Talk:MitoEAGLE preprint 2017-09-21

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» Manuscript phases and versions

Manuscript phases and versions - an open-access apporach

COST Action MitoEAGLE
This manuscript on ‘Mitochondrial respiratory states and rates’ is a position statement in the frame of COST Action CA15203 MitoEAGLE. The list of coauthors evolved beyond phase 1 in the bottom-up spirit of COST.
The global MitoEAGLE network made it possible to collaborate with a large number of coauthors to reach consensus on the present manuscript. Nevertheless, we do not consider scientific progress to be supported by ‘declaration’ statements (other than on ethical or political issues). Our manuscript aims at providing arguments for further debate rather than pushing opinions. We hope to initiate a much broader process of discussion and want to raise the awareness on the importance of a consistent terminology for reporting of scientific data in the field of bioenergetics, mitochondrial physiology and pathology. Quality of research requires quality of communication. Some established researchers in the field may not want to re-consider the use of jargon which has become established despite deficiencies of accuracy and meaning. In the long run, superior standards will become accepted. We hope to contribute to this evolutionary process, with an emphasis on harmonization rather than standardization.
  • Phase 1: The protonmotive force and respiratory control
» The protonmotive force and respiratory control - Discussion
» MitoEAGLE preprint 2017-09-21 - Discussion
  • Phase 2: Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1
» MitoEAGLE Task Group States and rates - Discussion
  • Phase 4: Journal submission
  • Target: CELL METABOLISM, aiming at indexing by The Web of Science and PubMed.
Coauthors
  • 2017-09-21 Version 01: 105 coauthors
  • 2017-10-15 Version 10: 131 coauthors
  • 2018-01-18 Version 20: 168 coauthors
  • 2018-02-26 Version 30: 225 coauthors
  • 2018-08-20 Version 40: 350 coauthors - EBEC Poster
  • 2018-10-17 Version 44: 426 coauthors - MiPschool Tromso-Bergen 2018
  • 2018-12-12 Version 50: 517 coauthors - Submission to the preprint server bioRxiv not successful
  • 2019-02-12 Preprint version 1: 530 coauthors
  • 2019-03-15 Preprint version 2: 533 coauthors
  • 2019-04-24 Preprint version 3: 533 coauthors
  • 2019-05-20 Preprint version 4: 542 coauthors
  • 2019-07-24 Preprint version 5: 612 coauthors
  • 2019-08-30 Preprint version 6: 622 coauthors - Preprint publication doi:10.26124/mitofit:190001.v6


Phase 2.1: Discussion

  • Please find attached the pdf with the references, and some comments (yellow highlights). What I also suggest is to put the concept of “where is the force in the pmf” and “where is the gradient” in the electrochemical gradient” in a rather prominent position in the manuscript.
  • Updated version reads very well. I agree with you that mitochondrial dynamics terminology is out of the context for the current report.
Here you go for the references:
Line 214: Please add reference for mitochondrial encoded small RNA "Sripada et al. 2012"
Sripada L, Tomar D, Prajapati P, Singh R, Singh AK, Singh R (2012) Systematic analysis of small RNAs associated with human mitochondria by deep sequencing: detailed analysis of mitochondrial associated miRNA. PLoS One. 7(9):e44873.
Line 229: Please add reference "Chan DC 2006"
Chan DC (2006) Mitochondria: dynamic organelles in disease, aging, and development. Cell 125(7):1241-52.
Line 852: Please add reference for distinct mitochondrial morphological structures "Collins et al. 2002"Collins TJ, Berridge MJ, Lipp P, Bootman MD (2002) Mitochondria are morphologically and functionally heterogeneous within cells. EMBO J 21(7):1616-27.
Gnaiger E : Sorry for the late response. There was great feedback, and I added many suggestions. Now the reference Chan (2006) is included in the newest version.
concerning the abbreviations IMM and OMM, I would insist not to replace what is already a common custom for the names of the mitochondrial membranes. Moreover, to my knowledge, the proper definition is "Inner/Outer Mitochondrial Membrane" and not "mitocondrial Inner/Outer Membrane" (mtIM/mtOM, according to your suggestion?).
  • 2017-11-26 Gnaiger E Version 19: New figure and updated Table 2
    Fig. 3. Mechanisms of respiratory uncoupling.
  • 2017-11-17 MitoEAGLE Working Group Meetings, 09:00 – 10:30 WG1: SOPs and user requirement document: Protocols – Terminology – Reporting
  • Instead of “In addition to mechanical permeabilization during homogenization of fresh tissue, saponin may be applied to ensure permeabilization of all cells.” Proposed: “In addition to mechanical permeabilization during homogenization of fresh tissue, either detergents (e.g. saponin) or toxins may be applied to ensure permeabilization of all cells.”
  • Ideal vs real mitochondrial preparations.
The manuscript discusses the theoretical case when
1. all mitochondria are functional in the preparation
2. in all mitochondria ATP production is under the control of proton motive force.
The significant role of mitochondrial purity and integrity should be emphasized.
1. Preparations with dead mitochondria – i.e. not functional.
2. Preparations with broken mitochondria, which may be enzymatically partly functional.
E.g. succinate may be still oxidized, but not under the control of proton motive force (noncoupled substrate oxidation).
  • Gnaiger E: one paragraph should be dedicated to purity and integrity.
  • Mitochondrial yield.
  • Hoppel C does not agree with the statement in line 302 “loss of a significant fraction of mitochondria.” In his laboratory 70-80% of mitochondria is recovered.
  • Gnaiger E: 20-30% is still significant.
  • Stankova P: according their data, 70% of rat liver mitochondria is lost in the nuclear fraction.
  • Gnaiger E: information on mitochondrial yield might be included in the supplement of the paper with direct link in the text.
  • Recommendations.
Conclusion section: the most important recommendations based on the paper should be summarized, e.g.
- report the yield after isolation of mitochondria
- report the concentrations of chemicals per amount of cells / mitochondria
  • UCP1: The terms “channel” and “facilitating” in the sentence "UCP1 is a proton channel of the mtIM facilitating the conductance of protons across the mtIM" are not precise enough.
Proposed alternative: “UCP1 is a member of mitochondrial carrier family which is involved in the translocation of protons across the mitochondrial inner membrane.”
  • Table 2.
“Loosely coupled” is a term which is generally accepted and used in the field of ROS production. Such a term implies something which does not exist (due to the potentially misleading terminology).
New alternative: “Partially coupled”.
  • Another proposed term: Acoupled respiration: respiration not associated with proton flux
  • 2017-11-16 Petit PX on mitochondrial markers (parts 4.3 and 4.4).
The part “4.4. Evaluation of mitochondrial markers” does not make much sense. Transmission electron microscopy or cardiolipin content measurement is not used routinely in normalization of respirometric data.
Since the manuscript is too long, it might be considered to remove / drastically reduce the mt markers part with reference to later parts of the series. Such a topic should be carefully elaborated and is especially important.
  • Hopefully these suggestions aren't too late or not helpful.
2017-11-09 MitoEAGLE preprint 2017-09-21 Version 15
  • Added new Eq. 3 and 4.
  • In my opinion the manuscript is well constructed and presented. The fundamentals of mitochondrial respiration are discussed in detail and at the same time in a clear manner, thus giving the possibility to investigators with different background and experience to follow the concepts. It is a useful reference text. I don't have additional comments.
  • There are only few comments from my side, my opinion is, that it is good and necessary paper.
  • I carefully read the paper. Indeed very nice and interesting; already forwarded to the people in the lab as a must-read article.
I took the opportunity to add some minor corrections in Box 1, Introduction and Conclusions parts mostly from the point of view of cellular-molecular biology (I have no added any References of ours but I can send them if you wish).
We are working on the crosstalk of mitostatic and proteostatic modules both in cells and in vivo in Drosophila or in cells isolated from patients treated with anti-tumor drugs (papers under revision). Apart from physiology measurements we also study in vivo mt dynamics, motility, colocalization with proteostatic modules and physiology by exploiting flies’ genetics and using specific fluorescent reporters.
In any case please feel free to use or reject these notes.
Again thanks for this excellent effort.
  • please find attached a few comments to manuscript.
It is great!! Excellent work!!
  • I fully support the manuscript MitoEAGLE preprint 2017-09-21(04). The protonmotive force and respiratory control: Building blocks of mitochondrial physiology Part 1.
  • I must say that I thoroughly enjoyed reading through this position-stand, albeit quite late in the review process so my apologies in advance!
It was full of interesting ideas/concepts/considerations, as to be expected, and it certainly didn't disappoint!
Bravo for having completed the paper and I appreciate how much hard work has gone into this; bravo also for pulling together so many outstanding investigators; the true measure of gentle(wo)manly science!
I have made a number of comments/modifications/suggestions that I hope may be of some use; if not, please feel free to disregard and I hope they're taken in the good spirit that they're intended!
From a conceptual standpont, I have sugegsted that we consider some more thought with regards:
[1] Evolutionary significance of oxygen/metabolism...and my personal interests hone the focus unabashedly towards the human brain...the gas guzzling energy hog that it is!
[2] Mitochondrial free radical formation and cell signalling...further justifying our interest in mitochondrial physiology!
[3] The emerging concept of quantum biology and its application to mitochondrial physiology.
  • I am happy with the treatment of Regulation and Control in the current version of the preprint, and I found the document as a whole gives a useful account of the issues involved in ensuring comparability and usefulness of measurements.
  • Please find attached an annotated version of the pre-print. I hope that my comments make sense and that they might be useful to some extent. Please let me know if you want more extensive details on specific comment(s).
As an general comment, the paper is probably a bit short of references in many places.
  • Here is the MitoEAGLE manuscript with my inputs.
Thank you for the opportunity to be part of this excellent initiative.
  • Congratulations to the revised manuscript which is very well written ! I carefully read the manuscript focusing mainly on the Introduction and 2. Respiratory coupling states including fig 1,2, 3 and 4. The figures are very clear and well described. From my side with my expertise I strongly agree with the methods described as well as with the outcome and statements of this paper. From my side I do not have any additional comments to add on this revised manuscript.
  • The manuscript has changed a lot, and reads very well in my opinion.
Attached are some very minor comments for you to consider.
Congratulations on this open project, and good luck with the reviews.
  • Please find attached some (minor) edits/suggestions on the joint MitoEAGLE manuscript. Overall, the manuscript is in great shape and I believe will be a very useful practice resource for both investigators who are new to mitochondrial energetics and those who are more experienced.
  • Thank you for your continuing work on this manuscript. We considered that expansion around the methodology used to determine mitochondrial content may be useful (section 4.3. Normalization for mitochondrial content). Attached is a prepared section for your consideration.
  • I have reviewed the 2017-09-21 version of the manuscript and have no substantial comments. I confirm that I have read and approved this version of the manuscript and I am happy for it to be submitted for publication. Nevertheless, below I have identified a few very minor items for your consideration. Please take or leave any of these comments as you like.
One question… perhaps you can explain why the switch in the manuscript from the original ETS to the new ETP? I very much liked the term ETS (system), as it invoked well the notion that there is more than one ‘path’ for electron transport. While a pathway can be convoluted, it is singular, which doesn’t seem to invoke (to my mind at least), a range of different ‘paths’ converging at the Q junction. This is just my simple take on it! I note that you still use the term ETS further down in the manuscript, so the usage of ETP and ETS should probably be reconciled throughout the manuscript.
Rossiter comments on 2017-09-21 version
Line 274 Delete “additionally” (it is repeated earlier in sentence).
Line 294 The oxidative and phosphorylation pathways comprise…
Line 447 Reference missing
Line 583 Here you revert to ETS (system). Above it was ETP (pathway)
Line 898 Above these “formats” were referred to as “parts”. Consider changing text to read: “…in either an electrical or chemical isomorphic parts (Table 4).”
Line 902 change “nominator” to “numerator”
Line 1171 I think “content” here should be “concentration”. The sentence alludes to “cell respiration”, which I assume means that some aspect of cell number or cell volume is being assessed, in which case the “amount” of mitochondria would be expressed as a concentration in that preparation (mitochondrial content per volume) and not as a total content… I think. Please check the correct use of content versus concertation here.


  • In the idea to add my experience, and participating as a co-author, I have carefully read the MitoEAGLE preprint, particularly focusing on the normalization issues which is what I thought my expertise could be, but it is so well written that I had nothing to add! The only small thing that I found was on page 18, line 446, there is a missing reference. Here is the paper (Greggio, Cell Metabolism, 2017) that I was mentioning in an earlier email where we have used the proposed nomenclature. While we were writing that manuscript, it was clear that a common language and protocols were needed. I struggled a fair amount with reviewers as I wanted to use unequivocal labels not referring to respiratory states. Still today, each time I present at a conference, I insist on the importance of having one language and relate to the MitoEAGLE guidelines. So as you see, even not being necessary active in the MitoEAGLE network and joined only later, I am one of the most fervent defenders!
  • This is a very mice initiative which I am glad to be part of.
I have only few minor suggestions that you find in the attached document.
2017-10-04 MitoEAGLE preprint 2017-09-21 Version 06
  • 'Electron transfer system' is changed to 'electron transfer pathways; ETS to ET pathways
  • 'Phosphorylation system' is changed to 'phosphorylation pathway'
  • 'OXPHOS system' is changed to 'OXPHOS pathway'
  • 'ETS capacity' is changed to 'ET capacity' (compare: OXPHOS capacity)
  • 'ETS state' is changed to 'ET state' (compare: OXPHOS state)
  • It’s becoming a very nice paper, thanks so much for the input and all the work! I don’t have any further suggestions to the current version. Looking forward to seeing the final version!
  • Here are a few more edits on the version preprint_2017-09-21
  • It looks quite good and detailed regarding OXPHOS. I would like to make some contribution to the paper, on aspect of "Is global assesment of cellular oxidative stress necessary for evaluation of mitochondrial oxidative stress.". Despite the evaluation of global oxidative stress and antioxidant capacity is closely related with mitochondrial oxidative stress, this part seems missing. So if you also find appropriate, I would like to prepare one short paragraph about this issue.
  • Gnaiger E
Thank you very much for your considerations. “Is global assesment of cellular oxidative stress necessary for evaluation of mitochondrial oxidative stress” is a highly relevant question. It should be on the list of one of our future manuscripts. Perhaps it is possible, but it may even be beyond the scope of our entire MitoEAGLE mission, since there are othe COST Actions addressing oxidative stress. What do you think about this level of thoughts?
  • after reading the new version of the manuscript, I feel that it has been further improved.
My minor comments is very brief.
p.6, line 181-182
For me sounds strange too write: As part of the OXPHOS system, these powerhouses of the cell...
Who are (besides mitochondria) the other part(s)?
My proposal is: In addition to the OXPHOS system, the powerhouses of the cell contain…
p.14, line 372
OXPHOS as abbreviation was already indroduced. Therefore, I propose to write: The capacity of OXPHOS….
p.12, line 405
I propose to substitute in F1F0, 0(zero) by O. Originally, O was used to indicate oligomycin-sensitivity.
p.50, line 1233
Sentence: Mitochondrial and cell respiration is the process of highly exothermic energy transformation….
According to my understanding, exothermic has to be substituted by exergonic.
  • Great effort.
  • On text of the MitoEAGLE manuscript, I like page 20~26 on OCR on different stages; Nowadays many scientists are using basal respiration, ATP turnover, maximum repiration to define the OCR. I wonder if you could put these definitions connected to your OCR, LEAK OXPHOS, ETS, ROX.
I like the manuscript very much and it is my honor to be a supporting co-author.

Many thanks for your kind comments. The appropriate corrections have been made.

  • Gnaiger E I have added the term ‘ATP turnover’ from your Email to our manuscript: “In general, it is inappropriate to use the term ATP production or ATP turnover for the difference of oxygen consumption measured in states P and L.”
‘Basal respiration’: Would you agree on the perspective provided in MitoPedia: http://www.bioblast.at/index.php/Basal_respiration
‘Maximum respiration’: This term is not sufficiently in the context of respiratory control. :::: Do you suggest that this should be stated explicitly, or is it sufficiently clear as an implicit key message of the report?


  • In my opinion the manuscripts reflects the current state of terminology very well and will serve excellently for teaching purposes. I would like to contribute by adding/challenging a couple of details from our experiences working with permeabilized muscle fibers.
Line 575: “In permeabilized muscle fibre bundles of high respiratory capacity, the apparent Km for ADP increases up to 0.5 mM (Saks et al. 1998), indicating that >90% saturation is reached only at >5 mM ADP.”
n our hands, performing two-step 2.5 - 5 mM ADP titrations has shown that 2.5 mM ADP is sufficient to achieve maximal OXPHOS (Malate+Glutamate+Pyurvate+Octanoyl carnitine+Succinate but also other substrate combinations) in permeabilized muscle fibers of both rat and human skeletal muscle. There is no additive respiratory effect beyond 2.5 mM ADP. However, for longer evaluation protocols it might be “safer” to use 5 mM ADP.
  • I focused on the sections that I felt are relevant to my level of knowledge. I think it's a very good and educational manuscript, very elaborate and structured. I don't have any input on the contents as such, only a few suggestions on minor text adjustments.
  • I appreciate your efforts to increase the visibility and aid harmonization within this field. I support the initiative and you may well add my name to the growing list of supporters.
  • I added two text sections on pages 10+11 of the manuscript plus references, concerning protein-driven mechanisms of respiration control. Please have a look at the attached pdf and find the two text boxes within the mansucript´s on pp. 10+11.
  • (6) Regulatory proteins, such as the inhibitory factor 1 (IF1), function in mitochondria of mammals challenged by severe O2 deprivation (hypoxia, anoxia, ischemia) to keep the reversal of proton pumping by complex V (ATP synthase), and, hence, the resulting ATP hydrolysis plus production of reactive oxygen species (ROS), at bay (Matic et al 2016; Esparza-Molto et al. 2017). HIF-1, on the other hand, coordinates access of mitochondrial substrates with hypoxia, in part through the transcriptional activation of the pyruvate dehydrogenase (PDH) inhibitor PDK1. This slow-down of the PDH reaction starves the tricarboxylic acid (TCA) cycle of pyruvate substrate, which, in turn, helps in decreasing respiration rates during conditions of low oxygen availability (Kim et al. 2006, Papandreou et al. 2006). As such, HIF-1 may well contribute in driving the switch from an oxy-regulated to an oxy-conforming mode of respiration (Gnaiger 2003; Gorr 2010; 2017).
  • References:
  1. Matic I, Cocco S, Ferraina C et al. Neuroprotective coordination of cell mitophagy by the F1Fo-ATPase inhibitory factor (IF1). Pharmacol Research 2016;103:56-68.
  2. Esparza-Molto PB, Nuevo-Tapioles C and Cuezva JM. Regulation of the H+-ATP synthase by IF1: a role in mitohormesis. Cell Mol Life Sci 2017;74:2151-66.
  3. Kim J-W, Tchernyshyov I, Semenza GL and Dang CV. HIF-1-mediated expression of pyruvate dehydrogenase kinase: A metabolic switch required for cellular adaptation to hypoxia 2006;3:177-85.
  4. Papandreou I, Cairns RA, Fontana L, Lim AL and Denko NC. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. 2006;3:187-97.
  5. Gnaiger E. Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. 2003;543:39-55.
  6. Gorr TA, Wichmann D, Hu J et al. Hypoxia Tolerance in Animals: Biology and Application. Physiol Biochem Zool 2010;83:733-52.
  7. Gorr TA. Hypometabolism as the ultimate defence in stress response: how the comparative approach helps understanding of medically relevant questions. Acta Physiol. 2017;219:409-440.
  • EG: Great I added it quickly.
  • I have provided specific comments in the attached document. It is extremely comprehensive and will be very useful for a range of researchers investigating mitochondrial respiration.
In publishing this manuscript we should think as much as possible about the researchers that are very interested, but maybe not experts, in mitochondrial research. With this in mind I have suggested the simplification of some sections and the include of some additional introductory sentences in some sections to help provide more of a basis for the information being provided.
  • I would like to introduce myself as an early career contributor in your excellent work to standardize the nomenclature for mitochondrial bioenergetics. Currently, I am working as a postdoctoral fellow at Temple University, Philadelphia USA and my work focused on the role of calcium in mitochondrial bioenergetics and functions. As a leading author, I discovered the molecular mechanism of one of mitochondrial calcium uniporter regulator (MCUR1) mediated regulation of uniporter function (Cell Reports, 2016). Recently, as a co-first author, I have shown that mitochondrial calcium uniporter senses the reactive oxygen species through the conserved cysteine residue (Molecular Cell, 2017). Besides these contributions in the field of mitochondrial biology, I was the part of the discovery of SPG7 as mitochondrial permeability transition pore from our group (Molecular Cell, 2015) and the association of small RNAs with mitochondria (PlosOne, 2012). I have thoroughly reviewed the preprint of the “The protonmotive force and respiratory control: Building blocks of mitochondrial physiology”. This is an excellent and comprehensive resource for the researchers working in the area of mitochondrial bioenergetics. I have three minor comments on Page 7 and Page 11. I hope these could be useful.
  • Thank you very much for your invitation. I would like to sign the article with you. It is very interesting and accurated article.
  • I think this reads very well. I have made a few comments that might be helpful.
2017-09-24 MitoEAGLE preprint 2017-09-21 Version 04
  • I reviewed the Preprint of the “Mitochondrial Respiratory Control” joint paper and found it to be quite a useful resource. There are minor editorial updates but overall the main theme and concepts are presented well and I have no major considerations at this point. Thank you for including us in the manuscript as it is very important for standardization of nomenclature.
2017-09-21 MitoEAGLE preprint 2017-09-21 Version 01

Phase 1: Work flow 44 versions until 2017-09-18

 » Talk:The protonmotive force and respiratory control