Bioblast alert 2024

Bioblast alert - 2024

Bioblast alert 2024(08) - Critical chemical concentrations: 2024-11-29

How do I determine the optimal oligomycin concentration for my assay?

Perform the oligomycin titration test, and adjust your oligomycin concentration to minimize the inhibition of ET capacity.
- »Inhibition of ET capacity by oligomycin«

Zdrazilova L, Hansikova H, Gnaiger E (2022) Comparable respiratory activity in attached and suspended human fibroblasts. »Bioblast link«

How can the uncoupler concentration affect my assay?

To measure ET capacity, uncouplers such as CCCP or FCCP must be titrated stepwise to reach optimum concentration, thus avoiding inhibition of respiration at higher concentrations.
- »Uncoupler«, »ET capacity«

What is the effective concentration of digitonin for complete plasma membrane permeabilization?

It needs to be determined for each cell type and digitonin batch. The SUIT protocol SUIT-010 was designed to evaluate the optimum digitonin concentration to permeabilize the plasma membrane.
- »Digitonin«, »SUIT-010«

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. »Bioblast link«

What concentration of palmitoylcarnitine should be used to support fatty-acid oxidation-linked respiration?

We suggest testing for the optimum concentration for your sample, as palmitoylcarnitine may inhibit respiration.
- »Palmitoylcarnitine«

Cardoso LHD, Cecatto C, Ozola M, Korzh S, Zvejniece L, Gukalova B, Doerrier C, Dambrova M, Makrecka-Kuka M, Gnaiger E, Liepinsh E (2025) Fatty acid β-oxidation in brain mitochondria: Insights from high-resolution respirometry in mouse, rat and Drosophila brain, ischemia and aging models. »Bioblast link«

Bioblast alert 2024(07) - Breast cancer: 2024-10-31

"Understanding the degree by which a specific cancer type utilizes mitochondria for processes such as macromolecule synthesis and energy transfer is required to ultimately determine targetability and therapeutic potential."

Zunica ERM, Axelrod CL, Gilmore LA, Gnaiger E, Kirwan JP (2024) The bioenergetic landscape of cancer. »Bioblast link«

from the O2k-Network »US LA Baton Rouge Noland RC« and »AT Innsbruck Oroboros«

"Etomoxir significantly and selectively impaired basal and maximal respiration (...), indicating that the catabolic contribution of lipids is higher in the more aggressive [breast cancer cell lines] models."

Lorito N, Subbiani A, Smiriglia A, Bacci M, Bonechi F, Tronci L, Romano E, Corrado A, Longo DL, Iozzo M, Ippolito L, Comito G, Giannoni E, Meattini I, Avgustinova A, Chiarugi P, Bachi A, Morandi A (2024) FADS1/2 control lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer. »Bioblast link«

from the O2k-Network »IT Florence Morandi A«

"Our findings support the notion that migratory and invasive characteristics of cancer cells are dependent on mitochondrial function."

Wang Y, Wang J, Chen L, et al (2023) PRRG4 regulates mitochondrial function and promotes migratory behaviors of breast cancer cells through the Src-STAT3-POLG axis »Bioblast link«

Bioblast alert 2024(06) - OXPHOS and other coupling control states: 2024-10-24

What are coupling-control states?

They are defined as LEAK respiration, OXPHOS and ET states, with corresponding respiration rates (L, P, and E respectively), in the various mitochondrial preparations.
- »Coupling-control state«

"The coupling sites are the respiratory Complexes CI, CIII, and CIV which pump protons towards the intermembrane space ... Pathways with a lower number of coupling sites are less likely to saturate the phosphorylation system capacity at identical rates of O₂ consumption."

Cardoso LHD, Gnaiger E (2024) OXPHOS coupling and uncoupling. Bioenerg Commun 2024.5. https://doi.org/10.26124/bec.2024-0005

“The bioenergetic characterization of liver mitochondria from IMT [inhibitor of mitochondrial transcription]-treated mice showed maintained fatty acid oxidation capacity despite markedly reduced OXPHOS capacity.”

Jiang S, Yuan T, Rosenberger FA, Mourier A, Dragano NRV, Kremer LS, Rubalcava-Gracia D, Hansen FM, Borg M, Mennuni M, Filograna R, Alsina D, Misic J, Koolmeister C, Papadea P, de Angelis MH, Ren L, Andersson O, Unger A, Bergbrede T, Di Lucrezia R, Wibom R, Zierath JR, Krook A, Giavalisco P, Mann M, Larsson NG (2024) Inhibition of mammalian mtDNA transcription acts paradoxically to reverse diet-induced hepatosteatosis and obesity. »Bioblast link«

from the O2k-Network »SE Stockholm Larsson NG« and »FR Bordeaux Mourier A«

"We demonstrated deficient platelet mitochondrial respiratory chain functions, oxidative phosphorylation (OXPHOS), and electron transfer (ET) capacity with complex I (CI)-linked substrates, and reduced [...] endogenous platelet coenzyme Q₁₀ (CoQ₁₀) concentration in UC [urothelial carcinoma] patients."

Palacka P, Gvozdjáková A, Rausová Z, Kucharská J, Slopovský J, Obertová J, Furka D, Furka S, Singh KK, Sumbalová Z (2021) Platelet mitochondrial bioenergetics reprogramming in patients with urothelial carcinoma. »Bioblast link«

from the O2k-Network »SK Bratislava Sumbalova Z«

Bioblast alert 2024(05) - SUIT reference protocols: 2024-10-18

Substrate-uncoupler-inhibitor titration (SUIT) reference protocols, RP1 and RP2, interrogate 20 mitochondrial pathway and coupling control states, including residual oxygen consumption for baseline correction, in different mitochondrial preparations.

- »SUIT-001«, »SUIT-002«

"Bioenergetic snapshots obtained by OXPHOS analysis gain a diagnostic perspective on the basis of a comparative mitochondrial database."

Timón-Gómez A, Doerrier C, Sumbalová Z, Garcia-Souza LF, Baglivo E, Cardoso LHD, Gnaiger E (2024) Analysis of mitochondrial respiratory pathway and coupling control by substrate-uncoupler-inhibitor titration reference protocols. MitoFit Preprints 2024.8. https://doi.org/10.26124/mitofit:2024-0008

With this MitoFit Preprint we invite feedback and comments, particularly on terms and abbreviations along the lines of the MitoEAGLE project and our critical discussion of ambiguities in the literature.

Living cells vs mitochondrial preparations

Information gained from simple respirometric protocols with living cells is limited compared to the analytical power of SUIT protocols with mitochondrial preparations, revealing the diversity of coupling and pathway control of mitochondrial respiration.
- »Electron-transfer-pathway state«

Bioblast alert 2024(04) - Data analysis: 2024-07-11

Can the S-pathway O₂ flux be derived by subtracting N-pathway O₂ flux from NS-pathway O₂ flux?

No, the sum of the O₂ fluxes measured in the presence of NADH-linked substrates alone and succinate as the single substrate is typically higher than using a combination of all these substrates. This effect is described as incomplete additivity:
- »Additive effect of convergent electron flow«

When should I analyze my data as flux control ratios (FCRs)?

FCRs enable the comparison of samples normalized with various mitochondrial content parameters or when sample concentration is unknown. This is achieved by internally normalizing O₂ fluxes in different respiratory states to the maximum flux in a common reference state.
- »Flux control ratio«

Internal normalization of rate – what is the difference between flux control ratio and flux control efficiency?

A flux control ratio is the ratio of oxygen flux in a specific respiratory control state, normalized for maximum flux in a common reference state. Flux control efficiency expresses the control of respiration as a fractional change of oxygen flux or flow between protocol steps. Both values vary between theoretical lower and upper limits of 0 and 1.
- »Gnaiger 2020 BEC MitoPathways - Chapter 3«

Did you know that if ET capacity is lower than OXPHOS capacity (P>E) in living cells or mitochondrial preparations, then you have encountered an experimental artifact? The possible triggers of this experimental artifact are:

- loss of oxidative capacity during the time course of the respirometric assay,
- insufficient uncoupler concentrations,
- over titration of an uncoupler, which inhibits ET capacity,
- inhibition of ET capacity due to the use of high oligomycin (inhibitor of the ATP synthase) concentrations employed to measure LEAK respiration prior ET capacity.

»Gnaiger - MitoEAGLE Task Group (2020) Mitochondrial physiology«

Bioblast alert 2024(03) - Respiratory states: 2024-03-06

Here is a system of terms and abbreviations for mitochondrial respiratory pathway and coupling control states.

Although states and rates are distinguished by different symbols for coupling control states, such a distinction is not suggested for pathway control states and rates to avoid handing and overwhelming number of different symbols.
- »MitoPedia: Pathway and coupling control states«

Do you know the difference between uncoupled, noncoupled and dyscoupled respiration?

Noncoupled respiration is induced experimentally for evaluation of ET capacity, while uncoupling and dyscoupling are respectively caused by physiological and pathological conditions that exert an influence on proton leak and slip.
- »Noncoupled respiration«, »BEC 2020.01«

What is ROUTINE respiration?

ROUTINE respiration is the respiratory activity of living (non-permeabilized) cells in the physiological coupling state, which is controlled by cellular energy demand, energy turnover and the degree of coupling to phosphorylation.
- »Gnaiger 2020 BEC MitoPathways - Chapter 2.4«

What is the limitation of State 3 respiration compared to OXPHOS capacity?

OXPHOS capacity is defined as the maximal respiratory capacity in the OXPHOS state in the presence of kinetically-saturating concentration of ADP, P_i, fuel substrates and O₂. However, State 3 as defined by Chance and Williams (1955) can lead to underestimation of OXPHOS capacity and overestimation of E-P excess capacity due to kinetic limitations of the phosphorylation system.
- »OXPHOS capacity«, »E-P excess capacity« -
-Gnaiger - MitoEAGLE Task Group (2020) Mitochondrial physiology - Table 3«

Bioblast alert 2024(02) - Bioenergetics Communications and EBEC2024: 2024-02-15

The tale of two “best-friends,” proteins BID & MTCH2, pivotal regulators of the apoptosis and metabolism programs at the mitochondria.

Gross A, (2024) A BID-MTCH2 love story: Energizing mitochondria until apoptosis do them part? Bioenerg Commun 2024.1. https://doi.org/10.26124/bec:2024-0001

EBEC2024 What is Life? Spotlights on Mito and Chlora:

Cutting-edge research on mitochondria & chloroplasts, metabolism, and disease
2024 August 26-31, Innsbruck, Austria
- »Registration and abstract submission open«

Rewarded Open Access - compensating authors..

Together with the MiPsociety, BEC is raising funds to reward the first 10 papers accepted for publication in BEC in 2024: 600 EUR to attend EBEC2024 and 2-year MiPmembership. -»Support open access in science«
- »Submit a manuscript«

Bioblast alert 2024(01) - Fatty acid oxidation: 2024-02-08

Measuring fatty acid oxidation (FAO)-linked respiration requires a low concentration of malate

In the absence of malate, accumulation of Acetyl-CoA inhibits FAO.
- »Fatty acid oxidation pathway control state«, »Mitochondrial Pathways: Chapter 5«
- communicated by »Cecatto Cristiane« and »Cardoso Luiza«

"The capacity of FA oxidative phosphorylation (F-OXPHOS) with palmitoylcarnitine was up to 4 times higher than respiration with octanoylcarnitine. The optimal concentration of palmitoylcarnitine was 10 μM which corresponds to the total concentration of LC acylcarnitines in the brain."

Cecatto C, Cardoso LHD, Ozola M, Korzh S, Zvejniece L, Gukalova B, Doerrier C, Dambrova M, Gnaiger E, Makrecka-Kuka M, Liepinsh E (2023) Fatty acid β-oxidation in brain mitochondria: Insights from high-resolution respirometry in mouse, rat and Drosophila brain, ischemia and aging models. MitoFit Preprints 2023.10. https://doi.org/10.26124/mitofit:2023-0010

from the O2k-Network »AT Innsbruck Oroboros« and »LV Riga Liepins E«

"... several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the β-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII."

Gnaiger E (2024) Complex II ambiguities ― FADH2 in the electron transfer system. »Bioblast link«

from the O2k-Network »AT Innsbruck Oroboros«

"Clinically approved cardiovascular and diabetes drugs reduce LC acylcarnitine concentrations in vivo and it would be worth investigating whether these compounds could be repurposed."

Dambrova M, Cecatto C, Vilskersts R, Liepinsh E (2022) Mitochondrial metabolites acylcarnitines: therapeutic potential and drug targets. Bioenerg Commun 2022.15. https://doi.org/10.26124/bec:2022-0015

from the O2k-Network »AT Innsbruck Oroboros« and »LV Riga Liepins E«