NextGen-O2k Technical developments

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


WP1: Technical developments

Abstract

In this workpackage the Q- and PB-Modules are developed for the NextGen-O2k.

• Development of the Q-Module and PB-Module and integration in the NextGen-O2k
• Testing of the NextGen-O2k in collaboration with the Early testers
• Testing and development of the software for the NextGen-O2k Modules
• Integration of the new protocols in the DatLab software

Achievements

Project Year 1: In the first year of the NextGen-O2k project, the new Q- and PB-Modules supported by the new architecture of the upgraded DatLab software have been developed and integrated. Additionally, we have successfully partnered with Key Opinion Leaders to begin testing the Q-Module prototypes. Working closely with our subcontractors WGT and H-Tech, key components such as cyclic voltammetry have been integrated into the system.

Project Year 2: During the second year of the project the testing of the PB-Module by algae KOLs began. Feedback prompted a change in the PB-Sensor that will allow a wider spectrum of research questions to be addressed.

Progress and next steps

See » Track record

• 

  Some of the Key Opinion Leader’s at the IOC141 Sept. 2019

• 

  Shaker (laboratory) for algae

• 

  Setting up our lab for algae culturing activities

MitoPedia: NextGen-O2k

» NextGen-O2k«

Term	Abbreviation	Description
Hydrogen peroxide	H2O2	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.
Light-enhanced dark respiration	LEDR	Light-enhanced dark respiration LEDR is a sharp (negative) maximum of dark respiration in plants in response to illumination, measured immediately after switching off the light. LEDR is supported by respiratory substrates produced during photosynthesis and closely reflects light-enhanced photorespiration (Xue et al 1996). Based on this assumption, the total photosynthetic oxygen flux TP is calculated as the sum of the measured net photosynthetic oxygen flux NP plus the absolute value of LEDR.
Mitochondrial membrane potential	mtMP, ΔΨp+, ΔelFep+ [V]	The mitochondrial membrane potential difference, mtMP or ΔΨp+ = ΔelFep+, is the electric part of the protonmotive force, Δp = ΔmFeH+. ΔelFep+ = ΔmFeH+ - ΔdFeH+ ΔΨp+ = Δp - ΔµH+·(zH+·F)-1 ΔΨp+ is the potential difference across the mitochondrial inner membrane (mtIM), expressed in the electric unit of volt [V]. Electric force of the mitochondrial membrane potential is the electric energy change per ‘motive’ charge or per charge moved across the transmembrane potential difference, with the number of ‘motive’ charges expressed in the unit coulomb [C].
NADH-Module		The NADH-Module, is a component of the NextGen-O2k for simultaneous measurement of oxygen consumption and NAD(P)H autofluorescence. NAD(P)H autofluorescence is used to evaluate the redox state of the NAD(P)H-pool. The NADH-Module incorporates an UV light and NADH-Sensors which include a photodiode and specific filters.
NADH-Sensor		The NADH-Sensor has been developed as a part of the NADH-Module for simultaneous monitoring of oxygen consumption and NADH redox state. The NADH-Sensor is composed of a photodiode and equipped with three supergel R370 Italian blue filters (Rosco, US).
NextGen-O2k
NextGen-O2k Technical developments
Oxygen kinetics		Oxygen 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 Light Source		The PhotoBiology Light Source (PBLS) has been designed as a part of the PB-Module to provide with an external source of light. This enables experiments for evaluating the production of O2 in the presence of light. The PBLS consists of one LED and one photodiode mounted on the PBLS head protected by a PMMA plastic cover. Three pairs of PBLS (white, blue, and red) are provided with the PB-Module. The light intensity can be regulated from 0 to 2750 µmol·s-1·m-2 (red PBLS), from 0 to 3000 µmol·s-1·m-2 (blue PBLS), from 0 to 3500 µmol·s-1·m-2 (white PBLS). An integrated photodiode provides real-time measurement of the light intensity allowing for continuous adjustment to the desired value.
PB-Module		The PB-Module has been developed for conducting measurements of PhotoBiology, including photosynthesis. It consists of the PB Light Source and electronic components which are an integral part of the NextGen-O2k. Measurements are recorded and evaluated with the DatLab 8 software.
PhotoBiology	PB	PhotoBiology is the science of the effect of light on biological processes. This includes photosynthesis, photochemistry, photophysics, photomorphogenesis, vision, bioluminescence, circadian rhythms and photodynamic therapy. Phototoxicity results from non-ionizing radiation (i.e. ultraviolet, visible and infrared radiation). Non-ionizing 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 interact with molecules, the molecules can absorb the photon energy and become excited, reacting with surrounding molecules and stimulating "photochemical" and "photophysical" changes. Respiration may be affected by light during photosynthesis or in dark respiration, with the transient response of light-enhanced dark respiration.
Photodecomposition	PD	Photodecomposition or photodegradation is the process of decay of organic material induced by increasing light intensity. Under aerobic conditions, the enhancement of photodecomposition by light intensity can be quantified by oxygen consumption in a controlled light regime.
Photosynthesis	PS	Photosynthesis is the process that converts light energy into chemical energy which is subsequently transformed to the physiological energy demand. Photosynthesis has a light-dependent and light-independent (dark) phase. In plants, algae, and cynobacteria, light energy is absorbed during the light phase by the pigment chlorophyll and used to split water and generate adenosine triphosphate (ATP) and reducing power - nicotinamide adenine dinucleotide phosphate (NADPH), with the net production of O2 as a waste product. During the dark phase ATP and NADPH are used to synthesize carbohydrates from CO2 through the metabolic pathway called Calvin-Benson cycle. Oxygenic photosynthesis is responsible for producing and maintaining the oxygen concentration of the Earth’s atmosphere. In bacteria such as cyanobacteria, photosynthesis involves the plasma membrane and the cytoplasm. In eukaryotic cells (plants and algae), photosynthesis takes place in the chloroplasts.
Q-Module	Q-Module	The Q-Module, developed for measuring the Q redox state and cyclic voltammetry, is supported by the NextGen-O2k and consists of the Q-Sensor, integrated electronic components in the O2k, and the DatLab software.
Q-Sensor		The 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).

Bioblast links: Q - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Coenzyme Q

» Coenzyme Q

» Quinone, Ubiquinone Q; oxidized

» Quinol, Ubiquinol QH₂; reduced

» Semiquinone

» Coenzyme Q2

» Q-redox state

» Q-pools

Mitochondrial pathways, respiratory Complexes, and Q

» Q-cycle

» Q-junction

» Convergent electron flow

» NS-pathway

» FNS

» FNSGp

» N-pathway

» Reverse electron flow from CII to CI

» CI

» Rotenone

» Amytal

» Piericidin

» S-pathway

» CII

» Malonate

» F-pathway

» CETF, Electron-transferring flavoprotein complex

» Gp-pathway

» CGpDH, Glycerophosphate dehydrogenase complex

» CIII

» Myxothiazol

» Choline dehydrogenase

» Dihydro-orotate dehydrogenase

NextGen-O2k and Q-Module

» NextGen-O2k

» Q-Module

» Q-Sensor

» Cyclic voltammetry

» Three-electrode system

General

» Categories of SUIT protocols

» Electron transfer pathway

» Electron-transfer-pathway state

» F-junction

» N-junction

Bioblast links: NADH - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Mitochondrial NADH and respiration

» Nicotinamide adenine dinucleotide

» NADH electron transfer-pathway state

» N-junction

» Complex I

» Rotenone

» Amytal

» Piericidin

Mitochondrial NADH production

» Tricarboxylic acid cycle

» Fatty acid oxidation

NextGen-O2k

» NextGen-O2k

Bioblast links: Hydrogen peroxide - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Specific

» Amplex UltraRed

» Discussion:Amplex UltraRed

» Horseradish peroxidase

» Hydrogen peroxide

» Resorufin

General

» Extrinsic fluorophores

» MiR05-Kit

» ROS

» Sensitivity

O2k-Procedures

» Komlodi 2021 MitoFit AmR-O2 — AmplexRed, O₂ dependence, yeast

» MiPNet20.14 AmplexRed H2O2-production

» MiPNet18.05 Amplex-Mouse-heart

Bioblast links: ATP production - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Phosphorylation pathway

» Phosphorylation pathway

» ATP synthase

» Adenine nucleotide translocase

» Phosphate carrier

Phosphorylation pathway substrates

» ADP

» ATP

» Inorganic phosphate

Phosphorylation pathway inhibitors

» Oligomycin

» Carboxyatractyloside

» Atractyloside

» Bongkrekik acid

Coupling control

» Oxidative phosphorylation

» OXPHOS capacity

» P-L control efficiency

» ROUTINE respiration

» R-L control efficiency

Respiratory complexes and coupling

» Complex I

» Complex III

» Complex IV

» Proton pump

» Electron-transfer-pathway state

ATP production measurement

» Magnesium Green

Bioblast links: pH and protons - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

pH and protons

» pH

» hydrogen ion H⁺

» hydron H⁺

» hydronium ion H₃O⁺

» hydride H^-

» proton p⁺

» pH buffering capacity

» proton flux

» proton pump versus hydrogen ion pump

» proton leak

» proton slip

» protonmotive force

O2k-pH

» O2k-Catalogue: O2k-pH ISE-Module

» O2k-Manual pH electrode: MiPNet23.15 O2k-pH ISE-Module

» O2k-SOP: MiPNet08.16 pH calibration

» File:PH-Calibration-List.xls

» NextGen-O2k, ratiometric: Carboxy SNARF 1

» NextGen-O2k, ratiometric: HPTS

» pH calibration buffers

O2k-Publications

» O2k-Publications: O2k-pH ISE-Module

HRFR - general

» O2k-Manual: MiPNet22.11 O2k-FluoRespirometer manual

» O2k signals and output

» O2k-SOP: MiPNet14.06 Instrumental O2 background

» MiPNet19.18A O2k-Series G: Start

» ESD

» O2k configuration

» O2k control

» O2k-FluoRespirometer

» O2k-Main Unit#O2k-Series

» Titration-Injection microPump

» Compare: O2k-TPP+_ISE-Module

DatLab

» DatLab 7

» MiPNet26.06 DatLab 7: Guide

» DatLab 6

» MiPNet19.18C DatLab 6: Guide

» MiPNet19.18D O2k-Series G and DatLab 6: Calibration

» Reference layouts for DatLab graphs

Bioblast links: Calcium - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Specific

» Calcium

» Calcium Green

» O2k-TPP+ ISE-Module

» Calcium retention capacity

» Permeability transition pore

Inhibitors

» Cyclosporin A

» Ruthenium red

» Bongkrekik acid

General

» Extrinsic fluorophores

» Smart Fluo-Sensor Blue

Bioblast links: PhotoBiology and plant physiology - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

PhotoBiology: photosynthesis

» PhotoBiology

» Photosynthesis

Plant physiology: respiration

» Photorespiration

» Chlororespiration

» Light-enhanced dark respiration

NextGen-O2k and PB-Module

» NextGen-O2k

» PB-Module

» PB-Sensor

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MitoPedia:NextGen-O2k