Difference between revisions of "Gnaiger 2021 MitoFit BCA"
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::::* [[Molina Anthony JA |Anthony JA Molina]] (version 0.4 2021-06-24): Take a look at this paper that just came out ([[Zhang 2021 PLOS ONE |attached]]) about the same dataset. | ::::* [[Molina Anthony JA |Anthony JA Molina]] (version 0.4 2021-06-24): Take a look at this paper that just came out ([[Zhang 2021 PLOS ONE |attached]]) about the same dataset. | ||
::::* [[Doerrier Carolina |Carolina Doerrier]] (version 0.4 2021-06-24): The most difficult part to follow was the ''OSI'' and the most stimulating sections those showing the bioenergetic clusters. Comments, suggestions and questions: | ::::* [[Doerrier Carolina |Carolina Doerrier]] (version 0.4 2021-06-24): The most difficult part to follow was the ''OSI'' and the most stimulating sections those showing the bioenergetic clusters. Comments, suggestions and questions: ''I''<sub>O2</sub> is not defined before Eq. 1. It is defined in line 145 (before Eq.4). Figure 1: (1) Sequence of the graphs a, b, c and d. In figure 1 and 3, graph “a” is on the upper part and “b” at the bottom, however in figure 4, 7 and 8 the sequence is different (“a” upper left, “b” upper right). I would suggest keeping the same structure to make it easy to read; (2) in Fig. 1, I would suggest the same scale for Fig 1b and Fig 1d. In lines 187 and 213 you have the first mention to tables 2 and 3. I went to tables 2 and 3 and ''OSI'' is shown there. At this point I did not know what ''OSI'' was. Eq. 10 (referred in the table 2) and the section for ''OSI'' comes later in the manuscript and I found this confuse and difficult to follow. Also Eq. 12 is shown later. I had to go to read more information about ''OSI'' and afterwards came back to table 2 and 3. In lines 265 and 266, the following sentence sounds weird to me: “LEAK respiration taken as the minimum in either L1, L2 or L3 is 24 %, 14 % and 8 % higher than flow in L1, L2 and L3, respectively.” At the beginning of section 3.2, I miss a brief explanation of why ''PSI'' was modified in the ''OSI'' index and, what is the ''OSI'' index. I find it easier to understand following the information from the website (https://www.bioblast.at/index.php/Outlier-skewness_index), maybe you could add a similar brief explanation in the manuscript (at the beginning of section 3.2, before line 277) and why this index is better than ''PSI''. Figure 2 was for me difficult to follow. The second time I went through it, it was much better. From where is coming the critical value indicative of outliers |''OSI''|>0.03? In Figure 5a you represented R3 as a function of E (shown in the graph). I would suggest to add it (''R''3) also in the figure legend. Maybe you could add “(bc)” in the label of the axis when it is baseline corrected for ''Rox''. Why the different measurements in ROUTINE, LEAK, and ET are not in italic (e.g. R1, R2 …)? Is there a reason? The cluster are in italic (e.g. ''L''C2), but not the different measurements (e.g. L1, L2, L3). In figure 6, are the measurements in LC3 the same than the measurements in ROXC2? By removing the ''r''C2 cluster (line 476, and figure 7d, e and f), the cluster LC3 is out. Do these measurements correspond to ROXC2 as well? In figure 7, is the cluster ''l''C2 is written like that just to distinguish it from ''L''C2? To understand Figure 7a, I need to go to figure 7b. It could help to see in Fig. 7a only ''L''C1 and ''L''C2, then figure 7b to identify ''l''C2 and afterwards a figure like the current Figure 7a (with ''l''C2). However, this will be too repetitive, then probably is better to keep it as it is. Once you read all figure it is very easy to understand and follow it. The values from the cluster ''l''C2 (with low coupling efficiency threshold (''E''-''L'')/''E''<0.8) are eliminated (Fig. 7b). In ROUTINE R3, the values which correspond to the same measurements (''l''C2) are not eliminated (Fig. 7b), however, we see that they differ from ''r''C1 (and the same measurements have lower coupling efficiency). Would not be correct to eliminate these measurements as well? How do you define the threshold values for outlier exclusion (Figure 7)? If people will apply the BCA for analysis of the own data, I think it will be useful to provide a short explanation. In all axis of the graphs (including ''FCR'' or coupling control efficiencies) maybe could be good to add which measurement is used, for example in Figure 7e, (''R''3-''L''min/''E''1) net control ratio. This could help the reader. In figure 8, maybe you could add in the graphs the instrument used, something similar like you have in Figure 1. Slopes and intercept are not clear for me in figure 8. | ||
::::* [[Torres-Quesada Omar |Omar Torres-Quesada]] (version 0.4 2021-06-24): Concerning the threshold values for detection of outliers (Figure 7, page 13), are these values specifically calculated for each data set or are they standard? This would be quite interesting to know if someone wants to use this analysis which own data. The BCA outlier level workflow shows a stepwise strategy to remove outliers from large-scale data. Would it be possible to apply this strategy with small-scale data? This would be relevant for example for BCA of O2k data (usually with lower population size). The excel file I find it quite useful when somebody wants to apply the same workflow with own data. | ::::* [[Torres-Quesada Omar |Omar Torres-Quesada]] (version 0.4 2021-06-24): Concerning the threshold values for detection of outliers (Figure 7, page 13), are these values specifically calculated for each data set or are they standard? This would be quite interesting to know if someone wants to use this analysis which own data. The BCA outlier level workflow shows a stepwise strategy to remove outliers from large-scale data. Would it be possible to apply this strategy with small-scale data? This would be relevant for example for BCA of O2k data (usually with lower population size). The excel file I find it quite useful when somebody wants to apply the same workflow with own data. |
Revision as of 14:16, 9 September 2021
Gnaiger 2021 MitoFit BCA
Gnaiger E (2021) Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts. MitoFit Preprints 2021.8. doi:10.26124/mitofit:2021-0008 (in preparation) |
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Bioenergetic cluster analysis – mitochondrial respiratory control in human fibroblasts
Gnaiger Erich (2021-##-##) MitoFit Preprints
Abstract: Cell respiration reflects mitochondrial fitness and plays a pivotal role in health and disease. Despite the rapidly increasing number of applications of cell respirometry to address current challenges in biomedical research, cross-references are rare between respirometric projects and platforms. Evaluation of accuracy and reproducibility between laboratories requires presentation of results in a common format independent of the applied method. When cell respiration is expressed as oxygen consumption rate in an experimental chamber, normalization is mandatory for comparability of results. Concept-driven normalization and regression analysis are key towards bioenergetic cluster analysis presented as a graphical tool to identify discrete data populations.
In a meta-analysis of human skin fibroblasts, high-resolution respirometry and polarography covering cell senescence and the human age range are compared with multiwell respirometry. The common coupling control protocol measures ROUTINE respiration of living cells followed by sequential titrations of oligomycin, uncoupler, and inhibitors of electron transfer.
Bioenergetic cluster analysis increases the resolution of outliers within and differences between groups. An outlier-skewness index is introduced as a guide towards logarithmic transformation for statistical analysis. Isolinear clusters are separated by variations in the extent of a quantity that correlates with the rate, whereas heterolinear clusters fall on different regression lines. Dispersed clusters are clouds of data separated by a critical threshold value. Bioenergetic cluster analysis provides new insights into mitochondrial respiratory control and a guideline for establishing a quality control paradigm for bioenergetics and databases in mitochondrial physiology. • Keywords: human dermal fibroblasts HDF, living cells ce, cell respiration, coupling control, oxidative phosphorylation OXPHOS, age, senescence, bioenergetic cluster analysis BCA, meta-analysis, normalization, high-resolution respirometry HRR, Oroboros O2k, Seahorse XF Analyzer, outlier-skewness index OSI, regression analysis • Bioblast editor: Gnaiger E • O2k-Network Lab: AT Innsbruck Oroboros
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Cited by
- Zdrazilova L, Hansikova H, Gnaiger E (2021) Comparable respiratory activity in attached and suspended human fibroblasts. MitoFit Preprints 2021.7. doi:10.26124/mitofit:2021-0007 - »Bioblast link«
- Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003
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Acknowledgements
- I thank Lucie Zdrazilova for collaboration and input from our parallel manuscript, and Hans Zischka, Pablo M Garcia-Roves, Omar Torres-Quesada, Anthony JA Molina, Philip A Kramer, Jenny L Gonzalez-Armenta, Mateus Grings, and the Oroboros team ― particularly Chris Donnelly, Sabine Schmitt, Timea Komlódi, and Carolina Doerrier ― for stimulating discussions and critical comments.
- To promote transparency, MitoFit Preprints encourages Open Access to relevant scientific contributions of acknowledged persons, even though it may not be possible to provide a fully balanced account of the correspondence and personal discussions.
- Lucie Zdrazilova: see Zdrazilova L, Hansikova H, Gnaiger E (2021) Comparable respiratory activity in attached and suspended human fibroblasts. MitoFit Preprints 2021.7. doi:10.26124/mitofit:2021-0007
- Chris Donnelly (v0.1 2021-06-15): One important point I believe is worthy to mention here is that in the Yépez paper they did not titrate Ama. I believe Rox is after Rot titration.
- Sabine Schmitt (v0.1 2021-06-15): The term concept-driven normalization requires explanation.
- Timea Komlódi (v0.1 2021-06-15): I found it very useful how you explain the different outlier levels and use these for excluding data. Regarding negative values, sometimes we observe negative O2 flux in the ROX state which is usually used for baseline correction. Can we use this “negative Rox” for correction which would lead to higher O2 fluxes? Or should we exclude those files where Rox is negative? Or in this case instead of baseline-corrected O2 flux we can only use flux control ratios in the whole study?
- Hans Zischka (v0.2 2021-06-16): I would separate the discussion and conclusions. The conclusions are at best unclear.
- Pablo M Garcia-Roves (v0.2 2021-06-16): I like the topic of the manuscript but I think it needs additional work to get the message deliver clearly. In my opinion it needs a different structure where you clearly define sequential steps in your rational and analysis. For example, Figure 1 address several issues at the same time and it could be confusing at the time to assess data representation. One part of the manuscript is dedicated to explain how data analysis has been performed (very important and informative: PSI, OSI, log-transformed; BCA, …). But explanations about the procedure to perform data analysis are intercalated with data representation and analysis. The work brings to my attention a way to analyze data sets that could be of interest, as you mentioned in the manuscript, for data collected during the MitoEAGLE COST action.
- Anthony JA Molina (version 0.4 2021-06-24): Take a look at this paper that just came out (attached) about the same dataset.
- Carolina Doerrier (version 0.4 2021-06-24): The most difficult part to follow was the OSI and the most stimulating sections those showing the bioenergetic clusters. Comments, suggestions and questions: IO2 is not defined before Eq. 1. It is defined in line 145 (before Eq.4). Figure 1: (1) Sequence of the graphs a, b, c and d. In figure 1 and 3, graph “a” is on the upper part and “b” at the bottom, however in figure 4, 7 and 8 the sequence is different (“a” upper left, “b” upper right). I would suggest keeping the same structure to make it easy to read; (2) in Fig. 1, I would suggest the same scale for Fig 1b and Fig 1d. In lines 187 and 213 you have the first mention to tables 2 and 3. I went to tables 2 and 3 and OSI is shown there. At this point I did not know what OSI was. Eq. 10 (referred in the table 2) and the section for OSI comes later in the manuscript and I found this confuse and difficult to follow. Also Eq. 12 is shown later. I had to go to read more information about OSI and afterwards came back to table 2 and 3. In lines 265 and 266, the following sentence sounds weird to me: “LEAK respiration taken as the minimum in either L1, L2 or L3 is 24 %, 14 % and 8 % higher than flow in L1, L2 and L3, respectively.” At the beginning of section 3.2, I miss a brief explanation of why PSI was modified in the OSI index and, what is the OSI index. I find it easier to understand following the information from the website (https://www.bioblast.at/index.php/Outlier-skewness_index), maybe you could add a similar brief explanation in the manuscript (at the beginning of section 3.2, before line 277) and why this index is better than PSI. Figure 2 was for me difficult to follow. The second time I went through it, it was much better. From where is coming the critical value indicative of outliers |OSI|>0.03? In Figure 5a you represented R3 as a function of E (shown in the graph). I would suggest to add it (R3) also in the figure legend. Maybe you could add “(bc)” in the label of the axis when it is baseline corrected for Rox. Why the different measurements in ROUTINE, LEAK, and ET are not in italic (e.g. R1, R2 …)? Is there a reason? The cluster are in italic (e.g. LC2), but not the different measurements (e.g. L1, L2, L3). In figure 6, are the measurements in LC3 the same than the measurements in ROXC2? By removing the rC2 cluster (line 476, and figure 7d, e and f), the cluster LC3 is out. Do these measurements correspond to ROXC2 as well? In figure 7, is the cluster lC2 is written like that just to distinguish it from LC2? To understand Figure 7a, I need to go to figure 7b. It could help to see in Fig. 7a only LC1 and LC2, then figure 7b to identify lC2 and afterwards a figure like the current Figure 7a (with lC2). However, this will be too repetitive, then probably is better to keep it as it is. Once you read all figure it is very easy to understand and follow it. The values from the cluster lC2 (with low coupling efficiency threshold (E-L)/E<0.8) are eliminated (Fig. 7b). In ROUTINE R3, the values which correspond to the same measurements (lC2) are not eliminated (Fig. 7b), however, we see that they differ from rC1 (and the same measurements have lower coupling efficiency). Would not be correct to eliminate these measurements as well? How do you define the threshold values for outlier exclusion (Figure 7)? If people will apply the BCA for analysis of the own data, I think it will be useful to provide a short explanation. In all axis of the graphs (including FCR or coupling control efficiencies) maybe could be good to add which measurement is used, for example in Figure 7e, (R3-Lmin/E1) net control ratio. This could help the reader. In figure 8, maybe you could add in the graphs the instrument used, something similar like you have in Figure 1. Slopes and intercept are not clear for me in figure 8.
- Omar Torres-Quesada (version 0.4 2021-06-24): Concerning the threshold values for detection of outliers (Figure 7, page 13), are these values specifically calculated for each data set or are they standard? This would be quite interesting to know if someone wants to use this analysis which own data. The BCA outlier level workflow shows a stepwise strategy to remove outliers from large-scale data. Would it be possible to apply this strategy with small-scale data? This would be relevant for example for BCA of O2k data (usually with lower population size). The excel file I find it quite useful when somebody wants to apply the same workflow with own data.
- Mateus Grings (v0.9, 2021-08-30): I really liked the ideas and data of the manuscript and agree that bioenergetic cluster analysis is a very interesting approach to analyze and compare bioenergetics data from different cells and performed with different instruments. Besides that, it was importantly pointed out that it is a great tool that can help in the evaluation of reproducibility of data. It is interesting that since it is a more visual approach, it is easier to observe differences among different data and also for the efficient detection of outliers. It was very easy and transparent to visualize the outliers and understand the problems related to them in the graphical analyzes performed for their characterization (Figures 9 and 10). I liked the general idea of normalization using internal experimental values for the comparison of data, excluding errors that may be induced by the addition of external values, such as differences in procedures to measure the external parameters in distinct laboratories. In addition, the use of normalization with flux control ratios and flux control efficiencies may show different aspects of the data. Although I do not have experience with cluster analysis and profound knowledge of all the mathematical concepts used to develop this methodology, the conceptual background was very clear and helpful for the understanding of the manuscript results. I did not profoundly understand all details in some of the results, but I could understand the main results and ideas of all of them, especially because of my previous knowledge on respirometry. I also found particularly interesting the models used in Figure 12 to observe the differentiation between the dyscapacity of fibroblasts from aged versus young donors and dyscoupling in senescent fibroblasts versus young proliferating and growth-arrested. Some problems pointed out about experiments performed with the Seahorse XF96 instrument were very relevant. Something that was mentioned and that I always think about is the use of a single uncoupler injection and the lack of uncoupler titration. Even though ideal concentrations of uncoupler for measuring ET capacity are tested in a pilot experiment for different cell types, the conditions of the cells or the assay may change in different experiments and situations, so that the uncoupler concentration needed for efficiently measuring ET capacity may vary (making it important to do a titration for each experiment). Another matter that I would like to comment about regards the normalization by cell number at the Seahorse XF96. I thought about it when I was reading the methodology used for NHDF in the Seahorse (normalization by seed count versus final cell count). When I performed some of my experiments in the Seahorse using fibroblasts from different patients with the same disease, I could not normalize the data using the seed count. This happened because the cells had very different proliferation rates and showed different adhesion patterns after seeding due to differences in patient genotypes and phenotypes. Therefore, I had to evaluate the cells after the assay to do the normalization. I think it is important to take this into account when performing normalization of experiments with attached cells.
Support
- This work was partially funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770, NextGen-O2k project. Contribution to the MitoEAGLE Task Group of the Mitochondrial Physiology Society.
Labels: MiParea: Respiration, Instruments;methods
Pathology: Aging;senescence
Organism: Human Tissue;cell: Fibroblast Preparation: Intact cells
Regulation: Coupling efficiency;uncoupling Coupling state: LEAK, OXPHOS, ET Pathway: ROX HRR: Oxygraph-2k
SUIT-003, MitoEAGLEPublication, MitoFit 2021 ace-sce, MitoFit 2021 CoQ
Term | Link to MitoPedia term | Symbol | Unit | Links and comments |
---|---|---|---|---|
catabolic rate of respiration | Cell respiration | JkO2; IkO2 | varies | flux J versus flow I |
catabolic reaction | Cell respiration | k | - | |
cell count | Count | Nce | [x] | see number of cells; countable object s=ce |
cell-count concentration | Concentration | Cce | [x∙L-1] | Cce = Nce∙V-1; count concentration C versus amount concentration c; subscript ce indicates the entity type: concentration of ce. But it does not signal 'per entity', which would be written as 'per cell' Xce. |
cell mass | Body mass | mce | [kg] | mass of cells m versus mass per cell (per single entity cell) MXce |
cell mass, mass per cell | Body mass | MXce | [kg∙x-1] | mass per single cell MXce; upper case M and subscript X signal 'per count', subscript ce signals the entity s=ce; in a context restricted to cells or molecules or a particular organism such as humans, the abbreviated symbol M [kg∙x-1] provides a sufficiently informative signal, particularly in combination with the explicit unit. |
cell-mass concentration in chamber | Concentration | Cmce | [kg∙L-1] | see Cms: Cmce = mce∙V-1; upper case C alone would signal 'count concentration' (CN is more explicit), whereas the signal for 'mass concentration' is in the combination Cm. |
concentration of O2, amount | Concentration | cO2 = nO2∙V-1 | [mol∙L-1] | [O2] |
concentration of s, count | Concentration | Cs = Ns∙V-1 | [x∙L-1] (number concentration Cohen 2008 IUPAC Green Book); the signal for count concentration is given by the upper case C in contrast to c for amount concentration. In both cases, the subscript X indicates the entity type, not to be confused with a number of entities. | |
count of Xs | Count | Ns | [x] | SI; see number of entities Xs |
coupling control | Coupling-control ratio | CCR | - | |
coupling control state | Coupling control state | CCS | - | |
electron transfer pathway | Electron transfer pathway | ET pathway | - | |
electron transfer, state | Electron transfer pathway | ET | - | (State 3u) |
electron transfer system | Electron transfer pathway | ETS | - | (electron transport chain) |
elementary entity | Entity | Xs | [x] | single countable object of sample type s |
ET capacity | ET capacity | E | varies | rate |
flow, for O2 | Flow | IO2 | [mol∙s-1] | system-related extensive quantity |
flux, for O2 | Flux | JO2 | varies | size-specific quantity |
flux control ratio | Flux control ratio | FCR | 1 | background/reference flux |
International System of Units | International System of Units | SI | - | Cohen 2008 IUPAC Green Book |
LEAK state | LEAK respiration | LEAK | - | (compare State 4) |
LEAK respiration | LEAK respiration | L | varies | rate |
living cells | Living cells | ce | - | (intact cells) |
mass concentration of sample s in chamber | Concentration | Cms | [kg∙L-1] | |
mass of sample s in a mixture | Mass | ms | [kg] | SI: mass of pure sample mS |
mass per single object | Body mass | MNX | [kg∙x1] | SI: m(X); compare molar mass M(X) |
mitochondria or mitochondrial | Mitochondria | mt | - | |
mitochondrial concentration | Mitochondrial marker, Concentration | CmtE = mtE∙V-1 | [mtEU∙L-1] | |
mitochondrial content per X | Mitochondrial marker | mtENX | [mtEU∙x-1] | mtENX = mtE∙NX-1 |
mitochondrial elementary marker | Mitochondria | mtE | [mtEU] | quantity of mt-marker |
mitochondrial elementary unit | Mitochondria | mtEU | varies | specific units for mt-marker |
MitoPedia | MitoPedia, MitoPedia: Respiratory states | |||
normalization of rate | Normalization of rate | - | - | |
number of cells | Count | Nce | [x] | total cell count of living cells, Nce = Nvce + Ndce |
oxidative phosphorylation | Oxidative phosphorylation | OXPHOS | - | |
OXPHOS-capacity | OXPHOS-capacity | P | varies | rate |
OXPHOS state | OXPHOS-capacity | OXPHOS | - | OXPHOS-state distinguished from the process OXPHOS (State 3 at kinetically-saturating [ADP] and [Pi]) |
oxygen concentration | Oxygen concentration | cO2 = nO2∙V-1 | [mol∙L-1] | [O2] |
oxygen solubility | Oxygen solubility | SO2 | [µmol·kPa-1] | |
oxygen flux, in reaction r | Oxygen flux | JrO2 | varies | |
quantities, symbols, and units | Quantities, symbols, and units | - | - | An explanation of symbols and unit [x] |
rate in ET state | Electron transfer pathway | E | varies | ET capacity |
rate in LEAK state | LEAK respiration | L | varies | L(Omy) |
rate in ROX state | Residual oxygen consumption | Rox | varies | |
residual oxygen consumption | Residual oxygen consumption | ROX; Rox | - | state ROX; rate Rox |
respiration | Respirometry | JrO2 | varies | rate of reaction r |
respiratory state | MitoPedia: Respiratory states | - | - | |
steay state | Steady state | - | - | |
substrate-uncoupler-inhibitor-titration | Substrate-uncoupler-inhibitor titration | SUIT | - | |
system | System | - | - | |
unit elementary entity | Entity | UX | [x] | single countable object |
uncoupling | Uncoupler titrations | - | - | |
volume of experimental chamber | Volume | V | [L] | liquid volume V including the sample s |