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|Short-Term Scientific Missions
COST Action CA15203 (2016-2021): MitoEAGLE
Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping
MitoPedia topics: EAGLE
|Zdrazilova Lucie, MSc.
First Faculty of Medicine,
Charles University, CZ
|Kateřinská 1660/32, 121 08
|CZ Prague Zeman J
MitoEAGLE Short-Term Scientific Mission
- Work Plan summary
- In the beginning of the year 2018 we performed an experimental comparison of 0.5 mL and 2.0 mL chambers in collaboration with Oroboros Instruments and optimized sV-Module by using different types of Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocols (SUIT1 and SUIT2) where we evaluated the possible deviations between different volume measurements. The following step was performed in Prague where we focused on methods, which measure mitochondrial respiration and created a platform comparison between O2k-FluoRespirometer (Oroboros) and Seahorse XFe24 Bioanalyzer (Agilent).
- The aim of this STSM is to prepare a manuscript giving an overview of a platform comparison of the O2k-FluoRespirometer and Seahorse XFe24 Bioanalyzer. The Seahose XFe24 Bioanalyzer is an instrument which provides multi-well plate measurement and analyzes two processes in real time. (1) Oxygen consumption rate (OCR) which is an indicator of mitochondrial respiration, and (2) extracellular acidification rate (ECAR) measuring extracellular acidification largely depending on glycolytic processes. Cellular oxygen consumption or also called respiration causes changes in concentration of dissolved oxygen and free protons in so called „transient microchamber “and this proton concentration is measured by solid state sensor probes. The Oroboros O2k is a high-resolution respirometer used in cell and mitochondrial research to measure respiration in various types of mitochondrial preparations and living cells. It has multisensor modules and measurement is performed in experimental chambers, where the suspension of cells or other types of mitochondrial preparations are constantly mixed. Oxygen consumption is calculated real-time from oxygen concentration measured by polarographic oxygen sensors. The O2k provides the option of unlimited titrations and, therefore, the possibility to apply various SUIT protocols designed specifically for a certain project.
- This publication should give an overview of the two broadly used methods, summarizing the pros and cons. The purpose of this STSM is to focus on the advantages of these two methods from various points of view ― for instance: amount of required biological material, reproducibility of the data, instrumental specifications) and provide a practical comparison between the O2k-FluoRespirometer (Oroboros) and Seahorse XFe24 Bioanalyzer (Agilent).
- The first part of this STSM will be focused on re-analyzing the data comparing the 0.5 mL (O2k-sV-Module) and 2.0 mL O2k-chambers. Next, the platform comparison between the O2k-sV-Module and the Seahorse XFe24 Bioanalyzer will be investigated. The main part will be focused on preparing the manuscript, in which mainly results from O2k-FluoRespirometer and Seahorse XFe24 Bioanalyzer will be compared and discussed.
- A platform comparison is one of the aims of the MitoEAGLE project defined in the MoU. Our publication should encourage more scientists to report comparative results. This focus on methodology will contribute to enhance reproducibility in mitochondrial respiratory physiology and to apply broadly used methods optimally, to resolve specific research questions in various fields of application.
- Work Plan summary
- Work Plan summary
- Congenital disorders of glycosylation (CDG) are rare inherited metabolic diseases caused by abnormal protein and lipid glycosylation. PMM2-CDG is the most abundant group of CDG patients and it is caused by mutation in PMM2 gene, which leads to defects in ezyme phosphomannomutase 2 and decreases its activity. This protein catalyzes isomerization of mannose-6-phosphate to mannose-1-phosphate, which is a precurzor to GDP-mannose necessary for the synthesis of dolichol-P-oligosaccharides. PMM2-CDG patients usually suffer from muscle weakness, developmental delay and have many other clinical features. Our preliminary data indicated some interconnection between glycosylation defects and mitochondrial function abnormalities. This study may help us investigate possible disturbances in detail in mitochondrial respiration.
- The aim of this study will be to analyze fibroblasts from 11 patients with PMM2-CDG and study their mitochondrial respiration under different conditions.
- This project should help us to find bioenergetic interconnection between defect in phospohomannomutase 2 and its secondary disturbances in bioenergetic metabolism, mainly with focus on all respiratory chain complexes and also on respiratory changes after glucose starvation.
- Results could reveal more information about pathomechanism in fibroblast cell lines from PMM2-CDG patients and give us new knowledge about mitochondrial physiology and energetic metabolism in those cells. Because measured enzyme activity of PMM2-CDG fibroblasts showed increased activity in Complexes I and II and reduced activity in Complex IV, another contribution of this project could reveal the connection between proven changed activity of respiratory chain complexes in PMM2-CDG fibroblasts and its connection to mitochondrial respiration.
- For detailed measurements of mitochondrial respiration will be used OROBOROS O2k-FluoRespirometer, where substrates, uncoupler and inhibitors of respiratory chain complexes will be added to the cell suspension and changes in oxygen consumption will be measured.
- First period will be focused on evaluating of the right protocol or two combinatory protocols and evaluate the right concentration of added cells and then observe the right oligolmycin and digitonin concentration for mitochondrial respiration measurements. Afterwards the detailed mitochondrial respiration in PMM2-CDG fibroblasts will be investigated by using OROBOROS O2k-FluoRespirometer.
- All data will be analyzed and statistically processed. The output of this study is planned to be published together with already obtained data from our laboratory in Prague.
- Work Plan summary
- Work Plan summary
- Congenital disorders of glycosylation (CDG) are rare inherited diseases caused by abnormal protein and lipid glycosylation. Recent published and our preliminary data indicated possible interconnection between glycosylation defects and mitochondrial function abnormalities. The study of mitochondrial metabolism in congenital disorders of glycosylation may contribute to the elucidation of pathomechanisms in unclear metabolic diseases.
- Aim of study will be to analyze fibroblasts from patients with CDG and their impact on cellular and energetic metabolism with help of OROBOROS with classical and new applications. The project will be focused on measuring mitochondrial respiration on fresh permeabilized fibroblast cell lines from patients with CDG. Our preliminary results indicate secondary functional abnormalities in mitochondria and glycolytic dysfunction due to a breakdown of the glycosylation pathway. We would like also to focus on measurements of membrane potential and ROS production in some of these CDG patients, because changes in ROS production and also membrane potential were found previously in CDG by other methods in our laboratory.
- In parallel, we will analyze fibroblasts lines from patients with proved or suspect for other rare metabolic diseases. For example, reduced level of ubiquinone was revealed in some patient’s fibroblasts by using HPLC method. This is the reason, why we would like to focus on measuring Q-redox changes in permeabilized fibroblasts to see how reduced Q level can involve in Q-redox changes.
- This project should help us to find cellular pathways interconnections with help of complex approach by investigation of mitochondrial respiration, ROS production, membrane potential and Q-redox changes in O2k-FluoRespirometer. The identical protocol will be used for all methods, which should give us more valid correlation between these different types of measurements.
- Results could reveal more information about pathomechanisms of CDG and other selected rare diseases, give us new knowledge about mitochondrial physiology and energetic metabolism in fibroblast cell lines and could show us cooperation between different metabolic pathways. This STSM gives us opportunity to consolidate and expand cooperation with OROBOROS Instrument laboratory as well.
- First period of this STSM will be focused on learning new types of measurements on O2k-FluoRespirometer. Digitonin test and toxicity test of some chemicals will be performed for optimizing the planned protocol. Afterwards pilot experiments will be performed on control fibroblast cell lines and then on very slowly growing fibroblasts from patients with diagnosed CDG or other fibroblasts from patients with rare inherited metabolic diseases. All data will be analyzed and statistically processed. The output of this study is planned to be finally published.
- Work Plan summary
- Work Plan summary
- My name is Lucie Zdrazilova and in October 2018 I started my PhD studies focused on Congenital disorders of glycosylation and their impact on cellular and energetic metabolism. The study of mitochondrial metabolism in congenital disorders of glycosylation may contribute to the elucidation of pathomechanisms in unclear metabolic diseases. We would like to focus on measuring mitochondrial respiration on permeabilized fibroblast cell lines from patients with congenital disorders of glycosylation, because our preliminary results indicate secondary functional abnormalities in mitochondria and glycolytic dysfunction due to a breakdown of the glycosylation pathway. The growth of fibroblast cell lines from patients with congenital disorders of glycosylation is the most limiting factor for our measurements to this date and smaller amount of cells would help us to get more data in shorter time. According to this fact, I would like to participate in optimizing new method using smaller chambers on O2k-FluoRespirometer during STSM in Prof. Gnaiger´s Laboratory. This new method with new type of SUIT protocol and smaller chambers would really help to our project, on which I am focused in my postgraduation studies. Furthermore STSM would be very helpful for me to achieve a lot of new experiences with measuring on O2k-FluoRespirometer in most specialized laboratory in the world for mitochondrial respiration measurements on this instrument and will be used a lot in our following-up project.
- These results could help also to other scientists working with cell cultures and measuring mitochondrial respiration in O2k-FluoRespirometer to get their results in shorter time because of reduced material requirements. Especially scientists using fibroblast cell lines from patients with mitochondrial disorders and with congenital disorders of glycosylation or other diseases could appreciate this reduced amount. Thus, STSM will be focused to target specific goals in MoU i.e. “Providing standardized measurements to link mitochondrial and physiological performance to understand the myriad of factors that play a role in mitochondrial physiology” and devoted to complete specific WG1 and WG4.
- This STSM will be focused on creating new methodological techniques with using additional equipment for O2k-FluoRespirometer, smaller chambers. We will also focus on creating protocol which will suit the best for our following up project. In this STSM I will improve my skills of O2k-FluoRespirometer use and working with different types of cells and get knowledge of different types of cell measurements. This STSM will have a purpose of getting more information about mitochondrial physiology and energetic metabolism in fibroblast cell lines and also in different types of cells. Working in different laboratory will improve my skills in team building, socializing and also help me to improve my English communication ability. This STSM gives us opportunities consolidate and expand cooperation with OROBOROS laboratory.
- First week of this STSM will be focused on training for measurement on O2k-FluoRespirometer and running a proficiency tests. Thereafter we will focus on comparing measurement results of the 0.5 and 2.0 mL chambers with HEK293 cells and thereafter with control fibroblast cell lines by using optimized SUIT protocol. Results of STSM will serve as background for additional experiments on Seahorse XFe24 Bioanalyzer and O2k-Respirometer provided in our laboratory in Prague. The identical SUIT-3 protocol and identical conditions will be used for quantitative comparison of results obtained by the Seahorse XFe24 Bioanalyzer with attached fibroblasts, and by the O2k-FluoRespirometer with suspended cells. The output of this study is planned to be finally published.
- Work Plan summary
Visiting scientist in the Oroboros O2k-Laboratory
- June 03 to September 28 2019
- December 10 to 2019 March 08 2018
|Zdrazilova 2022 PLOS ONE
|Zdrazilova L, Hansikova H, Gnaiger E (2022) Comparable respiratory activity in attached and suspended human fibroblasts. https://doi.org/10.1371/journal.pone.0264496
|BEC 2020.1 doi10.26124bec2020-0001.v1
|Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1
|Leo 2023 ESCI Prague
|Gnaiger 2019 NatureConference
|Gnaiger E, Passrugger M, Gallee L, Felipe F Garcia-Souza, Zdrazilova L, Doerrier C (2019) High-resolution respirometry and innovations in the Oroboros O2k-technology.
|Zdrazilova 2018 MiPschool Tromso C4
|Danhelovska 2018 MiP2018
|Zdrazilova 2018 MiP2018