeletal muscle mitochondrial function have been reported to occur in in aging and in many other pathological conditions. This review focuses on the state of the art ''in vivo'' and ''in vitro'' methodologies for assessment of muscle mitochondrial function.'''Recent findings''': Classic studies of isolated mitochondria have measured function from maximal respiratory capacity. These fundamental methods have recently been substantially improved and novel approaches to asses mitochondrial functions ''in vitro'' have been emerged. Noninvasivemethods based on magnetic resonance spectroscopy (MRS) and near-infraredspectroscopy (NIRS) permit ''in vivo'' assessment of mitochondrial function and are rapidly becoming more accessible to many investigators. Moreover, it is now possible to gather information on regulation of mitochondrial content by measuring the ''in vivo'' synthesis rate of individual mitochondrial proteins.'''Summary: High-resolution respirometry has emerged as a powerful tool for ''in vitro'' measurements of mitochondrial function in isolated mitochondria and permeabilized fibers.''' Direct measurements of ATP production are possible by bioluminescence. Mechanistic data provided by these methods is further complimented by ''in vivo'' assessment using MRS and NIRS and the translational rate of gene transcripts.he translational rate of gene transcripts. +
'''REASONS FOR PERFORMING STUDY:''' Limite … '''REASONS FOR PERFORMING STUDY:''' Limited information exists about the muscle mitochondrial respiratory function changes that occur in horses during an endurance season.'''OBJECTIVES:''' To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ET-pathway) capacities in horses with high resolution respirometry (HRR).'''METHODS:''' Mitochondrial respiration was measured in microbiopsies taken from the triceps brachii (tb) and gluteus medius (gm) muscles in 8 endurance horses (7 purebred Arabians and 1 crossbred Arabian) before training (T0), after two 10 week training periods (T1, T2) and after 2 CEI** endurance races (R1, R2). Muscle OXPHOS capacity was determined using 2 titration protocols without (SUIT 1) or with pyruvate (SUIT 2) as substrate. Electrons enter at the level of Complex I, Complex II or both complexes simultaneously (Complexes I+II). Muscle ET capacity was obtained by uncoupling Complexes I+II sustained respiration.'''RESULTS:''' T1 improved OXPHOS and ET capacities in the tb as demonstrated by the significant increase of oxygen fluxes vs. T0 (Complex I: +67%; ET-pathway: +37%). Training improved only OXPHOS in the gm (Complex I: +34%). Among horses that completed the race, a significant decrease in OXPHOS (Complex I: ∼ -35%) and ET-pathway (-22%) capacities was found in the tb with SUIT 2 indicating a reduced aerobic glycolysis. Significant correlations between CK activities and changes in OXPHOS were found suggesting a relationship between exercise-induced muscle damage and depression of mitochondrial respiration.'''CONCLUSIONS:''' For the first time, OXPHOS and ET capacities in equine muscle at different steps of an endurance season have been determined by HRR. Significant alterations in mitochondrial respiratory function in response to endurance training and endurance racing have been observed although these changes appeared to be muscle group specific.nges appeared to be muscle group specific. +
'''Reck M, Wyss M, Lassnig B, Gnaiger E (1 … '''Reck M, Wyss M, Lassnig B, Gnaiger E (1997) DatLab 2. High time resolution. Mitochondr Physiol Network 02.04:1-11.''' »[http://www.bioblast.at/index.php/File:MiPNet02.04_DatLab2_TimeConstant.pdf Versions]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue ]][Oroboros O2k-Catalogue ]] +
'''Research to Practice 2016, Melbourne, Victoria, AU; [http://researchtopractice2016.com.au Research to Practice 2016].''' +
'''SFRR Australasia 2016, Gold Coast, AU; [http://www.sfrra2016.org/overview.php SFRR Australasia 2016].''' +
'''Satellite symposium and workshop "Skeletal Muscle Research – from Cell to Human"'''. Ljubljana, Slovenia (2024 Sep 26). +
'''Summary:''' The Oroboros O2k provides t … '''Summary:''' The Oroboros O2k provides the instrumental basis for high-resolution respirometry. Compared to any of its competitors, the Oroboros O2k is a high-performance instrument, and high-resolution is distinguished from conventional approaches by a combination of unique features and specifications. These set a new standard in bioenergetics, mitochondrial physiology, clinical research and diagnosis of mitochondrial pathologies.nd diagnosis of mitochondrial pathologies. +
'''Summary:''' Whereas isolated mitochondr … '''Summary:''' Whereas isolated mitochondria remain one of the gold-standards in studies of bioenergetics and mitochondrial physiology, permeabilized tissues and cells have become an alternative with several advantages. But some disadvantages have to be considered, too, for optimum experimental design and critical evaluation of results.design and critical evaluation of results. +
'''The Cold Spring Harbor Asia conference on Mitochondria'''. Suzhou, China; 2017 October. +
'''The [[Oroboros O2k]] … '''The [[Oroboros O2k]] with [[DatLab]] software is the sole-source instrument for [[high-resolution respirometry]] (HRR), with the option of modular [[O2k-MultiSensor]] extension and electronically controlled [[Titration-Injection microPump]] (TIP2k), and accessories including the [[ISS-Integrated Suction System\230 V\EU]] (ISS) and titration syringes.'''[[ISS-Integrated Suction System\230 V\EU]] (ISS) and titration syringes.''' +
'''The aim of this research:''' To investi … '''The aim of this research:''' To investigate an effect of short time ischemia/reperfusion ''in vivo'' on rat kidney mitochondria oxidative phosphorylation.'''Objectives:''' To evaluate the effect of 20 min ischemia and 30 min reperfusion on mitochondria oxidative phosforilation system and investigate rat mitochondrial respiration chain complex I, II and II + III activity.'''Object of this research:''' Wistar breed rats males were used to perform this research.'''Methods:''' Warm ischemia (37 ° C) to rat kidneys was induced by clamping renal arteries using vascular clamps. Ischemia was induced for 20 min and after that reperfusion lasted for 30 min. Kidneys were removed and mitochondria were isolated by using differential centrifugation method. The amount of proteins was measured via Buret method. Mitochondrial respiration rates were measured by Oxygraph-2k system and using glutamate/malate and succinate as substrates. Mitochondrial respiration chain complexes activity was measured spectrophotometrically.'''Results:''' This research results show that short time (20 min) ischemia and reperfusion (30 min) does not affect the respiration rates when mitochondrial respiration chain complex I substrate glutamate/malate is being oxidized. This research shows that oxidizing mitochondrial respiration chain complex II substrate succinate evaluates respiration rate in state two after short-time ischemia 1.47 times but didn’t affect state three. Oxidizing succinate respiration control index decreases by 22 % which show that even after short-time ischemia mitochondrial membrane is getting damaged. Complex I activity decreased by 67% after 20 min ischemia and 30 min reperfusion.'''Conclusions:''' Research showed that even short time of ischemia damages mitochondrial oxidative phosphorylation system. Short-time ischemia decreases mitochondrial respiration chain complex I.mitochondrial respiration chain complex I. +
'''This manuscript was withdrawn by the au … '''This manuscript was withdrawn by the author!'''The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins ''in vitro''. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10 Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10 increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 ''in vitro'' and ''in vivo''. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1, display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10-proteasome mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.hondrial damage and programmed cell death. +
'''We summarize 10 compelling reasons for choosing the Oroboros O2k, for collaborating in the Oroboros Ecosystem, and for spreading our reproducibility committment. ‘Top 10’ reflects our corporate goals.''' +
'''Wolfgang Wieser (1924–2017) – a central … '''Wolfgang Wieser (1924–2017) – a central force in Austrian biology.'''The most important stages in Wolfgang Wieser’s life and scientific career are illustrated in this paper. Wolfgang Wieser was a central personality in Austrian biology. His contributions to the development of an eco-physiological approach are outlined, including his books on evolutionary biology, especially in context of the cultural development of mankind.xt of the cultural development of mankind. +
'''Workshop at the 5th Meeting of ASMRM Jo … '''Workshop at the 5th Meeting of ASMRM Jointly with Chinese Mit'2008 Tianjin University of Sport.''' Tianjin , China; 2008 November 09.:>> O2k-Workshop: [[Oroboros Events| Current dates]]:>> Product: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]][[Oroboros O2k-Catalogue | O2k-Catalogue]] +
'''[[File:Sunpoint Hsu Gnaiger Tsai Lu.JPG|right|500px|thumb|[[Hsu A| Ari Hsu]] … '''[[File:Sunpoint Hsu Gnaiger Tsai Lu.JPG|right|500px|thumb|[[Hsu A| Ari Hsu]], [[Gnaiger E| Erich Gnaiger]], [[Tsai S| Sunny Tsai]] and [[Lu A| Amelia Lu]] (left to right) in the Sunpoint Office at IOC98.''']][[Image:O2k-Workshops.png|left|130px|link=http://www.oroboros.at/?O2k-Workshops]]'''98th OROBOROS O2k-Workshop on high-resolution respirometry and O2k-Fluorometry'''lution respirometry and O2k-Fluorometry''' +
'''[[High-resolution respirometry]]''' (HRR) provides a quantitative approach to bioenergetics and mitochondrial physiology with the [[Oroboros O2k]] (Oroboros Instruments) offering several sole-source features. +
'''[[Karabatsiakis 2017 MitoFit Open Seminar|MitoFit Open Seminar on immune cell bioenergetics]]'''. Innsbruck, AT +
''AIMS'' Intracellular amyloid beta (Aβ) o … ''AIMS'' Intracellular amyloid beta (Aβ) oligomers and extracellular Aβ plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aβ production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aβ generation and thereby initiate a vicious cycle further impairing mitochondrial function.''RESULTS'' Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aβ. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aβ, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aβ showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aβ was partly reduced by an antioxidant, indicating that Aβ formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aβ levels ''in vivo''.''INNOVATION'' We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aβ production ''in vitro'' and ''in vivo''.''CONCLUSION'' Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aβ itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.ibutes to the pathogenesis of sporadic AD. +
''AIMS'': Heart failure (HF) with left ven … ''AIMS'': Heart failure (HF) with left ventricular systolic dysfunction (LVSD) is associated with a shift in substrate utilization and a compromised energetic state. Whether these changes are connected with mitochondrial dysfunction is not known. We hypothesized that the cardiac phenotype in LVSD could be caused by reduced mitochondrial oxidative phosphorylation (OXPHOS) capacity and reduced mitochondrial creatine kinase (miCK) capacity. The study aim was to test mitochondrial OXPHOS capacity in LVSD myocardium compared with OXPHOS capacity in a comparable patient group without LVSD.''METHODS AND RESULTS'': Myocardial biopsies were obtained from the left ventricle during cardiac valve or left ventricular assist device (LVAD) surgery. Patients were stratified according to left ventricular ejection fraction (LVEF) into LVSD (LVEF <45%, n = 14) or CONTROL (LVEF >45%, n = 15). Mitochondrial respiration was measured in muscle fibres with addition of non-fatty acid substrates or octanoyl-l-carnitine, a medium chain fatty acid (MCFA). The ''in situ'' enzyme capacity of miCK was determined from APD titrations in the presence or absence of creatine. Maximal OXPHOS capacity with non-fatty acid substrates was lower in the LVSD group compared with the CONTROL group (P ≤ 0.05). ADP sensitivity always increased significantly (P ≤ 0.05) with the addition of creatine, after which the sensitivity was highest (P ≤ 0.05) in LVSD compared with CONTROL. The stimulation of OXPHOS from octanoyl-l-carnitine titrations elicited ∼40% lower respiration in LVSD compared with CONTROL (P ≤ 0.05).''CONCLUSION'': Human LVSD is associated with markedly diminished OXPHOS capacity, particularly in MCFA oxidation. This offers a candidate mechanism for a compromised energetic state and decreased reliance on fatty acid utilization in HF.reased reliance on fatty acid utilization in HF. +
''AIMS'': Infarct-remodelled hearts are le … ''AIMS'': Infarct-remodelled hearts are less amenable to protection against ischaemia/reperfusion. Understanding preservation of energy metabolism in diseased vs. healthy hearts may help to develop anti-ischaemic strategies effective also in jeopardized myocardium.''METHODS AND RESULTS'': Isolated infarct-remodelled/sham Sprague-Dawley rat hearts were perfused in the working mode and subjected to 15 min of ischaemia and 30 min of reperfusion. Protection of post-ischaemic ventricular work was achieved by pharmacological conditioning with sevoflurane. Oxidative metabolism was measured by substrate flux in fatty acid and glucose oxidation using [(3)H]palmitate and [(14)C]glucose. Mitochondrial oxygen consumption was measured in saponin-permeabilized left ventricular muscle fibres. Activity assays of citric acid synthase, hydroxyacyl-CoA dehydrogenase, and pyruvate dehydrogenase and mass spectrometry for acylcarnitine profiling were also performed. Six weeks after coronary artery ligation, the hearts exhibited macroscopic and molecular signs of hypertrophy consistent with remodelling and limited respiratory chain and citric acid cycle capacity. Unprotected remodelled hearts showed a marked decline in palmitate oxidation and acetyl-CoA energy production after ischaemia/reperfusion, which normalized in sevoflurane-protected remodelled hearts. Protected remodelled hearts also showed higher β-oxidation flux as determined by increased oxygen consumption with palmitoylcarnitine/malate in isolated fibres and a lower ratio of C16:1+C16OH/C14 carnitine species, indicative of a higher long-chain hydroxyacyl-CoA dehydrogenase activity. Remodelled hearts exhibited higher PPARα-PGC-1α but defective HIF-1α signalling, and conditioning enabled them to mobilize fatty acids from endogenous triglyceride stores, which closely correlated with improved recovery.''CONCLUSIONS'': Protected infarct-remodelled hearts secure post-ischaemic energy production by activation of β-oxidation and mobilization of fatty acids from endogenous triglyceride stores.acids from endogenous triglyceride stores. +
''Acanthamoeba castellanii'' is a free-liv … ''Acanthamoeba castellanii'' is a free-living amoeba and the etiological agent of granulomatous amoebic encephalitis and amoebic keratitis. ''A. castellanii'' can be present as trophozoites or cysts. The trophozoite is the vegetative form of the cell and has great infective capacity compared to the cysts, which are the dormant form that protect the cell from environmental changes. Phosphate transporters are a group of proteins that are able to internalize inorganic phosphate from the extracellular to intracellular medium. Plasma membrane phosphate transporters are responsible for maintaining phosphate homeostasis, and in some organisms, regulating cellular growth. The aim of this work was to biochemically characterize the plasma membrane phosphate transporter in ''A. castellanii'' and its role in cellular growth and metabolism. To measure inorganic phosphate (Pi) uptake, trophozoites were grown in liquid PYG medium at 28 °C for 2 days. The phosphate uptake was measured by the rapid filtration of intact cells incubated with 0.5 μCi of 32Pi for 1 h. The Pi transport was linear as a function of time and exhibited Michaelis-Menten kinetics with a Km = 88.78 ± 6.86 μM Pi and Vmax = 547.5 ± 16.9 Pi × h-1 × 10-6 cells. ''A. castellanii'' presented linear phosphate uptake up to 1 h with a cell density ranging from 1 × 105 to 2 × 106 amoeba × ml-1. The Pi uptake was higher in the acidic pH range than in the alkaline range. The oxygen consumption of living trophozoites increased according to Pi addition to the extracellular medium. When the cells were treated with FCCP, no effect from Pi on the oxygen flow was observed. The addition of increasing Pi concentrations not only increased oxygen consumption but also increased the intracellular ATP pool. These phenomena were abolished when the cells were treated with FCCP or exposed to hypoxia. Together, these results reinforce the hypothesis that Pi is a key nutrient for ''Acanthamoeba castellanii'' metabolism.her, these results reinforce the hypothesis that Pi is a key nutrient for ''Acanthamoeba castellanii'' metabolism. +
''Acer pseudoplatanus'' contains toxins re … ''Acer pseudoplatanus'' contains toxins responsible for poisoning in various species [1], including humans [2]. In equids, this intoxication induces an often fatal rhabdomyolysis syndrome known as atypical myopathy (AM); [3]. Blood analysis reveals a severe metabolic disturbance characterised by hyperglycaemia, high triglycerides, and lipid intermediates [4]. Toxins inhibit several steps of the fatty acid β-oxidation cycle that leads to the accumulation of acyl-CoAs in the mitochondria, which are scavenged into acylcarnitines. Also, competitive inhibition of long-chain fatty acid transport into mitochondria results into their accumulation conjugated with carnitine. In addition, inhibition of the catabolic pathway of branched-chain amino acids, particularly leucine, leads to the accumulation of branched acylcarnitines [2; 5]. Acylcarnitines in tissues may explain parts of the pathophysiological process, such as the cardiac myopathy occurring in AM. Also, acylcarnitines accumulation could promote muscle insulin resistance and contribute to the hyperglycaemia observed in AM horses [4]. The disease also results from severe impairment of mitochondrial bioenergetics [6; 7]. In AM, the serum acylcarnitines profile contributes to the diagnosis of the disease, its prognosis and is also a valuable aid in monitoring ongoing metabolic disturbances. In search of new therapeutic approaches for this environmental intoxication, we are currently designing toxicity assays with cultured cells [7] and zebrafish larvae. These models will help us to test different drugs by exploring their ability to prevent metabolic disturbances as indicated by the acylcarnitines profile. Indeed, in both models, the alteration of the acylcarnitine profile can be followed.# Renaud B et al, (2022) Acer pseudoplatanus: A Potential Risk of Poisoning for Several Herbivore Species. https://doi.org/10.3390/toxins14080512# Tanaka K, Isselbacher KJ, Shih V (1972) Isovaleric and -methylbutyric acidemias induced by hypoglycin A: mechanism of Jamaican vomiting sickness. https://doi.org/10.1126/science.175.4017.69 # Votion DM, Serteyn D (2008) Equine atypical myopathy: a review. https://doi.org/10.1016/j.tvjl.2008.02.004# Boemer F, Detilleux J, Cello C, Amory H, Marcillaud-Pitel C, Richard E, van Galen G, van Loon G, Lefere L, Votion DM (2017) Acylcarnitines profile best predicts survival in horses with atypical myopathy. https://doi.org/10.1371/journal.pone.0182761# Wouters CP et al, (2021) Metabolomic Signatures Discriminate Horses with Clinical Signs of Atypical Myopathy from Healthy Co-grazing Horses. https://doi.org/10.1021/acs.jproteome.1c00225# Lemieux H et al, (2016) Mitochondrial function is altered in horse atypical myopathy. https://doi.org/10.1016/j.mito.2016.06.005 # Kruse CJ, Stern D, Mouithys-Mickalad A, Niesten A, Art T, Lemieux H, Votion DM (2021) In Vitro Assays for the Assessment of Impaired Mitochondrial Bioenergetics in Equine Atypical Myopathy. https://doi.org/10.3390/life11070719e Atypical Myopathy. https://doi.org/10.3390/life11070719 +
''Ad libitum'' high-fat diet (HFD) induces … ''Ad libitum'' high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.Copyright © 2020. Published by Elsevier B.V.right © 2020. Published by Elsevier B.V. +
''Aedes aegypti'' females are natural vect … ''Aedes aegypti'' females are natural vectors of important arboviruses including Dengue, Zika and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, which target the pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, there are phenotypic costs that ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on ''A. aegypti'' flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin expression in fat bodies in a time-dependent manner and ultimately affecting induced flight activity. Although oxidant levels in flight muscle were hardly altered, [[P-L net OXPHOS capacity |''P''-''L'' net OXPHOS capacity]] ([[OXPHOS capacity |OXPHOS capacity ''P'']] minus [[LEAK respiration |LEAK respiration ''L'']]; ADP→ATP-linked) and [[ET capacity |electron transfer capacity ''E'']] (maximal mitochondrial oxygen consumption rates) supported by pyruvate & proline were significantly reduced at 24 h upon zymosan injection. These effects were parallel to significant and specific reductions in Complex I activity upon zymosan treatment. Finally, the magnitude of defensin up-regulation negatively correlated with maximal, ATP-linked, and NADH&proline-linked respiratory rates ''P'' and ''E'' in flight muscles. Despite strong reductions were observed in proline and [[E-P excess capacity |''E''-''P'' excess capacity]] 24 h upon zymosan injection, this effect was not correlated to the magnitude of innate immune response activation. Collectively, we demonstrate that activation of innate immunity in fat body strongly associates to reduced flight muscle Complex I activity with direct consequences on mitochondrial physiology and dispersal. Remarkably, our results indicate that a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism on arbovirus transmission.drial energy metabolism on arbovirus transmission. +
''Aedes aegypti'' is the most important an … ''Aedes aegypti'' is the most important and widespread vector of arboviruses, including dengue and zika. Insect dispersal through the flight activity is a key parameter that determines vector competence, and is energetically driven by oxidative phosphorylation in flight muscle mitochondria. Analysis of mitochondrial function is central for a better understanding of cellular metabolism, and is mostly studied using isolated organelles. However, this approach has several challenges and methods for assessment of mitochondrial function in chemically-permeabilized tissues were designed. Here, we described a reliable protocol to assess mitochondrial physiology using mechanically permeabilized flight muscle of single ''A. aegypti'' mosquitoes in combination with high-resolution respirometry. By avoiding the use of detergents, high respiratory rates were obtained indicating that substrate access to mitochondria was not limited. This was confirmed by using selective inhibitors for specific mitochondrial substrates. Additionally, mitochondria revealed highly coupled, as ATP synthase or adenine nucleotide translocator inhibition strongly impacted respiration. Finally, we determined that pyruvate and proline induced the highest respiratory rates compared to other substrates tested. This method allows the assessment of mitochondrial physiology in mosquito flight muscle at individual level, and can be used for the identification of novel targets aiming rational insect vector control.Copyright © 2019. Published by Elsevier Inc.right © 2019. Published by Elsevier Inc. +
''Aims:'' Infarct-remodeled hearts are les … ''Aims:'' Infarct-remodeled hearts are less amenable to protection against ischemia-reperfusion. Understanding preservation of energy metabolism in diseased versus healthy hearts may help to develop anti-ischemic strategies also effective in jeopardized myocardium.''Methods and Results:'' Isolated infarct-remodeled/sham Sprague-Dawley rat hearts were perfused in the working mode and subjected to 15 min of ischemia and 30 min of reperfusion. Protection of postischemic ventricular work was achieved by pharmacologic conditioning with sevoflurane. Oxidative metabolism was measured by substrate flux in fatty acid and glucose oxidation using [(3)H]palmitate and [(14)C]glucose. Mitochondrial oxygen consumption was measured in saponin-permeabilized left ventricular muscle fibers. Activity assays of citric acid synthase, hydroxyacyl-CoA dehydrogenase, and pyruvate dehydrogenase and mass spectrometry for acylcarnitine profiling were also performed. Six weeks after coronary artery ligation, hearts exhibited macroscopic and molecular signs of hypertrophy consistent with remodeling and limited respiratory chain and citric acid cycle capacity. Unprotected remodeled hearts showed a marked decline in palmitate oxidation and acetyl-CoA energy production after ischemia/reperfusion, which normalized in sevoflurane-protected remodeled hearts. Protected remodeled hearts also showed higher β-oxidation flux as determined by increased oxygen consumption with palmitoylcarnitine/malate in isolated fibers and a lower ratio of C16:1+C16OH/C14 carnitine species, indicative of a higher long-chain hydroxyacyl-CoA dehydrogenase activity. Remodeled hearts exhibited higher PPARα-[[PGC-1α]] but defective HIF-1α signaling and conditioning enabled them to mobilize fatty acids from endogenous triglyceride store, which closely correlated with improved recovery.''Conclusions:'' Protected infarct-remodeled hearts secure postischemic energy production by activation of β-oxidation and mobilization of fatty acids from endogenous triglyceride stores.acids from endogenous triglyceride stores. +
''Araucaria angustifolia'' is listed as cr … ''Araucaria angustifolia'' is listed as critically endangered by International Union for Conservation of Nature (IUCN) red list of threatened species. The development and propagation of this species is strongly affected by abiotic stress, such as the temperature variation. We previously shown the activation of plant uncoupling mitochondrial protein (PUMP) in embryogenic ''A. angustifolia'' cells submitted to cold stress, an effect associated to oxidative stress. In this work, we advanced in these studies by submitting these cells to cold stress (4 ± 1°C for 24h or 48h) and evaluating the cellular and mitochondrial response associated to oxidative stress, namely: the H2O2 levels, the activity of antioxidant enzymes and lipid peroxidation. In mitochondria from these cells were evaluated the activity of NAD(P)H alternative dehydrogenases and mitochondrial permeability transition (MPT). The cold stress did not affect the morphology and viability of embryogenic ''A. angustifolia'' cells; however, increased the H2O2 levels by ~35% (at 24h and 48h) and lipid peroxidation by ~15% and 30% after 24h and 48h of stress, respectively. The activity of catalase was decreased by ~20% after 48h of cold stress while ascorbate peroxidase (APx) and dehydroascorbate redutase (DHAR) activities were increased by ~100% and ~64%, respectively. For the cells exposition to cold stress by 24h only dehydroascorbate redutase (MDHAR) had the activity increased by ~172%. Glutathione reductase (GR) and superoxide dismutase activities remained unchanged under both stress conditions. In mitochondria, the cold stress promoted a significant inhibition of external alternative NAD(P)H dehydrogenases (~40% at 24h of stress and ~65% at 48h of stress) while the mitochondrial permeability transition (MPT) was slightly inhibited in both, 24h and 48h of stress. The cold stress induces the oxidative stress in embryogenic ''A. angustifolia'' cells, which result in up-regulation of the enzymatic defense mainly the activation of gluthatione-ascorbate cycle in a compensatory way to the inhibition of catalase and external NAD(P)H dehydrogenases. These results contribute to understanding the pathway to overcoming the cold in this gymnosperm and are important for the development of conservation methods of this species such as ''in vitro'' micropropagation.ies such as ''in vitro'' micropropagation. +
''BACKGROUND AND AIM'' Acetaminophen overd … ''BACKGROUND AND AIM'' Acetaminophen overdose is the most frequent cause of acute liver failure. Non-alcoholic fatty liver disease is the most common chronic condition of the liver. The aim was to assess whether non-alcoholic steatosis sensitizes rat liver to acute toxic effect of acetaminophen.''METHODS'' Male Sprague-Dawley rats were fed a standard diet (ST-1, 10% kcal fat) and high-fat gelled diet (HFGD, 71% kcal fat) for 6 weeks and then acetaminophen was applied in a single dose (1 g/kg body weight). Animals were killed 24, 48 and 72 h after acetaminophen administration. Serum biochemistry, activities of mitochondrial complexes, hepatic malondialdehyde, reduced and oxidized glutathione, triacylglycerol and cholesterol contents, and concentrations of serum and liver cytokines (TNF-α, TGF-β1) were measured and histopathological samples were prepared.''RESULTS'' The degree of liver inflammation and hepatocellular necrosis were significantly higher in HFGD fed animals after acetaminophen administration. Serum markers of liver injury were elevated only in acetaminophen treated HFGD fed animals. Concentration of hepatic reduced glutathione and ratio of reduced/oxidized glutathione were decreased in both ST-1 and HFGD groups at 24 h after acetaminophen application. Mild oxidative stress induced by acetaminophen was confirmed by measurement of malondialdehyde. Liver content of TNF-α was not significantly altered, but hepatic TGF-β1 was elevated in acetaminophen treated HFGD rats. We did not observe acetaminophen-induced changes in activities of respiratory complexes I, II, and IV and activity of caspase-3.''CONCLUSION'' Liver from rats fed HFGD is more susceptible to acute toxic effect of acetaminophen, compared to non-steatotic liver.minophen, compared to non-steatotic liver. +
''Background & Purpose'': T1AM is a th … ''Background & Purpose'': T1AM is a thyronamine derivative of thyroid hormone acting as a signalling molecule via non-genomic effectors and can reach intracellular targets. In light of the importance of F0F1-ATPsynthase as a target in drug development, T1AM interaction with the enzyme is demonstrated by its effects on the activity and a model of binding locations is depicted.''Experimental Approach'': Kinetic analyses were performed on F0F1-ATPsynthase in sub-mitochondrial particles and soluble F1-ATPase. Activity assays and immunodetection of the inhibitor protein IF1 were used and combined with molecular docking analyses. ''In situ'' respirometric analysis of T1AM effect was investigated on H9c2 cardiomyocytes.''Key Results'': T1AM is a non-competitive inhibitor of F0F1-ATPsynthase whose binding is mutually exclusive with that of the inhibitors IF1 and aurovertin B. Distinct T1AM binding sites are consistent with results from both kinetic and docking analyses: at low nanomolar concentrations, T1AM binds to a high affinity-region likely located within the IF1 binding site, causing IF1 release; at higher concentrations, T1AM binds to a low affinity-region likely located within the aurovertin binding cavity and inhibits enzyme activity. Low nanomolar concentrations of T1AM elicit in cardiomyocytes an increase in ADP-stimulated mitochondrial respiration indicative for an activation of F0F1-ATPsynthase consistent with displacement of endogenous IF1, thereby reinforcing the ''in vitro'' results.''Conclusions & Implications'': The T1AM effects upon F0F1-ATPsynthase are twofold: IF1 displacement and enzyme inhibition. By targeting F0F1-ATPsynthase within mitochondria T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low endogenous concentration. T1AM putative binding locations overlapping with IF1 and aurovertin binding sites are depicted.lt;/sub>-ATPsynthase within mitochondria T1AM might affect cell bioenergetics with a positive effect on mitochondrial energy production at low endogenous concentration. T1AM putative binding locations overlapping with IF1 and aurovertin binding sites are depicted. +
''Background and Goal of Study'': Anesthet … ''Background and Goal of Study'': Anesthetics have been demonstrated to inhibit mitochondrial function in animal models, an effect that could be related to neurological sequelae of prolonged or excessive anesthesia in man. It has been proposed that toxicity of anesthetic agents could be caused by inhibition of the electron transport system. In this study, using high-resolved respirometry of human blood cells, the objective was to evaluate the influence of commonly used anesthetic agents in a wide concentration range on mitochondrial oxygen consumption in platelets.''Materials and Methods'': Platelets samples were isolated from healthy volunteers and were rapidly analyzed by [[high-resolution respirometry]] using an Oroboros-2k Oxygraph. Platelets were exposed to propofol (5-150 μg/mL), sevoflurane (0.4-8 mmol/L) and midazolam (0.1-20 μg/mL). Mitochondria were stimulated with complex-specific substrates and inhibitors. Statistical analysis were performed using one way ANOVA with post hoc Dunnett’s test and were compared to a separate control group (''N''＝20). Informed consent was received from all participants and the study was approved by the ethical committee of Tokyo Medical University.''Results and Discussion'': Within the therapeutic concentration-range of the investigated agents, no apparent inhibition of respiratory capacity was noted. Rather, at therapeutic concentrations, significant increases in mitochondrial respiratory parameters were detected for sevoflurane and propofol. Dose-dependent inhibition of respiration was found in the presence of high doses of propofol (30 μg/mL and above) and sevoflurane (1.6 mmol/L and above). The respiratory inhibition was more prominent for Complex I respiration as compared to Complex II-supported respiration. For midazolam no significant effects were noted at the concentration range investigated.''Conclusion'': In freshly isolated and permeabilized human platelets, the commonly used anesthetics sevoflurane and propofol stimulate mitochondrial respiratory capacity at clinically relevant concentrations. At higher concentrations, these agents displayed a dose-dependent inhibition of Complex I and II-supported respiration. The increased respiratory capacity induced by sevoflurane and propofol might be beneficial and the inhibition of respiration could be relevant to situations of prolonged or excessive exposure, especially in situations of tissue accumulation of these anesthetics. tissue accumulation of these anesthetics. +
''Background'': Hematophagy poses a challe … ''Background'': Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energytransducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito ''Aedes aegypti'', a vector of dengue and yellow fever.''Methodology/Principal Findings'': Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, anevent that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak ofblood digestion, when oxygen consumption was inhibited by 68%. Cytochromes ''c'' and ''a+a3 '' levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H2O2 formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed.''Conclusion'': Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which mayrepresent an important adaptation to blood feeding.ct mitochondria, which may represent an important adaptation to blood feeding. +
''Background'': Results from both animal a … ''Background'': Results from both animal and human being studies provide evidence that inhalation of concentrations of carbon monoxide (CO) at around 100 ppm has antiinflammatory effects. These low levels of CO are incriminated in ischemic heart diseases experienced by cigarette smokers and, in some cases, from air pollution. Although neurologic mechanisms have been investigated, the effects of CO on cardiovascular function are still poorly understood.''Methods and Results'': The effects of CO (250 ppm; 90 min) inhalation on myocardial function were investigated in isolated heart of rats killed immediately, and 3, 24, 48, and 96 h after CO exposure. CO exposure at 250 ppm resulted in an arterial carboxyhemoglobin (HbCO) level of approximately 11%, which was not associated with changes in mean arterial pressure and heart rate. CO exposure induced coronary perfusion pressure increases, which were associated with endothelium-dependent and -independent vascular relaxation abnormalities. CO-induced coronary vascular relaxation perturbations were observed in the presence of increased heart contractility. Spontaneous peak to maximal Ca2+-activated left ventricular pressure ratio was markedly increased in CO-exposed rats, indicating increases in myofilament calcium sensitivity. Heart cyclic guanosine monophosphate/cAMP ratio and myocardial permeabilized fiber respiration (complex intravenous activity) were reduced in CO-exposed rats, which lasted after 48 h of reoxygenation in air.''Conclusions'': These findings suggest that CO deteriorates heart oxygen supply to utilization and potentially may induce myocardial hypoxia through mechanisms that include increased oxygen demand due to increased contractility, reduced coronary blood flow reserve, and cardiomyocyte respiration inhibition.low reserve, and cardiomyocyte respiration inhibition. +
''Background'': Within the animal kingdom, … ''Background'': Within the animal kingdom, horses are among the most powerful aerobic athletic mammals. Determination of muscle respiratory capacity and control improves our knowledge of mitochondrial physiology in horses and high aerobic performance in general.We applied high-resolution respirometry and multiple [[substrate-uncoupler-inhibitor titration]] protocols to study mitochondrial physiology in small (1.0 – 2.5 mg) permeabilized muscle fibres sampled from triceps brachii of healthy horses. Oxidative phosphorylation ([[OXPHOS]]) capacity [pmol O2∙s-1∙mg-1 wet weight] in the NADH&succinate-pathway (NS, combined [[CI&II]]-linked substrate supply: glutamate&malate&succinate) increased from 77±18 in overweight horses to 103±18, 122±15, and 129±12 in untrained, trained andcompetitive horses (''N'' = 3, 8, 16, and 5, respectively). Similar to human muscle mitochondria, equine OXPHOS capacity was limited by the phosphorylation system to 0.85±0.10 (''N'' = 32) of electron transfer capacity, independent of fitness level. In 15 trained horses, OXPHOS capacity increased from 119±12 to 134±37 when pyruvate was included in the NS-substrate cocktail. Relative to this maximum OXPHOS capacity, NADH-linked OXPHOS capacities (N) were only 50 % with glutamate&malate, 64 % with pyruvate&malate, and 68 % with pyruvate&glutamate&malate, and ~78 % with succinate&rotenone (S). OXPHOS capacity with glutamate&malate increased with fitness relative to NS-supported ET capacity from a flux control ratio of 0.38 to 0.40, 0.41 and 0.46 in overweight to competitive horses, whereas the S/NS substrate control ratio remained constant at 0.70. Therefore, the apparent deficit of the N- over S-pathway capacity was reduced with physical fitness. The scope of mitochondrial density-dependent OXPHOS capacity and the density-independent (qualitative) increase of N-respiratory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology.tory capacity with increased fitness open up new perspectives of integrative and comparative mitochondrial respiratory physiology. +
''Blastocladiella emersonii'' is an early … ''Blastocladiella emersonii'' is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a ''B. emersonii'' cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in ''B. emersonii''.Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.ed by Elsevier Ltd. All rights reserved. +
''Brevibacillus massiliensis'' strain phR … ''Brevibacillus massiliensis'' strain phR is an obligately aerobic microbe that was isolated from human feces. Here, we show that it readily takes up tungsten (W), a metal previously associated only with anaerobes. The W is incorporated into an oxidoreductase enzyme (BmWOR) that was purified from native biomass. BmWOR consists of a single 65 kDa subunit and contains a single W-pyranopterin cofactor and a single [4Fe-4S] cluster. It exhibited high aldehyde-oxidizing activity with very high affinities (apparent ''K''m < 6 μM) for aldehydes common in the human gut and in cooked foods, including furfural, propionaldehyde, benzaldehyde and tolualdehyde, suggesting that BmWOR plays a key role in their detoxification. ''B. massiliensis'' converted added furfural to furoic acid when grown in the presence of W, but not in the presence of the analogous element molybdenum. ''B. massiliensis'' ferredoxin (BmFd) served as the electron acceptor (apparent ''K''m < 5 μM) for BmWOR suggesting it is the physiological electron carrier. Genome analysis revealed a Fd-dependent rather than NADH-dependent Complex I, suggesting that WOR not only serves a detoxification role but its aldehyde substrates could also serve as a source of energy. BmWOR is the first tungstoenzyme and the first member of the WOR family to be obtained from a strictly aerobic microorganism. Remarkably, BmWOR oxidized furfural in the presence of air (21 % O2, v/v) but only if BmFd was also present. BmWOR is the first characterized member of the Clade 83 WORs, which are predominantly found in extremely halophilic and aerobic archaea (Clade 83A), with many isolated from food sources, while the remaining bacterial members (Clade 83B) include both aerobes and anaerobes. The potential advantages for microbes found in foods and involved in human gut health that harbor O2-resistant WORs, including in ''Bacillus'' and ''Brevibacillus'' based-probiotics, are discussed.Brevibacillus'' based-probiotics, are discussed. +
''Callinectes sapidus'' and ''C. similis'' … ''Callinectes sapidus'' and ''C. similis'' co-occur in estuarine waters above 15 salinity. ''Callinectes sapidus'' also inhabits more dilute waters, but ''C. similis'' is rarely found below 15 . Previous work suggests that ''C. sapidus'' may be a better hyperosmoregulator than ''C. similis''. In this study, energy metabolism and the levels of transport-related enzymes in excised gills were used as indicators of adaptation to low salinity. Oxygen consumption rates and mitochondrial cytochrome content of excised gills increased in both species as acclimation salinity decreased, but to a significantly greater extent in ''C. similis'' gills. In addition, ''C. similis'' gills showed the same levels of carbonic anhydrase and Na+/K+-ATPase activities and the same degree of enzyme induction during low-salinity adaptation as has been reported for ''C. sapidus'' gills. However, hemolymph osmolality and ion concentrations were consistently lower in ''C. similis'' at low salinity than in ''C. sapidus''. Therefore, although gills from low-salinity-acclimated ''C. similis'' have a higher oxygen consumption rate and more mitochondrial cytochromes than ''C. sapidus'' gills and the same level of transport-related enzymes, ''C. similis'' cannot homeostatically regulate their hemolymph to the same extent as ''C. sapidus.''ymph to the same extent as ''C. sapidus.'' +
''Campylobacter jejuni'' is a widespread p … ''Campylobacter jejuni'' is a widespread pathogen responsible for most of the food-borne gastrointestinal diseases in Europe. The use of natural antimicrobial molecules is a promising alternative to antibiotic treatments for pathogen control in the food industry. Isothiocyanates are natural antimicrobial compounds, which also display anti-cancer activity. Several studies described the chemoprotective effect of isothiocyanates on eukaryotic cells, but the antimicrobial mechanism is still poorly understood.We investigated the early cellular response of ''C. jejuni'' to benzylisothiocyanate by both transcriptomic and physiological approaches. The transcriptomic response of ''C. jejuni'' to benzylisothiocyanate showed upregulation of heat shock response genes and an impact on energy metabolism. The oxygen consumption was progressively impaired by benzylisothiocyanate treatment as revealed by high-resolution respirometry, while the ATP content increased soon after benzylisothiocyanate exposition, which suggests a shift in the energy metabolism balance. Finally, benzylisothiocyanate induced intracellular protein aggregation. These results indicate that benzylisothiocyanate affects ''C. jejuni'' by targeting proteins, resulting in the disruption of major metabolic processes and eventually leading to cell death.sses and eventually leading to cell death. +
''Chlamydomonas reinhardtii'' is a photoau … ''Chlamydomonas reinhardtii'' is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris-acetate-phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris-acetate-phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less (1)O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of (1)O2 originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA(-)) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB(-)) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to (1)O2 production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting photosystem II charge recombination events and photoinhibition. recombination events and photoinhibition. +
''Diabetes mellitus'' is a metabolic disor … ''Diabetes mellitus'' is a metabolic disorder characterized by hyperglycemia. We investigated the effect of a prior 30 days voluntary exercise protocol on STZ-diabetic CF1 mice. Glycemia, and the liver and skeletal muscle glycogen, mitochondrial function, and redox status were analyzed up to 5 days after STZ injection. Animals were engaged in the following groups: Sedentary vehicle (Sed Veh), Sedentary STZ (Sed STZ), Exercise Vehicle (Ex Veh), and Exercise STZ (Ex STZ). Exercise prevented fasting hyperglycemia in the Ex STZ group. In the liver, there was decreased on glycogen level in Sed STZ group but not in EX STZ group. STZ groups showed decreased mitochondrial oxygen consumption compared to vehicle groups, whereas mitochondrial H2O2 production was not different between groups. Addition of ADP to the medium did not decrease H2O2 production in Sed STZ mice. Exercise increased GSH level. Sed STZ group increased nitrite levels compared to other groups. In quadriceps muscle, glycogen level was similar between groups. The Sed STZ group displayed decreased O2 consumption, and exercise prevented this reduction. The H2O2 production was higher in Ex STZ when compared to other groups. Also, GSH level decreased whereas nitrite levels increased in the Sed STZ compared to other groups. The PGC1 α levels increased in Sed STZ, Ex Veh, and Ex STZ groups. In summary, prior exercise training prevents hyperglycemia in STZ-mice diabetic associated with increased liver glycogen storage, and oxygen consumption by the mitochondria of skeletal muscle implying in increased oxidative/biogenesis capacity, and improved redox status of both tissues. J. Cell. Biochem. 9999: 1-8, 2016. © 2016 Wiley Periodicals, Inc.© 2016 Wiley Periodicals, Inc.edox status of both tissues. J. Cell. Biochem. 9999: 1-8, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc. +
''Drosophila melanogaster'' is a popular r … ''Drosophila melanogaster'' is a popular research model organism thanks to its powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates "the background advantage", but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail. of the GS system are discussed in detail. +
''Drosophila'' fruit flies have been used … ''Drosophila'' fruit flies have been used as a valuable, cheap, and powerful organism model to understand fundamental biological processes for many years. However, standardized methodologies specifically designed to assess mitochondrial physiology in this model are not available. Rodríguez and colleagues provided a detailed analysis of publicly available protocols to assess mitochondrial physiology in ''Drosophila melanogaster'' while performed experiments in flight muscles to address three technical parameters to define the optimal conditions for respirometry. The authors show that oxygen diffusion is not limited to sustaining respiratory capacity in either isolated mitochondria or chemically permeabilized fibers. In addition, chemical permeabilization revealed the best approach to assess mitochondrial physiology in fruit flies. Finally, the authors demonstrate that magnesium green is the only fluorescent probe that caused no effects on respiratory rates. Methodological standardization to study ''Drosophila'' mitochondrial physiology, as presented by Rodríguez and colleagues, represents a critical step towards more reproducible and comparative metabolic research in this important organism model. arch in this important organism model. +
''Drosophila'' melanogaster is undoubtedly … ''Drosophila'' melanogaster is undoubtedly one of the most useful model organisms in biology. From a bioenergectics and metabolism point-of-view, its four discrete life cycle stages, each with particular nutritional and energetic demands, represent multiple powerful experimental systems in a single organism. Extensive resources are available for the community of ''Drosophila'' researchers worldwide, including an ever-growing number of mutant, transgenic and genomically-edited lines currently being developed and carried by stock centers in North America, Europe and Asia. Here, we provide evidence for the importance of stock centers in sustaining the substantial increase in the output of ''Drosophila'' mitochondrial research worldwide in recent decades. We also argue that the difficulties in transporting fly lines into South America has stalled the progression of related ''Drosophila'' research areas in the continent. Establishing a local stock center is the first step towards building a strong local ''Drosophila'' community that will contribute to the general field of mitochondrial research. neral field of mitochondrial research. +
''Eugenia uniflora'' L(Myrtaceae family) h … ''Eugenia uniflora'' L(Myrtaceae family) has demonstrated several properties of human interest, including insecticide potential, due to its pro-oxidant properties. These properties likely result from the effects on its mitochondria, but the mechanism of this action is unclear. The aim of this work was to evaluate the mitochondrial bioenergetics function in ''Drosophila melanogaster'' exposed to ''E. uniflora'' leaf essential oil. For this, we used a high-resolution respirometry (HRR) protocol. We found that ''E. uniflora'' promoted a collapse of the mitochondrial transmembrane potential (ΔΨm). In addition the essential oil was able to promote the disruption of respiration coupled to oxidative phosphorylation (OXPHOS) and inhibit the respiratory electron transfer-pathway (ET-pathway) established with an uncoupler. In addition, exposure led to decreases of respiratory control ratio (RCR), bioenergetics capacity and OXPHOS coupling efficiency, and induced changes in the substrate control ratio. Altogether, our results suggested that ''E. uniflora'' impairs the mitochondrial function/viability and promotes the uncoupling of OXPHOS, which appears to play an important role in the cellular bioenergetics failure induced by essential oil in ''D. melanogaster''.d by essential oil in ''D. melanogaster''. +
''From'' [http://www.annualreviews.org/doi … ''From'' [http://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem-081009-125448 Schatz G (2012) The fires of life. Annu Rev Biochem 81: 34–59.]:This retrospective recounts the hunt for the mechanism of mitochondrialATP synthesis, the early days of research on mitochondrial formation,and some of the colorful personalities dominating these oftendramatic and emotional efforts. The narrative is set against the backdropof postwar Austria and Germany and the stream of young scientistswho had to leave their countries to receive postdoctoral trainingabroad. Many of them—including the author—chose the laboratory ofa scientist their country had expelled a few decades before. The articleconcludes with some thoughts on the uniqueness of U.S. research universitiesand a brief account of the struggles to revive science in Europe.Illustriert von P. Leslie Dutton Europe. Illustriert von P. Leslie Dutton +
''Geotrichum citri-aurantii'' is a posthar … ''Geotrichum citri-aurantii'' is a postharvest phytopathogenic fungus of lemons. We studied the mode of action of antifungal metabolites from ''Bacillus sp.'' strain IBA 33 on arthroconidia of ''G. citri-aurantii''. These metabolites are lipopeptides belonging to the iturin family. Membrane permeabilization of ''G. citri-aurantii'' was analyzed and mitochondrial respiratory rate was evaluated. Disturbance of the plasma membrane promotes the leakage of many cellular components into the surrounding media, and mitochondrial membrane disorganization promotes the inhibition of the respiratory rate. Our findings provide insights into the ability of lipopeptides to suppress plant fungal pathogens and their possible agronomical applications.d their possible agronomical applications. +
''Giardia intestinalis'' is the microaerop … ''Giardia intestinalis'' is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. ''Giardia'' possesses an O2 -scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O2 -detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O2 to H2O rapidly (∼3 μM O2 × min × 106 cells at 37 °C) and with high affinity (C50 = 3.4 ± 0.7 μM O2). Following a short-term (minutes) exposure to H2O2 ≥ 100 μM, the O2 consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome-inhibitor MG132. Instead, H2O2 does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of ''Giardia'' to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.e that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress. +
''Heterotheca inuloides'', traditionally e … ''Heterotheca inuloides'', traditionally employed in Mexico, has demonstrated anticancer activities. Although it has been proven that the cytotoxic effect is attributed to cadinane-type sesquiterpenes such as 7-hydroxy-3,4-dihydrocadalene, the mechanism of action by which these agents act in tumor lines and their regulation remain unknown. This study was undertaken to investigate for first time the cytotoxic activity and mechanism of action of 7-hydroxy-3,4-dihydrocadalene and two semi-synthetic cadinanes derivatives towards breast cancer cells.Cell viability and proliferation were assayed by thiazolyl blue tetrazolium bromide (MTT) assay and Trypan blue dye exclusion assay. Cell migration measure was tested by wound-healing assay. Moreover, the reactive oxygen species (ROS) and lipid peroxidation generation were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay and thiobarbituric acid reactive substance (TBARS) assay, respectively. Furthermore, expression of caspase-3, Bcl-2 and GAPDH were analyzed by western blot.The results showed that 7-hydroxy-3,4-dihydrocadalene inhibited MCF7 cell viability in a concentration and time dependent manner. The cytotoxic potency of semisynthetic derivatives 7-(phenylcarbamate)-3,4-dihydrocadalene and 7-(phenylcarbamate)-cadalene was remarkably lower. Moreover, ''in silico'' studies showed that 7-hydroxy-3,4-dihydrocadalene, and not so the semi-synthetic derivatives, has optimal physical-chemical properties to lead a promising cytotoxic agent. Further examination on the action mechanism of 7-hydroxy-3,4-dihydrocadalene suggested that this natural product exerted cytotoxicity via oxidative stress as evidenced in a significantly increase of intracellular ROS levels and in an induction of lipid peroxidation. Furthermore, the compound increased caspase-3 and caspase-9 activities and slightly inhibited Bcl-2 levels. Interestingly, it also reduced mitochondrial ATP synthesis and induced mitochondrial uncoupling.Taken together, 7-hydroxy-3,4-dihydrocadalene is a promising cytotoxic compound against breast cancer via oxidative stress-induction.ast cancer via oxidative stress-induction. +
''Homo deus'' shows us where we're going. … ''Homo deus'' shows us where we're going. Yuval Harari envisions a near future in sihch we face a new set of challenges. ''Homo deus'' exlores the projects, dreams and nightmares that will shape the twendty-first century and beyond - from overcoming death to creating artificial life. It asks the fundamental questions: how can we protect this fragile world from our own desctructive power? And what does our future hold?tive power? And what does our future hold? +
''In utero'' overnutrition can predispose … ''In utero'' overnutrition can predispose offspring to metabolic disease. Although the mechanisms are unclear, increased oxidative stress accelerating cellular aging has been shown to play a role. Mitochondria are the main site of reactive oxygen species (ROS) production in most cell types. Levels of ROS and the risk for oxidative damage are dictated by the balance between ROS production and antioxidant defense mechanisms. Originally considered as toxic species, physiologic levels of ROS are now known to be essential cell signaling molecules. Using a model of maternal overnutrition in C57BL6N mice, we investigate the mechanisms involved in the development of insulin resistance (IR) in muscle. In red and white gastrocnemius muscles of offspring, we are the first to report characteristics of oxidative phosphorylation, H2O2 production, activity of mitoflashes, and electron transport chain supercomplex formation. Results demonstrate altered mitochondrial function with reduced response to glucose in offspring of mice fed a high-fat and high-sucrose diet, increases in mitochondrial leak respiration, and a reduction in ROS production in red gastrocnemius in response to palmitoyl carnitine. We also demonstrate differences in supercomplex formation between red and white gastrocnemius, which may be integral to fiber-type specialization. We conclude that in this model of maternal overnutrition, mitochondrial alterations occur before the development of IR.ion, mitochondrial alterations occur before the development of IR. +
''In vitro'' studies of muscle mitochondri … ''In vitro'' studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome. with defects of the mitochondrial genome. +
''In vivo'' studies suggest that intestina … ''In vivo'' studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to provide mechanistic support, using an ''in vitro'' model mimicking the oxidative ''in vivo'' situation.Human Caco-2 cells were cultured for 10 days in culture flasks orfor 14 days on transwell inserts in either glucose-containing or galactose-containingmedium. Mitochondria were visualized and cellular respiration and levels of oxidativephosphorylation (OXPHOS) proteins were determined. Mitochondrial ATP depletionwas induced using CCCP, rotenone, or piericidin A (PA). Monolayer permeability wasassessed using transepithelial electrical resistance (TEER) and fluorescein flux. Geneexpression and cellular distribution of tight junction proteins were analyzed.Caco-2 cells cultured in galactose-containing, but not in glucose-containing,medium showed increased mitochondrial connectivity, oxygen consumption rates andlevels of OXPHOS proteins. Inhibition of mitochondrial ATP production using CCCP,rotenone or PA resulted in a dose-dependent increase in Caco-2 monolayer permeability.In-depth studies with PA showed a six fold decrease in cellular ATP and revealedincreased gene expression of tight junction proteins (TJP) 1 and 2, occludin, and claudin1, but decreased gene expression of claudin 2 and 7. Of these, claudin 7 was clearlyredistributed from the cellular membrane into the cytoplasm, while the others were not(TJP1, occludin) or slightly (claudin 2, actin) affected. ''In vivo'' studies suggest that intestinal barrier integrity is dependent on mitochondrial ATP production. Here, we aim to providemechanistic support, using an ''in vitro'' model mimicking the oxidative ''in vivo'' situation.Well-functioning mitochondria are essential for maintaining cellularenergy status and monolayer integrity of galactose grown Caco-2 cells. Energydepletion-induced Caco-2 monolayer permeability may be facilitated by changes in thedistribution of claudin 7. changes in the distribution of claudin 7. +
''In vivo'' ubiquinone (UQ) reduction leve … ''In vivo'' ubiquinone (UQ) reduction levels were measured during the development of the inflorescences of ''Arum maculatum'' and ''Amorphophallus krausei''. Thermogenesis in ''A. maculatum'' spadices appeared not to be confined to a single developmental stage, but occurred during various stages. The UQ pool in both ''A. maculatum'' and ''A. krausei'' appendices was approximately 90% reduced during thermogenesis. Respiratory characteristics of isolated appendix mitochondria did not change in the period around thermogenesis. Apparently, synthesis of the required enzyme capacity is regulated via a coarse control upon which a fine control of metabolism that regulates the onset of thermogenesis is imposed.tes the onset of thermogenesis is imposed. +
''Leishmania amazonensis'' is one of leish … ''Leishmania amazonensis'' is one of leishmaniasis' causative agents, a disease that has no cure and leads to the appearance of cutaneous lesions. Recently, our group showed that heme activates a Na+/K+ ATPase in these parasites through a signaling cascade involving hydrogen peroxide (H2O2) generation. Heme has a pro-oxidant activity and signaling capacity, but the mechanism by which this molecule increases H2O2 levels in ''L. amazonensis'' has not been elucidated. Here we investigated the source of H2O2 stimulated by heme, ruling out the participation of mitochondria and raising the possibility of a role for a NADPH oxidase (Nox) activity. Despite the absence of a classical Nox sequence in trypanosomatid genomes, ''L. amazonensis'' expresses a surface ferric iron reductase (LFR1). Interestingly, Nox enzymes are thought to have evolved from ferric iron reductases because they share same core domain and are very similar in structure. The main difference is that Nox catalyses electron flow from NADPH to oxygen, generating reactive oxygen species (ROS), while ferric iron reductase promotes electron flow to ferric iron, generating ferrous iron. Using ''L. amazonensis'' overexpressing or knockout for LFR1 and heterologous expression of LFR1 in mammalian embryonic kidney (HEK 293) cells, we show that this enzyme is bifunctional, being able to generate both ferrous iron and H2O2. It was previously described that protozoans knockout for LFR1 have their differentiation to virulent forms (amastigote and metacyclic promastigote) impaired. In this work, we observed that LFR1 overexpression stimulates protozoan differentiation to amastigote forms, reinforcing the importance of this enzyme in ''L. amazonensis'' life cycle regulation. Thus, we not only identified a new source of ROS production in Leishmania, but also described, for the first time, an enzyme with both ferric iron reductase and Nox activities.Copyright © 2019 Elsevier Inc. All rights reserved.o described, for the first time, an enzyme with both ferric iron reductase and Nox activities. Copyright © 2019 Elsevier Inc. All rights reserved. +
''Leishmania'' species are responsible for … ''Leishmania'' species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic ''Leishmania'' spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the ''Leishmania'' proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing ''L. braziliensis'', ''L. panamensis'' and ''L. guyanensis'' species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. "Total Protein Approach" and "Proteomic Ruler" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete ''Leishmania'' proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas ''L. braziliensis'' relies the more on glycolysis, ''L. panamensis'' and ''L. guyanensis'' seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.ailable via ProteomeXchange with identifier PXD017696. +
''MitoCom'' closing event and perspectives. Innsbruck, Austria; 2014 October 16 +
''Moniliophthora perniciosa'' is a fungal … ''Moniliophthora perniciosa'' is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against ''M. perniciosa'', an important outcome for the chocolate industry.ortant outcome for the chocolate industry. +
''Moringa oleifera'' seeds, which are used … ''Moringa oleifera'' seeds, which are used for water clarification, contain a lectin named WSMoL which has shown ''in vitro'' antibacterial and immunomodulatory activity. Due to their nutritional value and therapeutic potential, the leaves and seeds of this tree are eaten in some communities. Some plant lectins are non-toxic to mammals, but others have been reported to be harmful when ingested or administered by other means. As one of the steps needed to define the safety of WSMoL, we evaluated possible cardiotoxic effects of this purified protein. WSMoL was administered for 21 consecutive days to mice by gavage. Electrophysiological, mechanical, and metabolic cardiac functions were investigated by ''in vivo'' and ''ex vivo'' electrocardiographic recordings, nuclear magnetic resonance, and high-resolution respirometry. The treatment with WSMoL did not induce changes in blood glucose levels or body weight in comparison with control group. Moreover, the heart weight/body weight and heart weight/tibia length ratios were similar in both groups. Lectin ingestion also did not modify glucose tolerance or insulin resistance. No alterations were observed in electrocardiographic parameters or cardiac action potential duration. The heart of mice from the control and WSMoL groups showed preserved left ventricular function. Furthermore, WSMoL did not induce changes in mitochondrial function (in all cases, p > 0.05). The administration of WSMoL demonstrated a cardiac safety profile. These results contribute to the safety evaluation of using ''M. oleifera'' seeds to treat water, since this lectin is present in the preparation employed by some populations to this end.ion employed by some populations to this end. +
''Mycobacterium tuberculosis'' (MTB) is th … ''Mycobacterium tuberculosis'' (MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 μM), while increasing the dose (50 μM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP ''in vitro'' and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion. autophagy activation, and TNFα secretion. +
''Mycobacterium tuberculosis'' (Mtb) exhib … ''Mycobacterium tuberculosis'' (Mtb) exhibits remarkable metabolic flexibility that enables it to survive a plethora of host environments during its life cycle. With the advent of bedaquiline for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated as an important target and a vulnerable component of mycobacterial metabolism. Exploiting the dependence of Mtb on oxidative phosphorylation for energy production, several components of this pathway have been targeted for the development of new antimycobacterial agents. This includes targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by DG70 and other compounds, terminal oxidase by imidazopyridine amides and ATP synthase by diarylquinolines. Importantly, oxidative phosphorylation also plays a critical role in the survival of persisters. Thus, inhibitors of oxidative phosphorylation can synergize with frontline TB drugs to shorten the course of treatment. In this review, we discuss the oxidative phosphorylation pathway and development of its inhibitors in detail.d development of its inhibitors in detail. +
''Mycobacterium tuberculosis'' (Mtb) regul … ''Mycobacterium tuberculosis'' (Mtb) regulates the macrophage metabolic state to thrive in the host. Yet, the responsible mechanisms remain elusive. Macrophage activation towards the microbicidal (M1) program depends on the HIF-1 α-mediated metabolic shift from oxidative phosphorylation towards glycolysis. Here, we asked whether a tuberculosis (TB) microenvironment changes the M1 macrophage metabolic state. We exposed M1 macrophages to the acellular fraction of tuberculous pleural effusions (TB-PE), and found lower glycolytic activity, accompanied by elevated levels of oxidative phosphorylation and bacillary load, compared to controls. The host-derived lipid fraction of TB-PE drove these metabolic alterations. HIF-1α stabilization reverted the effect of TB-PE by restoring M1 metabolism. As a proof-of-concept, Mtb-infected mice with stabilized HIF-1α displayed lower bacillary loads and a pronounced M1-like metabolic profile in alveolar macrophages. Collectively, we demonstrate that host-derived lipids from a TB-associated microenvironment alter the M1 macrophage metabolic reprogramming by hampering HIF-1α functions, thereby impairing control of Mtb infection.hereby impairing control of Mtb infection. +
''OBJECTIVE'': The link between a reduced … ''OBJECTIVE'': The link between a reduced capacity for skeletal muscle mitochondrial fatty acid oxidation (FAO) and lipotoxicity in human insulin resistance has been the subject of intense debate. The objective of this study was to investigate whether reduced FAO is associated with elevated acyl CoA, ceramide, and diacylglycerol (DAG) in severely obese insulin resistant subjects.''DESIGN AND METHODS'': Muscle biopsies were conducted in lean (L, 22.6 ± 0.5 kg/m2, ''n'' = 8), Class I (CI, 32.1 ± 0.4 kg/m2, ''n'' = 7) and Class II&III obese (CII&III, 45.6 ± 1.1 kg/m2, ''n'' = 15) women for acyl CoA, sphingolipid and DAG profiling. Intramyocellular triglyceride (IMTG) content was determined by histology. FAO was assessed by incubating muscle homogenates with [1-C]palmitate and measuring CO2 production. Cardiolipin content was quantified as an index of mitochondrial content. Lipid metabolism proteins, DGAT1, PLIN5, and PNPLA2 were quantified in biopsy samples by western blot.''RESULTS'': CII&III were more insulin resistant (HOMA-IR: 4.5 ± 0.5 vs. 1.1 ± 0.1, ''P'' < 0.001), and had lower FAO (∼58%, ''P'' = 0.007) and cardiolipin content (∼31%, ''P'' = 0.013) compared to L. IMTG was elevated in CI (''P'' = 0.04) and CII&III (''P'' = 0.04) compared to L. Sphingolipid content was higher in CII&III compared to L (13.6 ± 1.1 vs. 10.3 ± 0.5 pmol/mg, ''P'' = 0.031) whereas DAG content was not different among groups. DGAT1 was elevated in CII&III, and PLIN5 was elevated in CI compared to L.''CONCLUSION'': Severe obesity is associated with reduced muscle oxidative capacity and occurs concomitantly with elevated IMTG, ceramide and insulin resistance.rs concomitantly with elevated IMTG, ceramide and insulin resistance. +
''Objective'' To culture equine myoblasts … ''Objective'' To culture equine myoblasts from muscle microbiopsy specimens, examine myoblast production of reactive oxygen species (ROS) in conditions of anoxia followed by reoxygenation, and assess the effects of horseradish peroxidase (HRP) and myeloperoxidase (MPO) on ROS production.''Animals'' 5 healthy horses (5 to 15 years old).''Procedures'' Equine skeletal myoblast cultures were derived from 1 or 2 microbiopsy specimens obtained from a triceps brachii muscle of each horse. Cultured myoblasts were exposed to conditions of anoxia followed by reoxygenation or to conditions of normoxia (control cells). Cell production of ROS in the presence or absence of HRP or MPO was assessed by use of a gas chromatography method, after which cells were treated with a 3,3′-diaminobenzidine chromogen solution to detect peroxidase binding.''Results'' Equine skeletal myoblasts were successfully cultured from microbiopsy specimens. In response to anoxia and reoxygenation, ROS production of myoblasts increased by 71%, compared with that of control cells. When experiments were performed in the presence of HRP or MPO, ROS production in myoblasts exposed to anoxia and reoxygenation was increased by 228% and 183%, respectively, compared with findings for control cells. Chromogen reaction revealed a close adherence of peroxidases to cells, even after several washes.''Conclusions and Clinical Relevance'' Results indicated that equine skeletal myoblast cultures can be generated from muscle microbiopsy specimens. Anoxia-reoxygenationtreated myoblasts produced ROS, and production was enhanced in the presence of peroxidases. This experimental model could be used to study the damaging effect of exercise on muscles in athletic horses.of exercise on muscles in athletic horses. +
''Oenothera biennis'' L. (OB), also common … ''Oenothera biennis'' L. (OB), also commonly known as evening primrose, belongs to the Onagraceae family and has the best studied biological activity of all the members in the family. In therapy, the most frequently used type of extracts are from the aerial part, which are the fatty oils obtained from the seeds and have a wide range of medicinal properties. The aim of this study was to evaluate the phytochemical composition and biological activity of OB hydroalcoholic extract and to provide directions for the antimicrobial effect, antiproliferative and pro-apoptotic potential against A375 melanoma cell line, and anti-angiogenic and anti-inflammatory capacity. The main polyphenols and flavonoids identified were gallic acid, caffeic acid, epicatechin, coumaric acid, ferulic acid, rutin and rosmarinic acid. The total phenolic content was 631.496 µgGAE/mL of extract and the antioxidant activity was 7258.67 μmolTrolox/g of extract. The tested extract had a mild bacteriostatic effect on the tested bacterial strains. It was bactericidal only against ''Candida spp.'' and ''S. aureus''. In the set of experimental conditions, the OB extract only manifested significant antiproliferative and pro-apoptotic activity against the A375 human melanoma cell line at the highest tested concentration, namely 60 μg/mL. The migration potential of A375 cells was hampered by the OB extract in a concentration-dependent manner. Furthermore, at the highest tested concentration, the OB extract altered the mitochondrial function ''in vitro'', while reducing the angiogenic reaction, hindering compact tumor formation in the chorioallantoic membrane assay. Moreover, the OB extract elicited an anti-inflammatory effect on the experimental animal model of ear inflammation.rimental animal model of ear inflammation. +
''Porphyromonas gingivalis'' (''P. gingiva … ''Porphyromonas gingivalis'' (''P. gingivalis''), a key pathogen in periodontitis, is associated with neuroinflammation. Periodontal disease increases with age; 70.1% of adults 65 years and older have periodontal problems. However, the ''P. gingivalis''- lipopolysaccharide (LPS)induced mitochondrial dysfunction in neurodegenerative diseases remains elusive. In this study, we investigated the possible role of ''P. gingivalis''-LPS in mitochondrial dysfunction during neurodegeneration. We found that ''P. gingivalis''-LPS treatment activated toll-like receptor (TLR) 4 signaling and upregulated the expression of Alzheimer's disease-related dementia and neuroinflammatory markers. Furthermore, the LPS treatment significantly exacerbated the production of reactive oxygen species and reduced the mitochondrial membrane potential. Our study highlighted the pivotal role of ''P. gingivalis''-LPS in the repression of serum response factor (SRF) and its co-factor p49/STRAP that regulate the actin cytoskeleton. The LPS treatment repressed the genes involved in mitochondrial function and biogenesis. ''P. gingivalis''-LPS negatively altered oxidative phosphorylation and glycolysis and reduced total adenosine triphosphate (ATP) production. Additionally, it specifically altered the mitochondrial functions in complexes I, II, and IV of the mitochondrial electron transport chain. Thus, it is conceivable that ''P. gingivalis''-LPS causes mitochondrial dysfunction through oxidative stress and inflammatory events in neurodegenerative diseases.tory events in neurodegenerative diseases. +
''Purpose'': Following the recent demonstr … ''Purpose'': Following the recent demonstration of increased mitochondrial DNA mutations in lymphocytes of POAG patients, the authors sought to characterize mitochondrial function in a separate cohort of POAG.''Methods'': Using similar methodology to that previous applied to Leber's hereditary optic neuropathy (LHON) patients, maximal adenosine triphosphate (ATP) synthesis and cellular respiration rates, as well as cell growth rates in glucose and galactose media, were assessed in transformed lymphocytes from POAG patients (n = 15) and a group of age- and sex-matched controls (n = 15).''Results'': POAG lymphoblasts had significantly lower rates of complex-I-driven ATP synthesis, with preserved complex-II-driven ATP synthesis. Complex-I driven maximal respiration was also significantly decreased in patient cells. Growth in galactose media, where cells are forced to rely on mitochondrial ATP production, revealed no significant differences between the control and POAG cohort.''Conclusions'': POAG lymphoblasts in the study cohort exhibited a defect in complex-I of the oxidative phosphorylation pathway, leading to decreased rates of respiration and ATP production. Studies in LHON and other diseases have established that lymphocyte oxidative phosphorylation measurement is a reliable indicator of systemic dysfunction of this pathway. While these defects did not impact lymphoblast growth when the cells were forced to rely on oxidative ATP supply, the authors suggest that in the presence of a multitude of cellular stressors as seen in the early stages of POAG, these defects may lead to a bioenergetic crisis in retinal ganglion cells and an increased susceptibility to cell death.an increased susceptibility to cell death. +
''SURF1'' is an assembly factor of mitocho … ''SURF1'' is an assembly factor of mitochondrial complex IV, and its mutations are the primary cause of Leigh syndrome in infants. To date, over 100 ''SURF1'' mutations have been reported worldwide, but the spectrum of the ''SURF1'' mutations in China remains unclear. Here, using next-generation sequencing targeting mitochondrial protein-coding sequences, we sequenced 178 patients suspected to have mitochondrial diseases. Fifteen ''SURF1'' mutations were identified in 12 Leigh syndrome patients, of which three, c.465_466delAA, c.532A > T, and c.826_827ins AGCATCTGCAGTACATCG, were newly described. The percentage of ''SURF1'' frameshift mutations (6/28, 21.4%) we detected in Chinese population is higher than other studies (21/106, 19.8%) with different populations, however, the percentage of missense mutations is lower in this study than others (4/28, 14.3% VS. 25/106, 23.6%). Since complex IV can be detected in cells carrying missense mutations (3/8) but not in cells carrying null mutations (0/4) by using cell model-based complementation assay, our results indicate that ''SURF1'' mutations may be associated with worse clinical outcome in Chinese patients than other populations. However, studies with larger sample size are needed to verify this conclusion. Additionally, we found that the frameshift mutations resulting in protein truncation closer to the C-terminus are not associated with better disease prognosis. Lastly, we found that determining the levels of complex IV assembly using cell models or lymphocyte analysis rather than invasive muscle and skin fibroblast biopsy, may help predict disease progression in Leigh syndrome patients.sease progression in Leigh syndrome patients. +
''Saccharomyces cerevisiae'' is a facultat … ''Saccharomyces cerevisiae'' is a facultative anaerobe devoid of mitochondrial alternative oxidase. In this yeast, the structure and biogenesis of the respiratory chain, on the one hand, and the functional interactions of oxidative phosphorylation with the cellular energetic metabolism, on the other, are well documented. However, to our knowledge, the molecular aspects and the physiological roles of the non-respiratory pathways that utilize molecular oxygen have not yet been reviewed. In this paper, we review the various non-respiratory pathways in a global context of utilization of molecular oxygen in S. cerevisiae. The roles of these pathways are examined as a function of environmental conditions, using either physiological, biochemical or molecular data. Special attention is paid to the characterization of the so-called 'cyanide-resistant respiration' that is induced by respiratory deficiency, catabolic repression and oxygen limitation during growth. Finally, several aspects of oxygen sensing are discussed.l aspects of oxygen sensing are discussed. +
''Schistosoma mansoni'', one of the causat … ''Schistosoma mansoni'', one of the causative agents of human schistosomiasis, has a unique antioxidant network that is key to parasite survival and a valuable chemotherapeutic target. The ability to detoxify and tolerate reactive oxygen species increases along ''S. mansoni'' development in the vertebrate host, suggesting that adult parasites are more exposed to redox challenges than young stages. Indeed, adult parasites are exposed to multiple redox insults generated from blood digestion, activated immune cells, and, potentially, from their own parasitic aerobic metabolism. However, it remains unknown how reactive oxygen species are produced by ''S. mansoni'' metabolism, as well as their biological effects on adult worms. Here, we assessed the contribution of nutrients and parasite gender to oxygen utilization pathways, and reactive oxygen species generation in whole unpaired adult ''S. mansoni'' worms. We also determined the susceptibilities of both parasite sexes to a pro-oxidant challenge. We observed that glutamine and serum importantly contribute to both respiratory and non-respiratory oxygen utilization in adult worms, but with different proportions among parasite sexes. Analyses of oxygen utilization pathways revealed that respiratory rates were high in male worms, which contrast with high non-respiratory rates in females, regardless nutritional sources. Interestingly, mitochondrial complex I-III activity was higher than complex IV specifically in females. We also observed sexual preferences in substrate utilization to sustain hydrogen peroxide production towards glucose in females, and glutamine in male worms. Despite strikingly high oxidant levels and hydrogen peroxide production rates, female worms were more resistant to a pro-oxidant challenge than male parasites. The data presented here indicate that sexual preferences in nutrient metabolism in adult ''S. mansoni'' worms regulate oxygen utilization and reactive oxygen species production, which may differently contribute to redox biology among parasite sexes.ute to redox biology among parasite sexes. +
''Spirulina platensis'' is a blue-green al … ''Spirulina platensis'' is a blue-green alga used as a dietary supplement because of its hypocholesterolemic properties. Among other bioactive substances, it is also rich in tetrapyrrolic compounds closely related to bilirubin molecule, a potent antioxidant and anti-proliferative agent. The aim of our study was to evaluate possible anticancer effects of ''S. platensis'' and ''S. platensis''-derived tetrapyrroles using an experimental model of pancreatic cancer. The anti-proliferative effects of ''S. platensis'' and its tetrapyrrolic components [phycocyanobilin (PCB) and chlorophyllin, a surrogate molecule for chlorophyll A] were tested on several human pancreatic cancer cell lines and xenotransplanted nude mice. The effects of experimental therapeutics on mitochondrial reactive oxygen species (ROS) production and glutathione redox status were also evaluated. Compared to untreated cells, experimental therapeutics significantly decreased proliferation of human pancreatic cancer cell lines ''in vitro'' in a dose-dependent manner (from 0.16 g•L-1 [''S. platensis''], 60 μM [PCB], and 125 μM [chlorophyllin], ''p''<0.05). The anti-proliferative effects of ''S. platensis'' were also shown ''in vivo'', where inhibition of pancreatic cancer growth was evidenced since the third day of treatment (''p''<0.05). All tested compounds decreased generation of mitochondrial ROS and glutathione redox status (''p''=0.0006; 0.016; and 0.006 for ''S. platensis'', PCB, and chlorophyllin, respectively). In conclusion, ''S. platensis'' and its tetrapyrrolic components substantially decreased the proliferation of experimental pancreatic cancer. These data support a chemopreventive role of this edible alga. Furthermore, it seems that dietary supplementation with this alga might enhance systemic pool of tetrapyrroles, known to be higher in subjects with Gilbert syndrome.roles, known to be higher in subjects with Gilbert syndrome. +
''Staphylcoccus epidermidis'' does not inv … ''Staphylcoccus epidermidis'' does not invade healthy tissues, however, it has been identified as a cause of nosocomial infections due to its ability to form biofilms on polymer surfaces [1]. ''S. epidermidis'' can be grown at different oxygen concentrations ([O2]), including mammalian skin where [O2] ranges from 3-5% and in anaerobic altered tissues [2,3]. Biofilm formation of ''S. epidermidis'' and its respiratory chain components grown in aerobic, microaerobic and anaerobic conditions were evaluated by in-gel activities, enzymatic activities, spectrophotometry and oxymetry. Varying [O2] modified both biofilm formation and the components in the respiratory chain: At high [O2], little tendency to form biofilms was observed. ''S. epidermidis'' expressed glycerol-3-phosphate, pyruvate, ethanol and succinate dehydrogenases; and cyt bo and aa3. Under micro-aerobiosis, biofilm formation increased slightly; pyruvate, ethanol, glycerol-3-phosphate and succinate dehydrogenase decreased; aa3 cyt was not detected; Under anaerobiosis high biofilm-formation and low ethanol and pyruvate dehydrogenase activities were found; anaerobic nitrate dehydrogenase activity was detected. Aerobic-grown cells with cyanide increased biofilm formation. Anaerobic-grown cells with methylamine decreased biofilm formation. Thus, either a decrease in [O2] or the inhibition of the aerobic chain led ''S. epidermidis'' to associate into biofilms. In contrast, high [O2] or inhibition of the anaerobic nitrate reductase prevented biofilm formation suggesting that the enzymes expressed at low to null [O2] are therapeutic targets against biofilm formation by ''S. epidermidis''. expressed at low to null [O2] are therapeutic targets against biofilm formation by ''S. epidermidis''. +
''Trichodesmium'' is a biogeochemically im … ''Trichodesmium'' is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual 'new' nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55-60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. ''Trichodesmium'' therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean.ical success of this species in the ocean. +
''Trypanosoma brucei'' is a parasitic flag … ''Trypanosoma brucei'' is a parasitic flagellate that causes devastating diseases of humans and lifestock. The infective form dwells in the glucose rich environment of mammalian blood and generate energy solely via glycolysis. In consequence, the bloodstream stage single mitochondrion is highly reduced lacking key Krebs cycle enzymes and traditional cytochrome mediated respiratory chain. Interestingly, the essential mitochondrial membrane potential (Δ''ψ''mt) is maintained by hydrolytic activity of the unique FoF1-ATPase, which contains several trypanosoma specific subunits of unknown function [1].We determined that one of the largest novel subunit, Tb2930 (43 kDa), is membrane-bound and localizes into monomeric and multimeric assemblies of the FoF1-ATPase. RNAi silencing of Tb2930 led to a significant decrease of Δ''ψ''mt and consequently to ''T. brucei'' growth inhibition, indicating that the FoF1-ATPase is not functioning properly even though its structural intergrity seems to be almost unchanged. To further explore the function of this protein, we employed naturally occuring trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) including subunit a, an essential component of the Fo-moiety and proton pore. These Dk cells maintain Δ''ψ''mt by electrogenic exchange of ATP4-/ADP3- by the ATP/ADP carrier (AAC) and hydrolytic activity of the soluble F1-ATPase [2]. So far, it has been assumed that only the F1-moiety subunits are present and will be essential for these parasites. Interestingly, glycerol gradient sedimentation and native electrophoresis of Dk mitochondria revealed the presence of high molecular weight ATPase complexes that correspond to the bloodstream stage monomeric and multimeric FoF1-ATPase. Furthermore, the Tb2930 subunit is expressed in Dk cells and co-sediments with these high molecular weight membrane bound complexes. The RNAi study demonstrated that Tb2930 subunit is essential for Dk trypanosoma cells and crucial for maintaining Δ''ψ''mt. Importantly, upon ablation of Tb2930 we observed a shift of the FoF1-ATPase complexes to the lower S-values on glycerol gradient, where the free F1-ATPase sediments, indicating changes in the structural integrity of the Dk FoF1-ATPase. In conclusion, we propose that Tb2930 is responsible for connecting the Dk F1-ATPase to the mitochondrial membrane in the absence of subunit a of the Fo-moiety, thus increasing the efficiency of the functional association between F1-ATPase and AAC.y, thus increasing the efficiency of the functional association between F1-ATPase and AAC. +
''Trypanosoma brucei'' undergoes a complex … ''Trypanosoma brucei'' undergoes a complex life cycle as it alternates between a mammalian host and the blood-feeding insect vector, a tsetse fly. Due to the different environments, the distinct life stages differ in their energy metabolism, i.e. insect stage (procyclic cells, PS) depends on mitochondrial oxidative phosphorylation (OXPHOS) for ATP production while the bloodstream stage (BS) gains energy by aerobic glycolysis. The dramatic switch from the OXPHOS to glycolysis happens during the complex development of the PS in the tsetse fly. This development differentiation is characterized by extensive remodeling of mitochondrion structure and changes in mitochondrial bioenergetics. Importantly, the molecular mechanism behind this process is completely unknown. We have established the ''in vitro'' differentiation system, in which the transition from PS to epimastigotes followed by differentiation to transmission-ready metacylic trypanosomes is triggered by RNA binding protein 6 (RBP6) expression. This ''in vitro'' induced differentiation of PF cells takes 8 days. The appearance of epimastigotes and metacyclic trypanosomes in the culture was mapped using light and fluorescent microscopy. The whole cell proteome of cell culture harvested every day after the RBP6 induction was identified by label-free quantitative mass spectrometry. This proteomic data serves as a resource for further detailed characterization of changes happening in the parasite mitochondrion as well as identification of possible candidates involved in the PS differentiation.idates involved in the PS differentiation. +
''Trypanosoma cruzi'' has a single mitocho … ''Trypanosoma cruzi'' has a single mitochondrion, the main site of reactive oxygen species (ROS) production. Moreover, ''T. cruzi'' epimastigotes proliferate in the presence of heme, which induces ROS formation (Nogueira et al 2011; Lara et al 2007). Therefore, we evaluated heme effect upon mitochondrial ROS formation and mitochondrial membrane potential (ΔΨmt). For that, epimastigotes were incubated with DHE or TMRM with or without heme. After this, FCCP and antymicin A (Ama) were added. Mitochondrial ROS production and ΔΨmt were analyzed by flow cytometry. Our results showed that heme duplicated ROS production and induced a 4-fold increase of ΔΨmt. The FCCP addition reversed heme effects upon ROS generation and ΔΨm. Additionally, Ama induced a 2-fold increase of ROS production and 46% increment in ΔΨmt, while co-incubation with heme and AA presented a 3-fold increase upon ROS formation and increase ΔΨmt in 70%. In order to corroborate the involvement of heme in mitochondrial ROS, we incubated the parasites with heme, in the absence or in the presence of mitoTEMPO, a mitochondrial antioxidant. Our results showed that in the presence of this antioxidant greatly decreased heme induced ROS generation. Afterwards, we incubated epimastigotes with heme for 30 min and then, performed a substrate-uncoupler-inhibitor-tritation protocol with rotenone, succinate, ADP, cytocrome c, FCCP and Ama. We were able to detect a decrease in several states, mainly ROUTINE, OXPHOS and reserve capacity, compared to control cells. Finally, we evaluated epimastigotes proliferation with or without heme, H2O2, FCCP, Ama or mitoTEMPO. We observed that low concentrations of H2O2 increased proliferation, while higher concentrations showed deleterious effects upon the cells. FCCP and mitoTEMPO also reversed heme-induced proliferation, whereas, Ama promoted a tripanostatic effect. Taken together, our results strongly suggest that heme modulates ''T. cruzi'' mitochondrial physiology since it promotes mitochondrial ROS production, decreasing mitochondrial states, and enhances the ΔΨmt.tochondrial states, and enhances the ΔΨmt. +
''Trypanosoma cruzi'' is a hemoflagellate … ''Trypanosoma cruzi'' is a hemoflagellate protozoan that causes Chagas’ disease. ''T. cruzi'' life-cycle is complex involving different evolutive forms that experience striking differences in their environmental condition. Here we carried out a functional assessment of mitochondrial function in two distinct ''T. cruzi'' forms: the insect stage, epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate electrons entry into the electron transport chain increasing Complex II-III activity. Curiously, cytochrome c oxidase (CIV) activity and the expression of CIV subunit IV were reduced in bloodstream forms, creating an “electron bottleneck” that favored increased electron leak and H2O2 formation. We propose that the oxidative preconditioning provided by this mechanism would confer a protection to the bloodstream trypomastigotes against host immune response. Thus, mitochondrial remodeling during the ''T. cruzi'' life-cycle can represent a key metabolic adaptation for parasite survival in different environments.rasite survival in different environments. +
''Trypanosoma cruzi'' is the etiologic age … ''Trypanosoma cruzi'' is the etiologic agent of Chagas disease, a disorder affecting thousands of people, for which an effective treatment is not available for the chronic phase. Calcium signaling is important for host cell invasion, differentiation, osmoregulation, cell death and flagellar function in trypanosomatids. The influx of calcium into the mitochondria, which is important for intracellular calcium homeostasis, occurs through a mitochondrial calcium uniporter complex (MCUC) and this complex consists of several components, including two regulatory proteins named mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2). In mammalian cells, these proteins are located in the mitochondrial intermembrane space and play a role in sensing cytosolic calcium levels and regulating the MCU opening. Although several MCUC components have been identified in trypanosomes, the mechanism by which it is regulated is still unknown. In this work, we aimed at studying the role of MICU1 and MICU2 in the mitochondrial calcium uptake of ''T. cruzi''. The predicted TcMICU1 and TcMICU2 proteins displayed a mitochondrial targeting signal and EF-hands domains that could be sensitive to changes in cytosolic calcium. We obtained TcMICU1 (MICU1-KO) and TcMICU2 (MICU2-KO) knockout cell lines using the CRISPR/Cas9 system by co-transfecting ''T. cruzi'' epimastigotes with the Cas9/pTREX-n vector (containing a specific sgRNA) and a DNA donor cassette with a blasticidin resistance marker to induce the DNA double-strand break repair by homologous recombination. Additionally, we generated a cell line of ''T. cruzi'' epimastigotes overexpressing TcMICU2 tagged with 2xHA (MICU2-OE) using pTREX-n vector. Such molecular constructs were used to analyze the mutant phenotypes and indicate the functions of these proteins. Our results show that MICU1-KO and MICU2-KO have a significant decrease in the capacity to take up calcium, showing a different regulation when we compared to what has already been described previously in mammals. In the absence of these proteins there is a decrease in the growth rate and respiration rates of epimastigotes, showing how important these two proteins are to this stage of ''T. cruzi''. In addition, MICU1-KO epimastigotes are able to differentiate to metacyclic trypomastigotes in a greater proportion than the control cells while the metacyclogenesis capacity was reduced in MICU2-KO cells. Using the MICU2-OE cell line we demonstrated by immunofluorescence microscopy the mitochondrial localization of MICU2 and that its overexpression does not alter the capacity to take up calcium, besides that it does not affect the mitochondrial membrane potential and parasite growth. We can conclude that the TcMICU1 and TcMICU2 proteins are essential for the regulation of mitochondrial calcium uptake by MCU in ''T. cruzi''. Likewise, the results suggest that both proteins play an important role in the growth and differentiation of epimastigotes.owth and differentiation of epimastigotes. +
''Trypanosoma cruzi'', the aetiological ag … ''Trypanosoma cruzi'', the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of ''T. cruzi'', suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in ''T. cruzi'', showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO2. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O2 consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host.oenergetics and parasite persistence within its invertebrate host. +
''Trypanosoma cruzi'', the causative agent … ''Trypanosoma cruzi'', the causative agent of Chagas disease, faces changes in redox status and nutritional availability during its life cycle. However, the influence of oxygen fluctuation upon the biology of ''T. cruzi'' is unclear. The present work investigated the response of ''T. cruzi'' epimastigotes to hypoxia. The parasites showed an adaptation to the hypoxic condition, presenting an increase in proliferation and a reduction in metacyclogenesis. Additionally, parasites cultured in hypoxia produced more reactive oxygen species (ROS) compared to parasites cultured in normoxia. The analyses of the mitochondrial physiology demonstrated that hypoxic condition induced a decrease in both oxidative phosphorylation and mitochondrial membrane potential (ΔΨm) in epimastigotes. In spite of that, ATP levels of parasites cultivated in hypoxia increased. The hypoxic condition also increased the expression of the hexokinase and NADH fumarate reductase genes and reduced NAD(P)H, suggesting that this increase in ATP levels of hypoxia-challenged parasites was a consequence of increased glycolysis and fermentation pathways. Taken together, our results suggest that decreased oxygen levels trigger a shift in the bioenergetic metabolism of ''T. cruzi'' epimastigotes, favoring ROS production and fermentation to sustain ATP production, allowing the parasite to survive and proliferate in the insect vector.vive and proliferate in the insect vector. +
''Trypanosoma cruzi'', the parasite causin … ''Trypanosoma cruzi'', the parasite causing Chagas disease, is a digenetic flagellated protistthat infects mammals (including humans) and reduviid insect vectors. Therefore, ''T. cruzi''must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard,it is well known that ''T. cruzi'' has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established hasbeen the capability of ''T. cruzi'' to use glucose and amino acids to support the differentiationprocess occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities ofusing externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in ''T. cruzi''. Herein, we show that during parasiteproliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can betaken up from the medium, leading to CO2 production. Additionally, we show that electronsare fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylicacid (TCA) cycle, which can be used to feed anabolic pathways such as the ''de novo'' biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization intothe mitochondrion diminishes the survival to severe starvation, and impairsmetacyclogenesis.s the survival to severe starvation, and impairs metacyclogenesis. +
''Wolbachia sp.'' has colonized over 70% o … ''Wolbachia sp.'' has colonized over 70% of insect species, successfully manipulatinghost fertility, protein expression, lifespan, and metabolism. Understanding and engineeringthe biochemistry and physiology of ''Wolbachia'' holds great promise for insectvector-borne disease eradication. ''Wolbachia'' is cultured in cell lines, which have longduplication times and are difficult to manipulate and study. The yeast strain''Saccharomyces cerevisiae'' W303 was used successfully as an artificial host for''Wolbachia'' wAlbB. As compared to controls, infected yeast lost viability early, probablyas a result of an abnormally high mitochondrial oxidative phosphorylation activityobserved at late stages of growth. No respiratory chain proteins from ''Wolbachia''were detected, while several ''Wolbachia'' F1F0-ATPasesubunits were revealed. After 5 days outside the cell, Wolbachia remained fully infective against insect cells.the cell, Wolbachia remained fully infective against insect cells. +
'Mitochondria, Metabolism and Energetics': [[Media:MiPNet18.14 IOC85 Mahabaleshwar.pdf|'''38th Mahabaleshwar Seminar''']], [http://www.tifr.res.in/~dbsconf/mito2014/Home.html mito2014], including '''[[MiPNet18.14 | 85th OROBOROS O2k-Workshop]]'''. +
'The Neanderthals live on in many of us to … 'The Neanderthals live on in many of us today' (p 199).Neanderthal Man tells the story of geneticist Svante Pääbo's mission to answer this question, and recounts his ultimately successful efforts to genetically define what makes us different from our Neanderthal cousins. Beginning with the study of DNA in Egyptian mummies in the early 1980s and culminating in the sequencing of the Neanderthal genome in 2010, Neanderthal Man describes the events, intrigues, failures, and triumphs of these scientifically rich years through the lens of the pioneer and inventor of the field of ancient DNA.We learn that Neanderthal genes offer a unique window into the lives of our hominin relatives and may hold the key to unlocking the mystery of why humans survived while Neanderthals went extinct. Drawing on genetic and fossil clues, Pääbo explores what is known about the origin of modern humans and their relationship to the Neanderthals and describes the fierce debate surrounding the nature of the two species' interactions. His findings have not only redrawn our family tree, but recast the fundamentals of human history—the biological beginnings of fully modern ''Homo sapiens'', the direct ancestors of all people alive today.A riveting story about a visionary researcher and the nature of scientific inquiry, Neanderthal Man offers rich insight into the fundamental question of who we are.to the fundamental question of who we are. +
(1) Endothelial cells are permanently chal … (1) Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH(i)) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH(i) regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH(i) was monitored by detecting the fluorescent changes. (2) The steady-state pH(i) of RBCE cells in HEPES-buffer (6.83 +/- 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 +/- 0.08). Cells were exposed to NH4CI to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na+ / H+ exchanger, HCO3- / Cl- exchange mechanism is also involved in the restoration of pH(i) after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na+ / H+ exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. (3) No pH(i) recovery was detected when the washout after an intracellular acid load occurred in nominally Na+ -free HEPES-buffered medium or in the presence of 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na+ / H+ exchanger. The new steady-state pH(i) were 6.37 +/- 0.02 and 6.60 +/- 0.02, respectively. (4) No detectable change was observed in the steady-state pH(i) in the presence of 100 microM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 microM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 is without effect on the activity of Na+ / H+ exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.xchanger after an intracellular acid load. +
(2020) Excel template for TPP data analys … (2020) Excel template for TPP data analysis. Mitochondr Physiol Network 25.14(01):1-8. <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>:» Product: [[DatLab]], [[Oroboros O2k]], [[Oroboros O2k-Catalogue |O2k-Catalogue]]oboros O2k-Catalogue |O2k-Catalogue]] +
* Amorphous membrane fragments depleted i … * Amorphous membrane fragments depleted in P-lipids and cytochrome oxidase were isolated from bovine heart mitochondria and were reconstituted with P-lipids and coupling factors to yield vesicular structures. These vesicles catalyzed a 32Pi—ATP exchange and showed an induced enhancement of anilinonaphthalene sulfonate fluorescence on addition of ATP* 32Pi—ATP exchange and fluorescence enhancement were abolished by uncouplers of oxidative phosphorylation and by energy transfer inhibitors. The ATPase activity was inhibited by energy transfer inhibitors, but stimulated by uncouplers or by the combined action of nigericin and valinomycin in the presence of K+. Both ATPase activity and 32Pi—ATP exchange were inhibited by a specific antibody against coupling factor 1.* It was shown that the reconstitution of vesicular structures with functional activity required several hours. Rapid reconstitution resulted in inactive vesicles. Evidence for the formation of new vesicles from solubilized P-lipids was obtained by demonstrating inclusion of macromolecules such as 14C-labeled inulin or ferritin which could not be removed by washing.itin which could not be removed by washing. +
*Saponin permeabilized skeletal muscle fi … *Saponin permeabilized skeletal muscle fiber bundles*'''Protocol''':#0.5mM Malate#50 µM palmitoyl-CoA + 2mM carnitine#5mM ADP#From here on out, various combinations for titration protocol*Coupling states:#LEAK_M+Palmitoylcarnitine#P_M+Palmitoylcarnitine#P_M+Palmitoylcarnitine+S#E_O+CCCP titrations#E_S+Rot#ROX_AntimycinA_O+CCCP titrations #E_S+Rot #ROX_AntimycinA +
.. From the start of my scientific career … .. From the start of my scientific career the galvanic cell, the first form of which, the Volta pile, popularized physics in a single stroke and at the same time presented us with so many problems, appeared to me to merit especially further study. ..It was particularly disillusioning to find a man like Helmholtz returning repeatedly throughout his scientific career to his first love, the galvanic cell, which he had courted in his great youthful work "Erhaltung der Kraft" (The conservation of energy), without however succeeding in finding a satisfactory solution.As often in natural science the picture changed quite suddenly. New fruitful concepts appeared, through the interplay and extension of which most of the darkness has been to a large extent dispelled in a single stroke. Such means were Van ’t Hoff’s theory of osmotic pressure, Arrhenius’ theory of electrolytic dissociation, and finally many new approaches to the treatment of chemical equilibria, which, brilliantly presented, are to be found scattered throughout the first edition of Ostwald’s "Lehrbuch der Allgemeinen Chemie" (Textbook of general chemistry). So there arose in 1889 the osmotic theory of galvanic current generation, which has not been seriously challenged since it was put forward more than thirty years ago and has undergone no appreciable elaboration since its acceptance, surely a clear sign that it has so far satisfied scientific needs. ..The osmotic theory of current generation stipulates moreover that when a metal ion concentration is higher than consistent with the solution tension of the particular metal, on immersion of the metal, ions of the relevant metal electrode must go into solution, while conversely they must settle on the electrode when the reverse is the case.he electrode when the reverse is the case. +
.. The convenience and simplicity of the p … .. The convenience and simplicity of the polarographic 'oxygen electrode' technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the invetigator's ingenuity and specific requirements of the system under investigation.rements of the system under investigation. +
0RGAN PRESERVATION under hypothermic ische … 0RGAN PRESERVATION under hypothermic ischemia is enhanced by storage solutions that protect the vascular endothelium from ischemia-reperfusion injury. Ischemia-reperfusion injury leads to primary graft failure and chronic rejection, and is commonly assessed by measuring endothelial activation and damage of the endothelial plasma membrane. However, corresponding primary intracellular events are little understood compared with the secondary cytokine/adhesion molecule cascade and inflammatory responses.1, 2 Because protection of intracellular and cell membrane function is fundamental for further improvement of organ preservation, we developed highresolution respirometry as a sensitive diagnostic test for mitochondrial and plasma membrane competence.3 Whereas the plasma membrane remained impermeable after clinically relevant cold storage times of 8 hours and 20 to 60 minutes of reoxygenation, mitochondrial function was impaired at several steps of the respiratory chain.l function was impaired at several steps of the respiratory chain. +
1,4-naphthoquinones, especially juglone, a … 1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function.Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k.Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC50 is 7.7 ± 0.28 μM and 9.6 ± 0.75 μM, respectively) and caused cell death by necrosis. Additionally, they increased the amount of intracellular ROS in a concentration-dependent manner. Moreover, even at very small concentrations (1-3 µM), these compounds significantly uncoupled mitochondrial oxidation from phosphorylation impairing energy production in cells. Lawsone had significantly lower viability decreasing and mitochondria-uncoupling effect, and exerted strong antioxidant activity.Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.unctions, and possesses strong antioxidant properties. +
1. As ATP has a higher affinity for Mg2+ t … 1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.hment of millimolar concentrations of ATP. +
1. Effects of 3-hydroxy-3-methylglutaryl c … 1. Effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, pravastatin and simvastatin, on the myocardial level of coenzyme Q10, and on mitochondrial respiration were examined in dogs. 2. Either vehicle (control), pravastatin (4 mg kg-1 day-1), or simvastatin (2 mg kg-1 day-1) was administered orally for 3 weeks. First, the myocardial tissue level of coenzyme Q10 was determined in the 3 groups. Second, ischaemia was induced by ligating the left anterior descending coronary artery (LAD) in anaesthetized open chest dogs, pretreated with the inhibitors. After 30 min of ischaemia, nonischaemic and ischaemic myocardium were removed from the left circumflex and LAD regions, respectively, and immediately used for isolation of mitochondria. The mitochondrial respiration was determined by polarography, with glutamate and succinate used as substrates. 3. Simvastatin significantly decreased the myocardial level of coenzyme Q10, but pravastatin did not. 4. Ischaemia decreased the mitochondrial respiratory control index (RCI) in both groups. Significant differences in RCI between nonischaemic and ischaemic myocardium were observed in the control and simvastatin-treated groups. 5. Only in the simvastatin-treated group did ischaemia significantly decrease the ADP/O ratio, determined with succinate. 6. The present results indicate that simvastatin but not pravastatin may cause worsening of the myocardial mitochondrial respiration during ischaemia, probably because of reduction of the myocardial coenzyme Q10 level.tion of the myocardial coenzyme Q10 level. +
1. In succinate oxidation reactivated by a … 1. In succinate oxidation reactivated by a low concentration of adenosine triphosphate, addition of small amounts of adenosine diphosphatc will lead to reestablishment of the inhibited state of succinate oxidation after a short burst of respiration.2. The inhibited state is not relieved by either phosphate or phosphate acceptors. 3. The inhibition is closely correlated with a high degree of oxidation of mitochondrial reduced diphosphopyridine nucleotide, which occurs immediately on addition of adenosine diphosphate and is followed by the inhibition of succinate oxidation after the oxidation or rather small amounts of succinate. 4. Oxidation of more than approximately two-thirds of the total diphosphopyridinc nuclcotide (DPN) reducible by succinate and adenosine triphosphate eventually leads to inhibition of succinate oxidation. 5. Based on independent evidence for a compartmentation of mitochondrial pyridine nucleotide (approximately one-third available to DPN-linked substrates), it is proposed that inhibi- tion occurs when oxidation of DPN in the compartment available to malate causes oxaloacetate formation sufficient to inhibit succinate oxidation. 6. The general possibility of DPN control of malate oxidation is considered with respect to the whole question of oxaloacetate regulation of the citric acid cycle.etate regulation of the citric acid cycle. +
1. It has been shown that the electron tra … 1. It has been shown that the electron transfer system in beef heart mitochondria may be reconstituted either totally or in any desired sequential segment by appropriate combinations of two or more of the four primary complexes that have been isolated in highly purified form in this laboratory. 2. The four enzyme systems that collectively comprise the complete machinery for transfer of electrons from reduced diphosphopyridine nucleotide (DPNH; =NADH) and succinate to oxygen re: I, DPNH-coenzyme Q reductase; II, succinic-coenzyme Q reductase; III, QH2-cytochrome ''c'' reductase; and IV, cytochrome ''c'' reductase. The specific inhibitors of each complex have been studied. 3. By appropriate combinations of the primary complexes the following secondary activities have been reconstituted: V, DPNH-cytochrome ''c'' reductase; VI, succinic-cytochrome ''c'' reductase; VII, DPNH, succinic-cytochrome c reductase; VIII, DPNH oxidase; IX, succinic oxidase; and X, DPNH, succinic oxidase activity. The general oxidation-reduction properties of the reconstituted systems, both in the presence and the absence of the usual specific inhibitors of the electron transfer system, are essentially the same as those found for the same activities in the intact mitochondria and in the integrated particles derived therefrom. 4. The reconstituted activities are quite stable to repeated freezing, thawing, and storage at -2O °C, and for the most part, when once formed, are not dissociated by dilution of the mixture or by centrifugation. The evidence supporting the conclusion that reconstitution necessarily involves a particle-particle interaction is discussed.article-particle interaction is discussed. +
1. M. iliotibialis (MIT) and M. pectoralis … 1. M. iliotibialis (MIT) and M. pectoralis (MP) of the BUT Big 6 and Kelly BBB turkey were characterised with respect to physical properties, mitochondrial function, metabolic state, morphology and meat quality.2. Mitochondrial enzyme activity and respiration rates in MP declined with increasing age while glycolytic enzyme activity remained nearly constant.3. There were no major differences between BUT Big 6 and Kelly BBB with respect to histological, histochemical or biochemical variables. In spite of the greater adult weight of BUT Big 6, body proportion was equal in both strains.4. In agreement with the histochemical findings MIT showed higher oxidative capacities, while glycolytic enzyme activity was higher in MP.5. Pyruvate was the best substrate for oxidative phosphorylation in MIT, but not in MP. Pyruvate dehydrogenase (PDH) activity was up to 15 times less in MP and blood lactate was correlated with intramuscular pH.6. Turkey breast muscle was restricted in its ability to oxidise pyruvate, especially in those animals that tended to develop intramuscular acidosis post mortem.7. It is concluded that the ''in vivo'' metabolic environment results in acidosis and impaired meat quality, at least in turkey M. pectoralis.quality, at least in turkey M. pectoralis. +
1. Materials and technical procedures invo … 1. Materials and technical procedures involved in the preparation of liver suspensions have been described and discussed. 2. Liver extracts prepared by the present method appear to contain almost exclusively elements of cytoplasmic origin and can be considered to represent, on a large scale, the cytoplasm of liver cells.large scale, the cytoplasm of liver cells. +
1. Oxidation of NADH by fumarate coupled t … 1. Oxidation of NADH by fumarate coupled to synthesis of ATP was found to occur in cyanide-poisoned rat heart submitochondrial particles. The reaction was inhibited by amytal, thenoyltrifluoroacetone and malonate, indicating the involvement of a portion of the electron transfer chain.2. Cytochrome b became oxidized (while the other cytochromes remained reduced) during the oxidation of NADH by fumarate, suggesting that cytochrome b is part of the reaction pathway.3. Succinate was recovered as the reaction product and accounted for the NADH oxidized.4. The P/2e ratios indicate that one ATP was produced for each pair of electrons transferred to fumarate.5. The reaction was also demonstrated to be present in liver and gastrocnemius muscle of rat. The reaction rate in heart was 2.0 times that of gastrocnemius and 3.3 times that of liver. These differences are not related to the activities of NADH or succinate dehydrogenase.6. The ubiquitous nature of this reaction suggests that it could serve as an important physiological mechanism for generating extra glycolytic energy during periods of anoxia.lycolytic energy during periods of anoxia. +
1. Pigeon heart mitochondria produce H(2)O … 1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20 nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26 nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic [[State 4]] has an optimum pH value of 7.5. In States 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in State 4, an increase in the ''p''O(2) up to 1.97 MPa (19.5 atm) increases H(2)O(2) formation 10-15-fold. Similar ''p''O(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97 MPa (19.5 atm). By increasing the ''p''O(2) to 1.97 MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic State 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent ''K''(m) of about 71 kPa (0.7-0.8 atm) and a ''V''(max) of 12 and 20 nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome ''b'' level.atory chain at the cytochrome ''b'' level. +
1. Pulses of acidity of the outer aqueous … 1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H(+) (or OH(-)) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and beta-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5.5 and 8.5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the -->H(+)/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of -->H(+)/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the -->H(+)/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na(+). These apparent changes of -->H(+)/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial -->H(+)/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial -->H(+)/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid-base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.xperimental support for a suggested configuration of loop 3. +
1. Rat tumor extracts, containing chiefly … 1. Rat tumor extracts, containing chiefly the cytoplasmic constituents of leukemic cells, were fractionated into three main portions, the different components separating in the centrifuge according to size. 2. Mitochondria were isolated by centrifugation at relatively low speed. Elementary composition of purified mitochondria was found to correspond to about 11.5 per cent nitrogen, 1.6 per cent phosphorus, and 27 per cent lipids. Phosphorus and nitrogen content of the lipid portion suggests that as much as 75 to 80 per cent of the lipids of mitochondria is represented by phospholipids. Tests for ribose nucleic acid were positive. 3. Microsomes were separated by means of centrifugation at 18,000 x g. A relation between the high phosphorus content of the microsomes and the marked basophilia of the cytoplasm of leukemic cells is suggested. 4. Phosphorus distribution in the tumor extract, and light absorption analysis of the third fraction, seem to demonstrate that nucleic acid was not present either in a free condition, or in the form of nucleoprotein of relatively low molecular weight. The nature of the results suggests that ribose nucleic acid occurs in the cytoplasm of leukemic cells only in association with formed elements of relatively large size, namely microsomes, and mitochondria.size, namely microsomes, and mitochondria. +
1. Submitochondrial particles were prepare … 1. Submitochondrial particles were prepared from beef‐heart mitochondria by sonication in the presence of EDTA. The particles were lyophilized and repeatedly extracted with pentane until no ubiquinone was found in the extract. Treatment of the ubiquinone‐depleted particles with pentane containing a suitable concentration of ubiquinone (ubiquinone‐50) and subsequent quick washing with ubiquinone‐free pentane resulted in a “re‐incorporation” of ubiquinone in an amount similar to that present in the original particles (3–6 nmoles/mg protein).2. The ubiquinone‐depleted particles exhibited very low or no succinate and NADH oxidase activities, which were restored upon the re‐incorporation of ubiquinone to the levels found in the lyophilized particles before extraction with pentane. Partial (about 50 %) extraction of ubiquinone resulted in markedly decreased succinate and NADH oxidase activities.3. Added cytochrome ''c'' did not replace ubiquinone in restoring the succinate or NADH oxidase activity of ubiquinone‐depleted particles. It stimulated the NADH oxidase, but not the succinate oxidase, activity of the “ubiquinone‐incorporated” particles, but the same stimulation occurred with the lyophilized particles before ubiquinone extraction. The normal, lyophilized, and “ubiquinone‐incorporated” particles contained equal amounts of both total and enzymatically reducible cytochromes.4. In the presence of KCN, NADH reduced the cytochromes, including cytochrome ''b'', only at insignificant rates in the ubiquinone‐depleted particles as compared to the normal and lyophilized preparations, and these rates were greatly stimulated upon the re‐incorporation of ubiquinone. Succinate caused a rapid partial (about 25 %) reduction of cytochrome ''b'', but not of the rest of the cytochromes, in the ubiquinone‐depleted particles. This reduction occurred also in the absence of KCN, and the fraction of cytochrome ''b'', so reduced was not reoxidized when succinate oxidation was inhibited by malonate. Evidence for the occurrence of such an enzymatically non‐oxidizable form of cytochrome ''b'' was also obtained in the normal, lyophilized and “ubiquinone‐incorporated” particles, but, in those cases, this cytochrome ''b'' was reduced by both succinate and NADH. In the presence of antimycin A, all cytochrome ''b'' in the ubiquinone‐depleted particles was rapidly reduced by succinate but not by NADH.5. The normal and lyophilized particles catalyzed a rotenone‐sensitive oxidation of NADH by fumarate. This reaction was completely absent from the ubiquinone‐depleted particles and was restored upon the re‐incorporation of ubiquinone.6. N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine catalyzed an NADH and succinate oxidase activity in antimycin A‐inhibited particles. This NADH oxidase activity was partially sensitive to rotenone in the normal, lyophilized and “ubiquinone‐incorporated” particles, but completely rotenone‐insensitive in the ubiquinone‐depleted particles. All four types of particles were active in catalyzing the antimycin A‐sensitive oxidation of menadiol.7. It is concluded that uniquinone is essential for the interaction of succinate dehydrogenase, NADH dehydrogenase and cytochrome ''b'', and that this interaction is a requisite for the normal function of the respiratory chain. Functionally modified forms of cytochrome ''b'', arising as a consequence of structural damage or antimycin A treatment, are discussed in relation to existing information and proposals concerning the role of cytochrome ''b'' and ubiquinone in electron transport.'b'' and ubiquinone in electron transport. +
1. The action of the antibiotics enniatin … 1. The action of the antibiotics enniatin A, valinomycin, the actin homologues, gramicidin, nigericin and dianemycin on mitochondria, erythrocytes and smectic mesophases of lecithin-dicetyl hydrogen phosphate was studied. 2. These antibiotics induced permeability to alkali-metal cations on all three membrane systems. 3. The ion specificity on each membrane system was the same. 4. Enniatin A, valinomycin and the actins did not induce permeability to protons, whereas nigericin and dianemycin rendered all three membrane systems freely permeable to protons. 5. Several differences were noted between permeability induced by nigericin and that induced by gramicidin. 6. The action of all these antibiotics on mitochondrial respiration could be accounted for by changes in passive ion permeability of the mitochondrial membrane similar to those induced in erythrocytes and phospholipid membranes, if it is assumed that a membrane potential is present in respiring mitochondria.tial is present in respiring mitochondria. +
1. The apparent binding constant (Kapp(Ca- … 1. The apparent binding constant (Kapp(Ca-G)) for GEDTA (ethylene glycol bis(β-aminoethyl ether)-N, N, N', N'-tetraacetic acid, EGTA) to calcium was determined under conditions of biological significance in the presence of various kinds of pH-buffering agents, using murexide or tetramethylmurexide as a Ca indicator.2. The value of Kapp(Ca-G) at pH 6.80 was 1.0×106M-1 at an ionic strength of 0.114 at 20°C, irrespective of the type of pH-buffering ions. This value is similar to that of Allen, Blinks and Prendergast (1977) (Science 196, 996-998), but still half that calculated from the results of Schwarzenbach, Senn and Anderegg (1957) (Helv. Chim. Acta 40, 1886-1900).3. The value of Kapp(Ca-G) varied according to the following equation as the ionic strength (I) was varied from 0.039 to 0.264:log Kapp(Ca-G)=6.460-[2_??_I/(1+_??_I)-0.4×I] (pH 6.80, 20°C)4. The discrepancy between the present results and previous ones (Ogawa, Y. (1968) J. Biochem. 64, 255-257) may have been due to inadequate regulation of the temperature of the reaction medium in the previous determinations, during which an increase in the temperature of the solution may have occurred.An increase of temperature causes a decrease in the pH of the solution in the presence of histidine, imidazole or Tris-maleate, but causes very little change of pH in the presence of phosphate or maleate.5. The association rate constant for GEDTA with calcium was determined by the stoppedflow method in solutions containing 100mM KCl and 20mM pH-buffering ions at 20°C: the values obtained were 1.4×106M-1s-1 in the presence of MOPS-KOH at pH 6.80; 3.0×106M-1s-1 with imidazole at pH 6.80; 1.0×106M-1s-1 with Tris-maleate at pH 6.80..0×106M-1s-1 with Tris-maleate at pH 6.80. +
1. The concentration-dependence of the int … 1. The concentration-dependence of the intramitochondrial accumulation of l-malate and succinate was measured and expressed in the form of adsorption isotherms. The accumulation, however, may arise because of an internal positive potential. 2. The competition for accumulation offered by some other anions, including phosphate, was measured and is expressed conventionally by additional terms in the adsorption equation. 3. The interactions between anions were also studied when one was acting as oxidized substrate. 4. In some examples there is a parallel between the effects of an added anion on both accumulation and oxidation; in other cases chemical participation of the added substance in metabolism is presumed to remove the correlation. 5. It is suggested that by combining kinetic data on penetration with stoicheiometric data on accumulation and specific reaction rates it may be possible to account for the rates of respiration obtained with intact mitochondria. 6. It is possible to show that there is a certain phosphate/substrate ratio for maximum phosphorylation rate with some substrates. This is to be expected when phosphate and substrate compete for accumulation.te and substrate compete for accumulation. +
1. The effects of succinate oxidation on p … 1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD(+) but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.milar in rat heart and liver mitochondria. +
1. The kinetics of the efflux of Pi and ma … 1. The kinetics of the efflux of Pi and malate as well as the relationship between Pi transport and intra- and extramitochondrial pH changes were studied in rat-liver mitochondria in the presence of rotenone and oligomycin at different pH's.2. At high pH a fast efflux of Pi from the mitochondria occurs in the first few seconds, followed by a slow re-entry of Pi into the mitochondria. Under the same conditions the exit of malate shows a time lag of 2–4 sec. The exit of malate coincides with the re-entry of Pi.3. In the presence of butylmalonate the exit of endogenous Pi is coupled with a concomitant alkalinization of the mitochondrial matrix space, as calculated from the distribution of 5,5-[14C]dimethyloxazolidine-2,4-dione.4. The stoicheiometry of the Pi-hydroxyl exchange was found to be 1:1.5. The kinetics of Pi transport are consistent with previous observations that there is a direct exchange between OH− and Pi, but not between OH− and malate. The equilibrium distribution of H2PO4− and OH− deviates from the Donnan distribution. This may be explained by assuming a pH-dependent binding of Pi in the mitochondria.pendent binding of Pi in the mitochondria. +
1. The present paper constitutes a prelimi … 1. The present paper constitutes a preliminary study of the morphology of mitochondria by means of electron microscopy.2. The mitochondria that were the subject of this investigation were obtained from a lymphosarcoma of the rat. They were separated from the other components of the leukemic cells by a method of differential centrifugation, and thus made available for direct examination in the electron microscope.3. In the purified form the mitochondria appeared as spherical bodies, the majority of them varying in size approximately from 0.6 to 1.3 µ in diameter.4. Certain aspects of mitochondria in the electron microscope suggest that these elements are surrounded by a differentiated membrane. In some cases the limiting membrane seemed to be responsible for maintaining the general shape of the mitochondria, even when most of the mitochondrial substance had been lost.5. By means of the electron microscope, it is possible to distinguish small elements, 80 to 100 mµ in diameter, within the body of certain mitochondria. Further work is suggested to establish whether these small granules are normal constituents of mitochondria, and what relation may exist between them and ordinary microsomes.6. The nature of mitochondria as morphological units is discussed. Present evidence indicates that mitochondria constitute definite physical entities which can persist in the absence of the cytoplasm.n persist in the absence of the cytoplasm. +
1. The yield of mitochondria isolated from … 1. The yield of mitochondria isolated from perfused hearts subjected to 30 min ischaemia followed by 15 min reperfusion was significantly less than that for control hearts, and this was associated with a decrease in the rates of ADP-stimulated respiration. 2. The presence of 0.2 microM cyclosporin A (CsA) in the perfusion medium during ischaemia and reperfusion caused mitochondrial recovery to return to control values, but did not reverse the inhibition of respiration. 3. A technique has been devised to investigate whether the Ca(2+)-induced non-specific pore of the mitochondrial inner membrane opens during ischaemia and/or reperfusion of the isolated rat heart. The protocol involved loading the heart with 2-deoxy[3H]glucose ([3H]DOG), which will only enter mitochondria when the pore opens. Subsequent isolation of mitochondria demonstrated that [3H]DOG did not enter mitochondria during global isothermic ischaemia, but did enter during the reperfusion period. 4. The amount of [3H]DOG that entered mitochondria increased with the time of ischaemia, and reached a maximal value after 30-40 min of ischaemia. 5. CsA at 0.2 microM did not prevent [3H]DOG becoming associated with the mitochondria, but rather increased it; this was despite CsA having a protective effect on heart function similar to that shown previously [Griffiths and Halestrap (1993) J. Mol. Cell. Cardiol. 25, 1461-1469]. 6. The non-immunosuppressive CsA analogue [MeAla6]cyclosporin was shown to have a similar Ki to CsA on purified mitochondrial peptidyl-prolyl cis-trans-isomerase and mitochondrial pore opening, and also to have a similar protective effect against reperfusion injury. 7. Using isolated heart mitochondria, it was demonstrated that pore opening could become CsA-insensitive under conditions of adenine nucleotide depletion and high matrix [Ca2+] such as may occur during the initial phase of reperfusion. The apparent increase in mitochondrial [3H]DOG in the CsA-perfused hearts is explained by the ability of the drug to stabilize pore closure and so decrease the loss of [3H]DOG from the mitochondria during their preparation.the mitochondria during their preparation. +
1. Unlike other known flavoproteins, in wh … 1. Unlike other known flavoproteins, in which the flavin is relatively loosely bound and is easily liberated by suitable methods of denaturation, in succinic dehydrogenase from beef heart the flavin component is so tightly held that neither treatment with strong acids nor thermal denaturation separates it from the protein.2. Extensive digestion of the purified dehydrogenase with suitable proteolytic enzymes liberates the flavin in an acid-soluble form, which is not, however, identical with known derivatives of riboflavin. The flavin appears in the digest in several chromatographically distinct forms, which may be separated from each other by purification on ion exchange resins or by chromatography on filter paper.3. The main flavin components have been extensively purified and degraded to the mononucleotide and dephosphorylated flavin levels. The dinucleotide contains 1 mole of 5’-adenylic acid, 2 atoms of phosphorus bound in pyrophosphate linkage and 1 mole of ribose. It differs from authentic flavin adenine dinucleotide (FAD) in numerous regards, including its inactivity in the n-amino acid oxidase test, shifted absorption spectrum, shifted pH-fluorescence curve, and in the presence of cationic group(s). After degradation to the mononucleotide and dephosphorylated flavin level, similar differences exist between the resulting compounds and authentic riboflavin 5’-phosphate and riboflavin, respectively. Irradiation in alkali degrades the flavin further, but the resulting compound is not identical with lumiflavin.4. These differences and the greater water solubility of the unphosphorylated compound as compared with riboflavin are best explained by the hypothesis that the flavin in the dehydrogenase is held to a peptide chain by a covalent linkage which survives proteolytic digestion. The compounds in the digest, therefore, would be peptides of FAD, representing fragments of the original enzyme.5. Evidence for the flavin peptide hypothesis has come from the finding that throughout very extensive purification by a variety of methods the flavin is always accompanied by peptide material. In the most purified fraction, believed to be free of contaminating peptides, alanine, serine, threonine, glutamic acid, and valine were present in molar ratio to the flavin and an additional mole of serine was present as N-terminal group. Similar amino acid compositions were found in 2 other samples, purified by different procedures.6. Evidence pertaining to the flavin peptide hypothesis and the possible structure of the flavin is discussed.ible structure of the flavin is discussed. +
10 years ago the uncoupling hypothesis was … 10 years ago the uncoupling hypothesis was presented for mitochondrial haplogroups of arctic populations suggesting that lower coupling of mitochondrial respiration to ATP production was selected for in favor of higher heat dissipation as an adaptation to cold climates [1,2]. Up to date no actual tests have been published to compare mitochondrial coupling in tissues obtained from human populations with regional mtDNA variations. Analysis of oxidative phosphorylation (OXPHOS) is a major component of mitochondrial phenotyping [3]. We studied mitochondrial coupling in small biopsies of arm and leg muscle of Inuit of the Thule and Dorset haplogroups in northern Greenland compared to Danes from western Europe haplogroups. Inuit had a higher capacity to oxidize fat substrate in leg and arm muscle, yet mitochondrial respiration compensating for proton leak was proportionate with OXPHOS capacity. Biochemical coupling efficiency was preserved across variations in muscle fiber type and uncoupling protein-3 content. After 42 days of skiing on the sea ice in northern Greenland, Danes demonstrated adaptive substrate control through an increase in fatty acid oxidation approaching the level of the Inuit, yet coupling control of oxidative phosphorylation was conserved. Our findings reveal that coupled ATP production is of primary evolutionary significance for muscle tissue independent of adaptations to the cold.ue independent of adaptations to the cold. +
10th Conference of the Asian Society of Mitochondrial Research and Medicine - [http://asmrm2013.com/common_files/mess.asp ASMRM 2013], Seoul KR +
10th MiP''conference'': Joint IUBMB/MiP Symposium on Mitochondrial Physiology - a Point/Counterpoint Meeting, Obergurgl, Austria; with post-conference workshop '''[[MiPNet19.10 | 95th Oroboros O2k-Workshop]]'''. +
10th Conference of the International Coenzyme Q10 Association, Hamburg, 2022 +
10th International Luebeck Conference on the Pathophysiology and Pharmacology of Erythropoietin and other Hemopoietic Growth Factors, Lübeck, DE, [https://www.physio.uni-luebeck.de/index.php?id=162 10th International Luebeck Conference] +
10th Translational Research in Mitochondri … 10th Translational Research in Mitochondria/Metabolism, Aging, and Disease (TRiMAD) Conference, Pennsylvania, United States, 2023 == General information ==:::: TRiMAD is a collaborative venture between The Pennsylvania State University, University of Pittsburgh Medical Center, The Children’s Hospital of Philadelphia (CHoP) Research Institute, and The University of Pennsylvania Perelman School of Medicine ([https://www.huck.psu.edu/node/15830 Website])== Venue == :::: University of Pittsburgh:::: Bridgeside Point 1, 5th Floor:::: 100 Technology Drive:::: Pittsburgh, PA 15219== Organizers ==:::: University of Pittsburgh:::: Aging Institute:::: Center for Metabolism & Mitochondrial Medicine== Program ==:::: Please find the programme [https://aging.pitt.edu/event/trimad-2023/ here]== Registration ==:::: [https://forms.office.com/pages/responsepage.aspx?id=ifT5nqDg606HzDpSYRL9DXg8U8hQ84RKssucFsBERrBURTU2T1lFR01DS0hYNlZGRjNDTzg2QVJRSC4u Register here] == Lecturers and tutors ==:::: The list of speakers can be found [https://aging.pitt.edu/event/trimad-2023/ here]ttps://aging.pitt.edu/event/trimad-2023/ here] +
10th World Congress on Targeting Mitochond … 10th World Congress on Targeting Mitochondria, Berlin, Germany, 2019 == General information == :::: Flyer available for [http://wiki.oroboros.at/images/7/7f/Berlin_2019.pdf download]== Venue == :::: INTERCONINENTAL BERLIN HOTEL:::: Budapester Str. 2, 10787:::: Berlin, Germany::::[https://targeting-mitochondria.com/venue Hotel and Travel]== Programme ==:::: [https://targeting-mitochondria.com/preliminary-program here]== Speakers == :::: List of speakers can be found [https://targeting-mitochondria.com/speakers-2019 here]== Registration ==:::: [https://targeting-mitochondria.com/registration Registration and more information]tration Registration and more information] +
115th ITC: Evolutionary mitochondrial biology: molecular, biochemical, and metabolic diversity, Titisee, Germany. +
11th Conference on Mitochondrial Physiology, 2015 Sep 07-11, Luční Bouda, Czech Republic. +
11th World Congress on Targeting Mitochond … 11th World Congress on Targeting Mitochondria, Virtual, 2020 == General information == :::: After a long and thorough discussion among the scientific and organizing committees, we have decided to organize our 11th Conference of Targeting Mitochondria, on October 29-30, 2020 as an ONLY Virtual Congress.== Programme ==:::: [https://targeting-mitochondria.com/preliminary-program here]== Speakers == :::: List of speakers can be found [https://targeting-mitochondria.com/speakers here]== Registration ==:::: [https://targeting-mitochondria.com/registration Registration and more information]tration Registration and more information] +
11th ÖGMBT Annual Meeting - Inside the world of biomolecules, Salzburg, Austria, 2019 +
12th Conference of the Asian Society of Mitochondrial Research and Medicine - [http://www.ig.zju.edu.cn/ASMRM/EN/ ASMRM 2015], Hangzhou CN +
12th International Conference on Obesity a … 12th International Conference on Obesity and Eating Disorders, Vienna, Austria, 2023 == General Information == :::: The theme of the conference is "New Emerging Challenges in Obesity and their Prevention"== Venue ==:::: [https://obesity.euroscicon.com/ How to get there]== Program ==:::: Program available [https://obesity.euroscicon.com/program-schedule here]== Organizers ==:::: The list of organizers can be found [https://obesity.euroscicon.com/organizing-committee here]== Registration ==:::: [https://obesity.euroscicon.com/registration Registration and more information]:::: Early registration deadline: 203-01-27:::: Late registration deadline: 2023-04-10::: Late registration deadline: 2023-04-10 +
12th International Phycological Congress - … 12th International Phycological Congress - IPC2021, Puerte Varas, Chile, 2021 == Venue == ::::[https://ipc2021.com/logistic-information/ Venue and how to get there]== Programme ==:::: [https://ipc2021.com/scientific-program/ here]== Speakers == :::: List of speakers can be found [https://ipc2021.com/invited-speakers/ here]== Organizers ==:::: The list of organizers can be found [https://ipc2021.com/local-organizing-committee-scientific-committee/ here]== Registration ==:::: [https://ipc2021.com/registration-and-registration-fees-submission-of-abstracts/ Registration and more information]tracts/ Registration and more information] +
12th ÖGMBT Annual Meeting - Biomolecules in/for 21st century, Virtual Event, 2020 '''''- Conference will be held via a virtual interactive meeting. Oroboros Instruments will be present with a virtual booth.''''' +
13th Life Sciences Meeting, Innsbruck, Aus … 13th Life Sciences Meeting, Innsbruck, Austria, 2018 __TOC__== General information== :::: The coming meeting will take place on the 5th and 6th of April, 2018 in the CCB (Center for Chemistry and Biomedicine) and offers all participants and young researchers the possibility to present their research work in the form of a posters or a short talk. At the end of the event the best presentation will be selected by a professional jury consisting of professors of the Medical University of Innsbruck and the winners will be awarded with a prize. The closure of the meeting will be made by the famous scientist Prof. Jannie Cracking of the Netherland Cancer Institute. The Medical University of Innsbruck is looking forward to welcoming Prof. Cracking as a „Key Note Speaker“. == Venue == :::: Center for Chemistry and Biomedicine (CCB):::: Innrain 80, 6020 Innsbruck:::: [http://biocenter.i-med.ac.at/ Location]== Organizers ==:::: Medical University of Innsbruck==Oroboros presentation ==:::: TALK: Marie Skłodowska-Curie Project '''[[TRANSMIT]]''' [[Bastos Sant'Anna Silva AC|Bastos Sant'Anna Silva Ana Carolina]]: [[Bastos Sant'Anna Silva AC 2018 Life Sciences Meeting 2018 Innsbruck AT|Effect of cell-permeable succinate and malonate prodrugs on mitochondrial respiration in prostate cancer cells]]:::: POSTER: Marie Skłodowska-Curie Project '''[[TRACT]]''' [[Chang Shao-Chiang]]: [[Chang 2018 Life Sciences Meeting 2018 Innsbruck AT|pH dependence of mitochondrial respiration and H2O2 production in oral cancer cells – a pilot study.]]:::: POSTER: K-Regio Project '''[[K-Regio_MitoFit|MitoFit]]''' [[Garcia-Souza LF|Garcia-Souza Luiz]]: [[Garcia-Souza 2018 Life Sciences Meeting 2018 Innsbruck AT|A respirometric cell viability test for peripheral-blood mononuclear cells and platelets]]-Souza 2018 Life Sciences Meeting 2018 Innsbruck AT|A respirometric cell viability test for peripheral-blood mononuclear cells and platelets]] +
14th Congress of the Federation of Asian and Oceanian Biochemists and Molecular Biologists (FAOBMB) - [http://www.ccmb.res.in/faobmb2015/ FAOBMB 2015], Hyderabad IN +
15th Annual Meeting: The Power of Metabolism - Linking energy supply and demand with contractile function, Weimar, +
15th Conference of the Asian Society of Mitochondrial Research and Medicine, Busan, South Korea, 2018. +
16th Chinese Biophysics Congress - Biophysics and human health , Chengdu, China, 2018 +
17th Annual Conference of Janpanese Society of Mitochondrial Research and Medicine, Kyoto, Japan +
17th International Biochemistry of Exercise Conference, Beijing, China, 2018 +
19th Annual Scientific Meeting of KSMRM , Seoul, Republic of Korea; [http://2014.ksmrm.org/congress/invitation.php KSMRM2014] +
19th Annual Meeting of the Society for Heart and Vascular Metabolism (SHVM), Seoul , South Korea, 2022 +
19th Beijing Conference and Exhibition on Instrumental Analysis, Beijing, China, 2021 +
19th Congress of the European Society for … 19th Congress of the European Society for Photobiology - ESP2021, Salzburg, Austria, 2021 == Venue == :::: Faculty of Natural Sciences (NAWI) of the Paris Lodron University Salzburg (PLUS):::: Venue address: Hellbrunnerstrasse 34, 5020 Salzburg, Austria.:::: [http://salzburg2021.photobiology.eu/congress-venue more information]== Program ==:::: [http://salzburg2021.photobiology.eu/ here]== Speakers == :::: List of speakers can be found [http://salzburg2021.photobiology.eu/ here]== Organizers ==:::: The list of organizers can be found [http://salzburg2021.photobiology.eu/organizing-committee here]== Registration ==:::: [http://salzburg2021.photobiology.eu/ Registration and more information]ogy.eu/ Registration and more information] +
19th International Conference on the Cell and Molecular Biology of Chlamydomonas, Ile des Embiez, France, 2021 +
1st 1st FEBS Workshop “Redox Medicine Workshop, Luso, Portugal, 2023 +
1st MiP''summer school'' on Mitochondrial Respiratory Physiology, 2007 July 12-18, Schroecken, AT. +
1st Myocardial Function Symposium: “Targets in cardiometabolic disease”, Graz, Austria, 2020 +
1st virtual meeting of the Society for Heart and Vascular Metabolism (SHVM), Virtual, 2021 +
1α,25-Dihydroxyvitamin D3</s … 1α,25-Dihydroxyvitamin D3 (1,25-D3) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D3 on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D3 stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active ''de novo'' protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D3 did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D3 stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.mportant contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis. +
2',7'-Dichlorofluorescein and dihydrorhoda … 2',7'-Dichlorofluorescein and dihydrorhodamine 123 were evaluated as probes for detecting changes in intracellular H2O2 in cultured endothelial cells. Stable intracellular levels of these probes were established within 15 min of exposure to the probe in culture medium. With continued presence of the probe in the medium, intracellular levels were unchanged for 1 h. However, if medium without the probes was used after intracellular loading had occurred, there was a greater than 90% loss of intracellular dichlorofluorescin, dichlorofluorescein, and dihydrorhodamine 123 while intracellular rhodamine 123 decreased by only 15%. Exposure of endothelial cells to exogenous 100 microM H2O2 for 1 h increased intracellular rhodamine 123 by 83%, but there was a reproducible decrease of 53% in intracellular dichlorofluorescein. Exposure to 0.05 mM BCNU plus 10 mM aminotriazole for 2 h increased intracellular rhodamine 123 by 111%. In vitro studies of dihydrorhodamine 123 oxidation were similar to previous reports of dichlorofluorescin oxidation. Oxidation of dihydrorhodamine 123 does not occur with H2O2 alone, but is mediated by a variety of secondary H2O2-dependent intracellular reactions including H2O2-cytochrome c and H2O2-Fe2+. Our results suggest that detection of increased oxidation of these probes in endothelial cells is most useful as a marker of a change in general cellular oxidant production.ge in general cellular oxidant production. +
2,4-Dinitrophenol greatly enhanced the … 2,4-Dinitrophenol greatly enhanced the liberation of inorganic phosphate from ATP by the nuclear and mitochondrial fraction of rat liver. The microsomal and supernatant fractions did not exhibit this effect. With mitochondria (Mw) the rate of phosphate liberation was proportional to the DNP concentration up to 6 X 10-5 M In the presence of excess DNP the rate was proportional to the quantity of Mw nd to time. With both fresh and preaged Mw, the response to DNP was much greater in mediums containing salt (either NaCl or KCl) than in isotonic sucrose. Magnesium salts in appreciable concentrations depressed the response of fresh Mw to DNP, but enhanced the response in preaged Mw. Calcium salts, which activate ATP hydrolysis by fresh Mw in the absence of DNP, also depressed the effect of DNP on phosphate liberation. Magnesium salts enhanced phosphate liberation by preaged Mw both in the presence and absence of DNP. Calcium was virtually without effect in preaged Mw. Oxalacetate enhanced phosphate liberation from ATP by fresh Mw. This dicarboxylic acid as well as succinate and L-malate depressed the effect of DNP on phosphate liberation. Fatty acids also depressed the effect of DNP. Caprylate enhanced phosphate liberation, probably be- cause of its surface activity. The thiol inhibitor, p-chloromercuribenzoate, strongly depressed the effect of DNP; iodoacetate and o-iodosobenzoate did not.''Continued in Free Text''ate did not. ''Continued in Free Text'' +
2,4-Dinitrophenol (DNP) is a neuroprotecti … 2,4-Dinitrophenol (DNP) is a neuroprotective compound previously shown to promote neuronal differentiation in a neuroblastoma cell line and neurite outgrowth in primary neurons. Here, we tested the hypothesis that DNP could induce neurogenesis in embryonic stem cells (ESCs). Murine ESCs, grown as embryoid bodies (EBs), were exposed to 20μM DNP (or vehicle) for 4days. Significant increases in the proportion of nestin- and β-tubulin III-positive cells were detected after EB exposure to DNP, accompanied by enhanced glial fibrillary acidic protein (GFAP), phosphorylated extracellular signal-regulated kinase (p-ERK) and ATP-linked oxygen consumption, thought to mediate DNP-induced neural differentiation. DNP further protected ESCs from cell death, as indicated by reduced caspase-3 positive cells, and increased proliferation. Cell migration from EBs was significantly higher in DNP-treated EBs, and migrating cells were positive for nestin, ß-tubulin III and MAP2, similar to that observed with retinoic acid (RA)-treated EBs. Compared to RA, however, DNP exerted a marked neuritogenic effect on differentiating ESCs, increasing the average length and number of neurites per cell. Results establish that DNP induces neural differentiation of ESCs, accompanied by cell proliferation, migration and neuritogenesis, suggesting that DNP may be a novel tool to induce neurogenesis in embryonic stem cells.duce neurogenesis in embryonic stem cells. +
2,4-Dinitrophenol (DNP) is classically kno … 2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupler and, at high concentrations, is toxic to a variety of cells. However, it has recently been shown that, at subtoxic concentrations, DNP protects neurons against a variety of insults and promotes neuronal differentiation and neuritogenesis. The molecular and cellular mechanisms underlying the beneficial neuroactive properties of DNP are still largely unknown. We have now used DNA microarray analysis to investigate changes in gene expression in rat hippocampal neurons in culture treated with low micromolar concentrations of DNP. Under conditions that did not affect neuronal viability, high-energy phosphate levels or mitochondrial oxygen consumption, DNP induced up-regulation of 275 genes and down-regulation of 231 genes. Significantly, several up-regulated genes were linked to intracellular cAMP signaling, known to be involved in neurite outgrowth, synaptic plasticity, and neuronal survival. Differential expression of specific genes was validated by quantitative RT-PCR using independent samples. Results shed light on molecular mechanisms underlying neuroprotection by DNP and point to possible targets for development of novel therapeutics for neurodegenerative disorders.rapeutics for neurodegenerative disorders. +
2014 Mitochondrial Disease Clinical Conference, Los Angeles, Ca US; [http://www.mitoaction.org/laconference 2014 Mitochondrial Disease Clinical Conference] +
2015 Spring PaduaMuscleDays: Translational Myology in Aging and Neuromuscular Disorders, Padova, IT; [http://www.pagepressjournals.org/index.php/bam/announcement/view/176 2015 Spring PaduaMuscleDays]. +
2016 Spring PaduaMuscleDays: Muscle Decline in Aging and Neuromuscular Disorders - Mechanisms and Countermeasures, Padua, IT +
2017 Annual Meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA), Kyoto, Japan +
2018 world conference on Movement and Cognition, Boston, Massachusetts, USA, 2018 +
2020 PaduaMuscleDays - 30 years of translational research, Vitual Event, 2020 +
20th European Bioenergetics Conference 2018, Budapest, Hungary, 2018 +
20th Annual Meeting of the Society for Heart and Vascular Metabolism (SHVM), Graz, Austria, 2023 +
21st European Bioenergetics Conference 2022, Aix-en-Provence, France, 2022. +
22st European Bioenergetics Conference 2024, Innsbruck, Austria, 2024 +
26th Meeting for the Society for Free Radical Research Australasia SFRR(A), Auckland, New Zeland, 2018 +
2nd FEBS Workshop on Ageing and Regeneration, Mutters, Austria, 2022 +
2nd Annual Conference of the Prevention and Control of Cardiovascular Metabolic Disease, Wuhan, CN; post-conference workshop '''[[MiPNet20.11_IOC102_Wuhan | 102nd Oroboros O2k-Workshop]]'''. +
3-BrPA (3-bromopyruvate) is an alkylating … 3-BrPA (3-bromopyruvate) is an alkylating agent with antitumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellularcarcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 μMfor 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 μM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium.Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were preincubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinatewas used as the oxidizable substrate. An increase in oligomycinindependentrespiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activityof succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3- BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.lay an important role in HepG2 cell death. +
3-Bromopyruvate (3BrPA) is an antitumor ag … 3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca2+-uptake activity was significantly inhibited by 80% with 150μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.hiolation mediated by GSH at a critical Cys residues of the SERCA. +
3-bromopyruvate (3BrPA) is an antitumor ag … 3-bromopyruvate (3BrPA) is an antitumor agent able to inhibit aerobic glycolysis and oxidative phosphorylation, therefore inducing cell death. However, cancer cells are also highly dependent of glutaminolysis and tricarboxylic acid cycle (TCA) regarding survival and 3BrPA action in these metabolic routes is poorly understood.The effect of 3BrPA was characterized in mice liver and kidney mitochondria, as well as in human HepG2 cells.Low concentration of 3-BrPA significantly affected both glutaminolysis and TCA cycle functions, through inhibition of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinate dehydrogenase. Additionally, 3-BrPA treatment significantly decreased the reduced status of thiol groups in HepG2 cells without proportional increase of oxidizing groups, suggesting that these chemical groups are the target of alkylation reactions induced by 3-BrPA.This work demonstrates, for the first time, the effect of 3-BrPA in glutaminolysis and TCA cycle. Our results suggest that the combined action of 3-BrPA in glutaminolysis, TCA and glycolysis, inhibiting steps downstream of the glucose and glutamine metabolism, has an antitumor effect.Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.John G. Delinassios), All rights reserved. +
3-hydroxy-3-methylglutaryl-coenzyme A (HMG … 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used drugs for lowering blood lipid levels and preventing cardiovascular diseases. However, statins can have serious adverse effects, which may be related to development of mitochondrial dysfunctions. The aim of study was to demonstrate the ''in vivo'' effect of high and therapeutic doses of statins on mitochondrial respiration in blood platelets. Model approach was used model in the study. Simvastatin was administered to rats at a high dose for 4 weeks. Humans were treated with therapeutic doses of rosuvastatin or atorvastatin for 6 weeks. Platelet mitochondrial respiration was measured using high-resolution respirometry. In rats, a significantly lower physiological respiratory rate was found in intact platelets of simvastatin-treated rats compared to controls. In humans, no significant changes in mitochondrial respiration were detected in intact platelets; however, decreased complex I-linked respiration was observed after statin treatment in permeabilized platelets. We propose that the small ''in vivo'' effect of statins on platelet energy metabolism can be attributed to drug effects on complex I of the electron transport system. Both intact and permeabilized platelets can be used as a readily available biological model to study changes in cellular energy metabolism in patients treated with statins.tabolism in patients treated with statins. +
32th Joint Annual Conference of Biomedical Science, Taipei, Taiwan. +
36th annual international congress of Czech Nutrition Society, Hradec Kralove, Czech Republic, 2020 +
3rd MiP''summer school'' on Mitochondrial Respiratory Physiology, 2009 June 17-23, Baton Rouge, Louisiana US. +
3rd Russian Congress with International Participation “Molecular Basis of Clinical Medicine: State-of-the-Art and Perspectives” dedicated to the memory of Eugeny I. Schwartz, St. Petersburg , Russia; +
3rd Edition of International Conference on Eye and Vision, Rome, Italy; 2018 +
4-hydroxy-2-oxoglutarate aldolase (HOGA) i … 4-hydroxy-2-oxoglutarate aldolase (HOGA) is a bi-functional mitochondrial enzyme, expressed predominantly in liver and kidney. HOGA is involved in the hydroxyproline degradation pathway (HOGglyoxylate+pyruvate), and mutations in HOGA result in primary Hyperoxaluria Type III, characterized by excessive oxalate production and kidney stone deposition [1]. We hypothesized that HOGA may also be involved in the TCA cycle as an oxaloacetate decarboxylase (oxaloacetatepyruvate; Fig. 1), which may allow the TCA cycle to turnover in the absence of pyruvate and/or excess oxaloacetate. The kinetics of HOGA with substrates HOG and oxaloacetate were investigated by measuring the ''K''’m and ''k''cat of recombinant human HOGA, using an LDH-coupled microplate assay. The role of HOGA in the TCA cycle was investigated using mitochondria, isolated from rat liver and kidney, where HOGA is highly expressed, and brain and heart, where expression is lower. ADP-stimulated malate respiration was measured relative to ADP-malate + pyruvate (M:PM), using oxygraphy (Oroboros Oxygraph-2k, note malate was used as oxaloacetate cannot cross the inner mitochondrial membrane). While HOGA was 75% less efficient at cleaving oxaloacetate than its other substrate, HOG (''K''’m/''k''cat), the ''K''’m for oxaloacetate was within range of that estimated for TCA intermediates (''K''’m,ox=129±8 µM, ''k''cat,ox=0.52±0.01 s-1; ''K''’m,HOG=55±5 µM, ''k''cat,HOG=1.01±0.03 s-1). Overall, HOGA appears to use the same catalytic mechanism to cleave both HOG and oxaloacetate substrates. Interestingly, the TCA cycle intermediate a-ketoglutarate was found to be a competitive inhibitor of HOGA oxaloacetate decarboxylase activity (''K''i=2.8 mM). Mitochondria from rat liver had the highest M:PM respiration relative to all other organs (0.46±0.05, ''P''<0.05). Though kidney had a higher M:PM respiration than heart (0.27±0.02 vs 0.15±0.02, ''P''<0.05 in kidney and heart, respectively), brain respired as well as kidney (0.33±0.04). In summary, HOGA cleaves oxaloacetate and HOG using the same catalytic mechanism but was less efficient with oxaloacetate. Liver and kidney have high HOGA expression, and mitochondria from both respire significantly better on malate relative to PM than heart mitochondria. The brain respires just as well with malate compared to kidney, and this may be due to high expression of malic enzyme, which can convert malate directly to pyruvate (Fig. 1). Malate supported respiration in HOGA overexpressing cells will confirm the direct role of HOGA in the TCA cycle.ession of malic enzyme, which can convert malate directly to pyruvate (Fig. 1). Malate supported respiration in HOGA overexpressing cells will confirm the direct role of HOGA in the TCA cycle. +
41st Annual Meeting of the Molecular Biology Society of Japan, Yokohama, Japan, 2018. +
42nd Annual Meeting of The Molecular Biology Society of Japan, Kurume, 2018 +
43rd Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT) +
46th All India Cell Biology Conference, Navi Mumbai, India, 2024 +
46th annual congres of the International Society of Oncology and Biomarkers, Athens, Greece, 2019 +
49th Annual Scientific Meeting of the European Society for Clinical Investigation, Cluj-Napoca, Romania; [http://www.esci.eu.com/meetings/ ESCI 2015] +
4th Annual Conference of the Society for Mitochondrial Research and Medicine, Kolkata, India. +
4th MiP''summer school'' on Mitochondrial Respiratory Physiology, 2010 June 10-16, Druskininkai, Lithuania. +
4th Regional Translational Research in Mitochondria, Aging, and Disease Symposium, Pittsburgh, PA, US. [http://www.upci.upmc.edu/trmad/ TrMAD2014] +
4th Global Chinese Symposium & The 8th Symposium for Cross-straits, Hong Kong and Macao on Free Radical Biology and Medicine, Macao, China, 2018 +
4th edition Metabolism & Cancer, Virtu … 4th edition Metabolism & Cancer, Virtual, 2021 == Program ==:::: [https://www.metabolism-cancer.com/program/ here]== Organizers ==:::: The list of organizers can be found [https://www.metabolism-cancer.com/under-construction/ here]== Registration ==:::: [https://www.metabolism-cancer.com/registration/ Registration and more information]== Oroboros at MetaboCancer 2021==:::: [[Gnaiger Erich]]: Oroboros Instruments innovations - NextGen-O2k and Bioenergetics Communications, ''May 28th at 11:25''=== Booth ===:::: The Oroboros team is looking forward to welcome you at our Oroboros booth which will be available at this conference.== Support ==[[File:Template NextGen-O2k.jpg|right|350px|link=NextGen-O2k]][[Category:NextGen-O2k]]:::: Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770. agreement No. 859770. +
5'-AMP-activated protein kinase (AMPK) is … 5'-AMP-activated protein kinase (AMPK) is activated as a consequence of lipolysis and has been shown to play a role in regulation of adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A (PKA)-targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, AMPK, and the β-adrenergic-mediated regulation of gene expression. In 3T3-L1 adipocytes, the adipose tissue triglyceride lipase (ATGL) inhibitor ATGListatin attenuated the Thr(172) phosphorylation of AMPK by a β3-adrenergic agonist (CL 316,243) independent of changes in PKA signaling. Similarly, CL 316,243-induced increases in the Thr(172) phosphorylation of AMPK were reduced in adipose tissue from whole body ATGL-deficient mice. Despite reductions in the activation of AMPK, the induction of PKA-targeted genes was intact or, in some cases, increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr(172) phosphorylation of AMPK. Taken together, our data provide evidence that AMPK is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release.Copyright © 2016 the American Physiological Society.© 2016 the American Physiological Society. +
5'-adenosine monophosphate-activated prote … 5'-adenosine monophosphate-activated protein kinase (AMPK) is considered central in regulation of energy status and substrate utilization within cells. In heart failure the energetic state is compromised and substrate metabolism is altered. We hypothesized that this could be linked to changes in AMPK activity and we therefore investigated mitochondrial oxidative phosphorylation capacity from the oxidation of long- and medium-chain fatty acids (LCFA and MCFA) in cardiomyocytes from young and old mice expressing a dominant negative AMPKα2 (AMPKα2-KD) construct and their wildtype (WT) littermates. We found a 35-45% (P < 0.05) lower mitochondrial capacity for oxidizing MCFA in AMPKα2-KD of both age-groups, compared to WT. This coincided with marked decreases in protein expression (19/29%, P < 0.05) and activity (14/21%, P < 0.05) of 3-hydroxyacyl-CoA-dehydrogenase (HAD), in young and old AMPKα2-KD mice, respectively, compared to WT. Maximal LCFA oxidation capacity was similar in AMPKα2-KD and WT mice independently of age implying that LCFA-transport into the mitochondria was unaffected by loss of AMPK activity or progressing age. Expression of regulatory proteins of glycolysis and glycogen breakdown showed equivocal effects of age and genotype. These results illustrate that AMPK is necessary for normal mitochondrial function in the heart and that decreased AMPK activity may lead to an altered energetic state as a consequence of reduced capacity to oxidize MCFA. We did not identify any clear aging effects on mitochondrial function. any clear aging effects on mitochondrial function. +
5-Hydroxydecanoate (5-HD) blocks pharmacol … 5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of β-oxidation. We have now analysed the complete β-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of β-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. ''V''max for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (l-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of d-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 μm 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via β-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for β-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial KATP channels in studies of preconditioning.TP channels in studies of preconditioning. +
50 years ago Peter Mitchell proposed the c … 50 years ago Peter Mitchell proposed the chemiosmotic hypothesis for which he was awarded the Nobel Prize for Chemistry in 1978. His comprehensive review on chemiosmotic coupling known as the first “Grey Book”, has been reprinted here with permission, to offer an electronic record and easy access to this important contribution to the biochemical literature. This remarkable account of Peter Mitchell's ideas originally published in 1966 is a landmark and must-read publication for any scientist in the field of bioenergetics. As far as was possible, the wording and format of the original publication have been retained. Some changes were required for consistency with BBA formats though these do not affect scientific meaning. A scanned version of the original publication is also provided as a downloadable file in Supplementary Information. See also Editorial in this issue by Peter R. Rich. Original title: CHEMIOSMOTIC COUPLING IN OXIDATIVE AND PHOTOSYNTHETIC PHOSPHORYLATION, by Peter Mitchell, Glynn Research Laboratories, Bodmin, Cornwall, England.h Laboratories, Bodmin, Cornwall, England. +
5th Targeting Mitochondria World Congress - [http://www.targeting-mitochondria.com/ Targeting Mitochondria], Berlin DE +
5th Academic Symposium of Metabolic Biology Branch of Chinese Biophysical Society, Zunyi, China, 2022 +
5th International Mitochondrial Medicine Conference Mitochondrial, Online, 2021 +
5th NHLBI Mitochondrial Biology Symposium, … 5th NHLBI Mitochondrial Biology Symposium, Bethesda, Maryland, USA, 2019 == General information == ::::On September 26-27, 2019, experts from around the world will gather on the NIH Campus in Bethesda, Maryland to review advances in our understanding of how mitochondrial structure, function, and interactions within the cell contribute to diseases and aging; and to highlight recent progress made with animal models and therapeutic interventions.== Venue == :::: William H. Natcher Conference Center – Building 45:::: National Institutes of Health:::: 45 Center Drive:::: Bethesda, MD 20814:::: [https://2019mbs.com/meeting-venue/ How to get there]== Organizer ==:::: [https://2019mbs.com/organizers/ Information available here]== Programme ==:::: [https://2019mbs.com/agenda/ Agenda]== Speakers == :::: List of speakers can be found [https://2019mbs.com/featured-speakers/ here]== Registration ==:::: [https://www.eventbrite.com/e/the-2019-nhlbi-mitochondrial-biology-symposium-registration-54765893261 Registration and more information]:::: The abstracts submission deadline is Friday, June 28, 2019, at 11:59PM EST. :::: All submissions must be made through the abstract submission portal. :::: Abstracts should be no longer than 500 words and include four clearly identifiable components: Background, Methods, Results, and Conclusion. :::: Abstracts will be reviewed by the Organizing Committee. Acceptance will be based on the quality of the abstract and availability of space. Four high-quality abstracts will be selected for oral presentation.ts will be selected for oral presentation. +
5th edition Metabolism & Cancer, Nice, … 5th edition Metabolism & Cancer, Nice, France, 2023 == Venue ==:::: [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 How to get there]== Program ==:::: Program available [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 here]== Organizers ==:::: The list of organizers can be found [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 here]== Registration ==:::: [https://www.metabolism-cancer.com/?utm_source=altemail&utm_medium=email&utm_campaign=2023-01-04%20METABO%202023%201 Registration and more information]utm_campaign=2023-01-04%20METABO%202023%201 Registration and more information] +
63rd Annual Meeting of the Biophysical Soc … 63rd Annual Meeting of the Biophysical Society, Baltimore, Maryland USA, 2019 == General information==:::: The Biophysical Society meeting is the only major scientific meeting in the United States that routinely includes bioenergetics and mitochondrial topics. The Bioenergetics, Mitochondria, and Metabolism Subgroup has its two symposia on the first day of the meeting, March 2nd, and these two symposia have a distinguished group of speakers who are leaders in the field of bioenergetics. == Venue == :::: Baltimore Convention Center:::: 1 W. Pratt Street:::: Baltimore, Maryland 21201::::[https://www.biophysics.org/2019meeting/hotel-travel Hotel and Travel]== Programme ==:::: [https://www.biophysics.org/2019meeting/program here]== Registration ==:::: [https://www.biophysics.org/2019meeting/registration Registration and more information]tration Registration and more information] +
64th Annual International Conference of the Associate of Microbiologists of India, Jhansi, India, 2023 +
67th Annual Meeting of the Biophysical Society, San Diego, California, USA, 2023 +
6th 45th Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT), Halle/Saale, Germany. +
6th Annual Conference of Chinese Society for Neurobiological Control of Metabolism, Quanzhou, China, 2024 +
6th Annual Conference of the Society for Mitochondrial Research and Medicine, New Delhi, India. +
6th MiP''summer school'' on Mitochondrial Physiology, 2013 August 26-30, Copenhagen, Denmark. +
6th Biannual Meeting on Mitochondria Apoptosis & Cancer, Prague, Czech Republic, 2019 +
6th EU-Cardioprotection WG Meeting CA16625 on mito and metabolism as targets for cardioprotection., Virtual Event, 2021 +
6th International Conference on Tumor Microenvironment and Cellular Stress: Signaling, Metabolism, Imaging and Therapeutic Targets, Chania, Crete, Greece, 2019 +
77th Annual Meeting of the Japanese Cancer Association at the Osaka International Convention Center and RIHGA, Osaka, Japan, 2018 +
77th Japanese Society of Physical Fitness and Sports Medicine, Tochigi, 2022 +
7th 46th Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT). Seoul, South Korea, 2018 +
7th Conference of the International Society for Applied Phycology - ISAP2021, Tsukuba, Japan, 2021 +
7th Molecular Mechanisms of Axon Degeneration Meeting, Loch Lomond, Scotland, Great Britain, 2019 +
7th World Congress on Targeting Microbiota … 7th World Congress on Targeting Microbiota, Krakow, Poland, 2019 == Venue == :::: Park Inn by Radisson Krakow Hotel:::: Ul. Monte Cassino 2 PL:::: 30337 - Krakow - Poland:::: [https://www.microbiota-site.com/venue.html More information]== Organizer ==:::: [https://www.microbiota-site.com/committee.html Information available here]== Programme ==:::: [https://www.microbiota-site.com/images/2019/PDF/Targeting_Microbiota_2019_Agenda_-_V7.pdf Agenda]== Speakers == :::: List of speakers can be found [https://www.microbiota-site.com/microbiota-2019-speakers.html here]== Registration ==:::: [https://www.microbiota-site.com/registrations.html Registration and more information]ns.html Registration and more information] +
8th MiP''school'' on Mitochondrial Physiology, 2015 Apr 20-24, London, UK. +
8th Annual Meeting of the Society for Mitochondria Research and Medicine-India , Virtual. +
8th SMRM and Mitochondria-Metabolism Netwo … 8th SMRM and Mitochondria-Metabolism Network Meeting, Pune, India, 2020 == General information == :::: Flyer available for [https://www.mitoeagle.org/images/b/b2/8th_SMRM_and_Mitochondria-Metabolism_Network_Meeting_Poster.pdf download]== Venue == :::: Indian Institute of Science Education and Research (ISER Pune):::: Dr. Homi Bhabha Road:::: Pashan, Pune 411 008:::: INDIA::::[http://www.iiserpune.ac.in/facilities/guesthouse-cum-convention-centre Hotel and Travel]== Programme ==:::: [https://indico.tifr.res.in/indico/internalPage.py?pageId=12&confId=7288 here]== Speakers == :::: List of speakers can be found [https://indico.tifr.res.in/indico/internalPage.py?pageId=0&confId=7288 here]== Organizers ==:::: The list of organizers can be found [https://indico.tifr.res.in/indico/internalPage.py?pageId=9&confId=7288 here]== Registration ==:::: [https://indico.tifr.res.in/indico/internalPage.py?pageId=6&confId=7288 Registration and more information]ageId=6&confId=7288 Registration and more information] +
8th Translational Research in Mitochondria … 8th Translational Research in Mitochondria, Aging, and Disease (TRiMAD) Symposium, Pennsylvania, United States, 2018 == General information ==:::: TRiMAD is a collaborative venture between The Pennsylvania State University, University of Pittsburgh Medical Center, The Children’s Hospital of Philadelphia (CHoP) Research Institute, and The University of Pennsylvania Perelman School of Medicine ([https://www.huck.psu.edu/node/15830 Website])== Venue == :::: The Pennsylvania State University:::: 100 Huck Life Sciences Building:::: University Park, Pennsylvania 16802:::: [http://www.cvent.com/events/8th-regional-translational-research-in-mitochondria-aging-and-disease-symposium/directions-16730cf0fe2c47a1b79f1a3b9ab0b364.aspx directions]== Organizers ==:::: Kateryna Makova, PhD - Penn State, University Park:::: Donna Korzick, PhD - Penn State, University Park[[File:Image001.jpg|right|550px]]== Programme ==:::: Please find the programme [http://www.cvent.com/events/8th-regional-translational-research-in-mitochondria-aging-and-disease-symposium/agenda-16730cf0fe2c47a1b79f1a3b9ab0b364.aspx here]== Registration ==:::: [https://www.cvent.com/events/8th-regional-translational-research-in-mitochondria-aging-and-disease-symposium/registration-16730cf0fe2c47a1b79f1a3b9ab0b364.aspx?fqp=true Register here] == Lecturers and tutors ==:::: The list of speakers can be found [http://www.cvent.com/events/8th-regional-translational-research-in-mitochondria-aging-and-disease-symposium/custom-18-16730cf0fe2c47a1b79f1a3b9ab0b364.aspx here]6730cf0fe2c47a1b79f1a3b9ab0b364.aspx here] +
92nd Annual Meet of The Society of Biological Chemists, Goa, India, 2023 +
95th Annual Meeting of the DPG, [http://www.dpg2016.de/ DPG 2016], Luebeck, DE +
9th Conference of t … 9th Conference of the Asian Society of Mitochondrial Research and Medicine and 5th Conference of Chinese Society of Mitochondrial Research and Medicine (Chinese-Mit), [http://asmrm2012.csp.escience.cn/dct/page/65540 ASMRM 2012], Bejing CN://asmrm2012.csp.escience.cn/dct/page/65540 ASMRM 2012], Bejing CN +
9th International Y-chromosome workshop & 6th International EMPOP meeting, Brussels, Belgium; DNA Forensics 2014 +
9th MiP''school'' f … 9th MiP''school'' for cellular bioenergetics and mitochondrial physiology students, 2015 Aug 10-14, Greenville, US.» [http://www.ecu.edu/cs-admin/news/mip.cfm '''Global conference highlights mitochondria expertise at ECU'''], by Kathryn Kennedy ECU News Services.tise at ECU'''], by Kathryn Kennedy ECU News Services. +
::: Version 2 ('''v2''') '' … ::: Version 2 ('''v2''') '''2019-06-27''' [https://www.mitofit.org/images/6/68/Crispim_2019_MitoFit_Preprint_Arch_doi_10.26124mitofitea19.MiPSchool.0007.v2.pdf doi:10.26124/mitofit:ea19.MiPSchool.0007.v2]; v1 2019-06-17 [https://wiki.oroboros.at/images/8/81/Crispim_2019_MitoFit_Preprint_Arch_doi_10.26124mitofitea19.MiPSchool.0007.pdf doi:10.26124/mitofit:ea19.MiPSchool.0007]::: Version 1 (v1) [https://wiki.oroboros.at/images/8/81/Crispim_2019_MitoFit_Preprint_Arch_doi_10.26124mitofitea19.MiPSchool.0007.pdf doi:10.26124/mitofit:ea19.MiPSchool.0007]== Although atovaquone is one of the newest antimalarial compounds discovered, resistant parasites have already been reported1. Atovaquone mechanism of action is established to be the competition with ubiquinol (UQH2) for the bc1 union at mitochondrial cytochrome bc1 complex and preventing the parasite from maintaining an oxidized ubiquinone (UQ) pool, essential for the DHODH activity and consequently for the pyrimidine's biosynthesis. In this sense, possible inhibitors of the ubiquinone biosynthesis pathway would be candidates by stimulating the effects of atovaquone. 4-nitrobenzoate (4-NB) is a well-known inhibitor of 4HPT (4-hydroxybenzoate polyprenyltransferase), the first enzyme of UQ biosynthesis. 4-NB also showed an important effect on reducing the UQs pool in P. falciparum. Herein is presenting the effect of atovaquone and 4-NB on parasitic respiration UQ biosynthesis. The purpose of this study was to better understand the atovaquone mechanism of action in a molecular scale, drug target potential of UQ biosynthesis. Oxygen consumption assays revealed 4-NB potentiates atovaquone mitochondrial effects and showed itself the ability to decrease the respiration rate. ==- ''Extended abstract''crease the respiration rate. == - ''Extended abstract'' +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2019-04-24''' [http://www.mitofit.org/images/d/d3/Gnaiger_2019_MitoFit_Preprint_Arch_doi_10.26124_mitofit_190002.v2.pdf doi:10.26124/mitofit:190002.v2]::: Version 1 (v1) 2019-04-01 [http://www.mitofit.org/images/archive/d/d3/20190424180311%21Gnaiger_2019_MitoFit_Preprint_Arch_doi_10.26124_mitofit_190002.v2.pdf doi:10.26124/mitofit:190002] - [http://www.mitofit.org/index.php/File:Gnaiger_2019_MitoFit_Preprint_Arch_doi_10.26124_mitofit_190002.v2.pdf#Links_to_all_versions »Link to all versions«]A manuscript in preparation for publication on ‘Mitochondrial states and rates’ is the first preprint posted on ''[[MitoFit Preprints]]'' (Gnaiger ''et al'' 2019). It actually triggered the initiation of a preprint server for mitochondrial physiology and bioenergetics. This editorial presents the story behind starting ''MitoFit Preprints'', to develop a vision of science communication beyond traditional journal and preprint publication. This is an open invitation to scientists of mitochondrial physiology and bioenergetics to join the preprint community by submitting manuscripts as preprints. We face the ''reproducibility crisis'' in the battle to separate doubtful data from relevant information. This is linked to the ''inflation crisis'' emanating from an exponential increase of scientific articles published per day. Unsustainable exponential growth leads to the ''value-impact crisis'' in the struggle to forge scientific innovation into knowledge and community benefits. forge scientific innovation into knowledge and community benefits. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2021-09-06''' [https://www.mitofit.org/images/4/4a/Gnaiger_MitoFit_Preprint_Arch_2020.4_doi_10.26214mitofit.200004.pdf doi:10.26124/mitofit:200004.v2]::: Version 1 ('''v1''') 2020-08-11 [https://wiki.oroboros.at/images/archive/4/4a/20210906072525%21Gnaiger_MitoFit_Preprint_Arch_2020.4_doi_10.26214mitofit.200004.pdf doi:10.26124/mitofit:200004] — [https://www.mitofit.org/index.php/File:Gnaiger_MitoFit_Preprint_Arch_2020.4_doi_10.26214mitofit.200004.pdf »Link to all versions«]“The International System of Units, the SI, has been used around the world as the preferred system of units, the basic language for science, technology, industry and trade since it was established in 1960.” This statement heralds the 9th edition of the SI released on 2019-May-20. An new approach was introduced by defining the SI base units ― and thus the abstract SI units in general ― by their relation to fixed numerical values of fundamental constants of nature. Previous definitions of abstract units relied on a reference to concrete individual things realized as material artefacts, such as the International Prototype of the Kilogram (IPK). The (general) abstract unit ‘kilogram’ had to be calibrated in balance against an (individual) ‘entetic’ unit defining “1 kg” as a reference for the unit of mass and the mole [mol] as the unit of amount. Now the SI defines the mole as the fixed number of entities given by the Avogadro constant ''N''A. The elementary charge ''e'' is a fixed number of charges per proton. Amount and charge are thus in a fixed relation to the count of elementary entities ''U''''X'' [x]. Count, amount, and charge are isomorphic elementary quantities. Amount and charge are linked to the count ''N''''X'' = ''N''∙''U''''X'' with elementary unit x by fixed conversion constants ''N''A-1 [mol∙x−1] and ''e'' [C∙x−1], respectively. The SI does not use the elementary unit x. This causes a number of formal inconsistencies as discussed in the present communication on Euclid’s unit, which is ''U''''X'', and Euclid’s number, which is a count ''N''''X''.sistencies as discussed in the present communication on Euclid’s unit, which is ''U''''X'', and Euclid’s number, which is a count ''N''''X''. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2022-05-09''' [https://wiki.oroboros.at/images/c/c8/Baglivo_2022_MitoFit-QC.pdf doi:10.26124/mitofit:2022-0018.v2]::: Version 1 (v1) 2022-05-05 [https://wiki.oroboros.at/images/archive/c/c8/20220506062726%21Baglivo_2022_MitoFit-QC.pdf doi:10.26124/mitofit:2022-0018.v1] - [https://wiki.oroboros.at/index.php/File:Baglivo_2022_MitoFit-QC.pdf »Link to all versions«][[File:Baglivo 2022 MitoFit QC graphical-abstract.png|right|300px|Graphical abstract]][[Baglivo 2022 Abstract Bioblast]]: Evaluation of instrumental reproducibility is a primary component of quality control to quantify the precision and limit of detection of analytical procedures. A pre-analytical instrumental standard operating procedure (SOP) is implemented in high-resolution respirometry consisting of: (''1'') a daily SOP-POS for air calibration of the polarographic oxygen sensor (POS) in terms of oxygen concentration ''c''O2 [µM]. This is part of the ''sensor test'' to evaluate POS performance; (''2'') a monthly SOP-BG starting with the SOP-POS followed by the ''chamber test'' quantifying the instrumental O2 background. The chamber test focuses on the slope d''c''O2/d''t'' [pmol∙s−1∙mL−1] to determine O2 consumption by the POS and O2 backdiffusion into the chamber as a function of ''c''O2 in the absence of sample. Finally, zero O2 calibration completes the sensor test. We applied this SOP in a 3-year study using 48 Oroboros O2k chambers. Stability of air and zero O2 calibration signals was monitored throughout intervals of up to 8 months without sensor service. Maximum drift over 1 to 3 days was 0.06 pmol∙s−1∙mL−1, without persistence over time since drift was <0.004 pmol∙s−1∙mL−1 for time intervals of one month, corresponding to a drift per day of 0.2 % of the signal at air saturation. Instrumental O2 background -d''c''O2/d''t'' was stable within ±1 pmol∙s−1∙mL−1 when measured at monthly intervals. These results confirm the instrumental limit of detection of volume-specific O2 flux at ±1 pmol∙s−1∙mL−1. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry. These results confirm the instrumental limit of detection of volume-specific O2 flux at ±1 pmol∙s−1∙mL−1. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2022-07-07''' [https://wiki.oroboros.at/images/5/54/Alencar_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0009.v2]::: Version 1 (v1) 2022-04-07 [https://wiki.oroboros.at/images/archive/5/54/20220707123437%21Alencar_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0009.v1] - [https://wiki.oroboros.at/index.php/File:Alencar_2022_MitoFit.pdf »Link to all versions«][[Oliveira 2022 Abstract Bioblast]]: The parasite ''Trypanosoma brucei'' is the causative agent of sleeping sickness and involves an insect vector and a mammalian host through its complex life-cycle. ''T. brucei'' mammalian bloodstream forms (BSF) exhibit unique metabolic features including: ''i)'' reduced expression and activity of mitochondrial enzymes; ''ii)'' respiration mediated by the glycerol phosphate shuttle (GPSh) and the ''Trypanosome'' alternative oxidase (TAO) that is intrinsically uncoupled from generation of mitochondrial membrane potential; ''iii)'' maintenance of mitochondrial membrane potential by ATP hydrolysis through the reversal of F1Fo ATP synthase activity; ''iv)'' strong reliance on glycolysis to meet their energy demands; ''v)'' high susceptibility to oxidants. Here, we critically review the main metabolic features of BSF and provide a hypothesis to explain the unusual metabolic network and its biological significance for this parasite form. We postulate that intrinsically uncoupled respiration provided by GPSh-TAO system would act as a preventive antioxidant defense by limiting mitochondrial superoxide production and complementing the NADPH-dependent scavenging antioxidant defenses to maintain parasite redox balance. Given the uncoupled nature of the GPSh-TAO system, BSF would avoid programmed cell death processes by maintaining mitochondrial membrane potential through the reversal of ATP synthase activity using the ATP generated by glycolysis. This unique “metabolic design” in BSF has no biological parallel outside of Trypanosomatids and highlights the enormous diversity of the parasite mitochondrial processes to adapt to distinct environments.parasite mitochondrial processes to adapt to distinct environments. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2022-07-19''' [https://wiki.oroboros.at/images/d/d6/Roach_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0023.v2]::: Version 1 (v1) 2022-06-03 [https://wiki.oroboros.at/images/archive/d/d6/20220719153447%21Roach_2022_MitoFit.pdf doi:10.26124/mitofit:2022-0023.v1] - [https://wiki.oroboros.at/index.php/File:Roach_2022_MitoFit.pdf »Link to all versions«][[Roach 2022 Abstract Bioblast]]: Tolerance of rapid changes in light intensity by photosynthetic organisms is facilitated by non-photochemical quenching (NPQ), a term with reference to quenching of chlorophyll fluorescence, the technique used in its discovery. Mechanisms of NPQ include dissipating excess light energy to heat (qE), the reversible attachment of light-harvesting complexes (LHC) to photosystems (state transition / qT) and photoinhibition (qI). Chlorophyll is a ubiquitous pigment of photosynthetic organisms, found in LHC and the reaction centres of photosystem II and I (PSII; PSI). At room temperature, pulse-amplitude modulated (PAM) chlorophyll fluorescence protocols provide insights into PSII efficiency, thus a reasonable proxy for photosynthetic activity (carbon fixation), at least under optimal conditions. NPQ has a major impact on chlorophyll fluorescence intensity and is also quantified by PAM. Since NPQ mechanisms can occur simultaneously, they cause complexities in deciphering the signal. In algae, the ability for chlorophyll fluorescence in determining photosynthetic rates is not perfect, but it can still provide valuable information of processes affecting light harvesting. The aim of this report is to provide an overview of how various NPQ mechanisms in the model unicellular chlorophyte alga, ''Chlamydomonas reinhardtii'', as well as environmental conditions, affect chlorophyll fluorescence. I also propose a PAM protocol enabling the kinetics associated with each of the NPQ phases to be semi-quantified in under 20 min. h of the NPQ phases to be semi-quantified in under 20 min. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2022-08-16''' [https://wiki.oroboros.at/images/5/5a/Gainutdinov_2022_MitoFit.pdf https://doi.org/10.26124/mitofit:2022-0015.v2]::: Version 1 (v1) 2022-04-21 [https://wiki.oroboros.at/images/archive/5/5a/20220816100352%21Gainutdinov_2022_MitoFit.pdf https://doi.org/10.26124/mitofit:2022-0015]- [https://wiki.oroboros.at/index.php/File:Gainutdinov_2022_MitoFit.pdf »Link to all versions«]Amyotrophic lateral sclerosis (ALS) is a progressive, devastating, neurodegenerative disorder affecting upper and lower motor neurons. Common mechanisms of ALS pathogenesis are believed to be the disturbance of calcium homeostasis in the cell and dysfunction of mitochondria. Both factors mutually influence each other. As a result, chronic mitochondrial energy stress impairs fine cellular signaling and transport processes, leading to degeneration of motor neurons. In the current study we comparatively evaluated the cytosolic Ca2+ in healthy and ALS fibroblasts. We found that the mitochondrial calcium capacity in fibroblasts obtained from patients with sporadic (sALS) and familial (fALS) ALS differs between two subtypes and from that in healthy individuals. The changes of [Ca2+]cyt dynamics in ALS fibroblasts could be almost completely rescued by treatment with antioxidants (Trolox and CoQ10). These data confirm an important role of oxidative stress as a causative factor of mitochondrial dysfunction in ALS.portant role of oxidative stress as a causative factor of mitochondrial dysfunction in ALS. +
::: Version 2 ('''v2''') ''' … ::: Version 2 ('''v2''') '''2022-11-10''' [https://wiki.oroboros.at/images/6/64/Ganguly_2022_MitoFit.pdf https://doi.org/10.26124/mitofit:2022-0013.v2]::: Version 1 (v1) 2022-04-19 [https://wiki.oroboros.at/images/archive/6/64/20221110103433%21Ganguly_2022_MitoFit.pdf https://doi.org/10.26124/mitofit:2022-0013]- [https://wiki.oroboros.at/index.php/File:Ganguly_2022_MitoFit.pdf »Link to all versions«]Ferroptosis has been identified as a type of regulated cell death triggered by a diverse set of agents with implications in various diseases like cancer and neurodegenerative diseases. Ferroptosis is iron-dependent and accompanied by an accumulation of reactive oxygen species (ROS) and lipid oxidation products, a depletion of reduced glutathione, mitochondrial morphological alterations and the rupture of cell membrane; the process is inhibited by specific antioxidants like ferrostatin-1 and liproxstatin-1 and by other general antioxidants like the iron-chelator deferoxamine, vitamin E and N-acetylcysteine. However, the mechanism of cell death in ferroptosis subsequent to the accumulation of ROS and lipid oxidation products is not clearly established. We show here that the classical mitochondrial Complex I inhibitor rotenone (0.5 µM) causes death of SH-SY5Y cells (a human neuroblastoma cell line) over a period of 48 h accompanied by mitochondrial membrane depolarization and intracellular ATP depletion. This is associated with an intracellular accumulation of ROS and the lipid oxidation product malondialdehyde or MDA and a decrease in reduced glutathione content. All these processes are inhibited very conspicuously by specific inhibitors of ferroptosis such as ferrostatin-1 and liproxstatin-1. However, the decrease in Complex I activity upon rotenone-treatment of SH-SY5Y cells is not significantly recovered by ferrostatin-1 and liproxstatin-1. When the rotenone-treated cells are analyzed morphologically by Hoechst 33258 and propidium iodide (PI) staining, a mixed picture is noticed with densely fluorescent and condensed nuclei indicating apoptotic death of cells (Hoechst 33258) and also significant numbers of necrotic cells with bright red nuclei (PI staining).ant numbers of necrotic cells with bright red nuclei (PI staining). +
::: Version 3 ('"v3"') '''20 … ::: Version 3 ('"v3"') '''2019-07-03''' [https://www.mitofit.org/images/1/15/Di_Marcello_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0005.v2.pdf doi:10.26124/mitofit:ea19.MiPSchool.0005.v2.pdf]::: Version 2 (v2) 2019-06-27 [https://www.mitofit.org/images/1/15/Di_Marcello_2019_MitoFit_Preprint_Arch_doi_10.26214mitofitea19.MiPSchool.0005.v2.pdf doi:10.26124/mitofit:ea19.MiPSchool.0005.v2.pdf]::: Version 1 (v1) 2019-06-15 [http://www.mitofit.org/images/0/09/Di_Marcello_2019_MitoFit_Preprint_Arch.pdf doi:10.26124/mitofit:ea19.MiPSchool.0005]Bioenergetics is the study of how living organisms acquire and transform energy to perform biological work. Energetic coupling between chloroplasts and mitochondria has been described in algae, demonstrating the good functionality and interaction between both organelles is necessary to maintain metabolic integrity. High-resolution respirometry (HRR) is widely used to assess mitochondrial respiration and other bioenergetics parameters in the biomedical field of mitochondrial research and its clinical applications. In our interdisciplinary study, we adapted the multimodal approach of the Oroboros O2k high-resolution respirometer to investigate algal bioenergetics for biotechnological purposes. - ''Extended abstract''gate algal bioenergetics for biotechnological purposes. - ''Extended abstract'' +
::: Version 3 ('''v3''') ''' … ::: Version 3 ('''v3''') '''2022-03-07''' [https://wiki.oroboros.at/images/4/42/Pallag_2022_MitoFit_Proline.pdf doi:10.26124/mitofit:2022-0001.v3]::: Version 2 (v2) 2022-03-03 [https://wiki.oroboros.at/images/archive/4/42/20220307085642%21Pallag_2022_MitoFit_Proline.pdf doi:10.26124/mitofit:2022-0001.v2]::: Version 1 (v1) 2022-03-02 [https://wiki.oroboros.at/images/archive/4/42/20220303104356%21Pallag_2022_MitoFit_Proline.pdf doi:10.26124/mitofit:2022-0001] - [https://wiki.oroboros.at/index.php/File:Pallag_2022_MitoFit_Proline.pdf »Link to all versions«]In mitochondria expressing proline dehydrogenase (ProDH), oxidation of proline to pyrroline-5-carboxylate (P5C) leads to transfer of electrons to ubiquinone supporting Complexes CIII and CIV, in turn generating the protonmotive force. Further catabolism of P5C forms glutamate that fuels the citric acid cycle yielding reducing equivalents sustaining oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirement. The extent of proline oxidation was established in isolated mitochondria of various mouse tissues by means of simultaneously measuring oxygen consumption, membrane potential, NADH and ubiquinone redox state using the NextGen-O2k (Oroboros Instruments) and correlated to ProDH activity and F1FO-ATPase directionality. In CI-inhibited mouse liver and kidney mitochondria exhibiting high levels of proline oxidation and ProDH activity, catabolism of proline generated a sufficiently high membrane potential maintaining F1FO-ATPase operation in forward mode. This was not observed when either CIII or CIV was inhibited, nor during anoxia. Fueling CIII and CIV with duroquinone partially reproduced the effects of proline. Excess glutamate could not reproduce the effects of proline, arguing that they are due to processes upstream of glutamate conversion from proline. The ProDH inhibitors L-tetrahydro-2-furoic acid and to lesser extent S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all effects conferred by proline. It is concluded that proline catabolism through ProDH generates sufficient CIII and CIV proton pumping, supporting ATP production by F1FO-ATPase even when CI is inhibited. production by F1FO-ATPase even when CI is inhibited. +
::: Version 1 ('''v1''') '''2021-09-21''' … ::: Version 1 ('''v1''') '''2021-09-21''' [https://www.mitofit.org/images/1/16/Gnaiger_2021_MitoFit_BCA.pdf doi:10.26124/mitofit:2021-0008][[File:Gnaiger 2021 MitoFit BCA-graphical abstract.png|right|300px|Graphical abstract]]Cell respiration reflects mitochondrial fitness and plays a pivotal role in health and disease. Despite the rapidly increasing number of applications of cell respirometry to address current challenges in biomedical research, cross-references are rare between respirometric projects and platforms. Evaluation of accuracy and reproducibility between laboratories requires presentation of results in a common format independent of the applied method. When cell respiration is expressed as oxygen consumption rate in an experimental chamber, normalization is mandatory for comparability of results. Concept-driven normalization and regression analysis are key towards bioenergetic cluster analysis presented as a graphical tool to identify discrete data populations.In a meta-analysis of human skin fibroblasts, high-resolution respirometry and polarography covering cell senescence and the human age range are compared with multiwell respirometry. The common coupling control protocol measures ROUTINE respiration of living cells followed by sequential titrations of oligomycin, uncoupler, and inhibitors of electron transfer.Bioenergetic cluster analysis increases the resolution of outliers within and differences between groups. An outlier-skewness index is introduced as a guide towards logarithmic transformation for statistical analysis. Isolinear clusters are separated by variations in the extent of a quantity that correlates with the rate, whereas heterolinear clusters fall on different regression lines. Dispersed clusters are clouds of data separated by a critical threshold value. Bioenergetic cluster analysis provides new insights into mitochondrial respiratory control and a guideline for establishing a quality control paradigm for bioenergetics and databases in mitochondrial physiology. bases in mitochondrial physiology. +
::: Version 2 ('''v2''') '''2022-07-15''' … ::: Version 2 ('''v2''') '''2022-07-15''' [https://wiki.oroboros.at/images/4/44/Donnelly_2022_MitoFit_Hypoxia.pdf The ABC of hypoxia – what is the norm https://doi.org/10.26124/mitofit:2022-0025.v2]::: Version 1 (v1) 2022-06-28 - [https://wiki.oroboros.at/index.php/File:Donnelly_2022_MitoFit_Hypoxia.pdf »Link to all versions«][[File:Oxia terms.png|right|250px]][[Donnelly 2022 Abstract Bioblast]]: Hypoxia is a condition of oxygen levels below normoxia and opposite to hyperoxia. We here define the normoxic reference state by three complementary precepts: ('''A''') ambient normoxia at sea level in the contemporary atmosphere and corresponding dissolved O2 concentration at air saturation of aqueous environments; ('''B''') biological compartmental O2 levels at ambient normoxia under physiological activity of healthy organisms in the absence of environmental stress (e.g. in a diving human, a stranded whale, a thermally stressed animal); and ('''C''') O2 levels above the control region, i.e., where the capacity for O2 consumption is not compromised by partial O2 pressure as evaluated by its kinetics. Conversely, the '''abc''' of hypoxia is concerned with deviations from these reference points caused by different mechanisms: ('''a''') ambient alterations of oxygen levels; ('''b''') biological O2 demand exceeding O2 supply under pathological or experimental limitations of convective O2 transport or O2 diffusion; and ('''c''') critical oxygen pressure in oxygen kinetics shifted by pathological and toxicological effects or environmental stress. The ABC of hypoxia may be of help in the design and interpretation of ''in vitro'' and ''in vivo'' experimental studies. ical effects or environmental stress. The ABC of hypoxia may be of help in the design and interpretation of ''in vitro'' and ''in vivo'' experimental studies. +
<big>'''Journal publication 2021-06- … <big>'''Journal publication 2021-06-30 in [https://www.bioenergetics-communications.org/index.php/bec/article/view/cardoso_2021_mgg »Bioenergetics Communications 2021.1«]'''</big>For the advanced study of mitochondrial function, high-resolution respirometry is extended by fluorometric measurement of ATP production using the fluorophore Magnesium Green™ (MgG). A common problem with several fluorescent dyes is the inhibition of mitochondrial respiration. In the present study, a coupling control protocol was applied in combination with MgG to measure ATP production simultaneously with respiration for calculation of P»/O2 ratios. MgG at 1.1 µM did not affect respiration through the NADH-linked and succinate-linked pathways. Respiration was not inhibited in any of the coupling control states, hence coupling control efficiencies were not affected by MgG.tes, hence coupling control efficiencies were not affected by MgG. +
<big>'''Journal publication 2021-10- … <big>'''Journal publication 2021-10-06 in [https://www.bioenergetics-communications.org/index.php/bec/article/view/krako_jakovljevic_2021_pd »Bioenergetics Communications 2021.2«]'''</big>Mitochondrial function is known to be an important factor in maintaining cellular homeostasis and its dysregulation has become a hallmark for multiple disease conditions. This review aims to synthesise the extent of this knowledge by analysing changes of mitochondrial physiology parameters in Parkinson’s disease (PD) and to evaluate the contribution of cellular models of PD in the field. The analysis provided here constitutes a platform for further elucidation of mitochondrial function parameters relative to factors that may potentiate disease progression.ve to factors that may potentiate disease progression. +
<big>'''Journal publication 2021-12- … <big>'''Journal publication 2021-12-08 in [[Vernerova 2021 Biomedicines |''Biomedicines'']]'''</big>[[File:Vernerova 2021 Mitofit PLT - graphical abstract.png|right|300px|Graphical abstract]] Multiple non-aggregatory functions of human platelets (PLT) are widely acknowledged, yet their functional examination is limited mainly due to a lack of standardized isolation and analytic methods. Platelet apheresis (PA) is an established clinical method for PLT isolation aiming at the treatment of bleeding diathesis in severe thrombocytopenia. On the other hand, density gradient centrifugation (DC) is an isolation method applied in research for the analysis of the mitochondrial metabolic profile of oxidative phosphorylation (OXPHOS) in PLT obtained from small samples of human blood. We studied PLT obtained from 29 healthy donors by high-resolution respirometry for comparison of PA and DC isolates. ROUTINE respiration and electron transfer capacity of living PLT isolated by PA were significantly higher than in the DC group, whereas plasma membrane permeabilization resulted in a 57 % decrease of succinate oxidation in PA compared to DC. These differences were eliminated after washing the PA cells with phosphate buffer containing 10 mmol·L-1 EGTA, suggesting that several components, particularly Ca2+ and fuel substrates, were carried over into the respiratory assay from the serum in PA. A simple washing step was sufficient to enable functional mitochondrial analysis in subsamples obtained from PA.The combination of the standard clinical PA isolation procedure with PLT quality control and routine mitochondrial OXPHOS diagnostics meets an acute clinical demand in biomedical research of patients suffering from thrombocytopenia and metabolic diseases. h of patients suffering from thrombocytopenia and metabolic diseases. +
<big>'''Journal publication 2021-12- … <big>'''Journal publication 2021-12-21 in [https://www.bioenergetics-communications.org/index.php/bec/article/view/komlodi_2021_amr »Bioenergetics Communications 2021.4«]'''</big>[[File:Komlodi 2021 MitoFit AmR-O2 graphical abstract.png|right|300px|Graphical abstract]]The fluorometric Amplex UltraRed AmR assay is frequently used for quantitative assessment of hydrogen peroxide production. It is specific to H2O2, can be calibrated accurately, and allows continuous real-time measurement. Without correction for the background fluorescence slope, however, H2O2-independent formation of the fluorescent product UltroxRed (or resorufin) leads to artefacts.We analysed (''1'') the medium specificity of the background fluorescence slope of the AmR assay, and (''2'') the oxygen dependence of H2O2 flux in baker´s yeast ''Saccharomyces cerevisiae''. Apparent H2O2 flux, O2 concentration and O2 flux were measured simultaneously by high-resolution respirometry equipped with the fluorescence module. The apparent H2O2 flux of yeast showed a maximum under hypoxia when incubated in Dulbecco´s Phosphate Buffered Saline DPBS or KCl-medium. This hypoxic peak increased with the sequential number of normoxic-anoxic transitions. Even in the absence of yeast, the fluorescence slope increased at low O2 levels as a function of fluorescence intensity. The hypoxic peak was not observed in mitochondrial respiration medium MiR05.Therefore, the hypoxic peak was a medium-specific background effect unrelated to cell physiology. In MiR05, H2O2 production of yeast decreased linearly from hyperoxia to hypoxia, with a steep decline towards anoxia. Respiration and oxygen dependence expressed as ''p''50 of yeast were higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range. The flux-dependence of oxygen affinity explained the higher ''p''50 in MiR05. /sub> of yeast were higher in MiR05 than DPBS. Respiration was a hyperbolic function of oxygen concentration in the low-oxygen range. The flux-dependence of oxygen affinity explained the higher ''p''50 in MiR05. +
<big>'''Peter Hochachka lecture'''&l … <big>'''Peter Hochachka lecture'''</big>Earth’s changing environment has been a major evolutionary force shaping the diversity of species both in the past and present. In particular, seasonal ice cover in northern latitudes has selected for hypoxia and anoxia tolerance in some species, such as freshwater turtles. At the northern reaches of their range North American western painted turtles spend 4 months or more buried in the mud bottom of ice covered lakes and ponds [1]. This offers a unique opportunity to understand how a vertebrate brain, an organ extremely sensitive to reduced oxygen availability in mammals, can function without oxygen [2]. Through oxidative phosphorylation mitochondria fuel the inherently high energetic demands of brain and in mammals mitochondria also play a key role in injury from hypoxic stress – including loss of calcium homeostasis and production of reactive oxygen species (ROS) leading to apoptosis and necrosis. Hypoxic or anoxic stress does not signal stress in turtle brain but rather protective mechanisms with the onset of anoxia. Indeed our data show that mitochondria play a key role in low oxygen signaling in turtle brain by a reduction in mitochondrial membrane potential and release of a relatively small but significant amount of calcium. The increase in cytosolic calcium signals a phosphatase based mechanism to decrease whole-cell glutamatergic (NMDA and AMPA) excitatory currents in pyramidal neurons. While in stellate neurons anoxia results in a large reduction in mitochondrial ROS production that increases the magnitude of GABAergic inhibitory neurotransmission. The increased GABA activity produces a chloride based shunting current that “arrests” action potentials in pyramidal cells resulting in metabolic depression and neuroprotection.resulting in metabolic depression and neuroprotection. +
<big>'''[[Johansen K|Kjell Johansen]] … <big>'''[[Johansen K|Kjell Johansen]] lecture'''</big>Vertebrate hemoglobins (Hb) are exquisitely designed to transport O2 from the respiratory organs to the tissues, thereby safeguarding mitochondrial O2 supply and aerobic metabolism in the face of wide and independent variations in O2 tensions and temperature at the sites for loading and unloading of O2 [1-3]. In transporting O2, vertebrate Hbs (composed of 2 α and 2 β globin chains) switch between the T (tense, low O2-affinity, deoxygenated) structure that predominates in the tissues, and the R (relaxed, high-affinity, oxygenated) structure that predominates in the lungs and gills. The T-R shift is basic to cooperativity between the O2-binding heme groups that increases O2 (un)loading for a given change in O2 tension - and is reflected in the sigmoid shape of O2 binding curves. Hb’s in vivo O2 binding properties are a product of its intrinsic O2 affinity and its interaction with red cell allosteric effectors that decreases Hb-O2 affinity by stabilizing the T-structure. Apart from protons and CO2 (that facilitate O2 unloading in the acid tissues via the “Bohr-effect”) these effectors include chloride ions and organic phosphates [ATP in lower vertebrates, IPP (inositol pentaphosphate) in birds and DPG (diphosphoglycerate) in mammals]. The interaction with effectors varies between and within individual species and plays a key role in adjusting O2 transport in response to changes in environmental conditions, metabolic requirements, and mode of life. The decrease in Hb-O2 affinity with rising temperature mandated by the exothermic nature of heme oxygenation, enhances O2 unloading in warm tissues that require more O2, but may become maladaptive – and thus commonly is reduced - in regional heterothermic species where it may hamper O2 unloading (in cold extremities of arctic mammals) or cause excessive O2 release (in warm muscles, brains or eyes of fast-swimming fish).Based on case studies (Hbs from estivating fish, fast-swimming gamefish, high-altitude Andean frogs, geese that scale the Himalayas, Rocky Mountain Deer mice and Hb recreated from extinct mammoths [4-6]) the treatise analyses the molecular mechanisms for Hb’s role in securing mitochondrial O2 supply under stressful conditions - illustrating the key significance of molecular interactions to understanding physiological ecology. of molecular interactions to understanding physiological ecology. +
<big>MiP2013 Keynote by Sir John Wal … <big>MiP2013 Keynote by Sir John Walker</big>The lecture will be devoted to the topic of how the biological world supplies itself with energy to make biology work, and what medical consequences ensue when the energy supply chain in our bodies is damaged or defective. We derive our energy from sunlight, which, via photosynthesis in green plants, provides high energy components in the foods that we ingest. We harvest that energy, effectively by “burning” (oxidising) the high energy components, releasing cellular energy in a controlled way to generate the fuel of life, in the form of the molecule known as adenosine triphosphate (or ATP for short). The key steps in this process take place in the mitochondria inside the cells that make up our tissues. They serve as biological “power stations” that contain millions of tiny molecular turbines, the ATP synthase, that rotate rather like man-made turbines churning out the cellular fuel in massive quantities, which is then delivered to all parts of our bodies to provide the energy to make them function. Each of us makes and expends about 60 kg of this fuel every day of our lives. Defects in the fuel supply process are increasingly being recognised as important components of complex human diseases such as cancer, neurodegeneration and neuromuscular diseases, and they may also be part of the process of ageing. The ATP synthases found in mitochondria, eubacteria and chloroplasts have many common features. Their overall architectures are similar, and they all consist of two rotary motors linked by a stator and a flexible rotor. When rotation of the membrane bound rotor is driven by proton motive force, the direction of rotation ensures that ATP is made from ADP and phosphate in the globular catalytic domain. When ATP serves as the source of energy and is hydrolysed in the catalytic domain, the rotor turns in the opposite sense and protons are pumped outwards through the membrane domain, and away from the catalytic domain. The lecture will describe the common features of their catalytic mechanisms. However, the ATP synthase from mitochondria, eubacteria and chloroplasts differ most fundamentally in the energy cost that is paid to make each ATP molecule. The most efficient ATP synthase is found in the mitochondria from multicellular animals. The ATP synthases in unicellular organisms, and chloroplasts, pay various higher costs that seem to reflect the supply of available energy in the biological niches that they inhabit. The ATP synthases also differ significantly in the way they are regulated. Eubacteria have evolved a range of mechanisms of regulation, and the chloroplast enzyme is rendered inactive by a redox mechanism in the hours darkness. Mitochondria contain an inhibitor protein, IF1, that inhibits ATP hydrolysis but not ATP synthesis. Its in vitro mechanism has been studied in great detail, but its in vivo role is mysterious, and suppression of expression of the protein appears not to influence respiration.In mitochondria the ATP synthase is organised in rows of dimers along the edges of the cristae, and as will be discussed, it has been suggested that the permeability transition pore involved in apoptosis resides in the dimeric enzyme.e involved in apoptosis resides in the dimeric enzyme. +
'''Lemieux H, Blier PU, Gnaig … '''Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8.''' - [[Lemieux 2017 Sci Rep |»Bioblast link«]]* Accepted for publication: 2017-04-18* [http://rdcu.be/tgpY Sci Rep Open Access]: 2017-06-06://rdcu.be/tgpY Sci Rep Open Access]: 2017-06-06 +
'''Oroboros Distributor Meeting'''. Innsbruck, Austria; 2019 Jul 01-03. +
'''Oroboros distributor training'''. Innsbruck, Austria; 2023 Apr 24-25. +
'''Oroboros distributor training'''. Innsbruck, Austria; 2023 Nov 07-09. +
: » [[MiP2015]] - all abstracts in alphabetical order and programme sessions. : » [[MiP2015 Abstracts in the MiPMap]] - sort by MiP''areas'', species, tissues, diseases, ... +
1H-NMR experiments … 1H-NMR experiments have determined intracellular O2 consumption (''V''O2) with oxymyoglobin (MbO2) desaturation kinetics in human calf muscle during plantar flexion exercise at 0.75, 0.92, and 1.17 Hz with a constant load. At the onset of muscle contraction, myoglobin (Mb) desaturates rapidly. The desaturation rate constant of approximately 30 s reflects the intracellular ''V''O2. Although Mb desaturates quickly with a similar time constant at all workload levels, its final steady-state level differs. As work increases, the final steady-state cellular ''P''O2 decreases progressively. After Mb desaturation has reached a steady state, however, ''V''O2 continues to rise. On the basis of current respiratory control models, the analysis in the present report reveals two distinct ''V''O2 phases: an ADP-independent phase at the onset of contraction and an ADP-dependent phase after Mb has reached a steady state. In contrast to the accepted view, the initial intracellular ''V''O2 shows that oxidative phosphorylation can support up to 36 % of the energy cost, a significantly higher fraction than expected. Partitioning of the energy flux shows that a 31 % nonoxidative component exists and responds to the dynamic energy utilization-restoration cycle (which lasts for only milliseconds) as postulated in the glycogen shunt theory. The present study offers perspectives on the regulation of respiration, bioenergetics, and Mb function during muscle contraction.ration cycle (which lasts for only milliseconds) as postulated in the glycogen shunt theory. The present study offers perspectives on the regulation of respiration, bioenergetics, and Mb function during muscle contraction. +
A new reaction which occurs in oxida … A new reaction which occurs in oxidative phosphorylation associated with the electron transport system has been observed in rat liver mitochondria with α-ketoglutarate, β-hydroxybutyrate, and succinate as substrates. This reaction manifests itself by a replacement of O18 with normal oxygen in inorganic phosphate labeled with O18 and parallels the phosphorylation which is associated with the oxidation. The number of molecules of inorganic phosphate which participate in this reaction, calculated on the basis that a monoester of phosphate is involved, is several times higher than the number of high energy phosphate bonds that can be formed. The reaction does not occur at the substrate level oxidation of α-ketoglutarate and the evidence suggests that it occurs at every step in the electron transport system. This phosphate turnover reaction occurs only when phosphorylation is proceeding. Dinitrophenol suppresses the reaction. The omission of Mg++ or adenylic acid also suppresses the reaction. The reaction is abolished when succinate oxidation is catalyzed by a succinic oxidase preparation containing no phosphorylating system. The possibility that the reaction is due to a direct reaction of ATP, hydrolytic or otherwise, is eliminated. Various mechanisms which are consistent with the findings are discussed.stent with the findings are discussed. +
A simplified procedure for preparing … A simplified procedure for preparing mitochondria suspensions from isotonic sucrose homogenates has been described. These preparations exhibit high rates of net 7 minute phosphorus formation from adenylic acid during the oxidation of α-ketoglutarate in the absence of inhibitors such as fluoride, and show very low dephosphorylating activities. It has been possible to study the complete phosphorylation of AMP in this system and to interpret the characteristics of this process on the basis of adenosinediphosphate as primary phosphate acceptor and the presence of a myokinase in mitochondria. The activity of this transphosphorylase has been directly determined and is of considerable magnitude. It has also been demonstrated that probably all of the myokinase is associated with the mitochondrial fraction. The changes in the characteristics of oxidative phosphorylation and adenosinetriphosphatase activity as the result of incubation of the enzyme at 28° in the absence of substrates have been studied. The inactivation of the phosphorylation system by aging has been considered in two phases, an initial lag phase which is completely reversible in short aging experiments and a permanent reduction in activity observed with more severely aged mitochondria. The initial very low ATPase activity of the mitochondria was increased to appreciable magnitudes by aging. All the characteristics of aging were prevented to a large extent by AMP, ADP, or ATP. a large extent by AMP, ADP, or ATP. +
A thermodynamically improbable reductio … A thermodynamically improbable reduction of pyridine nucleotide caused by the addition of succinate to isolated mitochondria has been demonstrated. The material so reduced exhibits kinetic responses, some of which can suggest its consideration as a member of the respiratory chain, but a quantitative examination of the kinetics of oxidation and reduction shows that only a small portion of the total respiratory activity in succinate oxidation passes through the diphosphopyridine nucleotide-linked pathway. The nature of the reduction product has been examined in heart, liver, and guinea pig kidney mitochondria and is found to be material absorbing at 340 mµ and having a fluorescence emission maximum at 440 mµ. Direct chemical assays on kidney mitochondria indicate that the reduced material is diphosphopyridine nucleotide. A preliminary evaluation of various hypotheses to explain this result leads us tentatively to reject hypotheses based upon a single pool of mitochondrial pyridine nucleotide in which diphosphopyridine nucleotide and succinate compete for oxidizing equivalents from the cytochrome chain. Further indication of the complexities of this reaction is that respiration can be initiated by succinate without measurable pyridine nucleotide reduction and that a transition from aerobiosis in state 3 to anaerobiosis (state 5) can lead to a higher oxidation level of pyridine nucleotide than was observed aerobically in state 4. These observations suggest that the presence of adenosine 5’-diphosphate inhibits pyridine nucleotide reduction under both aerobic and anaerobic conditions and support the possibility that an energy-linked reaction may be involved.nergy-linked reaction may be involved. +
A 28-year-old former amateur cyclist demon … A 28-year-old former amateur cyclist demonstrated a sudden exercise intolerance and impairment in muscle function since March 2008 without clinical explanation. The main symptom was a decreased ergometric aerobic capacity by 50%. A specific defect of mitochondrial glutamate dehydrogenase (GDH) was indicated by lack of ADP stimulation in the presence of glutamate and subsequent rescue of respiration after addition of malate.e of respiration after addition of malate. +
A 2×2 factorial design was used to evaluat … A 2×2 factorial design was used to evaluate possible preservation of mitochondrial functions in two cardioprotective experimental models, remote ischemic preconditioning and streptozotocin-induced ''diabetes mellitus'', and their interaction during ischemia/reperfusion injury (I/R) of the heart. Male Wistar rats were randomly allocated into four groups: control (C), streptozotocin-induced diabetic (DM), preconditioned (RPC) and preconditioned streptozotocin-induced diabetic (DM+RPC). RPC was conducted by 3 cycles of 5-min hind-limb ischemia and 5-min reperfusion. DM was induced by a single dose of 65 mg/kg streptozotocin. Isolated hearts were exposed to ischemia/reperfusion test according to Langendorff. Thereafter mitochondria were isolated and the mitochondrial respiration was measured. Additionally, the ATP synthase activity measurements on the same preparations were done. Animals of all groups subjected to I/R exhibited a decreased state 3 respiration with the least change noted in DM+RPC group associated with no significant changes in state 2 respiration. In RPC, DM and DM+RPC group, no significant changes in the activity of ATP synthase were observed after I/R injury. These results suggest that the endogenous protective mechanisms of RPC and DM do preserve the mitochondrial function in heart when they act in combination.ion in heart when they act in combination. +
A 3-year-old girl presented with severe ep … A 3-year-old girl presented with severe epilepsy in the context of ''Borrelia'' infection. After ceftriaxone/lidocaine administration, she showed secondarily generalized focal crises that led to neurological and motor sequelae. Genetic studies identified in the patient two heterozygous POLG mutations (c.2591A>G; p.Asn864Ser and c.3649G>C; p.Ala1217Pro). Through analysis of POLG activity in cultured fibroblasts, we confirmed that the mutations altered the mtDNA turnover. Moreover, patient fibroblasts were more sensitive than controls in the presence of a mitochondrial replication-affecting drug, the antiretroviral azidothymidine. To test if ceftriaxone treatment could worsen the deleterious effect of the patient mutations, toxicity assays were performed. Cell toxicity, without direct effect on mitochondrial respiratory function, was detected at different antibiotic concentrations. The clinical outcome, together with the different ''in vitro'' sensitivity to ceftriaxone among patient and control cells, suggested that the mitochondrial disease symptoms were hastened by the infection and were possibly worsened by the pharmacological treatment. This study underscores the benefit of early genetic diagnosis of the patients with mitochondrial diseases, since they may be a target group of patients especially vulnerable to environmental factors. especially vulnerable to environmental factors. +
A Kinase Interacting Protein 1 (AKIP1) is … A Kinase Interacting Protein 1 (AKIP1) is a signalling adaptor that promotes mitochondrial respiration and attenuates mitochondrial oxidative stress in cultured cardiomyocytes. We sought to determine whether AKIP1 influences mitochondrial function and the mitochondrial adaptation in response to exercise ''in vivo''. We assessed mitochondrial respiratory capacity, as well as electron microscopy and mitochondrial targeted-proteomics in hearts from mice with cardiomyocyte-specific overexpression of AKIP1 (AKIP1-TG) and their wild type (WT) littermates. These parameters were also assessed after four weeks of voluntary wheel running. In contrast to our previous ''in vitro'' study, respiratory capacity measured as state 3 respiration on palmitoyl carnitine was significantly lower in AKIP1-TG compared to WT mice, whereas state 3 respiration on pyruvate remained unaltered. Similar findings were observed for maximal respiration, after addition of FCCP. Mitochondrial DNA damage and oxidative stress markers were not elevated in AKIP1-TG mice and gross mitochondrial morphology was similar. Mitochondrial targeted-proteomics did reveal reductions in mitochondrial proteins involved in energy metabolism. Exercise performance was comparable between genotypes, whereas exercise-induced cardiac hypertrophy was significantly increased in AKIP1-TG mice. After exercise, mitochondrial state 3 respiration on pyruvate substrates was significantly lower in AKIP1-TG compared with WT mice, while respiration on palmitoyl carnitine was not further decreased. This was associated with increased mitochondrial fission on electron microscopy, and the activation of pathways associated with mitochondrial fission and mitophagy. This study suggests that AKIP1 regulates the mitochondrial proteome involved in energy metabolism and promotes mitochondrial turnover after exercise. Future studies are required to unravel the mechanistic underpinnings and whether the mitochondrial changes are required for the AKIP1-induced physiological cardiac growth.KIP1-induced physiological cardiac growth. +
A Mitochondrial Festival in the Spirit of … A Mitochondrial Festival in the Spirit of [[Gentle Science]] <div style="padding:0px;border: 1px solid #aaaaaa;margin-bottom:0px;margin-right:10px"><div style="font-size:100%;font-weight:bold;padding:0.2em;padding-right: 0.4em;padding-left: 0.4em;background-color:#eeeeee;border-bottom:1px solid #aaaaaa;text-align:left;">[[Image:O2k-support system.jpg|right|150px|link=http://wiki.oroboros.at/index.php/O2k-technical_support_and_open_innovation|O2k-technical support and open innovation]]: <big>Open the '''pdf document''' above.</big></div><div style="background-color:#ffffff;padding-top:0.2em;padding-right: 0.4em;padding-bottom: 0.2em;padding-left: 0.4em;">::::» Current O2k-series: '''[https://www.oroboros.at/index.php/product-category/products/o2k-packages/ NextGen-O2k Series XB and O2k Series J]'''::::» Current software versions DatLab 8.0: [[MitoPedia: DatLab]]::::* ''Further details:'' '''» [[MitoPedia: O2k-Open Support]]'''</div></div>itoPedia: O2k-Open Support]]''' </div> </div> +
A WHO expert consultation addressed the de … A WHO expert consultation addressed the debate about interpretation of recommended body-mass index (BMI) cut-off points for determining overweight and obesity in Asian populations, and considered whether population-specific cut-off points for BMI are necessary. They reviewed scientific evidence that suggests that Asian populations have different associations between BMI, percentage of body fat, and health risks than do European populations. The consultation concluded that the proportion of Asian people with a high risk of type 2 diabetes and cardiovascular disease is substantial at BMIs lower than the existing WHO cut-off point for overweight (> or =25 kg/m2). However, available data do not necessarily indicate a clear BMI cut-off point for all Asians for overweight or obesity. The cut-off point for observed risk varies from 22 kg/m2 to 25 kg/m2 in different Asian populations; for high risk it varies from 26 kg/m2 to 31 kg/m2. No attempt was made, therefore, to redefine cut-off points for each population separately. The consultation also agreed that the WHO BMI cut-off points should be retained as international classifications. The consultation identified further potential public health action points (23.0, 27.5, 32.5, and 37.5 kg/m2) along the continuum of BMI, and proposed methods by which countries could make decisions about the definitions of increased risk for their population.tions of increased risk for their population. +
A better understanding of the molecular ba … A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.ry functionality and mitochondrial health. +
A blue diode PAM (Pulse Amplitude Modulati … A blue diode PAM (Pulse Amplitude Modulation) fluorometer was used to measure rapid Photosynthesis (P) versus Irradiance (E) curves (P vs. E curves) in ''Synechococcus'' (classical cyanobacteria), ''Prochlorothrix'' (prochlorophyta), ''Chlorella'' (chlorophyta), ''Rhodomonas'' (cryptophyta), ''Phaeodactylum'' (bacillariophyta), ''Acaryochloris'' (Chl d/a cyanobacteria) and Subterranean Clover (''Trifolium subterraneum'', Papilionaceae, Angiospermae). Effective quantum yield (Phi(PSII)) versus irradiance curves could be described by a simple exponential decay function (Phi(PSII) = Phi(PSII, maxe(-kE)) although Log/Log transformation was sometimes found to be necessary to obtain the best fits. Photosynthesis was measured as relative Electron Transport Rate (rETR) standardised on a chlorophyll basis. P versus E curves were fitted to the waiting-in-line function (an equation of the form P = P(max) x k x E x e(-kE)) allowing half-saturating and optimal irradiances (E(optimum)) to be estimated. The second differential of the equation shows that at twice optimal light intensities, there is a point of inflection in the P versus E curve. Photosynthesis is inhibited 26.4% at this point of inflection. The waiting-in-line model was found to be a very good descriptor of photosynthetic light saturation curves and superior to hyperbolic functions with an asymptotic saturation point (Michaelis-Menten, exponential saturation and hyperbolic tangent). The exponential constants (k) of the Phi(PSII) versus E and P versus E curves should be equal because rETR is directly proportional to Phi(PSII) x E. The conventionally calculated Non-Photochemical Quenching (NPQ) in Synechococcus was not significantly different to zero but NPQ versus E curves for the other algae could be fitted to an exponential saturation model. The kinetics of NPQ does not appear to be related to the kinetics of Phi(PSII) or rETR.ated to the kinetics of Phi(PSII) or rETR. +
A burgeoning literature has attributed var … A burgeoning literature has attributed varied physiological effects to hydrogen sulfide (H2S), which is a product of eukaryotic sulfur amino acid metabolism. Protein persulfidation represents a major focus of studies elucidating the mechanism underlying H2S signaling. On the contrary, the capacity of H2S to induce reductive stress by targeting the electron transport chain (ETC) and signal by reprogramming redox metabolism has only recently begun to be elucidated. Recent Advances: In contrast to the nonspecific reaction of H2S with oxidized cysteines to form protein persulfides, its inhibition of complex IV represents a specific mechanism of action. Studies on the dual impact of H2S as an ETC substrate and an inhibitor have led to the exciting discovery of ETC plasticity and the use of fumarate as a terminal electron acceptor. H2S oxidation combined with complex IV targeting generates mitochondrial reductive stress, which is signaled through the metabolic network, leading to increased aerobic glycolysis, glutamine-dependent reductive carboxylation, and lipogenesis. Critical Issues: Insights into H2S-induced metabolic reprogramming are ushering in a paradigm shift for understanding the mechanism of its cellular action. It will be critical to reevaluate the physiological effects of H2S, for example, cytoprotection against ischemia-reperfusion injury, through the framework of metabolic reprogramming and ETC remodeling by H2S. Future Directions: The metabolic ramifications of H2S in other cellular compartments, for example, the endoplasmic reticulum and the nucleus, as well as the intersections between hypoxia and H2S signaling are important future directions that merit elucidation. future directions that merit elucidation. +
A by-product of mitochondrial substrate ox … A by-product of mitochondrial substrate oxidation and electron transfer to generate cellular energy (ATP) is reactive oxygen species (ROS). Superoxide anion radical and hydrogen peroxide (H2O2) are the proximal ROS produced by the mitochondria. Because low levels of ROS serve critical regulatory roles in cell physiology while excessive levels or inappropriately localized ROS result in aberrant physiological states, mitochondrial ROS need to be tightly regulated. While it is known that regulation of mitochondrial ROS involves balancing the rates of production and removal, the effects of stressors on these processes remain largely unknown. To illuminate how stressors modulate mitochondrial ROS homeostasis, we investigated the effects of temperature and cadmium (Cd) on H2O2 emission and consumption in rainbow trout liver mitochondria. We show that H2O2 emission rates increase with temperature and Cd exposure. Energizing mitochondria with malate-glutamate or succinate increased the rate of H2O2 emission; however, Cd exposure imposed different patterns of H2O2 emission depending on the concentration and substrate. Specifically, mitochondria respiring on malate-glutamate exhibited a saturable graded concentration-response curve that plateaued at 5 μM while mitochondria respiring on succinate had a biphasic concentration-response curve characterized by a spike in the emission rate at 1 μM Cd followed by gradual diminution at higher Cd concentrations. To explain the observed substrate- and concentration-dependent effects of Cd, we sequestered specific mitochondrial ROS-emitting sites using blockers of electron transfer and then tested the effect of the metal. The results indicate that the biphasic H2O2 emission response imposed by succinate is due to site IIF but is further modified at sites IQ and IIIQo. Moreover, the saturable graded H2O2 emission response in mitochondria energized with malate-glutamate is consistent with effect of Cd on site IF. Additionally, Cd and temperature acted cooperatively to increase mitochondrial H2O2 emission suggesting that increased toxicity of Cd at high temperature may be due to increased oxidative insult. Surprisingly, despite their clear stimulatory effect on H2O2 emission, Cd, temperature and bioenergetic status did not affect the kinetics of mitochondrial H2O2 consumption; the rate constants and half-lives for all the conditions tested were similar. Overall, our study indicates that the production processes of rainbow trout liver mitochondrial H2O2 metabolism are highly responsive to stressors and bioenergetics while the consumption processes are recalcitrant. The latter denotes the presence of a robust H2O2 scavenging system in liver mitochondria that would maintain H2O2 homeostasis in the face of increased production and reduced scavenging capacity.Copyright © 2019 Elsevier B.V. All rights reserved.The latter denotes the presence of a robust H2O2 scavenging system in liver mitochondria that would maintain H2O2 homeostasis in the face of increased production and reduced scavenging capacity. Copyright © 2019 Elsevier B.V. All rights reserved. +
A causal link between non-ischaemic heart … A causal link between non-ischaemic heart failure (HF) and humoral autoimmunity against G-protein-coupled receptors (GPCR) remains unclear except for Chagas' cardiomyopathy. Uncertainty arises from ambiguous reports on incidences of GPCR autoantibodies, spurious correlations of autoantibody levels with disease activity, and lack of standardization and validation of measuring procedures for putatively cardio-pathogenic GPCR autoantibodies. Here, we use validated and certified immune assays presenting native receptors as binding targets. We compared candidate GPCR autoantibody species between HF patients and healthy controls and tested associations of serum autoantibody levels with serological, haemodynamic, metabolic, and functional parameters in HF.Ninety-five non-ischaemic HF patients undergoing transcatheter endomyocardial biopsy and 60 healthy controls were included. GPCR autoantibodies were determined in serum by IgG binding to native receptors or a cyclic peptide (for β1AR autoantibodies). In patients, cardiac function, volumes, and myocardial structural properties were assessed by cardiac magnetic resonance imaging; right heart catheterization served for determination of cardiac haemodynamics; endomyocardial biopsies were used for histological assessment of cardiomyopathy and determination of cardiac mitochondrial oxidative function by high-resolution respirometry.Autoantibodies against β1 adrenergic (β1AR) , M5-muscarinic (M5AR), and angiotensin II type 2 receptors (AT2R) were increased in HF (all P < 0.001). Autoantibodies against α1 -adrenergic (α1 AR) and angiotensin II type 1 receptors (AT1R) were decreased in HF (all P < 0.001). Correlation of alterations of GPCR autoantibodies with markers of cardiac or systemic inflammation or cardiac damage, haemodynamics, myocardial histology, or left ventricular inflammation (judged by T2 mapping) were weak, even when corrected for total IgG. β1 AR autoantibodies were related inversely to markers of left ventricular fibrosis indicated by T1 mapping (r = -0.362, P < 0.05) and global longitudinal strain (r = -0.323, P < 0.05). AT2R autoantibodies were associated with improved myocardial mitochondrial coupling as measured by high-resolution respirometry in myocardial biopsies (r = -0.352, P < 0.05). In insulin-resistant HF patients, AT2R autoantibodies were decreased (r = -.240, P < 0.05), and AT1R autoantibodies were increased (r = 0.212, P < 0.05).GPCR autoantibodies are markedly altered in HF. However, they are correlated poorly or even inversely to haemodynamic, metabolic, and functional markers of disease severity, myocardial histology, and myocardial mitochondrial efficiency. These observations do not hint towards a specific cardio-pathogenic role of GPCR autoantibodies and suggest that further investigations are required before specific therapies directed at GPCR autoantibodies can be clinically tested in non-ischaemic HF.rected at GPCR autoantibodies can be clinically tested in non-ischaemic HF. +
A cellular model of cardiomyocytes (H9c2 c … A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.the mitochondrial electron-transfer chain. +
A central characteristic of many types of … A central characteristic of many types of cancer is altered energy metabolism processes such as enhanced glucose uptake and glycolysis and decreased oxidative metabolism. The regulation of energy metabolism is an elaborate process involving regulatory proteins such as HIF (pro-metastatic protein), which reduces oxidative metabolism, and some other proteins such as tumour suppressors that promote oxidative phosphorylation. In recent years, it has been demonstrated that signal transducer and activator of transcription (STAT) proteins play a pivotal role in metabolism regulation. STAT3 and STAT5 are essential regulators of cytokine- or growth factor-induced cell survival and proliferation, as well as the crosstalk between STAT signalling and oxidative metabolism. Several reports suggest that the constitutive activation of STAT proteins promotes glycolysis through the transcriptional activation of hypoxia-inducible factors and therefore, the alteration of mitochondrial activity. It seems that STAT proteins function as an integrative centre for different growth and survival signals for energy and respiratory metabolism. This review summarises the functions of STAT3 and STAT5 in the regulation of some metabolism-related genes and the importance of oxygen in the tumour microenvironment to regulate cell metabolism, particularly in the metabolic pathways that are involved in energy production in cancer cells.lved in energy production in cancer cells. +
A characteristic pattern of organization w … A characteristic pattern of organization was found with the help of the electron microscope in sectioned animal mitochondria irrespective of the species providing the specimen and of the cell type examined.Each mitochondrion was found to possess:# A limiting membrane.# A mitochondrial matrix that appears structureless at present levels of resolution.# A system of internal ridges (cristae mitochondriales) that protrude from the inside surface of the membrane towards the interior of the organelles. In many mitochondria the cristae are perpendicular to the long axis of the organelles and occur in series within which they lie parallel to one another at more or less regular intervals.In favorable electron micrographs the mitochondrial membrane appears to be double and the cristae appear to be folds of a second, internal mitochondrial membrane.a second, internal mitochondrial membrane. +
A chronic high fat diet results in hepatic … A chronic high fat diet results in hepatic mitochondrial dysfunction and induction of peroxisomal fatty acid oxidation (FAO); whether specific inhibition of peroxisomal FAO benefits mitochondrial FAO and reactive oxygen species (ROS) metabolism remains unclear. In this study a specific inhibitor for the rate-limiting enzyme involved in peroxisomal FAO, [[acyl-CoA oxidase]]-1 (ACOX1) was developed and used for the investigation of peroxisomal FAO inhibition upon mitochondrial FAO and ROS metabolism. Specific inhibition of ACOX1 by 10,12-tricosadiynoic acid increased hepatic mitochondrial FAO via activation of the SIRT1-AMPK (adenosine 5'-monophosphate-activated protein kinase) pathway and proliferator activator receptor α and reduced hydrogen peroxide accumulation in high fat diet-fed rats, which significantly decreased hepatic lipid and ROS contents, reduced body weight gain, and decreased serum triglyceride and insulin levels. Inhibition of ACOX1 is a novel and effective approach for the treatment of high fat diet- or obesity-induced metabolic diseases by improving mitochondrial lipid and ROS metabolism.ng mitochondrial lipid and ROS metabolism. +
A close link between Ca2+</s … A close link between Ca2+, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca2+ may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+ in cytosol. In differentiation process plasma membrane Ca2+ ATPase (PMCA) is considered as one of the major players for Ca2+ homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca2+]c resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.soforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation. +
A common metabolic change in cancer is the … A common metabolic change in cancer is the acquisition of glycolytic phenotypes. Increased expression of glycolytic enzymes is considered as one contributing factor. The role of mitochondrial defects in acquisition of glycolytic phenotypes has been postulated but remains controversial. Here we show that functional defects in mitochondrial respiration could be induced by oncogenic H-RasQ61L transformation, even though the mitochondrial contents or mass was not reduced in the transformed cells. First, mitochondrial respiration, as measured by mitochondrial oxygen consumption, was suppressed in NIH-3T3 cells transformed with H-RasQ61L. Second, oligomycin or rotenone did not reduce the cellular ATP levels in the H-RasQ61L transformed cells, suggesting a diminished role of mitochondrial respiration in the cellular energy metabolism. Third, inhibition of glycolysis with iodoacetic acid reduced ATP levels at a much faster rate in H-RasQ61L transformed cells than in the vector control cells. The reduction of cellular ATP levels was reversed by exogenously added pyruvate in the vector control cells but not in H-RasQ61L transformed cells. Finally when compared to the HRasQ61L transformed cells, the vector control cells had increased resistance toward glucose deprivation. The increased resistance was dependent on mitochondrial oxidative phosphorylation since rotenone or oligomycin abolished the increased survival of the vector control cells under glucose deprivation. The results also suggest an inability of the H-RasQ61L transformed cells to reactivate mitochondrial respiration under glucose deprivation. Taken together, the data suggest that mitochondrial respiration can be impaired during transformation of NIH-3T3 cells by oncogeneic H-RasQ61L.can be impaired during transformation of NIH-3T3 cells by oncogeneic H-RasQ61L. +
A complete kinetic analysis of the forward … A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: ''K''ib = 29 mM; ''K''b = 5.3 mM, and the maximum velocity of the reaction was unchanged: ''V''1 = 1 μmol/min/mg. Slight differences were noted for the dissociation constant (''K''ia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 mM in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant (''K''a) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 mM with exogenous ATP and 0.014 mM with oxidative phosphorylation. Since ''K''ia × ''K''b does not equal ''K''a × ''K''ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.its coupling to oxidative phosphorylation. +
A complete understanding of the role of th … A complete understanding of the role of the liver in burn-induced hypermetabolism is lacking. We investigated the acute effect of severe burn trauma on liver mitochondrial respiratory capacity and coupling control as well as the signaling events underlying these alterations.Male BALB/c mice (8-12 weeks) received full-thickness scald burns on ∼30% of the body surface. Liver tissue was harvested 24 hours post injury. Mitochondrial respiration was determined by high-resolution respirometry. Citrate synthase activity was determined as a proxy of mitochondrial density. Male Sprague-Dawley rats received full-thickness scald burns to ∼60% of the body surface. Serum was collected 24 hours post injury. HepG2 cells were cultured with serum-enriched media from either sham or burn treated rats. Protein levels were analyzed via western blot.Mass-specific (p = 0.01) and mitochondrial-specific (p = 0.01) respiration coupled to ATP production significantly increased in the liver after burn. The respiratory control ratio for ADP (p = 0.04) and the mitochondrial flux control ratio (p = 0.03) were elevated in the liver of burned animals. Complex III and Complex IV protein abundance in the liver increased after burn by 17% and 14%, respectively. Exposure of HepG2 cells to serum from burned rats increased the pAMPKα:AMPKα ratio (p < 0.001) and levels of SIRT1 (p = 0.01), Nrf2 (p < 0.001), and PGC1α (p = 0.02).Severe burn trauma augments respiratory capacity and function of liver mitochondria, adaptations that augment ATP production. This response may be mediated by systemic factors that activate signaling proteins responsible for regulating cellular energy metabolism and mitochondrial biogenesis. energy metabolism and mitochondrial biogenesis. +
A complex was reconstituted with hydrophob … A complex was reconstituted with hydrophobic proteins from bovine heart mitochondrial membranes, cytochrome c, cytochrome oxidase, phospholipids, and coupling factors. These vesicular structures catalyzed oxidative phosphorylation with reduced N-methylphenazinium methyl sulfate as substrate.ylphenazinium methyl sulfate as substrate. +
A compound with promising anticancer prope … A compound with promising anticancer properties, 3-bromopyruvate (3-BP) is a synthetic derivative of a pyruvate molecule; however, its toxicity in non-malignant cells has not yet been fully elucidated. Therefore, we elected to study the effects of 3-BP on primary hepatocytes in monolayer cultures, permeabilized hepatocytes and isolated mitochondria. After a 1-h treatment with 100 μM 3-BP cell viability of rat hepatocytes was decreased by 30 % as measured by the WST-1 test (p < 0.001); after 3-h exposure to ≥200 μM 3-BP lactate dehydrogenase leakage was increased (p < 0.001). Reactive oxygen species production was increased in the cell cultures after a 1-h treatment at concentrations ≥100 μmol/l (p < 0.01), and caspase 3 activity was increased after a 20-h incubation with 150 μM and 200 μM 3-BP (p < 0.001). This toxic effect of 3-BP was also proved using primary mouse hepatocytes. In isolated mitochondria, 3-BP induced a dose- and time-dependent decrease of mitochondrial membrane potential during a 10-min incubation both with Complex I substrates glutamate + malate or Complex II substrate succinate, although this decrease was more pronounced with the latter. We also measured the effect of 3-BP on respiration of isolated mitochondria. ADP-activated respiration was inhibited by 20 μM 3-BP within 10 min. Similar effects were also found in permeabilized hepatocytes of both species.so found in permeabilized hepatocytes of both species. +
A comprehensive assessment of skeletal mus … A comprehensive assessment of skeletal muscle ultrastructure and mitochondrial bioenergetics has not been undertaken in individuals with type 1 diabetes. This study aimed to systematically assess skeletal muscle mitochondrial phenotype in young adults with type 1 diabetes.Physically active, young adults (men and women) with type 1 diabetes (HbA1c 63.0 ± 16.0 mmol/mol [7.9% ± 1.5%]) and without type 1 diabetes (control), matched for sex, age, BMI and level of physical activity, were recruited (''n'' = 12/group) to undergo vastus lateralis muscle microbiopsies. Mitochondrial respiration (high-resolution respirometry), site-specific mitochondrial H2O2 emission and Ca2+ retention capacity (CRC) (spectrofluorometry) were assessed using permeabilised myofibre bundles. Electron microscopy and tomography were used to quantify mitochondrial content and investigate muscle ultrastructure. Skeletal muscle microvasculature was assessed by immunofluorescence.Mitochondrial oxidative capacity was significantly lower in participants with type 1 diabetes vs the control group, specifically at Complex II of the electron transport chain, without differences in mitochondrial content between groups. Muscles of those with type 1 diabetes also exhibited increased mitochondrial H2O2 emission at Complex III and decreased CRC relative to control individuals. Electron tomography revealed an increase in the size and number of autophagic remnants in the muscles of participants with type 1 diabetes. Despite this, levels of the autophagic regulatory protein, phosphorylated AMP-activated protein kinase (p-AMPKαThr172), and its downstream targets, phosphorylated Unc-51 like autophagy activating kinase 1 (p-ULK1Ser555) and p62, was similar between groups. In addition, no differences in muscle capillary density or platelet aggregation were observed between the groups.Alterations in mitochondrial ultrastructure and bioenergetics are evident within the skeletal muscle of active young adults with type 1 diabetes. It is yet to be elucidated whether more rigorous exercise may help to prevent skeletal muscle metabolic deficiencies in both active and inactive individuals with type 1 diabetes.rigorous exercise may help to prevent skeletal muscle metabolic deficiencies in both active and inactive individuals with type 1 diabetes. +
A computational model for the ATP-ADP stea … A computational model for the ATP-ADP steady-state exchange rate mediated by adenine nucleotide translocase (ANT) versus mitochondrial membrane potential dependence in isolated rat liver mitochondria is presented. The model represents the system of three ordinary differential equations, and the basic components included are ANT, F(0)/F(1)-ATPase, and the phosphate carrier. The model reproduces quantitatively the relationship between mitochondrial membrane potential and the ATP-ADP steady-state exchange rate mediated by the ANT operating in the forward mode, with the assumption that the phosphate carrier functions under rapid equilibrium. Furthermore, the model can simulate the kinetics of experimentally measured data on mitochondrial membrane potential titrated by an uncoupler. Verified predictions imply that the ADP influx rate is highly dependent on the mitochondrial membrane potential, and in the 0-100 mV range it is close to zero, owing to extremely low matrix ATP values. In addition to providing theoretical values of free matrix ATP and ADP, the model explains the diminished ADP-ATP exchange rate in the presence of nigericin, a condition in which there is hyperpolarization of the inner mitochondrial membrane at the expense of the mitochondrial Delta pH gradient; the latter parameter influences matrix inorganic phosphate and ATP concentrations in a manner also described.concentrations in a manner also described. +