Mechanisms and physiological function of daily haemoglobin oxidation rhythms in red blood cells

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Abstract

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox‐sensitive covalent haem‐Hb linkage that forms during SDS‐mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro , or taken from humans in vivo , and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

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    Response to reviewers

    We thank all reviewers for their comments and suggestions. In line, below, are our responses, marked in Bold. Textural changes in the manuscript are also marked in Bold.

    Reviewer #1__ (__Evidence, reproducibility and clarity (Required)):

    **Summary:**

    Anuculeate red blood cell (RBC) is one of the interesting biological models that indicate the presence of eukaryotic circadian system independent of transcription-translation feedback. In this manuscript, the authors set up a new method for quantifying the circadian rhythmicity in RBC. The method called "Bloody Blotting" was developed through the careful and insightful investigation of …

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    The manuscript "Clock gene-independent daily regulation of haemoglobin oxidation in red blood cells" describes a new assay for quantification of haemoglobin oxidation status (bloody blotting) in anucleate red blood cells". This study furthers our understanding of the role of a post-translational oscillator (PTO) in generating circadian rhythms in biology. The authors first describe how earlier work demonstrated 24h rhythms in the intensity of chemiluminescent bands on membranes blotted with protein from …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The study aims to provide a new tool for detecting the hemoglobin oxidative status named "Bloody blotting". It is based on redox-sensitive covalent linkage between the haem and the haemoglobin. This linkage is a consequence of an artifactual reaction provoked by the protein extraction, due to the lysis buffer's properties. In addition, using an in vitro (red blood cells) or in vivo (patients' blood) model the authors provide insight in the oscillating nature in the oxygen-carrying and nitrite reductase capacity of the blood, which is unaffected by the mutation of CK1εtau/tau and Fbxl3aafh/fh

    Major comments:

    In my honest …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Anuculeate red blood cell (RBC) is one of the interesting biological models that indicate the presence of eukaryotic circadian system independent of transcription-translation feedback. In this manuscript, the authors set up a new method for quantifying the circadian rhythmicity in RBC. The method called "Bloody Blotting" was developed through the careful and insightful investigation of "non-specific band" observed in the western blotting of peroxiredoxin, which has been used for the circadian monitoring of RBC. The authors characterized that the "non-specific' circadian-fluctuating signals, which can be observed by ECL …