Talk:MitoEAGLE Task Group States and rates

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» Gnaiger 2019 MitoFit Preprint Arch

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  • Friday, 2018-12-14

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BioRxiv_preprint_server_for_biology - From FAQ: "bioRxiv is intended for rapid sharing of new research. Some review articles contain new data/analyses and may therefore be deemed appropriate. Reviews that solely summarize existing knowledge are not appropriate and neither are term papers, book excerpts, and undergraduate dissertations." - However, we find the following statement on the bioRxiv website: "Some review articles contain new data/analyses and may therefore be deemed appropriate. Reviews that solely summarize existing knowledge are not appropriate and neither are term papers, book excerpts, and undergraduate dissertations."

Manuscript phases and versions towards the Preprint


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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


Circulars


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» 2018-11-30 MIG circular
Your message dated Fri, 30 Nov 2018 02:17:25 with subject "MitoEAGLE manuscript submission" has been successfully distributed to the MITOCHONDRIA-L list (1840 recipients).
Dear MIG members:
We send you an update on the MitoEAGLE manuscript ‘Mitochondrial respiratory states and rates’ as a final invitation to join as coauthors by contributing to this manuscript. At the same time this is a big thanks to those MIG members who contributed already among the 489 coauthors so far.
“As the knowledge base and importance of mitochondrial physiology to human health expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. .. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes.”
MIG circulars on this manuscript have been distributed previously since April 2017. Now we are initiating the process of submission to a preprint server and journal (Cell Metabolism as an option). This will take a few weeks, during which time final corrections and comments from additional co-authors are welcome.
The newest version 48 is available for download:
» www.mitoeagle.org/index.php/MitoEAGLE_preprint_States_and_rates
The coauthor invitation is as follows (page 38 of the pdf file):
S2. Authors

This manuscript developed as an open invitation to scientists and students to join as co-authors in the bottom-up spirit of COST, to provide a balanced view of mitochondrial respiratory control and a consensus statement on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes.
Co-authors are added in alphabetical order based upon a first draft written by the corresponding author, who edited all versions. Co-authors confirm that they have read the final manuscript, possibly have made additions or suggestions for improvement, and agree to implement the recommendations into future manuscripts, presentations and teaching materials.
We continue to invite comments and suggestions, particularly if you are an early career investigator adding an open future-oriented perspective, or an established scientist providing a balanced historical basis. Your critical input into the quality of the manuscript will be most welcome, improving our aims to be educational, general, consensus-oriented, and in practice be helpful to students working in mitochondrial respiratory physiology.
To join as a co-author, please feel free to focus on a particular section, providing direct input and references, and contributing to the scope of the manuscript from the perspective of your expertise. Your comments will be considered as appropriate in the manuscript and will be largely posted on the discussion page of the MitoEAGLE preprint website.

It will be of tremendous help for the submission process, if you can send us your ORCID identifier (orcid.org/ ) together with any corrections and suggestions, to
» mitoeagle@i-med.ac.at
We hope that many MiG members will join as additional coauthors. With many thanks for your cooperation,
Erich
Erich Gnaiger
Chair
COST Action CA15203 MitoEAGLE
T +43 512 566796 15, F +43 512 566796 20
mitoeagle@i-med.ac.at | www.mitoeagle.org


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» 2018-11-29 MitoEAGLE coauthor circular
Dear MitoEAGLE preprint co-authors:
The MitoEAGLE manuscript on ‘Mitochondrial respiratory states and rates’ has undergone a systematic development with new co-authors added in the process of many revisions (presently 485 co-authors). I hope that your suggestions have been properly implemented. I would like to thank you once again for your contributions or support, and for your understanding that elaboration towards journal submission has taken longer than expected.
The current version 47 of the MitoEAGLE preprint includes several improvements and simplifications. Now we are initiating the process of submission to a preprint server and journal (Cell Metabolism as an option). This will take a few weeks, during which time your final comments will be welcome.
Please make sure that your name and initials are correctly placed in alphabetical order on the website and in the manuscript (pdf), and that your current institutional address is correctly presented on the Bioblast (MitoEAGLE) website (follow the hyperlink by clicking on your name)
» www.mitoeagle.org/index.php/MitoEAGLE_preprint_States_and_rates
It will be of tremendous help for the submission process, if you can send us your ORCID identifier, e.g., orcid.org/0000-0003-3647-5895 (orcid.org/ ), together with any corrections to mitoeagle@i-med.ac.at.
If we do not receive any answer from you, we will use the current entry for submission.
With best wishes,
Erich
Erich Gnaiger
Chair
COST Action CA15203 MitoEAGLE
T +43 512 566796 15, F +43 512 566796 20
mitoeagle@i-med.ac.at | www.mitoeagle.org


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» 2018-02-22 MitoEAGLE preprint co-authors with link
Dear Gourlay,
Many thanks for the tremendous feedback received so far on the previous circular related to "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology".
Complementary information is provided here, giving the link to the preprint page with the Open Access pdf file and the updated list of co-authors.
http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_States_and_rates
As mentioned in the previous Email: If you are listed as supporting co-author, we ask you to inform us if you do not want to be upgraded as a co-author. In a journal submission the classification as a supporting co-author is not an option.
Again, we would appreciate to receive your final answer until February 28. If you have sent us your message already, please ignore the present Email.
With many thanks for your collaboration,
Erich


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» 2018-02-20 MitoEAGLE preprint supporting co-authors
Dear all,
Thank you for having joined as a co-author of the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology".
The updated version is now close to submission to CELL METABOLISM. The editor-in-chief recommended to send the full paper to this journal.
We continue to aim at incorporating your improvements into the final manuscript, if you have further suggestions.
Should this not be the case, please confirm to have read the newest version of the manuscript, possibly to have made additions or suggestions for improvement, or to agree to implement the recommendations into your future manuscripts, presentations and teaching materials.
This is necessary for our submission; the invitation is extended to contributing and supporting co-authors.
If you are are listed as a supporting co-author we ask you to inform us if you do not want to be upgraded as a contributing co-author. In a journal submission the classification as a supporting co-author is not an option.
We would appreciate to receive your final answer until February 28.
With many thanks for your collaboration,
Erich


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» 2018-02-11 MIG-List circular
Dear MIG-List members:
We would like to invite you to join the network - http://www.mitoeagle.org/index.php/MitoEAGLE_network -, and to join as a co-author of a manuscript prepared for publication (updated version 2018-02-10):
“Mitochondrial respiratory states and rates” http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_States_and_rates
If you are a journal editor, a member of the editorial board, or in contact with publication stakeholders, then we kindly ask you to consider the Open Letter to the Editors below and forward it to the relevant executive editors.
With many thanks for your cooperation,
Erich
Chair COST Action MitoEAGLE - http://www.mitoeagle.org
Disclosure: Erich Gnaiger is founder and CEO of Oroboros Instruments – www.oroboros.at
Open Letter to the Editors
Dear Editors:
We would like to ask you for your opinion about the increasingly urgent issue of nomenclature in mitochondrial physiology. With your collaboration we can contribute to making publications with data on mitochondrial respiration more generally comprehensible and improve a more uniform standard in the field.
As the knowledge base and importance of mitochondrial physiology to human health expand, the necessity for harmonizing nomenclature concerning mitochondrial respiratory states and rates has become increasingly apparent. In the frame of COST Action MitoEAGLE, we endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration. Uniform standards for evaluation of respiratory states and rates will ultimately support the development of databases of mitochondrial respiratory function in species, tissues, and cells.
A Working Group of the COST Action MitoEAGLE prepares a document on ‘Mitochondrial respiratory states and rates’, with Open Access as a ‘MitoEAGLE preprint’ and the ultimate aim of publication in a scientific journal:
We would like to include your opinion as editor. We aim at providing a list of journals, from whom we received valuable feedback:
  1. Do you recognize a general need for a consensus on nomenclature and standards of reporting in the field of mitochondrial respiratory physiology?
  2. Can you provide comments and suggestions for the MitoEAGLE preprint: ‘Mitochondrial respiratory states and rates’ from your point of view as an editor?
  3. Which further steps do you suggest towards implementing a harmonized terminology on mitochondrial states and rates in your editorial policy?

I thank you for your considerations and feedback.

With kind regards,
Erich Gnaiger, Ao.Univ.-Prof., Ph.D.
Chair Mitochondrial Physiology Society - http://www.mitophysiology.org
Chair COST Action MitoEAGLE - http://www.mitoeagle.org
Medical University of Innsbruck
Department of Visceral, Transplant and Thoracic Surgery
D. Swarovski Research Laboratory
A-6020 Innsbruck, Austria
Disclosure: Erich Gnaiger is founder and CEO of Oroboros Instruments – www.oroboros.at


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» 2018-02-08 Editors circular
Dear Editors:
We would like to ask you for your opinion about the increasingly urgent issue of nomenclature in mitochondrial physiology. With your collaboration we can contribute to making publications with data on mitochondrial respiration more generally comprehensible and improve a more uniform standard in the field.
As the knowledge base and importance of mitochondrial physiology to human health expand, the necessity for harmonizing nomenclature concerning mitochondrial respiratory states and rates has become increasingly apparent. In the frame of COST Action MitoEAGLE, we endeavour to provide a balanced view on mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration. Uniform standards for evaluation of respiratory states and rates will ultimately support the development of databases of mitochondrial respiratory function in species, tissues, and cells.
A Working Group of the COST Action MitoEAGLE prepares a document on ‘Mitochondrial respiratory states and rates’, with Open Access as a ‘MitoEAGLE preprint’ and the ultimate aim of publication in a scientific journal:
http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_States_and_rates
We would like to include your opinion as editors of e.g. FEBS J. We aim at providing a list of journals, from whom we received valuable feedback:
• Do you recognize a general need for a consensus on nomenclature and standards of reporting in the field of mitochondrial respiratory physiology?
• Can you provide comments and suggestions for the ‘MitoEAGLE preprint: Mitochondrial respiratory states and rates’ from your point of view as an editor?
• Which further steps do you suggest towards implementing a harmonized terminology on mitochondrial states and rates in your editorial policy?
We thank you for your considerations.
With kind regards,
Erich Gnaiger


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» 2018-02-08 MitoEAGLE coauthor circular
Change of title: Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology. Part 1.
Dear co-authors and MitoEAGLE Network Members:
  • New manuscript version 22: www.mitoeagle.org/index.php/M
  • Change of title: Mitochondrial respiratory states and rates
  • Section 3 removed
  • Your feedback will be appreciated until Feb 20
Our MitoEAGLE position statement originally entitled 'The protonmotive force and respiratory control' has been discussed in many meetings and working groups. Many opinions and concerns have been raised about: (1) the exploding length; (2) the heterogeneity from respiratory states, to protonmotive force, to normalization of respiratory flow and flux; (3) the complex thermodynamic treatment in Section 3 of compartmental systems, power and entropy production, exergy, forces, electric and chemical advancement, fluxes, molecular-molar-electrical formats, and motive units; and (4) corresponding concerns about the meaning of co-authorship, if an entire section of the manuscript remains a challenge rather than becoming a familiar piece of collaborative work for most participants.
In the attempt to give a more detailed introduction and provide clarification of some basic principles related to the protonmotive force, I tried to stick to the conventional presentations of electric potential differences, ΔΨ, and chemical potential differences, ΔμH+, up to the point of recognizing a physicochemical incompleteness in the formal representation of potential differences. I am highly motivated to share a very simple but fundamental solution of this generally unrecognized problem with you, and want to talk about this at EBEC2018. The physicochemical formalism of potential differences is incomplete and terminologically inconsistent. The protonmotive force is not an electrochemical potential difference, but a difference of 'stoichiometric potentials'. A generalized concept of 'isomorphic forces' is suggested to describe the incomplete although mathematically correct equation defining the protonmotive force more properly. Since this discussion appears to be presently beyond the scope of a MitoEAGLE position statement, Section 3 (The protonmotive force, proton flux, and respiratory control) was removed from the new Version 22, the manuscript was updated (see improved Figure 8; extended Table 5), and the title was changed to 'Mitochondrial respiratory states and rates'.
Many co-authors asked about the state of submission and possibilities to further contribute and improve our MitoEAGLE position statement. In this phase 3 towards journal submission, we are asking you and a wider range of experts in the field for input preferentially with corresponding references. This should allow us without too much further delay (deadline: Feb 20) to incorporate your feedback and contact relevant journal editors for their opinion on implementing our recommendations into their journal policy. We want to proceed with submission to a preprint server and final journal submission. BBA was discussed at MiP2017 as a potentially suitable journal, and we are open for further suggestions.
With many thanks for your collaboration, Erich


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» 2017-09-26 MitoEAGLE preprint: The protonmotive force and respiratory control
Dear all,


As a follow-up on the joint MitoEAGLE manuscript, I want to thank for the excellent feedback received so far, with the following update on a significantly edited new version:
MitoEAGLE preprint 2017-09-21(04). The protonmotive force and respiratory control: Building blocks of mitochondrial physiology Part 1. - http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2017-09-21.
Phase 2 - until October 12: We continue to invite comments and suggestions on the MitoEAGLE preprint. To join as a co-author, please feel free to focus on a particular section in terms of direct input and references, contributing to the scope of the manuscript from the perspective of your expertise.
Your comments will be largely posted on the discussion page of the MitoEAGLE preprint website. If you prefer to submit comments in the format of a referee's evaluation rather than a contribution as a co-author, I will be glad to distribute your views to the updated list of co-authors for a balanced response.
Newest versions of the manuscript (pdf) can be found here:
 » http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2017-09-21
In cooperation with the MiPsociety we organize a MitoEAGLE session linked to our series of reviews at the MiPconference Nov 2017 in Hradec Kralove (where you hopefully will attend) and at EBEC 2018 in Budapest.
 » http://www.mitoeagle.org/index.php/MiP2017_Hradec_Kralove_CZ
I thank you in advance for your feedback.
With best wishes,
Erich


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» 2017-08-22 MIG-List circular
Dear MIG-List members:
A multi-author manuscript on ‘Mitochondrial respiratory control’ is being prepared as a position statement in the frame of our COST Action MitoEAGLE.
Up to now, the list of co-authors is mainly based on previous MitoEAGLE Working Group Meetings. In the bottom-up spirit of COST, this is an open invitation to scientists and students to join as co-authors, to provide a balanced view on mitochondrial respiratory control, a fundamental introductory presentation of the concept of the protonmotive force, and a critical discussion on reporting mitochondrial respiration in terms of metabolic flows and fluxes. We plan a series of follow-up publications by the MitoEAGLE Terminology Group, to increase the scope of consensus-oriented recommendations and facilitate global communication and collaboration.
  • It would be great to receive your comments and suggestions by 2017-Sep-18, particularly if you are an early career investigator adding an open future-oriented perspective, or an established scientist providing a balanced fundamental basis. Your critical input into the quality of the manuscript will be most welcome, improving our aims to be educational, general, consensus-oriented, and practically helpful for students working in mitochondrial respiratory physiology.
  • Please feel free to focus on a particular section in terms of direct input and references, while evaluating the entire scope of the manuscript from the perspective of your expertise.
Newest versions of the ms (pdf) can be found here:
 » http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MitoEAGLE_recommendations
MitoEAGLE is organized in close association with the MiPsociety. We organize a MitoEAGLE session linked to our series of reviews at the MiPconference Nov 2017 in Hradec Kralove (where you hopefully will attend) and at EBEC 2018 in Budapest.
 » http://www.mitoeagle.org/index.php/MiP2017_Hradec_Kralove_CZ
I thank you in advance for your feedback,
with best wishes,
Erich
Erich Gnaiger, Ao.Univ.-Prof., Ph.D.
Chair Mitochondrial Physiology Society - http://www.mitophysiology.org
Chair COST Action MitoEAGLE - http://www.mitoeagle.org
Medical University of Innsbruck
Department of Visceral, Transplant and Thoracic Surgery
D. Swarovski Research Laboratory
A-6020 Innsbruck, Austria
Disclosure: Erich Gnaiger is founder and CEO of Oroboros Instruments – www.oroboros.at


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» 2017-08-15 Feedback on review MITOEAGLE
Thank you for your input on the COST Action CA15203 MITOEAGLE review on ‘Mitochondrial respiratory control’.
We welcome you as a co-author, and need your feedback on the latest version to finalize the list of contributing co-authors and, alternatively, supporting co-authors.
As a contributing co-author, we have stated on the website:
Contributing co-authors: Confirming to have read the final manuscript, possibly to have made additions or suggestions for improvement, and to agree to implement the recommendations into future manuscripts, presentations and teaching materials.
The present version has been updated according to numerous comments and questions raised by various co-authors. We cannot extend the length, but your critical input into the quality of our statements will be most welcome. The final definitions of our review may appear to be quite trivial, yet they should be educational, basic and consensus-oriented to provide a balanced view on the mitochondrial respiratory states, a fundamental introductory presentation of the concept of the protonmotive force, and a critical discussion on reporting mitochondrial respiration in terms of metabolic flows and fluxes.
Updated versions of the ms (pdf) can be found here:
http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MITOEAGLE_recommendations
Details on “work in progress”:
http://www.mitoeagle.org/index.php/Talk:Mitochondrial_respiratory_control:_MITOEAGLE_recommendations
The project MITOEAGLE is organized in close association with the MiPsociety. We intend to organize a MITOEAGLE session linked to our review at the MiPconference Nov 2017 in Hradec Kralove and at EBEC 2018 in Budapest in contact with Laszlo Tretter.
http://www.mitoeagle.org/index.php/MiP2017_Hradec_Kralove_CZ
I thank you in advance for your feedback,
with best wishes,
Erich


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» 2017-08-09 MITOEAGLE joint review on respiratory control
Dear all:
Our COST Action CA15203 MITOEAGLE aims at publishing a joint review on ‘Mitochondrial respiratory control’.
We would be honored if you would be willing to join us as a co-author of this review. We cannot extend the length, but your critical input into the quality of our statements and a key reference will be most welcome. The final definitions of our review may appear to be quite trivial, yet they should be educational, basic and consensus-oriented.
Newest versions of the ms (pdf) can be found here:
http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MITOEAGLE_recommendations
Details on “work in progress”:
http://www.mitoeagle.org/index.php/Talk:Mitochondrial_respiratory_control:_MITOEAGLE_recommendations
At present, the list of co-authors is still quite arbitrary, mainly based on MITOEAGLE Working Group Meetings. As a final goal, we hope to bring a large number of contributing co-authors on board on a global scale, to provide a balanced view on the mitochondrial respiratory states, a fundamental introductory presentation of the concept of the protonmotive force, and a critical discussion on reporting mitochondrial respiration in terms of metabolic flows and fluxes.
The project MITOEAGLE is organized in close association with the MiPsociety. We intend to organize a MITOEAGLE session linked to our review at the MiPconference Nov 2017 in Hradec Kralove and at EBEC 2018 in Budapest in contact with Laszlo Tretter.
http://www.mitoeagle.org/index.php/MiP2017_Hradec_Kralove_CZ
I thank you in advance for your feedback,
with best wishes,
Erich


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» 2017-04-26 MIG-List circular
Dear MIG-List members:
  1. Mitochondrial respiratory control: a conceptual perspective on coupling states.

A manuscript on MITOEAGLE recommendations in preparation - we invite additional members to join the MITOEAGLE Terminology Committee to secure a wide scope of the review:

» http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MITOEAGLE_recommendations_1
Best regards,
Erich
Erich Gnaiger, Ao.Univ.-Prof., Ph.D.
Chair Mitochondrial Physiology Society - http://www.mitophysiology.org
Chair COST Action MitoEAGLE - http://www.mitoeagle.org
Medical University of Innsbruck
Department of Visceral, Transplant and Thoracic Surgery
D. Swarovski Research Laboratory
A-6020 Innsbruck, Austria
Disclosure: Erich Gnaiger is founder and CEO of Oroboros Instruments – www.oroboros.at


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» 2017-04-21 MitoEAGLE today circular
COST Action CA15203 MITOEAGLE
Evolution - Age - Gender - Lifestyle – Environment: mitochondrial fitness mapping
Dear Dr Iyer,
1. Mitochondrial respiratory control: a conceptual perspective on coupling states.
A manuscript on MITOEAGLE recommendations in preparation. We invite additional members to join the MITOEAGLE Terminology Committee to secure a wide scope of the review:
 » http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MITOEAGLE_recommendations_1
2. Minutes of MITOEAGLE meeting Barcelona March 2017
We are pleased to send you the minutes of the Barcelona meeting:
 » http://www.mitoeagle.org/index.php/MITOEAGLE_Barcelona_2017#Minutes_MITOEAGLE_Barcelona_2017
3. Training School Obergurgl (AT): 2017 July 23-30
The MitoEAGLE Training School is organized in collaboration with the MiPsociety:
 » http://www.mitoglobal.org/index.php/MiPschool_Obergurgl_2017
• Preliminary programme
• Early Career Investigators and students - prepare your abstracts
• 42 MitoEAGLE scholarships and more
Please foward the training school announcement to your students. Many thanks for your collaboration.
Best regards,
Erich


Updates

Fig. 9. Mitochondrial recovery, YmtE, in preparation of isolated mitochondria.
Towards a shorter version: Old Fig. 9 was removed.
  • 2018-02-06 Gnaiger E Version 21: Note: Subscript ‘§’ indicates throughout the text those parts, where potential differences provide a mathematically correct but physicochemically incomplete description and should be replaced by stoichiometric potential differences (Gnaiger 1993b). A unified concept on vectorial motive transformations and scalar chemical reactions will be derived elsewhere (Gnaiger, in prep.). Appreciation of the fundamental distinction between differences of potential versus differences of stoichiometric potential may be considered a key to critically evaluate the arguments presented in Section 3 on the protonmotive force. Since this discussion appears to be presently beyond the scope of a MitoEAGLE position statement, Section 3 will be removed from the next version and final manuscript. This section should become a topic of discussion within Working Group 1 of the MitoEAGLE consortium, following a primary peer-reviewed publication of the concept of stoichiometric potential differences.

Further references


Pre-submission correspondence with editors

  • Dear Prof. Gnaiger
it is with great interest that I read your email. Indeed a more harmonised and well explained terminology in mitochondrial research would be excellent. I, as Editor in chief of Chemico-Biological Interactions, would support such a open source publication.
If we at CBI can support you, e.g. in publishing this terminology review paper as an open source publication in CBI, please do contact me directly.
Sincerely, Prof. Dr. Daniel Dietrich, Ph.D., FATS, DGPT, ERT Editor-in-Chief Chemico-Biological Interactions http://ees.elsevier.com/chembioint
  • Dear Dr. Gnaiger,
Thank you for forwarding us a copy of your manuscript under preparation. Since that the editors have already invited a submission, we recommend you submit the full paper via Editorial Manager for in-depth editorial evaluation when you are ready.
Thank you for your support of Cell Metabolism.
Best, Jennifer Estrompa - Journal Associate Cell Metabolism, Structure, and Cell Chemical Biology - Cell Press
  • Dear Dr. Sharma,
Thank you for asking us to consider your paper, “Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology”, for Cell Metabolism. In principle, we find the topic to be relevant and the work to be of interest. It is, however, somewhat difficult for us, based on the information you sent, to determine whether the paper would be a strong candidate for Cell Metabolism. I would therefore recommend you send us the full paper to be evaluated as part of our in-house editorial review system.
Before submission of the article online at our EM site, please make sure that the article conforms to the format guidelines for the journal. Please mention this correspondence in the cover letter.
We look forward to reading the full paper.
Sincerely, Nikla Emambokus, Ph.D. - Editor-in-Chief, Cell Metabolism
Discussion see 2018-02-15 Sharma P
  • Dear Dr. Sokolova
I have given this some thought and, after some delay for which I apologize, would like to offer a response. It is a personal one that in no way represents the views of editors of the JEB.
I can understand why some would wish to standardize nomenclature in mitochondrial physiology. Over the years, I have come across submissions and published papers that make claims about rates measured in "state 4" when the conditions actually did not fit the definition of state 4 established decades ago and widely accepted by mitochondrial biochemists.
However, there are terms in the list provided that are not generally accepted and I doubt whether these should be 'made standard by official declaration'. Adopting standard nomenclature would mean that deviation from it would be frowned upon by reviewers and editors. I would be more comfortable with the idea of having authors refer to (cite) your document should they wish to use any or all the terms in it. ::::: If, over time, people in the field choose to use the terms and cite the document, then the terms become accepted by consensus, rather than by declaration. This would be consistent with our wish for clarity and precision in the use of language in papers concerning mitochondrial physiology and biochemistry.
I hope this feedback shall be of some use.
With best wishes, Raul - Editor 'Journal of Experimental Biology'


Contacted editors (updated 2018-02-12)
  • Biochem J
  • Cell Metabolism: J. Estrompa, N. Emambokus
  • Chemico-Biological Interactions: D. Dietrich
  • Elsevier
Life Sciences: L. Wold
BBA Bioenergetics - Journal: A. Ruban and S. Arnold
Platelets: P. Harrison, S.P. Watson
Pharmacological Research: E. Clementi
Journal of Physiology and Biochemistry: M.J. Moreno Aliaga
  • FEBS Journal
  • FEBS Lett
  • Int. J. Biol. Macromol.: A. Dong
  • JIMD
  • Journal of Experimental Biology: I. Sokolova, H. Hoppeler, A.A. Biewener, R. Suarez, franklinlab@uq.edu.au
  • J Physiol: M.C. Hogan,
  • Mitochondrion: K.Singh, C. Thorn
  • Mitochondrial Research: P. Bernardi
  • PLOSOne: J. Heber
  • Redox Biology: S. Lamas

Phase 3.1: Discussion

  • This is a great idea and an impressive piece of work. I have some comments/suggestions that I hope will be useful. Please see these comments contained within the attached PDF.
  • Gnaiger E'
Many thanks - they are implemented into the new version, except the following:
mtPT: the identity is a highly controversial issue, and selective reference to a particular point of view in this controversy is not appropriate for our review
Semiquantitative measures used for normalization would be counterproductive – it renders the result semiquantitative. Why convert a quantitative measurement into a semiquantiative one? :::: Or use statistics based on linear regressions for pooled samples?
  • , I have read the manuscript and it is impressively well written and structured. However, I have a few comments that I think could add value to the manuscript.
  • Line 1236 - I consider that this sentence 'Stereological determination of mitochondrial content via two-dimensional transmission electron microscopy can have limitations due to the dynamics of mitochondrial size (Meinild Lundby et al. 2017)' is not well constructed and/or valid.
  • Stereological determination of mitochondrial content has been clasically considered as the gold standard for mitochondrial volume and/or content. Mitochondrial size does not interfere in this determination, as it would only interfere in the mitochondrial morphological determination (size and shape). Thus, changes in mitochondrial size can not be a limitation for stereological determination of mitochonrdial content. I would suggest re-phrasing or deleting.
  • Line 1238 - 'Accurate determination of three-dimensional volume by two-dimensional microscopy can be both time consuming and statistically challenging (Larsen et al. 2012)'
I would add to this sentence that despite being time consuming, it is considered as the gold-standard for mitochondrial content/volume determination and should always be considered when possible. Furthermore, I would merge it with the next paragraph (line 1240).
  • I have a proposition concerning the role of cardiolipin. Because cardiolipin is known to play a crucial role in cristae biogenesis, I would suggest adding this information at the line 281 (here in bold). “…in particular, it plays an essential role in cristae biogenesis (Ikon and Ryan, 2017b; Khalifat et al., 2008) and it stabilizes and promotes the formation…”.
• Ikon, N., and Ryan, R.O. (2017b). Cardiolipin and mitochondrial cristae organization. Biochim. Biophys. Acta BBA - Biomembr. 1859, 1156–1163.
• Khalifat, N., Puff, N., Bonneau, S., Fournier, J.-B., and Angelova, M.I. (2008). Membrane deformation under local pH gradient: mimicking mitochondrial cristae dynamics. Biophys. J. 95, 4924–4933.
I also have a proposition concerning the description of dyscoupled respiration.
After a PhD in the lab of Renée Ventura-Clapier and Vladimir Veksler, I’m currently working on the demonstration of the existence of a membrane property that was hypothesized by another group in 2016 (Yoshinaga MY et al. Prog Lipid Res 2016) and called “hyducton”. ::::: Based on data from comparative lipidomics and biochemical ecology and on theoretical calculation, they proposed that phospholipids form proton transport circuitry at the membrane's surface. At the mitochondrial level, “hyducton” could facilitate proton transfer on the inner membrane from respiratory chain to ATP synthase.
In our lab, using a KO mice model, we obtained data that strongly support this hypothesis and suggest a crucial role of cardiolipin in this process (paper in revision in Cell Metabolism). In collaboration with biophysicists, we are now developing approaches to experimentally demonstrate the existence of “hyducton”. As a preliminary data, we were able to measure ATP production on very pure preparation of mitoplast and using a method to modify the phospholipids composition of the membrane, we obtained evidence that cardiolipin is necessary for proton-binding-dependent ATP production.
I know that it’s still hypothetic, but we are convinced that this property will emerge as an important regulator of mitochondrial coupling.
It's probably too far from the purpose of the article and I know that you have to keep the length but maybe a bullet point could be added at the end of the paragraph 2.4.3 to present this property? Or in the Box 1: In brief-Mitochondria and Bioblasts?
• Yoshinaga MY, Kellermann MY, Valentine DL, Valentine RC. Phospholipids and glycolipids mediate proton containment and circulation along the surface of energy-transducing membranes. Prog Lipid Res. 2016 Oct;64:1-15. doi: 10.1016/j.plipres.2016.07.001. Epub 2016 Jul 20.
  • Gnaiger E:Towards preparation of Version 2, I would like to ask you if your paper in prep has now been published and can be cited. Otherwise we should add the Yoshinaga 2016 reference.
  • Enclosed please find my few comments to the MitoEAGLE manuscript, and I would like to join as co-author.
  • Gnaiger E: Thank you for your carefully considered comments and joining as a coauthor. Many of your suggestions are now included in the new version
  • Dalgaard LT
Regarding your question in item 8 below: I don’t think it will improve reading or understanding of the abbreviations for oxygen concentration or flux to change them into ‘concentration of’ or ‘flux of’, as these, at least to me, are more universally used abbreviations. But I agree that it is important to be consistent when defining the abbreviations.
  • Here attached some comments to suggestion to manuscript. Its a terrific work, congrats!
  • Gnaiger E: Thank you for your feedback and comments.
Tab 2 - Line up: Uncoupled actually incorporates the indented terms below.
Line 1139: Thank you for the references on mt subpopulations. We will include them in the website of the MitoEAGLE preprint. Since the section is on mt-markers, these important references on mt subpopulations may belong to a different review – perhaps in the future.
  • I have gone through the paper till page 11, attached please find the PDF for my comments and suggestions with references (as PMID) at: Line no 275, 285 and 313. I thought I just start posting my comments so that there should be no delay because of me. I will keep posting as I move more forward to this paper.
  • Gnaiger E: Here are my comments:
Line 275: Will there ever be a unique abbreviation (mt) for mitochondria, if we continue with MIM and MOM?
Line 285 and 313: Any of the sentences in this ‘In brief’ section can be expanded in detail and with references, but then it is not ‘brief’ any longer. But your valuable additional details and references will be included on the discussion page of the preprint website.
  • In the Executive Summary, under 1. the phrase “as the counterpart of cellular core energy metabolism” is potentially confusing. I suggest removing this phrase because it is not clear whether it refers to the subject of the sentence, "vectorial oxidative phosphorylation”, or the object "glycolytic fermentation”. Either way it is not helpful to refer to either as "cellular core energy metabolism” since glycolytic energy production is essential for survival of all cells and is “core energy metabolism” from an evolutionary perspective, whereas coupled mitochondrial ATP production is “core” for cellular function but can be dispensed with in rapidly proliferating tumour cells (we have now shown using ATP5B nuclear gene knockouts, see our recent Cell Metabolism paper published online 15 Nov 2018, attached below). However, the mitochondrial electron transport chain is essential for cell proliferation because it is required for de novo pyrimidine biosynthesis where the 4th enzyme in the pyrimidine biosynthetic pathway requires DHODH (dihydroorotate dehydrogenase) activity which is dependent on CoQ redox cycling via mitochondrial electron transport. As shown with DHODH gene deletion, DHODH respiration is required for tumour growth in breast cancer and melanoma models. So in summary, although mitochondrial ATP production is used to support tumour growth, it is not essential, whereas DHODH respiration is essential for tumour growth.
DHODH respiration is about 10% of mitochondrial O2 consumption in proliferating tumour cells, is ETC/CoQ dependent, is not coupled to ATP production through OXOHOS, and is required for anabolic metabolism.
Hope this is helpful and that this new information about mitochondrial respiration and its involvement in de novo pyrimidine biosynthesis can be included in the MitoEagle manuscript.
  • I was reviewing the final version of the preprint, and found that figure 4 was changed as I proposed from UCP1 to UCP, because UCPs 2 and 3 could act as uncouplers as well (I already sent the references to Erich in two previous e-mails). Nevertheless, on lines 610, 699, 700 and table 2 (line 692), UCP1 still appears. In my opinion, it should be changed.
  • I have the following comment to make:
  • Line 530-531:
‘β-oxidation of fatty acids 531 (FA) generates FADH2 as the substrate of electron transferring flavoprotein complex (CETF).’
  • This could be a bit misleading as it implies that fatty acid oxidation only supplies reducing equivalents via CETF to the Q pool. In fact, fatty acid oxidation supplies reducing equivalents via (i) acetyl-CoA generated by chain shortening; (ii) FADH2 via CEFT; (iii) NADH generated via 3-hydroxyacyl-CoA dehydrogenases. Quantitatively, the acetyl-CoA route will generate by far the most ATP, although this is dependent on whether acetyl-CoA enters the TCA cycle, or is for example used in ketogenesis.
  • Although a detailed explanation of fatty acid oxidation is clearly outside the scope of the document, maybe a brief clarification would be helpful.
  • I have attached an edited version of version 44. I had trouble editing the PDF and so converted the PDF to word so I could more easily edit with the marks shown clearly. In the references I highlighted one PLOS ONE, which should be PLos One. Also the city is not given for many of the books used. Probably check Cell Metabolism for the instructions about this.
  • I believe the manuscript is looking good and look forward to seeing it submitted for publication.
  • I have had a look through this MS and I think it is an excellent piece.
  • I have a couple of very minor points-
  • Line 137- write IUPAC in full.
  • Line 158- …’commonly accepted and understood terminology’….
  • Line 173- terminal oxygen acceptor.
  • Line 328- worth mentioning that mtDNA maternally inherited?
  • Line 827- worth including a ‘typical’ trace for isolated mitochondria showing the different metabolic states graphically?
  • I really enjoyed while reading the MitoEAGLE preprint. It makes mitochondrial biology so clear bur uncompromising the specificity and science. I’m still beginner at the mitochondrial biology and don’t have anything to contribute to the paper but I’ll have a metabolism course to teach on spring and I definitely will use this paper to come as background material for the oxidative phosphorylation part of the course.
  • as you probably remember, I was skeptic about the pork silhouette of Fig. 1 (and other figures of the manuscript with a similar silhouette) because, in my perception, it riminds of a species specific cell type whereas the cell scheme should be more general.
  • As I suggested, a different silhouette with at least more than one animal might be more appropriate.
  • I would love to read it and provide some comments for you.
  • In my opinion the manuscript is reads well and is clear. Just two minor things I picked up.
  • Line 127: small typo on specific
  • Line 142: should IUPAC be defined?
  • I fully endorse the need to provide experimental guidelines and standards of reporting.
  • Together with colleagues from the COST Action CardioRNA CA17129 which has recently started and which I am chairing, we will refine guidelines and standards for RNA studies related to heart disease.
  • I hope we can have further links between our Actions in the near future. A manuscript on the importance of mitochondrial RNA in cardioprotection could be a starting point to link our 3 Actions MitoEAGLE, CARDIOPROTECTION and CardioRNA. This is open for discussion.
  • There were 2 points i commented. The first one was about citing Altmann's publication.  ::::* I did not read the original publication of Altmann but I saw that some records say it was published in 1890 (not in 1894). (Example publication: Front. Physiol, 1:7, 2010.). I am not sure which is true but you may want to check.
  • The second one was about the definition of cellular respiration at lines 239-241 in previous versions. But in the the final version, it is not cell respiration anymore and changed to mitochondrial respiration. So the second point is not important anymore.
  • I have read the pre-print closely over the last few days. I think it reads very well indeed and will be a timely addition to the field. Congratulations on putting this together!
  • Attached are a few small suggestions for you to consider.
  • I went through the paper you gave me (and by the way: thank you for involving me in this). ::::* I am pretty sure it will be an important tool for all the bioenergetics community. I have just a few suggestions you may want to consider.
  • 1) Page 3 line 254: Cardiolipin does not only stabilizes supercomplexes, but has a number of additional functions, including the regulation of carriers, ATPase, and fission/fusion machinery.
  • 2) Page 11, line 498: FADH2 is described, but you may want to consider to include it in figure 2B (NADH is represented but not FADH2)
  • 3)Page 12 paragraph "Control and regulation": I found htis a bit difficult to read, especially starting from line 564: you underline the differences between respiratory control and regulation, but you put together the mechanisms. i suggests to split the mechanisms of respiratory control from those of regulation.
  • 4)Page 22 lines 978: The first three line of the paragraph sounds not to clear to me.
  • Andrew Murray suggested that myself and Alice read the MitoEagle terminology paper draft with a view to getting on top of the terminology. We thought it was tremendously helpful review and a great contribution to the field. We noted you are encouraging younger investigators to get involved as co-authors and wondered if it would be possible for us to join at this late stage? If so, we have identified possible ways in which the manuscript should be improved and thought these might be useful coming from the perspective of those relatively new to the field who are the intended audience. comments in docx comments in pdf
  • Please find our observations, comments and suggested additions to your manuscript. As promised, we are sending our comments within the three weeks deadline established by common agreement in Tallinn.
Let's hope our suggestions may be of some help.
Rafael and Sara
  • The manuscript is almost perfect, however, I found typewriting mistakes which are listed below:
Line 203 is ….lipd…. should be ..lipid, Line 279 is …1,200.. should be …1200 Line 376 is …ade nosine… should be.. adenosine Line 483 is Coenzyme Q, Q, and.. should be once Q


  • I would be happy and honored to join this publication.
I have only a few comments (in order, mixed science, presentation, and writing). Take or leave as you choose.
Line 265; replace "which" with "that"
line 468: replace "or glutamate" with "or to glutamate"
line 539: consider italicizing "regulation"
line 541: consider italicizing "control"
line 625: this point of differing nomenclature for different aspects of coupling is really well handled in Table 2, I would note here (line 625) that we will be returning to it below
Line 674 and Table 1: in the text, you are careful to note that you are talking about a minimal backflux of cations, but the table is less clear. I was confused when looking at the table at first because i think about cation cycling in the context of pathology such as permeability transition or uncontrolled calcium degregulation. Given that the audience for this manuscript/proposal is to try to loop in the broadest possible community, I would try to clarify this in the Table.
Longer term (i.e., not this manuscript), the other issue that this raises is whether the proposal should add another state (or qualifier), e.g., "Damaged" to characterize states that are limited by non-physiological (e.g., pathological, pathophysiological, toxicological issues). You touch on one aspect of this issue as "dyscoupled" beginning in line 691, but it's really not defined there, and that doesn't cover situations where, for example, reactive species damage has limited oxygen consumption. Similarly, , it is unclear how "physiological" inhibition, e.g., by H2S or NO, would fit into the schemes, as, although technically literally true, it is arguably somewhat (unintentionally) deceptive to say that low flux in the presence of ADP and substrate is OXPHOS-limited, when it is really limited by inhibition due to H2S, NO, etc. Another option would be to denote as something like *OXPHOS-D [damaged] or OXPHOS-I (inhibited) etc. In the context of the field historically, one can simply look at what is reported and figure it out. if you are trying to create a useful database you may need to build something like this into an ontology to facilitate searching and retrieval
Line 699: replace "which" with "that"
line 839 -- add comma after stoichiometry
lines 904-912 and table 3: Consider adding the alternative State 2 and State 5 to the Table 3, with notes for clarity
Gnaiger E : Many thanks for your feedback. I added all of your comments to the new version (37), except for not including the alternative State 2 into Table 3, which is strictly the original table of Britton Chance.
  • I have read the manuscript. Well done.
One reference may be added in the section Electron transfer-state (Figure 6C) in line 802. Inhibition of respiration is observed above optimum uncoupler concentrations.
Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.
Chowdhury SR, Djordjevic J, Albensi BC, Fernyhough P. - Biosci Rep. 2015 Dec 8;36(1):e00286. doi: 10.1042/BSR20150244.
Gnaiger E : Thank you for your feedback. I have added the reference in the newest version (37).
  • First, thank you for including me in this nice piece of work. The manuscript is very informative and I see no major changes needed.


  • Here are my few comments and suggestions on the manuscript. I also noticed one mistake in the references.
This is a master piece of work and will be more than welcome.
I also think that it will be quite useful for comparative and evolutionary physiologists.  ::::: I therefore made some suggestions of easy additions to convince colleagues in the comparative field that they are also concerned. Again these are all and only suggestions.
I should say that it has been difficult to try to improve a manuscript that has been polished by so many experts.
Gnaiger E : Version 37 includes almost all of your suggestions. I particularly like the emphasis on comparative physiology and evolutionary biology.
  • Thank you for applying me to the society of MitoEAGLE.
Please find an enclosed file with my comments.
In my opinion manuscript is very well and extremely polished.
Gnaiger E Many thanks for your feedback. I tried to accommodate your comments in the new version (37).
  • I`ve read through the manuscript which is very detailed and contains all the relevant information on mitochondrial physiology and function. The manuscript is very well written and I could find no errors or information that was absent. I concur with John Land and wonder if at all possible that Dr. Simon J R Heales could be included amongst the authors of the manuscript.
  • I have been thru it as my staff will tell you with my Eagle eye and not found anything I would wish to change.
  • for figure 2 do you think that we need to have something that talks about the mtCK and the creatine shuttle (Theo Wallimann?) - the inner MM has ANT and the OM has VDAC and mtCK catalyzes the exchange in the IMS?
  • . A manuscript like this is definitely needed in order to achieve uniformity in the way in which data are reported in this field.
The background of the topic has been eloquently described, using terminology that is both clear and succinct. I did, however, wonder if MitoEAGLE itself required a little more explanation to the reader. To someone who has not come across it before, it could be difficult to determine if this is a concept or a group of people (i.e. a scientific collective) from what is written. I also wonder if the term gender is being used correctly, it tends to denote a more social or cultural reference to sex rather than a biological one, and I am assuming that chosen gender (as opposed to allocated sex) has no influence on mitochondrial function. I realise this a only a small point and removing the word gender would cause the EAGLE acronym to become EASLE but in this ever-changing world we live in, sex and gender are slowly drifting further apart.
In the background it states that robust mitochondrial function is supported by physical exercise (line 300) - I wonder if a reference would be useful here.
I think the figures are extremely useful and along with the text provide one of the best descriptions of oxidative phosphorylation I have ever come across.
The sections of the main body of the text go through matters very clearly. Like Helen, I agree that tacking the difficult and controversial territory of normalisation is important. We certainly spent a lot of time thinking about this when putting together our recent ICU patient data. So a guide like this would have been very helpful.
  • -p4: why the pig? The pig is distracting (sensitive from animal ethics Point of view) and not necessary
  • p5: introduction could be shortened

Quotes: how relevant? Would delete the Hofstadter quote (for some prob. Too intellectual) P>> direction or flux?

  • P10: figure very busy.
  • Figures in general: avoid red-green due to prevalent red-green blindness (->grayscale for these individuals)
  • Toxicological: shouldn’t it read “toxic”?
  • Figure 5: not really a “compartment model” as it does not show interaction
  • Table1: examples would be very helpful
  • Table3: already published, maybe simply cite and refer to original publication
  • Chapter: normalization per sample – could go into a separate follow-up publication Difference between permeabilized tissue and permeabilized fiber is unclear as fiber=tissue??
  • Would replace “mito dysfunction” with different term “altered mito fnxn” or so… “Subjects” should be replaced by diff term “individuals”, “participants”, etc… Conclusions should not be in bullet-point format for Cell Metab (rather free text). Maybe compile into info box?
  • Journal: In general, Cell Metab is good with good impact and readership; but why not consider Physiological Reviews?
  • My comments are rather editorial, see pdf attached The blue “tube” in figure 2A should be the electron transport system? Rather unclear


  • Suggested changes and comments:
  • line 159, shouldn't "b)" be "(b)"?
  • line 246 suggest changing the sentence to, "Mitochondria form dynamic networks within eukaryotic cells and are morphologically ..."
  • on line 250, I think we need to be careful defining the intermembrane space as a positively charged space. It is only positive with respect to the matrix. It would be negative if compared to the extra-cellular compartment.
  • on lines 320-322, consider changing the sentence to, "In this way, researchers from a variety of disciplines can compare their findings using clearly defined and accepted international standards."
  • on line 358, it is said succinate and phosphate are impermeable to the cell membrane. In many cases this may be true; however, there are solute carriers that transport these metabolite across the cell membrane, e.g. SLC13A3 and SLC20A2.
  • on line 380, suggest changing the sentence to, "Using Percoll or sucrose density gradients to maximize the purity of isolated mitochondria may compromise the mitochondrial yield or structural and functional integrity."
  • text on lines 416-425 highlight an extremely important point to consider regarding mitochondrial studies. I think we should emphasize this a little more. For example, there are many studies out there looking at FCCP-uncoupled respiration but do not mention that they titrated FCCP to yield the optimal mitochondrial response. This is especially true if the study is exploring a treatment or disease that alters the mitochondrial content, but they use the same FCCP concentration to measure maximum respiration in the noncoupled state.
  • on line 704, suggested change "Calcium influx is balanced by mitochondrial Na+/Ca2+ and/or H+/Ca2+ exchange..."
  • In Table 2, the fully coupled state needs to be carefully calculated. It's not simply JP - JL, since JL will be different during JP because of the strong membrane potential-dependence of JL. During JP, the membrane potential will be lower than that during JL. Therefore, JP-L = JP - JL(@membrane potential during JP). I just noticed that this is discussed on line 829.
  • On line 822, a very important concept is presented, and, at least to my knowledge, it is not emphasized in the literature. The times that respiratory states are measured are an extremely important experimental variable to consider and disclose. Both the leak and oxphos capacity decrease with time at 37 C with the latter having a more pronounced decrease. These are likely due to endogenous proteases and overall instability of isolated mito preps when incubated at body temp. We tested whether or not the protease we use to isolate the mitos from heart tissue could be the culprit but found no difference between preps obtained from a single spin and a double spin for the initial crude homogenate pelleting step during differential centrifugation. The double spin was presumed to remove more exogenous protease at the cost of lowering the mito yield.
  • On line 826, I like the fact that it is mentioned that oligo can inhibit E. One must be careful not to poison one's mitos when trying to measure respiration in the presence of oligomycin.
  • For the discussion on lines 1253-1269 about mito markers, I suggest adding the following sentence at the end of the paragraph, "With no single best mitochondrial marker, a good strategy is to quantify several different biomarkers to minimize the decorrelating effects caused by diseases, treatments, or other factors.
  • I found the paper very important, congratulations for the great efforts.
The attached version contains very few edits.
Good luck with this milestone publication.
Gnaiger E: Thank you for your enthusiastic feedback and helpful suggestions. I particularly like your re-arrangement in the abstract. And I added more of your suggestions to the new version. A donkey would be nice, but It would have to be a very fat individual.
  • I read the most recent version of the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology" with the great interest. I have some comments and proposals for small additions in the text.
Gnaiger E: Thank you for your comments, which help to provide a broader perspective on the background information of the mitochondria (we should call them bioblasts!). ::::: I added your input and references into the new version (same version number, date updated


  • We think the MitoEAGLE manuscript will be a highly valuable compilation of facts on mitochondrial respiration. It provides a hugh amount of information and helps harmonizing not only nomenclature but also experimental set-ups and the interpretation of the results concerning mitochondrial respiration.
  • I read the manuscript. Yes, it will be very helpful for the community and those that will enter the field of research on mitochondria. The manuscript is at an advanced / final stage and ready for submission. I have however, made a few, rather marginal comments on it. You may think about taking them into account.
Thank you very much for bringing me on board.
P.S. I find it great that you make clear that the ‚power plants' of the cell do not PRODUCE energy! I read it again and again even in papers of 'great shots‘. I also like your quote 'Making data available without making it understandable may be worse than not making it available at all’ (National 1411 Academies of Sciences, Engineering, and Medicine 2018)'
Gnaiger E: Thank you so much for your helpful comments, which are now included with the reference on aging in the updated version.


  • I highly agree that uniform definitions and standardized methodology is highly desirable and a major impediment to progress in the field – particularly in my own clinical research realm. To use an analogy from clinical thrombosis research, I feel like the field is where bleeding and prothrombin time measurements were before the development of the INR – in fact, that might be an apt comparison to add to your introduction section. Some additional suggestions to consider adding if space limits permit:
1.On page 6, sentence ending 283 or 286. I would suggest adding a sentence noting the role of acetylation as a post-translational modification capable of influencing the bioenergetic response, which clinically significant implications for health and disease (some cites below, can provide others if needed). Alternatively or in addition, this should be included on page 12 when discussing mechanisms of respiratory control (particularly the 2nd citation)
a.Cell Metab. 2018 Mar 6;27(3):497-512. doi: 10.1016/j.cmet.2018.01.016. The Mitochondrial Acylome Emerges: Proteomics, Regulation by Sirtuins, and Metabolic and Disease Implications. Carrico C1, Meyer JG2, He W3, Gibson BW2, Verdin E4.
b.JCI Insight. 2016 Feb;2(1). pii: e84897. Epub 2016 Feb 25. Mitochondrial protein hyperacetylation in the failing heart. Horton JL1, Martin OJ1, Lai L1, Riley NM2, Richards AL2, Vega RB1, Leone TC1, Pagliarini DJ3, Muoio DM4, Bedi KC Jr5, Margulies KB5, Coon JJ6, Kelly DP1.
2.Also on page 6, I don’t know if you would like to mention that, consistent with the symbiotic hypothesis, that mtDNA is recognized as “other” by the immune system, and systemic release can activate inflammation via TLR9 activation (PMID 20203610).
3.Page 7, following the sentence ending on line 326, consider adding something along the lines of “Standardization and homogenization of terminology, methodology, and data sets could lead to the development of open-access databases such as those that have been developed for National Institutes of Health sponsored research in genetics, proteomics, and metabolomics.” ::::: This bridges nicely into the statement related to meta-analyses
4. No specific additions, but the section on normalization is excellent.
Gnaiger E: Thank you for your feedback and detailed comments. Welcome on board of MitoEAGLE. I added your sentences on pages 6 and 7, and your ref.
  • I am delighted to be a part of this paper. I will agree to implement these changes into our future communications – I really like that we are trying to reach consensus terminology.
  • I just had a quick glance and it looks interesting.
I will get back to you by the end of the week about it.
  • It’s fantastic to see this come together. I think it is going to be an important consensus paper working toward consistent nomenclature. I read the manuscript and agree wholeheartedly. I do not have any comments or edits to offer
  • As a clinical researcher with experience in using the Oxygraph, I found it to be an extremely useful and comprehensive reference for many of the important conceptual questions regarding its use.
The discussion of the important area of normalisation is particularly helpful - and I personally have found the concept of ET- and OXPHOS-capacity as an integrative functional marker to be valuable in my own work and understanding.
The only thing that comes to my mind, which I discussed in our group sessions in Obergurgl, was whether any consensus exists for using the wet or dry weight for mass correction (for a tissue such as permeabilised muscle fibres). I realise this may be very small print for such a comprehensive - but wondered what your own thoughts were, even if not relevant for the manuscript.
Gnaiger E: Your question on wet versus dry weight in permeabilized fibers is important. We prefer wet weight measurements for a series of arguments, with emphasis on more carefully standardized procedures compared to previous descriptions. With Carolina (Cc) and several colleagues, therefore, we prepare a complete documentation including video illustrations. In addition, the MitoEAGLE Working Group 2 (muscle tissue) has initiated interlaboratory studies for standardization and experimental validation, to be published separately.
  • I have read your manuscript, and I think it is based on your concept “Gentle science” and is essential for further advance in mitochondrial research.
I guess this article is read by a lot of scientists who engage in mitochondrial research and is cited in many future articles included mine.
I always appreciate your effort.


  • I appreciate the invitation and thankful for the opportunity to be part of this great team.
I had previously read the draft and it looked excellent. However, I will give another read and if I have any further comments I will make sure to send it to you.
  • I have read the manuscript with great interest. I confirm my support and willingness to serve s a co-author on this manuscript.
Thank you for your leadership in mitochondrial research field!
Gnaiger E I thank you for your confirmation, and will keep you in the loop on further steps of manuscript submission


  • I really like it, particularly the thermodynamic contexts – nice job. I read it closely and there was nothing that struck me as needing editing. The thermodynamics material will be challenging for readers, but there is not enough attention given to the basics elsewhere, so it is good that you have taken that approach.
My only minor comment is the use of E, L and P as abbreviations. They are abbreviations of abbreviations, which I find unnecessarily confusing. All of the other abbreviations are logical and related to function, so would prefer that a similar approach be used with these. That’s just my opinion of course.
Gnaiger E: Many thanks for taking the time to check our paper, I really appreciate your feedback.
Let me clarify the concept on the abbreviations L, P, and E: You are correct – these have been abbreviations of abbreviations as used in our previous publications, where no terminological distinction has been made between OXPHOS state and OXPHOS capacity, when P was used for both. In the MitoEAGLE manuscript, states and rates are carefully distinguished:
The three coupling states, ET, LEAK and OXPHOS, are shown schematically with the corresponding respiratory rates, abbreviated as E, L and P, respectively (Figure 5). We distinguish metabolic pathways from metabolic states and the corresponding metabolic rates; for example: ET-pathways (Figure 5), ET-states (Figure 6C), and ET-capacities, E, respectively (Table 1).
Still, you may consider as L, P, and E as abbreviations of LEAK-rate, OXPHOS-capacity, and ET-capacity. But then we need short abbreviations. For instance: OXPHOS- and ET-capacity with combined NADH-linked substrates and succinate (NS) are distinguished as NSP and NSL.
We have used this system of abbreviations, e.g. in http://wiki.oroboros.at/index.php/Lemieux_2017_Sci_Rep
Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8. - Table 1 on page 6
Along these lines, I hope that I could adequately resolve your concerns about L, P, and E.
  • I read with enthusiasm your paper (the final version) that I have found very well writte. ::::: I have only minor comments on the therminology.
I think the word "flux" is generally used for metabolic fluxes (ie glucose, lipidss, amino acids) as it is intended for mass transfer; here I would say is more a flow of ions (protons) or Molecules (O2)
Table 4 uses kg as unit for sample mass : I would consider grams instead of kg, since kg are generally used for total body while here it is mainly referred to an organ or cells
Gnaiger E: Many thanks for your enthusiastic response and comments. I am glad to reply:
Flux and flow: You are raising an important point, which shows that use of these terms is very area-specific. Mass transfer, molecular transfer, vectoral versus vectorial ion translocation, scalar reactions, … have to be covered. To come to a general recommendation in an interdisciplinary context, IUPAC may be considered as a guideline. Not even IUPAC is completely helpful, but it provides the basics. This is explained in detail in Box 2. Fig. 2A has been conceptually refined over many discussions. I think, we are breaking into a new generalization with a conceptually based terminology, that cannot accommodate various less concerned traditions. Progress is not a linear process.
Use of kg versus g: You are absolutely right with reference to what is used in practice. We may even argue, that mg would be more appropriate than g in our context. The units, however, are not given according to practice (which varies from amol to mmol, etc.) but according to the metre–kilogram–second-ampere SI system of units, as opposed to the centimetre–gram–second system of units. Your comment makes me aware that this has not been explained. Therefore, I added in Box 2: We use the metre–kilogram–second–ampere (MKSA) international system of units (SI) for general cases ([m], [kg], [s] and [A]), with decimal SI prefixes for specific applications (Table 4).


  • It was a pleasure to read the paper, congratulations for all the work and to involve so many people.
I propose to add a comment on mitochondria proteomics as it is becoming a reality and relevant to tackle novel mitochondria diseases.
My suggestion goes for page 27 on the 3rd paragraph inserted with the mtDNA concept.
I am sending the comment and the 2 references:
Only lately it is possible to systematically identify the complement of over 1,000 proteins that contain the mammalian mitochondrial proteome, which is used to discover the genetic basis of respiratory chain disorders as well as to characterize novel mitochondrial disease. (refs bellow)
1) Systems proteomics of liver mitochondria function
Evan G. Williams, Yibo Wu, Pooja Jha, Sébastien Dubuis, Peter Blattmann, Carmen A. Argmann, Sander M. Houten, Tiffany Amariuta, Witold Wolski, Nicola Zamboni, Ruedi Aebersold, Johan Auwerx
Science 10 Jun 2016: Vol. 352, Issue 6291, aad0189
DOI: 10.1126/science.aad0189
2) Proteomics of human mitochondria.
Palmfeldt J, Bross P.
Mitochondrion. 2017 Mar;33:2-14. doi: 10.1016/j.mito.2016.07.006. Epub 2016 Jul 19.
Gnaiger E: Many thanks for your suggestion, and welcome as a co-author.
I added your comment upfront in Box 1, where a reference was already made to the human proteome. Your two suggested references fit there, too.


  • Martin und ich haben das Manuskript gelesen und sind damit einverstanden.
  • The article is very well written. I have no comments.
  • I confirm to have read the latest version of the manuscript with a few comments. I will do my best to implement the recommendations and terminology in my own work.
Comments:
Line 174: typing error of ‘preparations’, an ‘e’ instead of an ‘a’
Line 625-626: Should the reference to Fig 4 instead be to Fig 2A?
  • Thank you very much for your email and update. I finally had the time to carefully check all demonstrations and equations. This version is very good but I would like to add just minor things. I am attaching a PDF with just a few comments.
  • Just wanted to confirm that I am very happy to be upgraded as author – if this is ok with you all.
Please let me know if there is anything more I can do at this stage.
  • I have read the manuscript and think it will be an important contribution.
Although a minor part of the manuscript overall, I found the discussion of the role of the role of cation cycling in respiratory uncoupling enlightening. In this context, I think a more explicit statement in Box 1 regarding mutual regulation of mitochondrial coupling and calcium homeostasis (as considered in more detail later in the manuscript) could be helpful for some readers whose primary research is in related fields, such as excitation-contraction coupling. For example, starting at line 250: "There is a constant crosstalk between mitochondria and the other cellular components. The crosstalk between mitochondria and endoplasmic reticulum is involved in the regulation of respiratory coupling as well calcium homeostasis, cell division, autophagy, differentiation, anti-viral signaling (Murley and Nunnari 2016)."
  • I confirm that I have read the latest version of the manuscript and was pleased with the newly added section 3.5 on the evaluation of mitochondrial markers. It is a topic I often address in my ongoing projects and look forward to future discussions by the group.
I agree and look forward to implementing the recommendations into all of my future manuscripts and educational materials
  • Thank you very much for information, I have read the newest version.
  • I hereby confirm that I have read the newest version of the manuscript, "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology". I also agree to implement the recommendations into future manuscripts, presentations and teaching materials.
Unfortunately, my limited biophysical competence prevents me from making serious improvements of the manuscript, but I have read it carefully and gained important insight into mitochondrial function, which will be useful in future scientific projects.
My comments below are merely suggestions
Line 194: Definition of FMN?
Line 217-222: Long sentence. I would prefer full stop after “level” in line 219. The next sentence could read: “In addition, crosstalk takes place through cell signalling……….”
Line 251: Replace “…will be positioned to…” with “can”
Line 252: Remove …”an agreed upon set of”…
Line 262: “are” instead of “is”
Line 281: ….bilayer with embedded proteins and attached….
Line 314: …”upper bound”... or …“upper boundary”…?
Line 414: The term “P»/O2 ratio” ratio (also JP») appears several places throughout the manuscript. When I first saw it (line 414), I thought the “»” should not be omitted, but I guess it is intentionally included.
Line 807: Replace …”does mean”… with …”means”…
Fig. 8: Part A and B (as mentioned in the figure legend) are missing in the figure.
  • Hereby I do confirm that I have read the newest version of the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology", and agree to implement the recommendations into your future manuscripts, presentations and teaching materials.
Gnaiger E: Here a quick response regarding our preprint. In Innsbruck we discussed mitochondria-containing non-cellular particles. In Version 31 I include a new figure, which refers to extracellular O2 consumption. Do you have any reference along these lines?
Michalak S: Please find enclosed reports and review about microvesicles/exosomes mitochondria and intercellular transfer of mitochondria.
  • I read the current version with deep interest and find the cumulative resulting work of authors systematic, comprehensive and clear. From my personal point I like very much that throughout the work the nature concept of organismal and cellular compartmentalization of products and functions is pointed.
Following my previous email I would like to stress on very important fact regarding the value of MitoEAGLE manuscript which excites me a great deal.
In my teaching practice similarly to many other tutors, I face a challenge of lack of systematic and detailed instructions for physiological experimentation with isolated mitochondria and permeabilized samples to be recommended to students.
This manuscript is going to be invaluable manual both for experienced researchers and beginners.
  • please find attached the .pdf of the most recent manuscript. I looked over again, specifically over the newer ‘block sections’ in lighter blue. I was only able to find very few writing mistakes and the manuscript overall reads very well and will help to move the field forward.
  • I have just read the latest version of the manuscript and I am happy that you decided to write it. I think it is a great guideline not only for people who start their adventure with mitochondria. It helps to understand mitochondrial respiratory physiology, parameters that can be measured, ways in which data should be presented and explains how important is the normalization of rates. The article gives us clear terminology related to mitochondrial respiration measurements. Therefore, we agree with the information in the manuscript and we will start to implement this knowledge into in our articles, documents and presentations.
  • The final manuscript version reads well – you have done a tremendous job. Good luck with the submission.
  • I read the manuscript. I think it is great. I don’t have any comments.
  • thank you very much for this great work on terminology. For the purpose of publication, I would like to confirm that I have read the newest version of the manuscript and agree to send it for publication.
  • Nevertheless, in our opinion Omics, by enabling an integrative picture of the functional organization of mitochondria and their adaptive and maladaptive responses to Evolutionary background, Age, Gender and sex, Lifestyle and Environment (EAGLE), are excellent approaches to be considered within the project. The integration of Omics data retrieved from text-mining focusing on mitochondria plasticity will certainly allow to increase the knowledge on the biological processes harbored in mitochondria and modulated by distinct stimuli. The network perspective that can be obtained from proteomics, metabolomics and transcriptomics might help to explain functional alterations reported in certain conditions. So, in our opinion, the relevance of this topic justifies its consideration by the MitoEAGLE project.
Such challenge should be carried out in different steps: i) exhaustive text-mining considering Omics data and functional data; ii) network analysis of Omics data with bioinformatics tools; iii) cross-validation with distinct bioinformatics approaches; iv) correlation with functional data; v) guidelines for biological validation of network data.
Gnaiger E: I just added your thoughts to the conclusions (Version 31):
Technical terms related to and defined with normal words can be used as index terms in databases, support the creation of ontologies towards semantic information processing (MitoPedia), and help in communicating analytical findings as impactful data-driven stories. ‘Making data available without making it understandable may be worse than not making it available at all’ (National Academies of Sciences, Engineering, and Medicine 2018). Success will depend on taking next steps: (1) exhaustive text-mining considering Omics data and functional data; (2) network analysis of Omics data with bioinformatics tools; (3) cross-validation with distinct bioinformatics approaches; (4) correlation with functional data; (5) guidelines for biological validation of network data. This is a call to carefully contribute to FAIR principles (Findable, Accessible, Interoperable, Reusable) for the sharing of scientific data.


  • Many thanks for your message from last week on the manuscript convering Mitochondrial respiratory states and rates. It took me a long time to read it since I am with a heavy teaching load these days. I have to tell you though that the manuscript reads well, and I have no significant comments on it. Congratulations for the great job.


  • I’ve reviewed the most recent version of the manuscript and have no objections to the content. I think it will serve the discipline well in not only standardizing nomenclature, but also compelling folks to actively contemplate the experimental conditions that form the basis of the nomenclature. Please continue to include me as a co-author.
  • I've reviewed the newest version of the manuscript and have no further comments or suggestions. I've read several versions of the manuscript by now, and I'm impressed with the progress made. I'm confident that this article will be a great resource for researchers in the bioenergetics field, and I'm glad to be part of it. I'll certainly incorporate the recommendations into future manuscripts, presentations and teaching materials.
  • thank you for your email. I confirm that I have read the newest version of the manuscript. I have no further comments or additions. Congratulations, it's great work!
  • I like the last versión of Box 1. Please find attached some comments related to the new Figure 1.
  • I have read the MS and found it much easier to understand than the previous version (from autumn 2017), because its structure is better to follow and the section on chemical and electrical part of the proton motive force is not included in this version.
I cannot add anything to the MS but would be honoured to be co-author and will try to contribute to future follow up manuscripts.
  • Thank you for your work on this publication. The only comments I have on the current version are to potentially change the word "trademarks" in the abstract to something less colloquial, for examples "features"; and also to expand the acronyms in the abstract (IUPAC, COST, EAGLE).
  • I read the last version and I am happy with it. Thanks
  • I think Cell metabolism would be ideal.
Check the type of submissions possible, but it could be something different from an editorial to a short report?
Certainly will contribute and also can ask Steffi if ok with you to contribute.
  • A few corrections and suggestions from me and Robert:
1) Line 160 (Fig. 1 legend) – change:” mtOM and mtIM – to mitochondrial outer membrane (mtOM) and mitochondrial inner membrane (mtIM)…
2) Line 268: Since bivalves were just introduced add ‘Human’ before mtDNA: “Human mtDNA is 16.5 kB…”.
3) Line 346: change ‘peremeabilization’ to ‘permeabilization’.
4) Line 512: (CHNO, 2 [H] in Fig. 2). I do not see CHNO in Fig. 2.
5) Line 522: Exergy – Should Gibbs Free Energy be introduced instead? Exergy is mostly used by engineers I think and the audience might be more familiar with Gibbs - I am not sure.
  • I read the last version of the manuscript with great interest. It really provides in depth and systematic overview.
I noticed just a few very minor things (typos etc.). Probably they are already corrected, but just in case I’m sending them in the attachment (highlighted in yellow).
  • I have read your article. This is an excellent initiative and this will indeed significantly improve the way to collect and report data for mitochondrial assay. And as you stated, this is the only avenue for getting data to run meta-analysis and in turn, figuring out big pattern of the role of mitochondria in life.
I do have some comments:
  • I would suggest re-organisation for the article such a naïve reader can understand it. Each paragraph with minimal definition, highlight of the problem in the literature (ie where 1 word has been used for several concept; or several words has been used for 1 concept, with references); and then the proposed solution to this confusion – how we think we should harmonise everything.
  • I think the suggestion of new terms should be only reserved where ambiguity there is/there was. ::::::For instance, paragraph 422-41, I think P” and “P is unlikely to be used by anyone because ADP/O; ATP/ and P/O are clearly defined concept and as far as I am aware, there haven’t been mis-used. JATP and JADP are usually used understood for ATP production and ADP production. If they have been mis-used, highlight where/how.
  • I took note all over the text of concepts that are not explain (irreversible thermodynamics, compartments) – so will bring confusion for many readers, or that might not be needed for our understanding (for instance, ETS and ET can be replaced by ET system and ET).
  • There is jumps between the mitochondrial physiology in vivo, and the experimental condition in vitro; this might be clarified to avoid confusion of the reader.
  • The word Coupling is constantly used, but up to the end, I don’t understand what does it mean precisely? For me coupling is a ratio between A and B, and what are A and B should be clearly stated.
This is an excellent news that Cell Metabolism has offered a submission of this article. Very exiting!
  • I am really grateful to be part of this work and thank you so much for this oppotunity!
  • I like the preprint manuscript
  • Thank you very much for your invitation and collaboration. I have read the article and it is very good work. I am in agreement with the last version of the manuscript.


  • Great job with this important paper, and congratulations on this “open science” approach.
I have only some very minor comments – attachment.
  • After reading the latest version of the article ready to send to Cell Metabolism, I write this email to confirm that I agree on its content; In fact, I have already considered the new terminology in recent articles that we have published.
  • I am truly glad that our paper is finally reaching the final submission.
I have read the final paper for submission, and I have no further comments. I am aware we are submitting the paper to CELL METABOLISM, and I agree to implement these recommendations on my upcoming work.
Congratulations on leading this immense task to its final goal.
  • I have read the manuscript and found it comprehensive and of a high scientific standard.
I am honored to be part of this important work.
  • I have carefully read the last version of the manuscript and agree to implement the recommendations in my future scientific and teaching activities.
  • This is a great overview paper MITOEAGLE prepared, I fully support the submission and if need I would be happy to contribute further to the manuscript preparation.
  • Great work so far, please find my comments below.
Line 223: This sentence is not very clear. Firstly, the meaning of ‘robust’ is not clear and does not help with understanding the link with exercise and caloric balance. Secondly, we need to clarify what we mean by ‘mitochondrial function’ – you could have normal function but decreasing quantity of mitochondria. Similarly, you could maintain content but have more, but smaller, mitochondria that are not as efficient at producing ATP (or other functions). We should reference the statement that mitochondrial function is ‘central for sustained metabolic health throughout life’.
Introduction: It would be helpful to contextual the manuscript in the face of the increased use of mitochondrial analysis by a broad range of researchers. A sentence similar to this could be used: ‘The widespread use of mitochondrial analysis by a broad range of researchers necessitates a re-evaluation of the nomenclature used and the establishment of terms that can be more generally accepted and applied’.
Lines 523-525: An example should be included here to help understand the point.
Lines 554-555: This sentence needs further clarification. There is no obvious link to ‘why’ and ‘how’ in the following sentences.
Lines 579-582: The use of the term OXPHOS is being defined at kinetically-saturating concentrations but OXPHOS itself can take place at sub-max substrate concentrations. A term like OXPHOS-capacity would be better and consistent with the term ‘ET-capacity’ in line 582.
Line 674-676: Other uncoupling proteins should be mentioned in addition to UCP1 – why not mentions UCP3 or ANT?
Lines 828-842: Fig. 6. In my opinion the text in the legend of the figure does not explain the figure. In addition, it should not be necessary to have to refer to Table 4 to further explain Fig. 6 – it should be self – explanatory.
Lines 914-933: This paragraph is overly technical and will lose the majority of readers. If the purpose of the paper is to start the process of standardisation then the majority need to understand the process. This paragraph needs to be explained in terms that biologists/physiologists would understand – or relevant examples provided to make it easier to understand. Right now it is too technical and too heavy on chemistry
Minor comments
Line 103: ‘expands’
Line 188: ‘energy-transformation’
Line 237: Should this be ‘exerts’
Line 444-446: I think this is the first time that C1, CIII, CIV and AOX have been used in the text – their full names should be provided at this point (if not explained earlier).
Line 447: Should complex I be used instead of, or in conjunction with, the NADH-pathway – the remainder of the sentence refers to CIII and CIV
Line 476: Insulin resistance is not a signalling pathway – should refer to the insulin signalling cascade in this case.
Line 519: Should be ‘lose’
  • Thank you for the opportunity to be part of this submission. Overall the manuscript is well structured and concisely written covering a broad range of areas. I had some minor comments
1.The influence of physiological substrates on mitochondrial function parameters in different preparations/cell types could be described further.
Gnaiger E:Very good idea. The physiological substrates and all the substrate-inhibitor states (=pathway control states) are as important as the coupling states. We emphasize (see Conclusions): The present recommendations on coupling control states and rates, linked to the concept of the protonmotive force, are focused on studies with mitochondrial preparations. These will be extended in a series of reports on pathway control of mitochondrial respiration, respiratory states in intact cells, and harmonization of experimental procedures.
Pulinilkunnil T: 2.Validation of mitochondrial function using oroboros in different genetically modified mouse models either using a table or a figure would be helpful.
Gnaiger E: Working Group 2 works on an inter-laboratory comparison of respiratory results obtained on a strictly defined control mouse model, and a separate manuscript is in preparation along these lines.
Pulinilkunnil T: 3.Normalization of mitochondrial function in lipid-laden tissue is a frequent issue and could be discussed in that section.
Gnaiger E: I fully agree. Working Group 3 should take up this topic (Adipose tissue). Presently, the blood cell group is active, and this will hopefully stimulate other actions, too: http://www.mitoeagle.org/index.php/MitoEAGLE_blood_cells_1
  • thank you for email and updates.
I confirm that I have read the latest updated version of the manuscript.
  • This is great manuscript!!! Compliments.
As I promised, please find the attached pdf of the manuscript where I included my comments and proposal of corrections.
In yellow I also marked something to remove (come characters/symbols that shouldn't appear).
Gnaiger E : I saw your corrections – the symbols that you did not like (the long - ) is a standard symbol in scientific writing. Perhaps more US than EU? The font will be a matter of the publisher.
Perhaps we may need an improved explanation here (Line 549): Cycling of other cations, strongly stimulated by permeability transition, or experimentally induced by valinomycin in the presence of K+;
Wieckowski MR
Yes, you are right, I am not used to that symbol.
However, can you explain me what is the mining of (long - ) in the case (line 330): i.g. „…. such as lactate dehydrogenase—and results in the complete loss of cell viability”?
I will think about the improved explanation of the line 549…..
  • I would like to confirm that I read the newest version of the manuscript. I do not have any further suggestions and I agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
Thank you for letting me participate as a contributing co-author.
  • I have looked at the latest version of the manuscript and have no further suggestions. Should the opportunity arise in the future, I would implement the recommendations into any future manuscripts, presentations and teaching materials.
  • With the rise of novel omics technologies devoted to mitochondria, this initiative results from the existence of several bioinformatic tools available, but small amount of information is provided considering “what (kind of information), when (which cases is useful), how (interpret and extract biological meaning)”. Thus, I wonder if you will be interested to create an issue devoted to Bioinformatic tools and mitochondria.
I am available to discuss in more detail this topic.
Gnaiger E: Thank you for your very interesting (and challenging) suggestion. ‘Bioinformatic tools and mitochondria’ is exactly what we need and what is badly missing. I guess it could be even more than an issue – it could be a full European Project Proposal.
We need this perspective in the entire MitoEAGLE project. Now we work full-power on a ‘guideline’ paper for blood cells: http://www.mitoeagle.org/index.php/MitoEAGLE_blood_cells_1
A similar manuscript on permeabilized muscle fibres is in the works.
We need to implement a data management plan and databank logistics. The very well established omics tools are not developed for mitochondrial physiology. It needs to be done.


  • I read very carefully the MS, and I find it fantastic. To say the truth I don´t have critiques, I think that nomenclature proposed is very clear, and definitions are very precise and will be extremely useful. I have a comment/suggestion:
I understand that most of the literature in which the text is based refers to mammalian cells mitochondria. However, it would be useful to suggest the proposed nomenclature for all mitochondrial studies (including those of plants, fungi and unicellular organisms. So I should mention this clearly in the Executive summary (for example in line 137 you restrict your statement to mammalian mitochondria, while it could be expanded to mitochondria of most of the eukaryotic cells). I think that a sentence explicitly "inviting" the readers working on mitochondria in non-mammal systems to incorporate these standards would reinforce the idea that this could be useful for the mitochondria community in general.
Gnaiger E: Great suggestion to replace mammalian by ‘most eukaryotic’ in line 137.
  • I have read the latest version of the manuscript;
I agree to implement the recommendations into your future manuscripts, presentations and teaching materials.
The manuscripts is excellent as it is and I have no further suggestions.


  • I confirm that I have reviewed the most recent version (ver. 22) of the manuscript. I will implement these recommendations into my future manuscripts. Thank you again for your invitation.
  • Thank you for your great effort with the manuscript and its a great honor to be a part of this to me. I was re-reading the manuscript and 2 points took my attention.
The first is a minor issue in the Box 1, lines 218-219, citing Altmann's publication. I did not read the original publication of Altmann but I saw that some records say it was published in 1890 (not in 1894). (Example publication: Front. Physiol, 1:7, 2010.). I am not sure which is true but you may want to check.
Second, the definition of cell respiration at lines 239-241. There is a definion named "non-mitochondrial respiration" in several articles and books (ie. Bioenergetics, 4th ed, Page 279. According to this definition, most cells have some non-mitochondrial oxygen uptake caused by cytoplasmic oxidases and related enzymes. In the current manuscript, cellular respirations sounds like it exludes this concept.
  • It is really my great honour to be a co-author of MitoEAGLE manuscript, the preprint is great. I would like to confirm that I have read the most recent version of the manuscript and I agree with the submission. I agree to participate as a contributing co-author and I fully agree to implement the recommendations into our future manuscripts, presentations and teaching materials.
  • After looking through it I really think this is an excellent piece of work that have the potential to bring order to this fragmented field. A few thoughts:
Consider not emphasizing the COST MitoEagle as much in the abstract. It is of course true that this initiative is the fundamental basis for this paper to take form, but at the same time it makes the article somewhat dated. Consider the possibility that COST MitoEagle does not get another term of funding, then it would be a policy statement from an organization that is no longer existing. By just making it a statement from key researchers in the field, it would be more resistant to the passing of time. I understand that you would like to promote COST MitoEagle, but it could prove short-sighted. As you are aware, there are many organization “competing” for being the world-wide network for mitochondrial research, and by making your paper more organization neutral, you would take the high road and soar above petty positioning. Just a thought, you do as you see best of course.
In the executive summary bullet 3, the sentence Selective removal or permeabilization of the plasma membrane yields mitochondrial preparations—including isolated mitochondria, tissue and cellular preparations—with structural and functional integrity might be somewhat provocative as it implies that the function of the mitochondrial is not altered by its removal from its cellular milieu. Could this maybe be softened somewhat?
To me, the expression concept-driven terminology that appears in the abstract and executive summary feels like a buzz-word with little actual meaning. The intention of this expression is unclear. Could it be clarified?
Figure 1A:
Try to harmonize the font sizes in the figure. As it is now, the figure appears unnecessarily messy. 1B is much better in that regard.
In the legend, consider writing out mtOM and mtIM, as the figure legend ideally should be self-instructive.
  • These are my final comments after reviewing the last version of the manuscript. As mentioned before ::::: I agree in implementing the recommendations (already done in our last manuscript submitted)
Comments:
Box 1: In brief – Mitochondria and Bioblasts
The paragraph related to mitochondrial genome could be move to line 239 to complete the description of mitochondria. This section will be followed by the description of mitochondrial structure and function, organelle interactions and it would end with the second paragraph related to mitochondrial dysfunction (now starting in line 222). In my opinion it has a more logical flow.
Minor comment:
Line 382. I would suggest change the term “biochemical reagents” by “chemical reagents” I am not sure if FCCP or CCP, among other reagents that we commonly use to assess mitochondrial respiration, could be considered biochemical reagents (yes, if we consider that do they have life science applications). ::::: Later on, in the same paragraph we use the term chemicals (line 390)
I really like how the manuscript has developed. It is a great achievement.
Gnaiger E: Many thanks for your final comments and great feedback. Box 1: We go from structural to functional perspectives including the proteome (I just moved a late structural section with fission and fusion up to the structural paragraph), continuing with communication within the cell (well: fission and fusion was here), cell cycle, and ending with the genome perspective. With the Ernster and Schatz 1981 reference, we return to structure-function relationships.
I full agree on the “chemical reagents”.
In response to good questions on the definition of cell respiration, I added a new Figure 1 for the executive summary, with a summary figure legend and link to peroxisomes. I may need some feedback on this.
  • I had a thorough look yesterday evening at the manuscript and did not find points of concern. I'm grateful to be included as a coauthor and thereby fully support the content of the manuscript.
  • Great work has been done, my compliments.
I confirm to have read the newest version of the manuscript. At this point I have no additional comments. I have learned a lot from this manuscript. Naturally, I do agree to implement the recommendations into my future manuscripts, presentations and teaching materials. I also agree with the submission of the manuscript to Cell Metabolism, as contributing co-author.
I wish you good luck and look forward to see it published.
  • It is difficult not to be in this really important paper.
  • Here are my last comments on the manuscript. I went through it a last time and made minor comments on it (attached). I agree with the submission, and I will incorporate these concepts as much as I can in all my future manuscripts, presentations and teaching material.
Thanks a lot, and have a great day,
  • I finished reading the final version of the manuscript last night and I do not have anything to add. Well done! Thank you very much for your hard work and thank you for sharing.
  • Great job!
I have only one correction:
p28 line 1215 - there should be GLUCOSE instead of glycogen.
Overall, I confirm that I have read the newest version of the manuscript, and agree to implement the recommendations into future manuscripts, presentations and teaching materials.
  • I am OK with the current version, which also apples for Josef.
  • Sorry for the delay, but I wanted to make sure I had read the manuscript thoroughly before replying. I noticed several parts have been improved, but I myself don’t have any other contribution to make at this moment. The manuscript seems to me ready for submission.
  • I have read the final manuscript and I agree to implement the recommendations into future manuscripts, presentations and teaching materials.
  • I confirm to have read the newest version of the manuscript and I agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
  • I read the mitoEAGLE preprint and wrote my comments. I hope they will be acceptable.
Gnaiger E: Thank you for your comments and corrections. The new version (30) incorporates the corrections that you pointed out, and takes into account many of your suggestions. You have studied this manuscript in detail and made highly valuable contributions.
  • For me, the manuscript is now in an excellent shape.
Nevertheless, I´m free to add a very short comment. First, I propose to add in keywords (p.3) the term "uncoupling".
Second, a very sophisticated paper was recently published by D. Speijer (from the Netherlands), proposing that mitochondrial ROS diminuation has contributed to shaping the eucaryotic metabolism. The underlying paper is entitled: Being right on Q: shapping eukaryotic evolution (Biochem. J. 4103-4127.
See the abstract below:
Effective combinations of metabolic pathways from the host and the endosymbiont (mitochondrion) allowed larger varieties of substrates (with different F/N ratios) to be oxidized, but high F/N ratios increase ROS formation. This might have led to carnitine shuttles, uncoupling proteins, and multiple antioxidant mechanisms, especially linked to fatty acid oxidation Recent data regarding peroxisome evolution and their relationships with mitochondria, ROS Formation by Complex I during ischaemia/reperfusion injury, and supercomplex formation adjustment to F/N ratios strongly support the model. I will further discuss the model in the light of experimental findings regarding mitochondrial ROS Formation.
My proposal is to examine, whether it is worth to consider this important paper at the end of the first segment of box 1 (after line 221).
  • I looked through this manuscript. I think it is great! I am honored to be associated with it in whatever capacity you identify.
  • I was reading the manuscript and it looks very well. I believe that this is a necessary paper describing and standardizing the mitochondrial bioenergetics. It would be fantastic that Cell Metabolism accept it for its publication, because everybody working in mitochondria will have a methodological reference.
  • I am drilled that you have received the OK from Nikla to submit the full paper to Cell Metabolism, what a great news!
I have read the newest version of the manuscript. As done in the last Cell Metabolism wrote by myself and co-workers, and the presentations done since then, I site regularly the MitoEAGLE COST action. So you can count of the fact that I will continue to do it in future manuscripts, presentations and teaching material.
  • I have read the newest version of the manuscript and I approve it
just a short comments I am deeply convinced that this was a wonderful chance to publish a comprehensive piece of work on respiratory control but I am also perfectly aware that some chapters/paragraphs might result actually awkward for many readers. Therefore, the new changes/eliminations may help also a no-specialist reader in the final understanding of the problems. I thereby approve the proposal to change the title into Mitochondrial respiratory states and rates, to eliminate section 3, fig. 9, etc.
I'll surely use these recommendations in future manuscripts and I'll diffuse them in all my teaching activity at pre- and post-doctorate level in all my courses.
  • I hereby confirm that I have read the last version of the manuscript and will surely implement the MS recommendations into my future manuscripts, presentations and teaching materials.
  • Yes, indeed many small things have been done to advance the work. Thank you.
I offer the following suggestions to consider. All are editorial, not scientific (MitoEAGLE preprint 2018-02-21(27)):
1.Lines 201-202: I would use a colon after “for” to indicate a list is coming, then semicolons to separate the three items in the list. This approach helps the reader and the points stronger.
2.Line 244: “which” may be better as “that” in this setting.
3.Line 365: the ; after O2 might be better as just a comma, ,.
4.Line 456: 2 electron is better as 2-electron
5.Line 1160: “which” would be better as “that” here
6.Lines 1184-1190: Because these are recommendations, the list should stand out better. I would make the list as:
For studies of cells, we recommend that respiration be expressed, as far as possible, as:
(1) O2 flux normalized for a mitochondrial marker, for separation of the effects of mitochondrial quality and content on cell respiration (this includes FCRs as a normalization for a functional mitochondrial marker);
(2) O2 flux in units of cell volume or mass, for comparison of respiration of cells with different cell size (Renner et al. 2003) and with studies on tissue preparations; and
(3) O2 flow in units of attomole (10-18 mol) of O2 consumed in a second by each cell [amol∙s1∙cell-1], numerically equivalent to [pmol∙s-1∙10-6 cells].
These conventions allow information to be easily used when designing experiments in which O2 flow must be considered.
Also, in the area of the kursiv highlight, I have made a wording or punctuation change.
7.Lines 1243-1249: Again, because the list in these lines are recommendations, I would do as in #6 above.
  • The manuscript is looking really good and thanks again for including me.
I have one suggestion regarding the following part of the introduction that I think could be modified before submission as follows.
from line 268:
mtDNA is compact (16.5 kb in humans) and encodes 13 protein subunits of the transmembrane respiratory Complexes CI, CIII, CIV and F-ATPase, 22 tRNAs and two rRNAs. Additional gene content has been suggested to include microRNAs, piRNA, smithRNAs, repeat associated RNA, and even additional proteins (Duarte et al. 2014; Lee et 272 al. 2015; Cobb et al. 2016). The mitochondrial genome requires nuclear-encoded mitochondrial targeted proteins for its maintenance and expression (Rackham et al. 2012).
Additional reference: Rackham, O., Mercer, T. R. & Filipovska, A. The human mitochondrial transcriptome and the RNA-binding proteins that regulate its expression. WIREs RNA 3, 675–695 (2012).
I hope this is ok to change for scientific accuracy.
  • I have read the newest version of the manuscript and agree to implement the recommendations into future manuscripts, presentations and teaching materials. I have no adittional suggestions for further improvements of the manuscript. Many thanks
  • I think this is a great initiative.
I will forward this to the journals I am working with.
Gnaiger E: Many thanks, and welcome as a co-author
  • I would like to suggest some more points of note in the MitoEAGLE preprint 2018-02-21(27), as follows:
1) line 431. I don't see that phosphorylation of ADP can be catalyzed by phosphoenolpyruvate carboxykinase, which indeed catalyses a reaction that is reversible under intracellular conditions but in such case still requires GDP to form GTP. Perhaps, in this line, "phosphoenolpyruvate carboxykinase" should be replaced with "pyruvate kinase" (whose activity in the glycolitic pathway allows to transfer the phospholyl group from phosphoenolpyruvate to ADP, thus forming ATP, as in the scope of this section of the manuscript). If, in addition, substrate-level phosphorylation as the reversible reaction by phosphoenolpyruvate carboxykinase would still be mentioned in the manuscript, then a separate sentence should properly state that GDP is the substrate.
2) line 431 BIS. Given that the sentence gives emphasis to substrate-level phosphorylation of ADP, then "phosphoglycerate kinase" can be mentioned as an additional example.
3) line 1311. as we discussed in a previous occasion, the mtIM is also widely referred to as "Inner Mitochondrial Membrane, IMM" therefore I suggest to also mention this other abbreviation as a comment in the in the fourth column of the table: "MIM and IMM, as for Inner Mitocondrial Membrane, are widely used; the first M - mitochondria - is replaced by mt"
4) line 1314. (same reasoning as in point #3) I suggest to also mention OMM as a comment in the in the fourth column of the table: "MOM and OMM, as for Outer Mitocondrial Membrane, are widely used; the first M - mitochondria - is replaced by mt"
5) line 1333. Insert the term "Repiratory Supercomplex", SC InIIInIVn used to indicate the supramolecular assemblies composed of variable copy numbers (n) of CI, CIII and CIV; Box 1
6) line 234. Add the abbreviation in parenthesis: "...formation of respiratory supercomplexes (SC InIIInIVn), which are...".
Hopefully this suggestions will be of help.
  • Having read the most recent manuscript I can confirm I am very happy to be upgraded to co author. ::::: It is an excellent and comprehensive account, I wish it was in place when we started our experiments with the Oxygraph many years ago now.
  • Many thanks for your email, the preprint is great!
  • Thank you for sharing this preprint and the generous invitation to contribute as a coauthor in this project.
I have read the revised version with great interest and accept the offer to contribute as co-author of this authoritative review.
  • You did a wonderful and great job. I have a big congratulations on you. I am a great honor and happy to be with you as a coauthor.
  • Thanks for your mail. I read the newest version of the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology". It looks good to me and I have no further addition. I would incorporate the recommendations into my future manuscripts, presentations and teaching materials.
  • today I finished reading the new version of the manuscript. I haven't other suggestions for improvement. I think it is now perfect! I agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
  • , i went through the manuscript. It reads very well. great project, great result ! no comment at this final stage. Thank you very much for including me in the (co)authors.
  • I have just read the latest version of our position statement (v26). Overall, I think it works well, and though some sections of text are rather dense (particularly for a general reader or a newcomer, but I had to take it slowly at times), ultimately this gives a thorough justification for the final conclusions and recommendations, which are themselves very clear. I do, however, wonder if they could be made even clearer if presented as a final, numbered list of recommendations rather than presenting them as free text. The recommendations are the most important part of this manuscript and they do get a little lost. They aren't really handled in the Executive Summary, which makes broad statements about how uniform nomenclature is important but doesn't make specific recommendations. I don't have very strong feelings about this, so do feel free to ignore this suggestion, but my preference would be that the specific recommendations would be listed in a section at the end of the Conclusion that begins "We recommend:" and follows with numbered points on a) the terminology to use for respiratory states and b) recommendations for normalisation for the different preparations (tissue, cells, isolated mitos)
I have some minor, specific comments (mainly very minor):
  • Line 103: Should read "expands"
  • Line 154: Should read "explicit"
  • Lines 222-227: I don't think this paragraph is necessary. It breaks the logical flow of this box.
  • Line 345: Should read "concentrations"
  • Line 519: Should read "lose"
Other than that, I am delighted to be part of this project. Many thanks to you for bringing this together - it's been a lot of work so fingers crossed the reviews. I give my approval to be included as a co-author and for you to submit the manuscript. I also agree to implement the recommendations in my teaching and research.
  • That's great news about the manuscript! I have read the latest version and agree to incorporate the recommendations in my future work.
Thanks again for the opportunity to be a part of this important undertaking!
  • This is indeed a great effort towards establishing unified terms for mitochondrial bioenergetics, although the task is not so easy. Submitting it to BBA would surely be a good option.
We have thoroughly read the article and have only few points to mention:
1. Apart from other factors, ROS production is an important mitochondrial phenomenon that describes the cell fate. Some text should be included on this topic as well. Page 17, line 734, here especially the term ROX could give an ambiguous correlation to ROS with respect to general readership.
Gnaiger E: Yes, but what could be the better solution?
2. Another important factor of mitochondrial respiration is ?pH which is an important part of mitochondrial proton motive force but unfortunately has rarely been discussed. It would be worthwhile to explain its context at the respective place in the text e.g. on page

10 before control and regulation part.

Gnaiger E: It is already included: “(5) Ca2+ and other ions including H+;”
3. It is also important for the general readership to understand the

differences of metabolic states and rates of normal mitochondria (relaxed), excited (active), excited but malfunctioned (unhealthy condition), excited but trained mitochondria (athletes).

4. For the general readers, especially the new comers into the mitochondrial field who are familiar with general mitochondrial terms used in the text books, it is important to describe and discuss the conventional terms of ?mitochondrial membrane potential? in parallel to any newly assigned terms so they can really relate the terms and understand the context better.
Gnaiger E: It was decided to shorten the present MS and move the detailed part on protonmotive force to another MS
5. Similar is the case of mentioning the terms in the text (where

necessary), the decrease or increase in mitochondrial membrane potential (polarization /depolarization /hyperpolarization).

Last but not at least. May I give a little very personal remark?
Page 10 Line 412 ? 426:
Yes, of course. Phosphorylation in the context of OXPHOS is defined as phosphorylation of ADP by Pi to ATP. The electron transfer induced phosphorylation has to include substrate- level phosphorylation, but attention should be paid for the consequence at the regulation level (1).
Isn't it better to introduce one sentence to pronounce the difference between oxidative phosphorylation and regulative phosphorylation?
Phosphorylation means also in detail PO3 transfer to three amino acids (Tyrosine, Serine and Threonine). This process is an essential part of regulation in the ETC (2). Multiple steps are involved in these circuits of signaling (3). This has value because respiratory kinetics are definitively changed, see the c-Src for instance (4).
Our groups had several times stressed the role of phosphorylation sites of complex IV in regard to its enzymatic activity and OXPHOS (5, 6). Even ATP level itself has a regulatory action (7).
Ramzan R, Weber P, Linne U, Vogt S. GAPDH: the missing link between glycolysis and mitochondrial oxidative phosphorylation? Biochem Soc Trans. 2013; 41(5):1294-1297
Vogt S, Rhiel A, Koch V, Kadenbach B. Regulation of oxidative phosphorylation by inhibition of its enzyme complexes via reversible phosphorylation. Current Enzyme Inhibition 2007; 3, 189- 206
Lim S, Smith KR, Lim ST, Tian R, Lu J, Tan M. Regulation of mitochondrial functions by protein phosphorylation and dephosphorylation. Cell Biosci 2016; 6:25
Ogura M, Yamaki J, Homma MK, Homma Y. Mitochondrial c-Src regulates cell survival through phosphorylation of respiratory chain components.

Biochem J. 2012; 447(2): 281-289

Hüttemann M, Helling S, Sanderson TH, Sinkler C, Samavati L, Mahapatra G, Varughese A, Lu G, Liu J, Ramzan R, Vogt S, Grossman LI, Doan JW, Marcus K, Lee I. Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation. Biochim Biophys Acta.

2012;1817(4): 598-609

Helling S, Hüttemann M, Ramzan R, Kim SH, Lee I, Müller T, Langenfeld E, Meyer HE, Kadenbach B, Vogt S, Marcus K. Multiple phosphorylations of cytochrome c oxidase and their functions. Proteomics. 2012

Apr;12(7):950-959

Ramzan R, Schaper AK, Weber P, Rhiel A, Siddiq MS, Vogt S.

Mitochondrial cytochrome c oxidase is inhibited by ATP only at very high ATP/ADP ratios. Biol Chem. 2017; 398(7): 737-750

Gnaiger E: A lot of this would fit, but how to keep the length and focus? We should keep that in mind for another context?
  • Hereby, I declare that I have read the final manuscript and I agree to implement the recommendations into my future presentations and manuscripts.
  • I have read the newest version of the manuscript and I agree to implement the recommendations into my future manuscripts, presentations and teaching materials. Great work!
  • Thank you for your great effort in integrating such an important paper to provide an accurate definition of mitochondrial respiration states and related terminologies. I am pleased to be included as one of the co-authors. I have nothing to add except expressing my appreciation of the tremendous work you have done for this paper. I believe that the manuscript should be more easily accepted by Chem.-Biol. Interact. as for Cell Metabolism, the contents may be too long for a review article, and condensation may improve the likability to get accepted.
  • I am reading the manuscript, and I will send you my comments and proposal of corrections (if any) till the end of this week.
  • Thank you very much for the update. I like the concept of the paper and think this is very much needed in the field. Recently at the Keystone meeting on bioenergetics I realised how much is currently rediscovered, so a paper like yours in a prominent journal is very important.
  • The manuscript is excellent and solid work. Great work!
This time I did just a few small comments.
Thank you for including me as a coauthor. Indeed, I fully support this publication and will use recommendations in future works and teaching materials.
  • In page 10, line 429, please remove “hexokinase”, as this enzyme does not perform a transphosphorylation, it performs only a phosphorylation reaction.
Otherwise, I confirm to have read the newest version of the manuscript and agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
Good luck with the submission!
  • Dear Erich, thank you for your kind proposal about co-authorship Clara and I will send in our contribution (if any) to an already excellent paper. And by next week I will confirm whether we can publish it in Pharmacol Res
  • Thanks for the newest version of the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology" that I read carefully.
I agree to be upgraded as contributing author for submission to Cell Metabolism
Many thanks for this paper that will represent the reference we are all expecting for mitochondrial physiology standards.
Gnaiger E: Thank you for your confirmation and positive feedback. The updated version is in preparation.
  • I confirm to have read the newest version of the manuscript and I agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
I checked the comments by the editor-in-chief of Cell metabolism and I also agree that it is a very good option. Let's hope for the best.
Thank you very much for the opportunity to collaborate on this manuscript.
  • I have read the latest version of the manuscript and it looks great. I have no additional comments and agree with the submission to Cell Metabolism.
Thank you for organising this beautiful work.
  • I have read the latest manuscript and think it is a great body of work and reads very well.
  • I have given a thorough read to the latest version of the manuscript as downloaded from the MitoEAGLE webpage.
Please find attached my feedback (you will see a series, not many, of comments, or words typed in RED font, to attract your attention to what I have changed/suggested to change).

It is very polished at this stage and it has come a long way from the first version I reviewed back in summer 2017.

With this in mind; "I confirm that I read the latest version of the manuscript, and that I have provided feedback/improvements by sending a .pdf revised copy to the corresponding author (Prof. Erich Gnaiger), and that I agree to implement the recommendations into future manuscripts, presentations and teaching materials"
I would be extremely happy and grateful if I could be upgraded as a "contributing co-author".
Gnaiger E : Many thanks for your further input into our MS. I have added practically all your very helpful suggestions and corrections into Version 29. This is really great teamwork. Welcome as a co-author
Granata C: Thanks Erich for acknowledging receipt of the feedback. It is a very well detailed and explained paper
  • I have read the manuscript. Again, congratulations for the good job, in my opinion everything is quite clear.
Just couple of small things, it could be interesting to mention that cardiolipin/phospholipids are necessary for the activity and stability of respiratory complexes and interact with subunits in all of them (i.e. PMID: 25038566; 16388600; 26300339). Also, I have noticed that in the manuscript is on UK english and on page 11 (line 480) the word “channeling” is in US english.
Gnaiger E: Many thanks for your feedback. We changed your signature. Do you suggest to add the three sentences?
Hernansanz-Agustin P: yes, I think it is worth mention this role of cardiolipin. My suggestion between quotation marks -Refs in PMID (line 232, page 6):…The mtIM contains the non-bilayer phospholipid cardiolipin, which is not present in any other eukaryotic cellular membrane. Cardiolipin "is necessary for the activity and stability of respiratory complexes and interact with subunits in all of them (i.e. PMID: 25038566; 16388600; 26300339). In addition, "promotes the formation of respiratory supercomplexes, which are supramolecular assemblies based upon specific, though dynamic, interactions between individual respiratory complexes (Greggio et al. 2017; Lenaz et al. 2017)
  • Paper looks perfect, I am happy to agree.
  • I will return my inputs (if any) on February 27 or 28.
  • I am very much honored to be an author of this historical paper. I firmly believe this paper will be a base of new mitochondria based medicine. I had suggested a little for this paper early on and read most of the paper, while I did not grasp some part of it.
  • Here I confirm that I have read the newest version of the manuscript, and agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
I would be happy to be a contributing author.
  • Thank you for your message and your work in finalizing this review.
I am happy to confirm that I have read the newest version of the manuscript, I am happy to be contributing co-author on the paper and I am happy to implement the recommendations in my future manuscripts presentations and teaching materials.
I have a few minor comments on the manuscript, please see below.
Line 237: ‘exerts’ instead of ‘exters’
Line 388: ‘processes’ instead of ‘process’
Line 388-390: This sentence is confusing; needs to be rephrased. ‘The amount of these chemicals…. being sought.’
Line 393-394: ‘are applied’ should go at the end of the phrase; comma may be needed after the paranthesis ‘…TMRM),’.
Line 738-739: Should be rephrased: ‘Cyanide and azide inhibit CIV and several additional peroxidases involved in Rox.’
Line 959: ‘optimized’ or ‘undergo optimization’ instead of ‘optimization’
Table 5: ‘Mtprep’ should this be with capital letter? Should it acctualy be ‘mt-prep’ or ‘mtprep’ ?
Also in the text we have ‘mt-markers’ and in the table ‘mt-Markers’
I guess we need to have them written in the same way.
General note: We use ‘z’ rather than ‘s’ in various situations like ‘normalization/ optimization’. I guess it needs to be checked with the editor.
Gnaiger E: Many thanks for your helpful comments, all of which are incorporated in the new version of the MS. ‘mt-Markers’ is a header in Tab. 5, hence the capital M. I guess, it should not be Mt-marker, but mt-Marker. It is mt-marker in normal text.
  • Thanks for sharing the progress of the manuscript.
  • In response to your questions, as co-author, I confirm that I have read the newest version of the manuscript, and together with my PhD student Tânia Dias (one of the co-authors) we have made some suggestions for improvement of the manuscript. Moreover, as asked, I state that I will implement the recommendations into future manuscripts, presentations and teaching materials.
Moreover, I confirm that I have been informed and agree withe the submission of the manuscript to Cell Metabolism, as co-author.
  • Stupendous piece of work. It ill be for sure a reference in the field.
I am sending the file with some comments and corrections. Please follow them only if you agree.
  • I will look over the manuscript by the designated deadline.
Thank you very much
  • It is my great honor to be a co-author of MitoEAGLE manuscript.
I like the sections of metabolic state of mitochondria and coupling state. I wondered how to and what portion of protonmotive force generate heat for a long time. If you can explain or comment this on section 2.2, it would be great.
Thank you for your effort and dedication to mitochondria research.
  • I confirm to have read the final manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology", possibly to have made additions or suggestions for improvement, and to agree to implement the recommendations into future manuscripts, presentations and teaching materials.
  • I totally agree with the last version of the maniuscript. I have read it carefully.
  • Congratulations on the profound and necessary work in our field.
I read a manuscript versioon 22.
I totally agree with the removal of the chapter about thermodynamics,as we dscussed it at the Mip2017 meeting in Czech Republic.
I have only minor notes. First, Figure 1 shows also AOX. This is correct, but i think that comment or remark that AOX is absent in mammals should be added to the legend of the figure.
Chapter 3 about normalization is proportionally too comprehensive and i see possibilities for shortering paper mainly in this chapter. Table 6 can be excluded, if nessesery.
  • I would like to confirm that I have read the most recent version of the manuscript and I agree to the submission.
I agree to participate as a contributing co-author.
  • Many thanks for your note. I confirm that I read and approve the ms. for submission. Thanks for the great and important work.
  • The revised version is definitively clearer for non-experts, and my previous comments on the manuscript have been addressed. I’ll implement the recommendations detailed in this paper into future publications and teaching material.
Therefore I am happy to be listed as co-author for the submission to Cell Metab.
  • First of all, I think that the manuscript gains a lot after splitting it into the two parts. Reader of the text is more focused and do not get tired before the end .
There are some suggestions and thoughts, when reading the article.
In Figure 1 - maybe it is worth to mention that AOX is not present in mammalian cells ?
Table 2 explains very well terms of different coupled states in Figure 3, which are maybe otherwise difficult to distinguish for people who are not familiar with them. I do not know how it appears in the journal but maybe it is better to sum Table 1 and Table 2 so that when you look Figure 3, it is easier to find definition of the a/un/Dys/de-coupled respiration?
It is great news that the editor-in-chief of Cell Metabolism considered our article to be suitable for his journal. If it is necessary to make the article even shorter, 3.6 may be the part in my opinion. Maybe Table 7 is not so necessary as the texts already explains the basic concept.
In general I think the article is with a great importance to have all the basic concepts and definitions in one article and I am honored to be part of it.
  • I have thoroughly gone through the recent version "MitoEAGLE preprint 2018-02-21(27)". It reads very well, and I would implement the recommendations into my future manuscripts/presentations.
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.
  • Many thanks for the note and well done for the choice to submit to CELL METABOLISM.
I confirm that I read the newest version of the paper and have no further additions.
Please let me know if anything else is needed.
  • I have read through the entire manuscript and I have no further comments or additions. I will certainly incorporate these recommendations into future studies and teaching materials. I think this will be great resource for future studies using bioenergetics applications.
  • I have fully read the latest version of the manuscript entitled "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology" I am excited and honored to be a contributing co-author to this critical work and I agree to implement all of the manuscripts suggestions into future publications. I have a couple of suggestions:
line 959 "Therefore, cell density must be optimization (changed to optimized), particularly in
The other comment is in regards to line 1017-1024 discussion of different mitochondrial sub populations. It may be out of the scope of this paper, but it also may be worth noting that with proper planning the selective isolation of different mitochondrial subpopulations can be an advantage and all different sub populations can be analyzed. Also, because permeabilized fibers contain all mitochondrial sub populations it is difficult to determine which sub population is being altered in a specific disease or intervention.
Gnaiger E Thank you for your feedback and critical checking. This is what we need.
On mt-subpopulations – do you think that this sentence is doing the job? “Different sizes of mitochondria are enriched at specific centrifugation speeds, which is used strategically for isolation of mitochondrial subpopulations.”
Valentine JM: Yes I like this sentence very much I just changed the word is to can be!
"Different sizes of mitochondria are enriched at specific centrifugation speeds, which can be used strategically for isolation of mitochondrial subpopulations.”
  • The manuscript looks good, and I agree to incorporate the recommendations into my work.
  • I confirm to have read the almost final version of the preprint publication, and agree to implement these recommendations.
  • I confirm to have read the newest version of the manuscript. I do not have any additional additions or suggestions for improvement. I agree to implement the recommendations into my future manuscripts, presentations and teaching materials.

This is great work and I hope it will accepted in your journal of choice.


  • I confirm that I have read the newest version of the manuscript and agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
Thank you very much
  • Everything looks and sounds for me.
  • In figure 1A. I believe there are typos in the protons pumped at complex III and IV.
What ends up on the innermembrane side of complex III is 4H+ (or for molecular oxygen 8H+). Some of which comes from UQH2 and some from the matrix.
What ends up on the innermembrane side of complex IV is 2H+ (or for molecular oxygen 4H+). Half the protons from the matrix side combine with O2 to form water and don’t make it into the innermembrane space. Please see the attached edit. Plus a Figure 4.2 from the Brand and Nicholson Text.
If this is not correct, please clarify this issue for me.
Otherwise I agree with the manuscript and to be a co-author. Thank you.
  • I'm happy with this version of the manuscript. I confirm to have read the newest version of the manuscript and i agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
  • It is great to hear that Cell Metabolism is interested. This work is a masterpiece of gentile science with rigorous attention to detail, and is sure to be broadly cited wherever it ends up. I am honored to have contributed as a co-author.
Best of luck with submission!
  • I am working at a couple of new short comments that I would like to submit to your attention as my additional contribution to the improvement of the current version of the manuscript. I hope that I will be able to send you my notes within a few days.
Meanwhile, as a contributing co-author, I confirm "to have read the newest version of the manuscript and to have made additions or suggestions for improvement". I also agree to implement the recommendations into my future manuscripts, presentations and teaching materials.
  • No further suggestions. I confirm to have read the newest version of the manuscript and to have made a suggestion for improvement
  • I have had an opportunity to review the contents of the manuscript and agree to implement the recommendations into our future manuscripts, presentations and teaching materials.
I am excited at the opportunity to present this article to the broader community and hope this gets accepted in ‘Cell Metabolism’. I once again thank you for your leadership in this regard, and look forward to our next meeting.
  • Thank-you for listing me. Happy to help in any other way
  • I´ve read the latest version and everything is to my satisfaction, my suggestion have been considered. I will implement this in my future papers, presentations and lectures. Best wishes and good luck with the submission.
  • I herby confirm that I have read the last version of the manuscript. I think the manuscript give a clear introduction to terms related to mitocondrial respiratory and I confirm to include them in my lab and in teaching.
  • Thanks for your letter re the manuscript. I’d be happy to be included as a co-author and will have a detailed read of it and get back to you in the next few days if i have any comments/suggestions.
...thanks for all the hard work.
  • I have read the newest version of the manuscript which it is far over my actual knowledge in the field. I have learned during the reading, and consequently I will be implement the concepts into our manuscripts, presentations, as well as teaching materials.
  • I am happy to be upgraded as contributing co-author and I will have a careful read over the current manuscript by Monday.
  • Thank you once more for the opportunity to participate in introducing concepts for the broad scientific community. I have read the newest version of the manuscript and I fully agree with nomenclature of mitochondrial respiratory states and rates. Also, I will try the best to implement the nomencalture in my (or my students) future papers, presentations and materials as I have been trying to do up to now.
  • I have read through the latest version of the manuscript. I do not have any additional suggestions for revision. Thank you for including me as a co-author.
I agree to implement the recommendations into my future manuscripts, presentations, and teaching materials. Please let me know if anything additional is needed.
  • This is fantastic to see the end of this first manuscript.
I hope this will go through.
  • Thank you for your note and update. I’ve been reading thoroughly and with great interest the latest version (#22) of the manuscript. I do not expect to have any further comments, but in case I have them you will receive them within the deadline
  • I have read the paper, it is very comprehensive. It struck me it might be perhaps informative to make a more head-on comparison of the new (leak, ET, ROX ) and old (state 1, 2 etc) definitions of the respiratory states, for example in one of the tables, or an addition table.
  • I am glad to have this opportunity and happy for selecting me as a Joint collaborator.
However, I go through the manuscript of all the concept of mitochondrial physiology, and previously that book I have already, also helps me a to understand the concept of Mitochondrial Physiology. Here, I am sending you a three answer of your question, I am bit unfit for reviewing all that concepts in this short span period. Here, I am sending all the explanations which I think, as per my understanding. If any feedback please let me know.
I will also share with my Supervisors and Colleague in further.
Gnaiger E: Do you recognize a general need for a consensus on nomenclature and standards of reporting in the field of mitochondrial respiratory physiology?
Sharma V: Respiratory rates and states would be possible to develop a researchers need to choose which effect size provides the best summary and specify which effect size they report. In the end, the choice of an Proton motive force, ETS, ET has play a significant role in effect size calculation depends on the research question and the experimental design. It is important to explicitly stated that which effect size is calculated, and to make a motivated choice about which effect sizes to report. With the current overview, The manuscript have provided a practical concept and consistency to assist researchers in choosing and calculating effect sizes by establishing the link between vectorial and scalar energy transformation and coupling in oxidative phosphorylation in both states.
Gnaiger E: Can you provide comments and suggestions for the ‘MitoEAGLE preprint: Mitochondrial respiratory states and rates’ from your point of view as an editor?
Sharma V: As per my suggestion the manuscript explained very well about to do the mechanisms behind observed oxygen consumption, and factors affecting the oxygen consumption rates and consequence of different electron pathway.
However, I would suggest the elaborate of any kind of procedure which will conduct as in vivo method determination of mitochondrial respiratory chain. I am not aware about how to perform such measurements in tissue does anybody have any experience in this topic kindly mentioned.
  • Short Classification of Mitochondrial biomarkers with respect to disorders in Respiratory states and Rates. (Healthy and Diseases States)
  • Emphasize the abnormal mitochondrial function and positioning alters multiple components of the specific organ physiological system.
Gnaiger E: Which further steps do you suggest towards implementing a harmonized terminology on mitochondrial states and rates in your editorial policy?
Sharma V:
  • Metabolic programming of immune cell differentiation and proliferation into anti and pro-inflammatory phenotypes, driven by the balance of oxidative phosphorylation (OXPHOS) vs. glycolysis and mitochondrial reactive oxygen species (mtROS).[1]
  • Mitochondrial epidemiological studies with reference to particular disease states and rates. [2]
  • Modulating the mitochondrial quality with diseases transmission with respect to mitochondrial biomarkers.[3][4]
  • Development of simulation studies, based on experimental method for predicting the future perspective of specific mitochondrial disorder.
  • Designing of multi-omics experiments and applying on large data sets to develop a precise medicine approach in mitochondrial dysfunction and also helps us to understand the physiological mechanism in different aspects. [3][4]
Reference
1. Picard, M., D.C. Wallace, and Y. Burelle, The rise of mitochondria in medicine. Mitochondrion, 2016. 30: p. 105-116.
2. Diot, A., et al., Modulating mitochondrial quality in disease transmission: towards enabling mitochondrial DNA disease carriers to have healthy children. Biochemical Society Transactions, 2016. 44(4): p. 1091-1100.
3. Haas, R., et al., Designing and interpreting ‘multi-omic’experiments that may change our understanding of biology. Current Opinion in Systems Biology, 2017. 6: p. 37-45.
4. Meng, C., et al., A multivariate approach to the integration of multi-omics datasets. BMC bioinformatics, 2014. 15(1): p. 162.
Gnaiger E: Many thanks for your detailed message. I am glad to include you in the list of co-authors. The next update is in preparation.
Sharma V: Warm thanks for replying. If you have any further question, please let me know.
Looking further for fruitful interaction.


  • I have read the manuscript and have no suggestions for further improvements. Let me know if you need any more information from me. I accept the upgrade to contributing co-author.
  • I read the last version and my group are all studying the manuscript to use it as a “bible” of mitochondria.
I am ok with the submission to a journal
  • I have not yet congratulated and thanked you for putting together this impressive document and for coordinating this whole effort, and I am doing so now. I attach the manuscript with a few comments, all minor and editorial. Good luck with the submission and thanks for involving me in the project!
  • Thank you. I have gone through the last version and will go through the latest version and get back asap.
I have read the manuscript again. It’s very solid. This time I only have a couple suggestions which may be implemented:
Page 11, line 480-482:
“…(4) mitochondrial density (enzyme concentrations and membrane area) and morphology (cristae folding, fission and fusion).”
Suggested reference to support this sentence: PMID: 24606803
Page 23, line 1018-1019:

“Mitochondria undergo dynamic fission and fusion cycles, and can exist in multiple stages and sizes which may be altered by a range of factors.”

I suggest to add something like this after the quoted sentence: “Changes in mitochondrial morphology is often associated with alterations in mitochondrial function, and In cultured cells this can be evaluated by quantitative analysis of mitochondrial shape using fluorescence microscopy and image analysis in 2 or 3 dimensions (Ref PMID: 24988307)”
Thanks.
  • Thank you for the opportunity to join as a co-author on the manuscript "Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology". I have taken the time to read the latest version, and am favourably impressed with the clarity of the writing and the content. This will be a most useful publication for many different research groups and teachers world-wide.
I did detect one typographical error: Box 1. Line 236/237 Membrane fluidity “exters”…, should I think read “extends”.
  • I have read the newest version of the manuscript and I think the manuscript looks excellent and I have no more further suggestions.


  • I'm glad to take part in the discussion of the MitoEAGLE preprint. The new version of the manuscript provides a significant step forward for the approach to mitochondrial metabolism concepts and the terminology harmonization, especially for the young scientists. For my purposes, I found very important the section on the Flux Normalization methods and principles; this point is crucial for the interpretation of the final outcomes and for the results comparison between different labs. The endeavour to find standard protocols and normalization requires time; nonetheless a statistical evaluation of the best mitochondrial marker for different mitochondrial preparation and tissues is needed. This issue is well pointed out in the conclusion section.
In Classical terminology for isolated mitochondria, the “classical states” of mitochondrial respiration are well presented; I have personally started the study of mitochondrial bioenergetic using the mitochondrial States as references for evaluating my respirometry experiments. Must we consider this terminology out of date or rather a co-existing complementary terminology?
Thank you very much for all your work.
here my last comments of the revised version of the manuscript:
Fig 1: In the figure might be useful to add peroxisome (or an abbreviation) directly in the picture.
159, 163: extra-mitochondrial instead of extramitochondrial
170, 171: Capacities of oxidative phosphorylation and electron transfer are measured..
212: Morphologically
223, 224: Mitochondria are the structural and functional elements of cell respiration consisting in the reduction of oxygen …
229: ..complexes (proton pumps…
242: cellular mitochondrial homeostasis (mitostatis)
245: , and through genome stability modules
246: stress cues.
249: ..network, leading to sharing of soluble factors and mitochondrial mtDNA molecules (see below),..
398: “where P indicates phosphorylation of ADP to ATP or GDP to GTP.”: Although GDP is no longer cited in the text, the parallelism between ADP and GDP might be misleading for not experienced scientists, since only ADP is involved in electrotransfer phosphorylation.
Fig 2: The capitol letters of the panels (A, B, C) might be in the upper left of the panels instead of the lower left.
489: , cytochrome c;
647: … adenylates, i.e., AMP, ADP, ATP.
700,701: Superoxide production by Complex CI and CIII of the ETS leads to…
For a better understanding of figure 4 and the relationship between the respiratory rates L, P and E, I was wondering whether the part ranging from line 614 and 641 could be shifted after the coupling states explanations and ROX description. (Just an idea).
982: ..number of cells..
1269: Residual O2 consumption (ROX)..
Gnaiger E : Many thanks for your detailed reading. I included your nice improvements.
  • please find attached our position statement manuscript - with one comment from me, right at the beginning (highlighted yellowed text).
Overall, the text is still not easy to digest. I had to read mnay sections two or three times to understand their intention. Maybe, a professional science writer should work on in to smoothen the tex as much as possible.
  • Thank you very much for inviting me to be a coauthor of your masterpiece of mitochondrial history! I will try my best to be qualified for being a contributing co-author
Gnaiger E: I would be very thankful for receiving your comments and critical evaluation of the present version of our manuscript.
  • Dear Anthony, Pushpa did a great job. Here is a first-level promising answer (see here).
Molina AJ: That is fantastic! I have worked with that editor before. A positive response from her is a promising sign.
  • I read the MitoEAGLE paper, and you've done a lot of work with it! It is more compact now, and it's much easier to read. The part of thermodynamics might be a separate article, this would be very important for young scientists and for the education in general in the field of bioenergetics.
May be you could advise me? I have problem with terminology, but I´m not sure, is it the topic of our paper (Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology). It is not clear at all, when the mitochondrial preparations are in vivo, in situ, ex vivo or in vitro. With isolated mitochondria no problem, this mitochondrial preparation is always described as in vitro. But with mitochondria in cell cultures the terminology is very messy. I met various variants like in vivo, in situ and ex vivo in different papers. The story is not better also with permebilized samples and tissue homogenates.
Gnaiger E: Thank you for your positive feedback. I agree that the thermodynamics part is important. I was reluctant to remove it, but the arguments to reduce the complexity of the present MS were valid.
You make a good point on the terminology of in vivo, in situ, ex vivo, .. I suggest that we ‘make it’ a topic of our paper, since we start with the definition of mitochondrial preparations. I suggest: all mitochondrial preparations are ‘in vitro’ (then we do not need ‘ex vivo’). In contrast to isolated mitochondrial and homogenate preparations, mitochondria can be considered as ‘in situ’ relative to the plasma membrane in permeabilized fibers and permeabilized cells. Do you think that everybody can agree on that? The text is included in the new version as a suggestion:
“Mitochondrial preparations are defined as either isolated mitochondria, or tissue and cellular preparations in which the barrier function of the plasma membrane is disrupted. Since this entails the loss of cell viability, mitochondrial preparations are not studied in vivo. In contrast to isolated mitochondria and tissue homogenate preparations, mitochondria in permeabilized tissues and cells are in situ relative to the plasma membrane.” (I will upload the new version in a few minutes)
Now we were invited by CELL METABOLISM to submit our manuscript for in-depth editorial evaluation.
  • Dear Prof. Liu Shusen, I would be very thankful for receiving your comments and critical evaluation of the present version of our manuscript. Below is the link to our updated version.
Liu SS: I will give my response as much as possible to you after my finishment of reading it. but, I am not sure every point I could agree with that noted in the manuscript, although, it is very good totally and generally! My main consideration is that biomembrane bioenergetics ,the study on mitochondrial oxidative phosphorylation is still in the rapid period of development and refinement, not only technically, but also theoritically/conceptionally. So, I need get time to learn and to read more recent scientific research achievements and progresses, including your manuscript!
  • I have received a response from "CELL METABOLISM". Looks like they are interested in our manuscript. Please read their response and advise me what to do next.
Gnaiger E: Thank you so much for your correspondence with CELL METABOLISM. This sounds like the door is not closed. I propose that I will add the Executive summar into the present pdf file and send the whole manuscript to Nikla Emambokus, Editor-in-Chief, Cell Metabolism. In any case, it will be interesting to receive his response.
Sharma P: Excellent idea to polish the manuscript and submit to CELL METABOLISM ASAP before editor changes his mind.
Gnaiger E: Dear Dr. Emambokus, we thank you for your interest in evaluating our manuscript ‘Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology’ in your in-house editorial review system.
A pdf file of the full manuscript is attached. A Task Group of the COST Action MitoEAGLE has been working on this manuscript with Open Access as a ‘MitoEAGLE preprint’ and the ultimate aim of peer-reviewed publication: » http://www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08
The global MitoEAGLE network made it possible to collaborate with more than 250 co-authors 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 the 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.
The manuscript has not yet been formatted for a specific journal. We will be glad to ensure that-before submission-an updated version conforms to the format guidelines for your journal, should you encourage us to proceed with submission at your EM site.
We are looking forward to hearing your opinion about the timeliness and potential impact of our manuscript, and possibly your suggestions for improvement.
With many thanks for your consideration, and kind regards, Erich Gnaiger
  • I have sent the enlosed letter to several jpournals, in which we published during the last 5 years, and which may have something in common with mitochondrial physiology. I have included you as the “to your information” addressee.
Gnaiger E: Many thanks for your great efforts!
  • Dr. Gnaiger, here are some suggestions or items that might need to be double checked. Most of the items are minor. The manuscript looks great
Thank you for your effort. This manuscript is timely, informative, and clearly sets forth the future: highly educational as well. No single author or smaller group could do what’s been accomplished here. Again, I must Thank you for orchestrating it so well!
I found a couple of things in the manuscript where some items need to be double checked, clarified, or verified that they are not typos or missing characters as posted (Version 22_2018-02-08 ). Hopefully, the below listed comments will aid in the final manuscript and publication. and editing.
Lines 210-211: might be better as “enzymes of the tricarboxylic acid and fatty acid oxidation”.
Lines 219-223: Possible to many “and’s, not sure if this is a run on sentence or not.
Line 404: (10 ug•10-6 cells) is this 10-6 cells or 106 cells, also is the dot the best way to separate the 2 different normalization values?
464, 524-525, Figure 3 Note 5 and through the whole manuscript: “superoxide anion radical” could be simply “superoxide” unless the intent is to educate people unfamiliar with this molecule and highlight that it’s a radical and an anion.
Line 741: is (CHNO; 2[H]) correct or a typo or missing a character?
Gnaiger E: Dear Brett, many thanks for your excellent comments and (already earlier) contributions. We have an interesting reply from CELL METABOLISM (see here). We will send them the MS.


  • Here are a few additinal comments on the review (pages 6, 13, 25). I'm still a little bit under the impression that it is very complex for general users to apply properly all the nomenclature.
About the editors, nobody comes into my mind but I will look if I can find some to send the letter of information too.
  • I think, the letter to the editor you sent is it's really nicely written, with all needed information.
  • Thank you very much for your collaboration adn invitation. I think that it is a very nice article.
I will send it to the editors.
In fact, this afternoon I have a meeting with Santiago Lamas who is an associate editor of redox Biology,and I can talk about the article.
Gnaiger E:May I ask you to send a letter with the information contained in the template (feel free to modify) to relevant editors whom you know.
  • Thanks, Erich. I will do so.
  • I deeply appreciate your efforts to make the text more affordable also for not-experts in the field: I agree with you and I am convinced that this help to better widespread the message inside and to gain much more visibility and an enhanced number of potential reader will be reached.
BTW, I am preparing to send the letter you suggested to some EiC but I think it would be useful to avoid any overlapping in this task: therefore I am suggesting you make available a list of EiC/journals already contacted in order it could be easier to resend the same letter to the same EiC.
Moreover, before sending the letter, the sender should advise you for a continuous update of the list.
You can also prepare a doodle/surveymonkey or similar where each contributor could add by his/her own the journal contacted.
  • I offer some suggestions.
Gnaiger E: Many thanks for your excellent comments and (already earlier) contributions. We have an interesting reply from CELL METABOLISM (see Dr.Sharma). We will send them the MS.
Buettner GR: This manuscript has had a rigorous “internal” review. Thus, a journal’s review would not be expected to add too much.
I think you are in the driver’s seat, as many journals would welcome this work. I would not bend too much to a specific journal’s demands. Bargain hard if the editor(s) want changes in format, length etc.
To ensure the work gets into PubMed Central (12 mo from publication), I assume an author(s) must have NIH grant support. I have such support. Others may as well. Thus, this may need consideration.  ::::: I have done this for other collaborative works; obviously it helps me justify my grant -- showing productively and progress, but most important it assists with long-term accessibility.
The goal is to get easy and wide distribution to all researchers whose research touches on this area.
Stocker R: I completely agree with Garry. Accessibility is going to be a key determinator whether this project will be a success and result in broad adherence to the guidelines provided.
  • Dear Erich, thanks a lot. I think this is a great idea. I sent the message contained in the attachment to three editors I’ve been in contact with and their colleagues.
  • I would suggest you to contact Prof. Liu Shusen for his comments. I copy this message to him so that he may be helpful.
Gnaiger E: Many thanks for reaching out to Prof. Liu Shusen. I have gladly added you to the list of co-authors of our MitoEAGLE Position Statement.
We recieved an interesting reply from CELL METABOLISM (see preprints main page). We will send them the MS.
  • 2018-02-12 Jadiya P
  • I have gone through the preprint version and its really nice compilation of our current understanding of the mitochondrial physiology as well as related terminology. In the Box1, Line 229, it would be great to add one sentence for the endoplasmic reticulum and mitochondrial contacts as these contact sites involved in metabolites transfer as well as mitochondrial dynamics regulation.
"The crosstalk between mitochondria and endoplasmic reticulum are involved in the regulation of various cellular functions, such as calcium homeostasis, cell division, autophagy, differentiation, anti-viral signaling, and others (Murley and Nunnari 2016)".
Ref: Murley A, Nunnari J (2016) The Emerging Network of Mitochondria-Organelle Contacts. Mol Cell 61(5):648-653.
Gnaiger E: Many thanks for your contribution, which I added according to your suggestion (line 217). Thanks for joining our initiative as a co-author.
  • I has become a balanced and high quality document. Nevertheless still some comments. Please find these attached (written in the scan and I added only the pages with comments). Please find also an alternative abstract, which is slightly altered (improved) over the one pasted in the scan.
Gnaiger E: Thank you very much, your input is much appreciated and largely included in the new ms version. We have an interesting reply from CELL METABOLISM (see here). We will send them the MS.
  • I believe there is strong need for such armonization procedure.
A detail regarding it. I would personally propose to redefine the electron transfer capacity as ETC rather than ETS –it is less confusing.
  • Thank you for your note. I have a question regarding the preparation of the manuscript. ::::: As a Member of the MitoEAGLE Management Committee, could I make suggestions and/or contributions to the manuscript, regarding mitochondrial physiology during ageing?
Gnaiger E: Many thanks for your offer to collaborate on our MS. To address various EAGLE issues (aging is of course important), might be a good idea in the context of a manuscript with a focus on various factors on mitochondrial function. The present MS is a position statement on general definitions of mitochondrial respiratory states and rates, and even with this specific title we have to focus on respiratory coupling states. Otherwise, the MS turns into an oversized document.


  • Thank you for the email and the attached paper.
I found the paper really very informative and as discussed before the initiative is very much needed and timely.
At this point I have no further input; we do have some papers in Revision and if we get them through I will send the citations for adding in this Review paper.
Gnaiger E: On ET- versus ETS-capacity. I also like ETS better. BUT: There is the OXPHOS system, and we do not say ‘OXPHOS system capacity’. There is the ET system – we should say ET-capacity.
  • It's a pleasure to participate in such interesting project for me, thank You for invitation!
  • I can send this letter to the editor of journals "Cell Metabolism,Mitochondrion, Nature, and Science
Gnaiger E: Thank you so much for your correspondence with CELL METABOLISM. This sounds like the door is not closed. I propose that I will add the Executive summar into the present pdf file and send the whole manuscript to Nikla Emambokus, Editor-in-Chief, Cell Metabolism. In any case, it will be interesting to receive his response.
  • A suggestion is to post something similar to relevant sections at the American Physiological Society. (http://connect.the-aps.org/home). You could probably sent this to all sections, but I’m pretty sure there would be interest from members of the endocrinology and metabolism and exercise physiology sections.
  • I would send it to the editor in Chief of Mitochondrion. Is it OK?
Gnaiger E:Excellent – thanks
  • the demand specifies to send the information to Editors of Journals that I know. I could do that for J. Cellular Physiology but do not you expect a higher and more poweful journal to publish this excellent paper ? In addition, the timing....would be bfore you submit or after...
  • I want to congratulate you and all the co-authors for the excellent work. The manuscript has highly improved since my last reading. I am sending some minor suggestions annotated in the attached pdf file. Thank you very much for the collaboration,
Gnaiger E: Thank you for your kind feedback and for the careful reading with excellent improvements – obviously you are a professional science writer. I have incorportated your suggestions in the new version 24.
  • I think Cell Metabolism is hardly possible. Did you consider JBC (with John Demu as editor for mitochondria) or Nature Communications? Maybe also Molecular Metabolism, but that may be too little physiological.
  • Will have a look.
  • Off course! I can prepare a letter and forward this message to actual editors whom I know.
  • Sure Erich. I will send it to a couple of editors. I will keep you informed.
  • OK- I sent the letter to an editor of JIMD
  • The manuscript looks fantastic and I am very excited about it. Just a couple of comments. In the section on standardization. It is well outlined how proper standardization should be conducted; however in the manuscript we do not say, which method of standardization to use. For instance, flux can be normalized to wet weight or dry weight of the tissue (for permeabilized fibers) but we do not say which to use and the flux per mass values are very different depending on which you use. There is a lot more variability in weight wet measurements due to how much drying researcher does. Also, most scales are not accurate enough for measuring 1-2mg pieces of tissue and weighing the sample three times can give much more consistent values. If measurements are off by just one or even less than one mg when weighing a 1mg piece of muscle, then values can have double or half the respiration rates when normalizing to mass. Also, I recommend in the paper suggesting all raw data should be published in a supplemental table. This way anyone can access and compare values. Also, while the limitations of the normalizing factors are discussed in detail, we should specify which marker of mitochondrial content (CS activity mtDNA copy number) to use for normalizing. It may be important to explain that when phosphorylation is not limiting oxygen consumption such as in mouse permeabilized muscle fibers the addition of uncouplers does not increase oxygen flux; therefore, when generating flux control ratios using CI+CII_E to normalize to becomes confusing because CI+CII_P will also equal 1.
As far as journals go, I think that diabetes would be a good journal to submit to if cell metabolism does not work out. Although it is limited to this one metabolic disorder nearly every researcher studying mitochondrial physiology have published in this journal.
Another possible journal is EMBO as the word mitochondria turns up over 1500 articles and is a well read journal.
Gnaiger E: Thank you for your valuable comments. I agree with you, that recommendations on wet weight versus dry weight should be given. In fact, a MitoEAGLE Task Group is working on this to provide a comparative experimental basis for evaluation and a corresponding recommendation. This will be summarized in a separate MitoEAGLE position paper. Similarly, a recommendation on the ‘best’ mt-marker would be helpful, if such a best marker does exist. The present MS may contribute to make us more aware on the importance of paying critical attention to mt-markers. A insufficiently substantiated recommendation, however, would not help and weaken the impact of our manuscript.
Ps: We are working on nomenclature of the pathway states. I regret to have introduced the CI+II nomenclature (but CI+CII is different), hence we replaced it with a less difficult terminology:
  • Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8. - http://www.bioblast.at/index.php/Lemieux_2017_Sci_Rep
Valentine JM: I agree and I am excited to contribute to this critical work in any way possible. Please let me know what I can do!!! Thank you for the reference paper.
Garcia-Roves P Most of your comments are being addressed in different tasks coordinated by Working Group 2 (MitoEAGLE data repository on muscle tissues): http://www.mitoeagle.org/index.php/MitoEAGLE_data:_muscle
We are currently interested on OXPHOS capacity in permeabilized myofibers, addressing the effect of oxygen, ADP and blebbistatin in MiR05Cr and Buffer Z; where wet and dry weight is also being discussed. Moreover, we are performing a study to supply mitochondrial respirometry reference values using permeabilized mouse soleus muscle fibers from C57Bl6J mice and same experimental procedures.
Valentine JM: This is very exciting!!!
Especially about the reference values for C57/BL6J mice. I am sure that you have seen the paper below, but incase you have not there seems to be a pretty substantial difference in respiration rates between C57BL/6J and C57/BL6N mice depending on the substrates used. It may be worth highlighting the differences in the two mouse lines at the working group2 because often times people do not mention or even know which C57/BL6 mouse they are using.(https://www.ncbi.nlm.nih.gov/pubmed/27495226)
  • I am happy to send a letter to editors. I came up with four editors from their Cell Metab website. They are Martin Brand, Michael Murphy, Nils-Goran Larsson, and Varnsi Mootha. I wanted to see if you suggest anyone else who I might include in the email as this will have an impact on our paper. I am happy with your template and will send it as is. I would also like to confirm that you want me to use the email address of metabolism@cell.com instead of individual emails of relevant editors.
  • I'll do it with pleasure
  • Of course that I will send the letter to the known editors. I would appreciate if you would confirm if the idea is to send to Cell Metabolism or any journal that you know that includes studies of mitochondria such as Sci report, Hepatology, BBA, etc.
  • As you requested, I will think of possible Journal Editors (e.g. I am familiar with U. Brandt-BBA bioenergetis, but I suppose that you already asked him; if not, please let me know). Meanwhile, I wonder if you would like to consider also the possibility of submitting the same inquiry to the "Scientific Committee of the World Mitochondria Society", whose annual conference will be held as usual in Berlin (next date: October 2018 - https://www.targeting-mitochondria.com/)
  • I have just looked at the preprint very quickly and I see many changes 😊 Wow! Very, very good job! During next days I will be reading it carefully. Now I am thinking about the journal where this review could be sent. So, in my opinion, this manuscript is excellent and you may consider to send it i.e. here ? :) ... Nature Methods, IF >25; of course earlier writing to editor
  • Will do and send to other editors. I am the editor of experimental gerontology. Have you considered to publish this in a methods journal?
Gnaiger E: It is not clear which journal will finally turn out to be most suitable for our multi-author paper. We received a letter from the Editor-in-Chief of CELL METABOLISM to submit our paper. ::::: We should give it a try. It is not a typical methods manuscript, although it might perhaps fit into this category.

Please, let me know your evaluation and feedback, and thanks in advance for joining and supporting our aim.


  • Thank you very much, I will send the input of the editors.
  • Great initiative. I will send the letter to the editors I know and will come back to you. Again thanks again for your efforts
  • Many thanks for the information. I will disseminate the information.
sent to : kksingh@uab.edu
  • I know Keshav K Singh, Editor-in-chief of “Mitochondrion”, but I guess he might have been contacted already?
  • Id be happy to forward the letter to Editors
  • Thank you very much for your great effort in revising the manuscript and for sending me the revised version of this article that we have co-authored last year! It has been greatly improved!
  • Yes, thanks for this and the preprint earlier. I will try to get it done by early next week.


  • MitoEAGLE preprint 2018-02-08 Version 22


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