MitoFit Open Seminars

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MitoFit Open Seminars

Time: Weekly on Thursday, 16:00 to 17:00
Location: Oroboros Instruments, Schöpfstr 18, MiPArt

2019-05-23 MitoFit Open Seminar

2019-05-29 MitoFit Open Seminar
  • 16:00-16:30

Click to expand or collaps
» MitoFit Open Seminars calendar archive
2019-05-16 MitoFit Open Seminar
2019-05-09 MitoFit Open Seminar
2019-05-02 MitoFit Open Seminar
  • 16:00-17:00 Enis Garipi: Phenotyping mitochondrial metabolism in oral and oesophageal cancer: respiratory capacity in cancer cell lines and human oesophagus biopsies » Abstract pdf « 
2019-04-24 MitoFit Open Seminar
  • 08:15-09:15 Carolina Doerrier: Fighting against reproducibility crisis: Inter-laboratory harmonization of protocols for mitochondrial function evaluation in permeabilized muscle fibers » Abstract pdf « 
2019-04-19 MitoFit Journal Club
  • 08:30-09:15 “Mitochondrial physiology in the major arbovirus vector Aedes aegypti: substrate preferences and sexual differences define respiratory capacity and superoxide production”, Soares et al., PLoS One, 2015 presented by Luiz Garcia-Souza
2019-04-12 MitoFit Friday
  • 08:30-09:15 Luiza Cardoso: Exploring the technique of using Mg Green to analyse mitochondrial ATP production » Abstract pdf « 
2019-04-05 MitoFit Friday
  • 08:30-09:15 Timea Komlodi: Facts and artefacts in measurements of H2O2 production using Amplex UltraRed assay as a function of oxygen pressure » Abstract pdf « 
2019-03-29 MitoFit Friday
  • 08:30-09:15 Karavaeva Iuliia: Overview of the results of my STSM. High-resolution respirometry of isolated brown fat mitochondria and permeabilized brown adipocytes
2019-03-22 MitoFit Friday
2019-03-15 MitoFit Friday
2019-03-08 MitoFit Friday
  • 08:30-09:15 Lucie Zdrazilova: Mitochondrial respiration in different cell lines measured in the 2.0 mL and 0.5 mL chambers of the O2k-FluoRespirometer (Oroboros): an experimental basis for platform comparison with the Seahorse XFe24 Bioanalyzer (Agilent) » Abstract pdf «

  • 13:00-13:30 Diana Antunes: Novel insights into mitochondrial phospholipid homeostasis in a disease-relevant yeast model » Abstract pdf «
2019-03-01 MitoFit Friday
  • 08:30-09:15 Timi Komlodi: ROS production under pathological condition at tissue normoxia: do the cells still survive? mtFOIE GRAS project report from Munich » Abstract pdf «
2019-02-22 MitoFit Friday
2019-02-08 MitoFit Friday
2019-02-08 MitoFit Friday
2019-02-01 MitoFit Friday
2019-01-25 MitoFit Friday
2019-01-18 MitoFit Friday
2019-01-11 MitoFit Friday
  • 08:30-09:15 Iuliia Karavaeva: Investigating the physiological substrate and role of the circadian mitochondrial transporter Slc25a34 in brown adipose tissue.
2018-12-21 MitoFit Friday
  • 08:30-09:15 Ondrej Sobotka: The effect of culture media composition on mitochondrial respiration of HEK and Huh7 cell lines.
2018-12-14 MitoFit Friday
2018-10-12 MitoFit Friday
  • 08:30-09:15 Yaschar Kabiri DE Munich Zischka H: Mitochondrial intermembrane space proteins in liver cancer. His secondment is supported by MtFOIE GRAS.
2018-09-28 MitoFit Friday
  • 08:30-09:15 Enis Garipi: Phenotyping mitochondrial metabolism in Barrett‘s metaplasia-dysplasia-adenocarcinoma sequence.
2018-09-21 MitoFit Friday
  • 08:30-09:15 Maria Huete-Ortega: Tackling the global challenges: microalgae bioenergy production.
2018-09-14 MitoFit Friday
  • 08:30-09:10: Luiza Cardoso: Influence of High-Fat Diet on Copper Metabolism in Mice: Involvement of the Cu(I)-ATPase ATP7B and Protein Kinase Cε (PKCε).
  • 09:10-09:40: Journal Club: Timea Komlodi: Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria [1]
Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.
2018-08-17 MitoFit Friday
  • 08:30-09:30 MitoEAGLE preprint
  • Javier Iglesias-Gonzalez: Brainstorming on normalization (Section 3). Any idea is a good and welcome idea!!!! Let's discuss!
  • Juan Perez Valencia: Reviewing for the figure 4, the inducible uncoupling is due to the activity of UCP1. Not only for the data in the literature (that grows fast) but for some experimental observations at Franklin’s lab in Rio, it is well known that this kind of uncoupling could be made not only by UCP1, but also for 2 and 3, depending on the conditions. As showed in figure 1 and 2, lipid catabolism fuels the TCA cycle and provides substrates for the ETS, to contribute to the protonmotive force. It has been shown that the mitochondrial proton conductance of UCPs (1, 2 and 3) increase by superoxide presence (Superoxide activates mitochondrial uncoupling proteins; Nature 415, 96–99 [2002]; A signalling role for 4‐hydroxy‐2‐nonenal in regulation of mitochondrial uncoupling; The EMBO Journal 22, 4103-4110 [2003]), also depends on fatty acids, at least for superoxide induced-uncoupling.
  • Let's discuss: there are serious critical issues related to the Nature 2002 paper. ~ Gnaiger Erich (talk) 07:56, 17 August 2018 (CEST)
2018-08-10 MitoFit Friday
  • 08:30-09:15 Timea Komlodi: Facts and artefacts in measurements of H2O2 production under different oxygen regime
There is a large body of literature deciphering the role of mitochondria in reactive oxygen species-mediated signalling, which is mainly based on alterations in superoxide generation rate (further dismutated to H2O2). At sea level, the partial pressure of oxygen (O2) is around 20 kPa (concentration ≈200 µM) in the ambient air, but only < 1 kPa (≈10 µM) in the vicinity of mitochondria [4]. Quite importantly, ambient oxygen levels, also often applied in vitro, are hyperoxic for mitochondria. When reducing O2 levels towards the physiological intracellular range, contrasting results were reported either with increasing or decreasing H2O2 production rates. Such differences might arise from variability in species, tissue, detection method, respiration medium and O2 levels used. To shed light on some of the above cofounding factors, we set out to investigate the effect of various O2 concentrations on H2O2 production and O2 consumption in isolated mitochondria under well controlled conditions.To this aim, isolated mouse brain and heart mitochondria respiring with the combination of NADH-linked substrates glutamate and malate and complex II-linked substrate, succinate in a closed-chamber respirometer combined with fluorimetry (Oroboros O2k High-Resolution FluoRespirometer). O2 concentration was varied from ≈170 µM to 15-20 µM with injection of N2 in the gas phase both in the LEAK and OXPHOS states. H2O2 production was determined by the highly specific Amplex UltraRed assay in MiR05 and KCl-based media. The rate of H2O2 generation was corrected for chemical background, changes of O2 level over time and for the sensitivity of the detection system.Here we reveal that upon decrease of the O2 concentration from ≈170 µM to ≈20 µM, the rate of H2O2 formation is reduced as compared with normoxic H2O2 production in the LEAK but not in the OXPHOS state. After re-oxygenation, the rate of H2O2 generation again increases. At the same time, reducing the O2 concentration to ≈ 20 µM did not compromise O2 consumption, which is in line with the high affinity of mitochondrial respiration for O2 (p50 ≈ 0.03-0.04 kPa in OXPHOS).Taken together, when reducing the O2 concentration to the physiological intracellular range in vitro, the rate of H2O2 production declines in a metabolic states-dependent manner. In contrast, respiration was not affected by the O2 concentration down to ≈ 20 µM neither in brain nor in heart. Based on this, we can assume that the H2O2 production is more sensitive to the changes of O2 concentration than the O2 consumption.
2018-08-03 MitoFit Friday
  • 08:30-09:15 Erich Gnaiger Is there a recommendable textbook on basic thermodynamics in bioenergetics? - "Thermodynamic ignorance is also responsible for some extraordinary errors found in the current literature, particularly in the field of mitochondrial physiology (see Part 3)." (Nicholls and Ferguson 2013 Bioenergetics4, Part 3: p 27)
This is a self-reference - for thermodynamic ignorance, see Part 3 (pp 27-32).
An introduction to critical reading and pitfalls in physical chemistry. - see Gibbs energy or Gibbs force?
  • 09:15-09:45 Juan Alberto Perez Valencia Angiogenesis and evading immune destruction are the main related transcriptomic characteristics to the invasive process of oral cancer [2]
Metastasis of head and neck tumors is responsible for a high mortality rate. Understanding its biochemistry may allow insights into tumorigenesis. To that end we carried out RNA-Seq analyses of 5 SCC9 derived oral cancer cell lines displaying increased invasive potential. Differentially expressed genes (DEGs) were annotated based on p-values and false discovery rate (q-values). All 292 KEGG pathways related to the human genome were compared in order to pinpoint the absolute and relative contributions to the invasive process considering the 8 hallmarks of cancer plus 2 new defined categories, as well as we made with our transcriptomic data. In terms of absolute contribution, the highest correlations were associated to the categories of evading immune destruction and energy metabolism and for relative contributions, angiogenesis and evading immune destruction. DEGs were distributed into each one of all possible modes of regulation, regarding up, down and continuum expression, along the 3 stages of metastatic progression. For p-values twenty-six genes were consistently present along the tumoral progression and 4 for q-values. Among the DEGs, we found 2 novel potentially informative metastatic markers: PIGG and SLC8B1. Furthermore, interactome analysis showed that MYH14, ANGPTL4, PPARD and ENPP1 are amenable to pharmacological interventions.
  • 09:45-10:00 DatLab Protocol Session: Timea Komlodi: Discussion of a DL-SUIT protocol for H2O2 production and DatLab 7.3.
2018-07-27 MitoFit Friday
2018-07-20 MitoFit Friday
2018-07-13 MitoFit Friday
  • 08:30-09:00 Gerhard Krumschnabel: Respiratory control in mitochondrial preparations: isolated mitochondria, tissue homogenate, and permeabilized fibres.
Abstract: The application of high-resolution respirometry (HRR) for assessing mitochondrial energy metabolism is well established. Experimental substrate-uncoupler-inhibitor protocols for OXPHOS analysis enable the dissection of functional modules of substrate utilization, converging branches of electron transfer and coupling control for elucidation of the pathways supporting mitochondrial performance and dysfunction. Here we applied substrate-uncoupler-inhibitor titration protocols to evaluate different mitochondrial preparations from two mammalian experimental models for their suitability in HRR. In mouse heart, a tissue homogenate micropreparation was compared to a permeabilized fiber preparation. In beef heart, with abundant tissue available, we extended this comparison to include the preparation of isolated mitochondria. The permeabilized fiber preparation has its advantages, but also has limitations, specifically the diffusion limitations imposed on mitochondria for oxygen and ADP, inorganic phosphate and energy substrates. To reduce these limitations, we evaluated a method for the preparation of cardiac homogenate which is based on low-shear mechanical homogenization using the PBI-Shredder, an auxiliary tool for HRR. For mouse cardiac tissue, paired OXPHOS analysis on fibers and Shredder-samples indicated similar mitochondrial coupling control and respiratory capacities. Use of the PBI-Shredder reduced processing time of homogenate preparation compared to permeabilized fiber preparation and improved reproducibility within samples. Application of the PBI-Shredder with homogenates from mouse brain and liver yielded high-quality samples displaying tissue-specific OXPHOS fingerprints. In beef heart samples, Shredder homogenates did not reduce variability between preparations, but enhanced the coupling control ratio compared to both permeabilized fibers and isolated mitochondria. Overall, Shredder homogenates provide a valuable mitochondrial preparation, reducing preparation time and sample requirement while preserving the quality of the mitochondrial preparation.
  • 09:00-09:20 ######: Discussion of a DL-SUIT protocol for the MitoPedia: SUIT and DatLab 7.3.
  • 09:20-09:45 Erich Gnaiger: O2k-Publications in brief
2018-07-06 MitoFit Friday
MitoEAGLE secondment report Lund
  • 09:00-09:45 Erich Gnaiger: Mitochondrial pathway control of OXPHOS- and ET-capacity: Additivity of NADH- and succinate-linked pathways - N+S versus NS.
Abstract: Measuring the upper limit of mitochondrial performance
2018-06-15 MitoFit Friday
  • Dr. Sabine Schmitt, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München: Impact of tigecyclin on mitochondrial respiration, H2O2 production, ATP production and membrane potential.
Final report MtFOIE GRAS secondment
  • Carolina Doerrier, Erich Gnaiger: Update on SUIT protocols and DL7