NextGen-O2k

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NextGen-O2k


NextGen-O2k         K-Regio MitoFit         O2k-Innovation         Houska-Award 2012         K-Regio MitoCom         Bioblast 2012


NextGen-O2k

The revolutionary all-in-one instrument to conquer mitochondrial disease

NextGen-O2k


NextGen-O2k: Horizon 2020 Framework Programme

  • EIC-SMEInst-2018-2020 — SME instrument phase 2, Grant Agreement No. 859770, Oroboros Instruments
  • Budget: € 2.35 Million
  • Duration: 24 months
  • Application: 2019-01-09; Pitch in Brussels: 2019-02-15; Granted: 2019-03-05; Start: 2019-06-01


About NextGen-O2k

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Oroboros - as a driving force in mitochondrial physiology - extends the analytical and diagnostic power of high-resolution respirometry by integration of NADH- and Q-redox monitoring in the NextGen-O2k. We aim at establishing the Oroboros quality control management for dissemination to our worldwide O2k-Network laboratories. This will become an effective contribution to address the acute reproducibility crisis of scientific investigation. In the spirit of Open Science and global networking, we will enable data sharing across projects and institutions in an Open Access database on mitochondrial physiology and pathology, to resolve the inflation crisis and ultimately the value-impact crisis of present academic publication. This will support key developments in mitochondrial medicine. In addition, we expand our business to algal biotechnology and ecology with the photobiology module of the NextGen-O2k, widening our focus from medicine to environment and climate.
- a partner in the Oroboros Ecosystem

Aims

Respiration and photosynthesis
Form the O2k to the NextGen-O2k
Between 1992 and 1994, Oroboros Instruments released its core technology for high-resolution respirometry (HRR), the Oxygraph-2k or O2k, for real-time monitoring of oxygen consumption of mitochondria, tissues, and living cells. Dissolved oxygen is measured using high-resolution polarographic oxygen sensors. Its main advantages are the unique signal stability and resolution of oxygen. HRR is the core technology present in every single of the devices and it will remain the basis of the NextGen-O2k.
In 2012, the updated version of the O2k was launched, the O2k-FluoRespirometer. The integrated Smart Fluo-Sensors combine fluorescence excitation and detection of defined wavelengths that in combination with fluorophores allow for the measurement of additional mitochondrial parameters, such as membrane potential, H2O2 production, calcium concentration, and ATP production.
The unique software DatLab has been developed for data acquisition and analysis. DatLab allows the researcher to perform calibrations (including automatic barometric and temperature adjustments), background correction, experimental SUIT (substrate-uncoupler-inhibitor-titration) protocols and data analysis.


With the O2k core technology, developed exclusively by Oroboros for high-resolution measurement of oxygen flux, the NextGen-O2k was developed.


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O2k
High-resolution respirometry (HRR) and unique combinations of the O2k with





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NextGen-O2k
Extend high-resolution respirometry (HRR) to the all-in-one device
The NextGen-O2k is a MultiSensor all-in-one instrument for in-depth studies of (1) the mitochondrial role in human diseases, and (2) mitochondria and chloroplasts ability in regulating algal growth and metabolite production rates.
(+2) Controlled wavelengths and light intensity: PhotoBiology-Module
  • Save on experimental time, reagents and samples
  • Minimize errors and ensure reproducible and accurate results
  • Highest oxygen sensitivity (5 nM detection limit)
  • Exceptional resolution (wide oxygen concentration range, between 0 - 1,000 μM)
  • Robustness (>10 years lifespan)



Objectives

The overall goal of this Phase 2 project is to complete the technical and scientific development of the new NextGen-O2k and prepare it for market launch.
WP1
» Technical developments
WP2
» Scientific developments
WP3
» Documentation
WP4
» Exploitation
WP5
» Dissemination
WP6
» Project management

Coordinator

Oroboros Instruments GmbH

Network

Partners

Our partners WGT-Elektronik and HTech have been essential in the development of the NextGen-O2k.
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Early testers

The influence of KOLs and Early testers/adopters will be used to build awareness of NextGen-O2k among researchers, academic and industrial partners. We have confirmed relevant mitochondrial and algal biotechnology researchers who want to collaborate as early testers:


Biomedical research Algal research
Prof. Anthony Moore Prof. Tom Bibby
Prof. Massimo Zeviani, Dr. Carlo Viscomi, Prof. Mervyn Singer Dr. Tomas Morosinotto
Prof. Pablo Garcia-Roves Prof. Yagut Allahverdiyeva
Prof. Christos Chinopoulos Prof. Ted Packard
Dr. Anthony Molina Prof. Claire Remacle
Dr. Marten Szibor
Prof. Howard Jacobs
Dr. Alexander Karabatsiakis

NextGen-O2k events

 WhenWhere
MiPNet24.01 IOC139 Schroecken AT2019-06-17
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Schroecken AT, 2019 June 17-22 Oroboros O2k-Workshop on high-resolution respirometry (HRR), IOC139.
MiPNet24.04 IOC140 Amsterdam NL2019-06-23
O2k-Network
Amsterdam NL, 2019 Jun 23 Pre-conference Oroboros O2k-Workshop on high-resolution respirometry (HRR), IOC140.
OroDM01 Innsbruck AT2019-07-01
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Innsbruck AT, 2019 Jul 01-03 Oroboros Distributor Meeting, OroDM01.
MiPschool Coimbra 20192019-07-08
COST Action MitoEAGLE
MiPsociety
Coimbra, PT. 2019 Jul 08-11, 12th MiP/MitoEAGLE Training School 2019. Mitochondrial respiratory physiology: Challenges on data sharing, reproducibility, and interpretation.
NextGen-O2k SME2 coaching Orily Pratt part 12019-07-22
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Innsbruck AT, 2019 Jul 22-23 NextGen-O2k SME2 coaching Orily Pratt (part 1), NextGen-O2k SME2.
Mitochondrial Physiology ‐ from Organelle to Organism 2019 Copenhagen DK2019-08-19
COST Action MitoEAGLE
Copenhagen DK , 2019 Aug 19-23. Mitochondrial Physiology ‐ from Organelle to Organism
FEBS Workshop Ageing 2019 Innsbruck AT2019-09-09
FEBS
Innsbruck, AT. 2019 Sep 09-12, FEBS Workshop “Ageing and Regeneration”.
NextGen-O2k SME2 coaching Samia Kappe part 12019-09-12
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Innsbruck AT, 2019 Sep 10-11 NextGen-O2k SME2 coaching Samia Kappe (part 1), NextGen-O2k SME2.
MiPNet24.02 IOC141 Schroecken AT2019-09-23
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Schroecken AT, 2019 Sep 23-28 Oroboros O2k-Workshop on high-resolution respirometry (HRR), IOC141.
MiPNet24.13 IOC142 Fukuoka JP2019-10-03
O2k-Network
ASMRM & J-mit 2019, Fukuoka JP, 2019 Oct 03 Oroboros Lunch Seminar at ASMRM & J-mit 2019 Fukuoka JP - Explore the Mito-World! (IOC142).
ASMRM & J-mit 2019 Fukuoka JP2019-10-03
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Fukuoka, JP, 2019 Oct 03-05. Joint ASMRM and J-mit Conference
MiP2019/MitoEAGLE Belgrade RS2019-10-13
COST Action MitoEAGLE
MiPsociety
Belgrade RS, 13-16 Oct 2019. 14th Conference on Mitochondrial Physiology: Mitochondrial function: changes during life cycle and in noncommunicable diseases - COST MitoEAGLE perspectives and MitoEAGLE WG and MC Meeting.
Neurocon 2019 Haryana IN2019-11-15
Neurocon
Haryana, IN. 2019 Nov 15-18, Neurocon 2019.
MiPNet24.15 IOC143 Haryana IN2019-11-15
O2k-Network
Haryana IN, 2019 Nov 15. Oroboros O2k-Workshop on high-resolution respirometry: Assessment of mitochondrial function in health and disease IOC143.
NextGen-O2k SME2 coaching Orily Pratt part 22019-12-03
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Innsbruck AT, 2019 Dec 03-04 NextGen-O2k SME2 coaching Orily Pratt (part 2), NextGen-O2k SME2.
OroDM02 Schroecken AT2020
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Schroecken AT, Oroboros Distributor Meeting, OroDM02. Originally scheduled for 2020 Jul 06-07 - Due to COVID-19 we change the strategy and will provide the O2k-Workshop in a virtual form - more information will follow.
1st Myocardial Function Symposium 2020 Graz AT2020-01-10Graz, AT, 2020 Jan 10-11. 1st Myocardial Function Symposium: “Targets in cardiometabolic disease”.
MiPNet25.03 IOC144 Innsbruck AT2020-01-20
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Innsbruck AT, 2020 Jan 20-22. O2k-Coaching Days - Basic IOC144.
MiPNet25.06 IOC145 Innsbruck AT2020-01-27
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Innsbruck AT, 2020 Jan 27-29. O2k-Coaching Days - Basic IOC145.
MiPNet25.10 Distributor engagement Tokyo JP2020-02-04
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Tokyo,JP, 2020 Feb 4-7. On-site O2k-Workshop on High-Resolution Respirometry and partner distribution engagement.
MitoEAGLE WG4 Innsbruck AT2020-02-26
COST Action MitoEAGLE
Innsbruck AT, 2020 Feb 26-28. WG 4 blood cell workshop and retreat - COST Action MitoEAGLE.
MitoEAGLE WG2 Innsbruck AT2020-03-09
COST Action MitoEAGLE
Innsbruck AT, 2020 Mar 9-13. WG 2 on human skeletal muscle fibers retreat - COST Action MitoEAGLE.
36th Congress Czech Nutrition Society 2020 Hradec Kralove CZ2020-09-10Hradec Kralove, CZ, 2020 Sep 10-12 . 36th annual international congress of Czech Nutrition Society
Long Night of Research 2020 Virtual Event2020-10-09
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Virtual Event, 2020 Oct 09. Oroboros at Long Night of Research, CCB. The diagnostic bioenergetic report – a milestone on the way to mitochondrial fitness and physical well-being.
NextGen-O2k Launch event 2021 Innsbruck AT2021-05-03
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Innsbruck AT, 2021 May 3-8. NextGen-O2k Launch event, NextGen-O2k SME2.


NextGen-O2k dissemination

NextGen-O2k press releases


Gentle Science

In addition to O2k-Workshops and on-site training, Oroboros will disseminate the not-for-profit initiatives on Mitochondrial Physiology and Bioenergetics Science Camps, specialized Mitochondrial Physiology Conferences, and Preprint publications:
» Oroboros is a key player in support of the COST Action MitoEAGLE
» MitoGlobal - the world-wide platform for societies and organizations supporting mitochondrial research and medicine
» MitoEAGLE with cooperation by Oroboros launched MitoFit Preprint Archives - the preprint server for mitochondrial physiology and bioenergetics
» A vision on preprints
» Mitochondrial respiratory states and rates : >530 coauthors join forces towards harmonization on nomenclature
» Gentle Science - recognizes the responsibility of the scientific community - for the quality of science, the quality of life in science, and its mission


MitoEAGLE Q-workshop

MitoFit



Support

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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.

MitoPedia: NextGen-O2k

» NextGen-O2k«
TermAbbreviationDescription
ChlororespirationChlororespiration is the phenomenon by which oxygen is consumed by a putative respiratory electron transfer chain (ETC) within the thylakoid membrane of the chloroplasts and ATP is produced. It is a process that involves the interaction with the photosynthetic ETC in which the NAD(P)H dehydrogenase enzyme transfers electrons to oxygen molecules with the assistance of the photosynthetic Plastoquinone (PQ), which acts as a non-photochemical redox carrier. Initially described in the unicellular alga Chlamydomonas reindhartdii, chlororespiration was highly disputed for years until the discovery of a NAD(P)H-dehydrogenase (Ndh) complex (plastidic encoded) and plastid terminal oxidase (PTOX) (nuclear encoded) in higher-plant chloroplasts. The PTOX, which is homologous to the plant mitochondrial alternative oxidase, has the role of preventing the over-reduction of the PQ pool while the Ndh complexes provide a gateway for the electrons to form the ETC and consume oxygen. As a result of this process there is a cyclic electron flow around the Photosystem I (PSI) that has been reported to be activated under stress conditions acting as a photoprotection mechanism and could be involved in protecting against any other stress that implies the increase of ROS formation.
Cyclic voltammetryCVCyclic voltammetry (CV) is a type of electrochemical measurement which is applied in the Q-Module as a quality control step to determine the redox potential of Coenzyme Q in the specific experimental conditions used. In cyclic voltammetry, the Q-Sensor with the three-electrode system is used to obtain information about the analyte (CoQ) by measuring the current (I) as the electric potential (V) between two of the electrodes is varied. In CV the electric potential between the glassy carbon (GC) and the Ag/AgCl reference electrode changes linearly versus time in cyclical phases, while the current is detected between GC and platinum electrode (Pt). The detected current is plotted versus the applied voltage to obtain the typical cyclic voltammogram trace (Figure 1). The presence of substances that are oxidized/reduced will result in current between GC and Pt, which can be seen as characteristic peaks in the voltammogram at a defined potential. The oxidation or the reduction peak potential values are used to set the GC (integrated into the Q-Sensor) for a separate experiment to measure the Q redox state of a biological sample. The oxidation and reduction peak potentials can be influenced by 1) the respiration medium, 2) type of CoQ, 3) polarization window, 4) scan speed, 5) number of cycles, 6) concentration of the analyte (CoQ),and 7) initial polarization voltage. For further information, see: MiPNet24.12 NextGen-O2k: Q-Module.
Hydrogen peroxideH2O2
Hydrogen peroxide
Hydrogen peroxide, H2O2 or dihydrogen dioxide, is one of several reactive oxygen intermediates generally referred to as reactive oxygen species (ROS). It is formed in various enzyme-catalyzed reactions (e.g., superoxide dismutase) with the potential to damage cellular molecules and structures. H2O2 is dismutated by catalase to water and oxygen. H2O2 is produced as a signaling molecule in aerobic metabolism and passes membranes more easily compared to other ROS.
Mitochondrial membrane potentialmtMP, Δψ [V]The mitochondrial membrane potential, mtMP, is the electric part of the protonmotive force, ΔpH+.

Δψ = ΔpH+ - ΔµH+ / F

mtMP or Δψ is the potential difference across the inner mitochondrial (mt) membrane, expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ electron or per electron moved across the transmembrane potential difference, with the number of ‘motive’ electrons expressed in the unit coulomb [C].
NextGen-O2k
NextGen-O2k InstrumentNextGen-O2k Instrument
NextGen-O2k Technical developments
Oxygen kineticsOxygen kinetics describes the dependence of respiration of isolated mitochondria or cells on oxygen partial pressure. Frequently, a strictly hyperbolic kinetics is observed, with two parameters, the oxygen pressure at half-maximum flux, p50, and maximum flux, Jmax. The p50 is in the range of 0.2 to 0.8 kPa for cytochrome c oxidase, isolated mitochondria and small cells, strongly dependent on Jmax and coupling state.
PB-ModulePB-ModuleThe PB-Module has been developed for conducting measurements of PhotoBiology, including photosynthesis. It consists of the PB-Sensor and electronic components which are an integral part of the NextGen-O2k. Measurements are recorded and evaluated with the DatLab 8 software.
PB-SensorThe PB-Sensor has been designed as a part of the PB-Module to provide with an external source of light, in the range of the blue (451 nm) or red (634 nm) wavelengths. This enables experiments for evaluating the production/consumption of O2 in the presence of light. The PB-Sensor consists on one LED and one photodiode mounted on the sensor tip behind a plastic cover. The LED emits with blue or red light on the sample and its intensity can be regulated from 0 to 5000 µEinsteins*m-2*s-1. The photodiode provides real-time measurement of the light intensity allowing for continuous adjustment to the desired value, which can also help compensate for the temperature rise due to the operation of the LED.
PhotoBiologyPBPhotoBiology is the scientific study of the beneficial or harmful effects of light, understood as non-ionizing radiation (i.e. ultraviolet, visible and infrared radiation) on living organisms. It includes topics such as the study of photosynthesis, photochemistry, photophysics, photomorphogenesis, vision, bioluminescence, circadian rhythms and photodynamic therapy. Non-ionizing (or non-ionising) radiation is any type of electromagnetic radiation that does not carry enough energy per quantum (photon energy below 10 eV) to completely remove an electron from an atom or molecule. When photons contact molecules, the molecules can absorb the photon energy and become excited, reacting with surrounding molecules and stimulating "photochemical" and "photophysical" changes.
PhotorespirationPhotorespiration is the process by which the enzyme RuBisCo oxygenates the Ribulose Biphosphate (RuBP) instead of carboxylating it as part of the Calvin-Benson cycle, thus wasting the energy produced by photosynthesis (in the form of a direct cost in ATP and NAD(P)H) and creating a product that cannot be used within this cycle, phosphoglycolate. It is estimated that approximately 25 % of RuBisCo reactions are photorespiration, meaning a potential 25 % reduction in photosynthetic output due to the carbon fixed by photorespiration being released as carbon dioxide and nitrogen as ammonia, while the other product, 3-phosphoglycerate (G3P), requires a higher metabolic cost. This process involves a complex network of enzyme and metabolite exchanges between the chloroplasts, peroxisomes and mitochondria. It is also known as the oxidative photosynthetic carbon cycle or C2 photosynthesis and abiotic conditions tend to affect it such as temperature and the atmospheric partial pressures of oxygen and carbon dioxide. Certain type of plants (C4 plants and CAM plants) and algae have biochemical and biophysical mechanisms to overcome the photosynthetic losses due to photorespiration making them more photosynthetically efficient than C3 plants. Recent plant biotechnology advances have been focused on increasing plant photosynthetic carbon fixation by reducing photorespiration loses.
PhotosynthesisPSPhotosynthesis is the process used by plants and other organisms that converts light (mostly solar) energy into chemical energy which is subsequently released to fuel organisms' activities. It has two phases: the light-dependent phase and the light-independent (dark) phase. In plants, algae, and cynobacteria, light energy is absorbed during the light phase by the pigment called Chlorophyll and used to split water and generate short-term stores of chemical energy - adenosine triphosphate (ATP), and reducing power - nicotinamide adenine dinucleotide phosphate (NADPH), with the net production of O2 gas as a waste product. And during the dark phase this chemical energy and reducing power are used to synthesize organic matter from the atmospheric CO2 in the form of carbohydrates or sugars through the metabolic pathway called Calvin-Benson cycle. The whole process is what is called oxygenic photosynthesis and is responsible for producing and maintaining the oxygen concentration of the Earth’s atmosphere. In bacteria such as the cyanobacteria photosynthesis involves the plasma membrane and the cytoplasm, and in Eukaryotic cells (plants and algae) photosynthesis takes place inside organelles called chloroplasts.
Q redox stateQr/QtThe Q redox state reflects the redox status of the Q-junction in the mitochondrial or chloroplast electron transfer system (ETS). Ubiquinones, also known as coenzyme Q, and plastoquinones are essential mobile components of the mitochondria and chloroplasts that transfer electrons between the respiratory or photosynthetic complexes of the ETS. The Q redox state is dependent on the relative activities of the ETS enzymes that reduce and oxidize the quinones. Therefore, deficiencies in the mitochondrial ETS, originating from e.g. the malfunction of respiratory enzymes (complexes), can be detected by measuring the changes of the Q redox state with respect to respiratory activity.
Q-ModuleQ-ModuleThe Q-Module, developed both for measuring the Q redox state and for cyclic voltammetry measurements, is an integral part of the NextGen-O2k and consists of the Q-Sensor, integrated electronic components in the O2k, and the DatLab software.
Q-SensorThe Q-Sensor has been designed as a part of the Q-Module for measurements with cyclic voltammetry and voltammetry, allowing for analysis of the Q redox state. The Q-Stopper with the reference electrode is called Q-Sensor, which is plugged in the NextGen-O2k. A three-electrode system is used to detect the Q redox state. Two of the three electrodes (glassy carbon and platinum electrode) are built into the Q-Stopper, while the reference electrode is removable (Reference-Electrode\2.4 mm).
Three-electrode systemA three-electrode system is the setup used in the Q-Sensor, which is an integral part of the Q-Module. This system is used in voltammetry (including cyclic voltammetry) to study the current as a function of the applied potential using three different electrodes: 1) the working electrode 2) the reference electrode, and 3) the counter electrode. The working or detecting electrode is a glassy carbon (GC) electrode that is set to a given potential and makes contact with the analyte. The potential of the working electrode is controlled by the constant potential of the a silver/silver chloride (Ag/AgCl) reference electrode, which does not pass any current. The applied potential on the surface of the GC should be sufficient to either oxidize reduced analyte (in our case Coenzyme Q) or to reduce oxidized CoQ. Thus, the counter electrode is a platinum electrode (Pt) that passes a current to counter these redox events by completing the circuit that is rate-limited by electron transfer on the GC. To determine the Q redox ratio the GC electrode is set at the oxidation peak potential, which can be determined with cyclic voltammetry.
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MitoPedia:NextGen-O2k