Category: Methodology

Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines

Abstract:

Cells require energy in the form of ATP to function. The two main ways in which cells generate energy in mammalian cells is through glycolysis and oxidative phosphorylation (OXPHOS). Glycolysis takes place in the cytosol and involves the breakdown of glucose molecules, generating ATP and pyruvate, while OXPHOS takes place in the mitochondria and is responsible for producing the majority of ATP for the cell. A dysregulation of these cellular processes has been reported in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In order to understand the mechanisms of the disease, it is imperative to understand how the bioenergetic pathways are altered in ME/CFS.

Here we describe a method for measuring mitochondrial function and glycolytic function using the Agilent Seahorse Extracellular Flux Analyzer. We have optimized these assays for use in actively proliferating lymphoblastoid cell lines that are generated from blood cells. This assay measures oxygen consumption rate and extracellular acidification rates providing an overview of mitochondrial function and efficiency and glycolytic rate and capacity, respectively. These assays are performed on live, intact cells, and enable us to view different components and measurements of energy metabolism through the injection of different compounds that stimulate or inhibit various sections of these pathways. The below method details an optimized glycolysis and mitochondrial assay for 96-well plates with modifications noted for use in 24-well plates.

Source: Katsaros T, Missailidis D, Annesley SJ. Real-Time Measurement of Mitochondrial Function and Glycolysis in Lymphoblastoid Cell Lines. Methods Mol Biol. 2025;2920:173-202. doi: 10.1007/978-1-0716-4498-0_11. PMID: 40372684. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_11

Mitochondrial Measures in Primary Cells Isolated from Patients with ME/CFS

Abstract:

Fibroblasts and peripheral blood mononuclear cells (PBMCs) are commonly utilized cell types for the analysis of mitochondrial function. Fibroblasts, derived from connective tissue, provide a reliable model for studying mitochondrial metabolism due to their active role in energy production and their accessibility for experimental manipulations. PBMCs, on the other hand, are a heterogeneous population of immune cells that include lymphocytes and monocytes. They offer the advantage of reflecting mitochondrial function in circulating cells and providing insights into systemic aspects of mitochondrial biology. Both cell types can be cultured and treated with various substrates or stressors to assess parameters of mitochondrial function.

Here we describe the use of fibroblasts and PBMCs isolated from patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to investigate mitochondrial abnormalities in the pathogenesis of this disease. Our techniques employ the use of fluorescent cellular dyes to measure mitochondrial mass, membrane potential and reactive oxygen species levels, luminescent measures of cellular NAD/NADH levels, and FRET-based measurements of the cellular and energy regulators, TORC1 and AMPK. These techniques are similarly useful for studying different physiological and pathological conditions.

Source: Allan CY, Katsaros T, Missailidis D, Fisher PR, Annesley SJ. Mitochondrial Measures in Primary Cells Isolated from Patients with ME/CFS. Methods Mol Biol. 2025;2920:203-223. doi: 10.1007/978-1-0716-4498-0_12. PMID: 40372685. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_12

Measuring Biomarkers of Oxidative Stress in ME/CFS Patients

Abstract:

Patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) have a deficiency in energy production as a result of dysfunctions in their mitochondrial metabolism, defects in the complexes of the electron transport chain, and in the regulation of reactive oxygen species (ROS). This can lead to an imbalance and excess of these species with subsequent modifications of proteins, lipids, and DNA.

Oxidative stress is defined as an accumulation of ROS due to a loss of regulation and the subsequent inability to detoxify them. The modifications to the cellular macromolecules by ROS can be used as biomarkers of oxidative stress and so have the potential to monitor the disease course of a condition like ME/CFS.

Proteins are especially vulnerable to oxidative stress as amino acid residues are naturally modified as part of cell signaling so, in an imbalance between ROS and antioxidants, proteins become modified at multiple sites potentially altering structure and function. Protein carbonyl modifications are stable and can be measured using 2,4-dinitrophenylhydrazine using a commercial ELISA assay. This has been applied here to immune cell proteins and plasma from ME/CFS patients who had moderate functional activity before and during an exercise protocol, and was shown to have potential as a marker of oxidative stress in these patients. The methods used to measure the DNA modification, 8-hydroxy-2′-deoxyguanosine (8-OHdG) are known to give varied results depending on the technology used.

Here, a commercial ELISA assay did not have the sensitivity to detect the modifications in the DNA before and during the exercise protocol of these ME/CFS patients.

Source: Walker M. Measuring Biomarkers of Oxidative Stress in ME/CFS Patients. Methods Mol Biol. 2025;2920:225-244. doi: 10.1007/978-1-0716-4498-0_13. PMID: 40372686. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_13

Measurement of Genetic Variations in ME/CFS Patients in the IDO2 Gene Encoding an Enzyme Metabolizing Tryptophan

Abstract:

Genetic variations in the indoleamine 2,3-dioxygenase (IDO2) gene that are commonly found in the general population have been assessed for their frequency in myalgic encephalomyelitis/chronic fatigue (ME/CFS) patients compared with healthy controls. They have potential for being genetic variations that lead to susceptibility to developing ME/CFS following exposure to a triggering stressor like a viral infection or other major stress events.

The IDO2 gene encodes an enzyme that is involved in the tryptophan-kynurenine pathway (TKP), and is activated if there are excessive amounts of tryptophan to prevent excessive serotonin production. The TKP pathway through production of NADH is involved in regulating the immune system and likely plays an important role in ME/CFS.

A simple method was developed to evaluate the 5 commonly occurring mutations in this gene in ME/CFS patients and to determine if one or more were present at higher frequency than in healthy controls. This might indicate a susceptibility factor for developing ME/CFS. In this chapter we describe the techniques used to isolate peripheral blood mononuclear cells (PBMCs), extract the DNA, and then do touchdown PCR and DNA sequencing for the analysis.

Source: Edgar CD, Blair A, Tate WP. Measurement of Genetic Variations in ME/CFS Patients in the IDO2 Gene Encoding an Enzyme Metabolizing Tryptophan. Methods Mol Biol. 2025;2920:247-256. doi: 10.1007/978-1-0716-4498-0_14. PMID: 40372687. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_14

Review of Neuroimaging Methods in ME/CFS

Abstract:

The brain is the most complex organ in the human body, and is involved in memory, speech, and movement, as well as regulating the functions of many other organs within the body. Various imaging techniques have detected subtle brain changes in vivo in ME/CFS. This chapter explores different neuroimaging studies used to investigate structural, functional, neurochemical, and tissue microstructural alterations in ME/CFS. These include magnetic resonance imaging (MRI), positron emission tomography (PET), and single photon emission computed tomography (SPECT).

Source: Thapaliya K, Inderyas M, Barnden L. Review of Neuroimaging Methods in ME/CFS. Methods Mol Biol. 2025;2920:257-277. doi: 10.1007/978-1-0716-4498-0_15. PMID: 40372688.  https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_15

Voice of the patient: people with myalgic encephalomyelitis/ chronic fatigue syndrome (ME/CFS) share in their own words

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, debilitating illness affecting millions of people worldwide. Patients with ME/CFS often feel misunderstood and report facing barriers to healthcare utilization.

Objective: We report on a Voice of the Patient (VOP) series that used tenets from photovoice and hermeneutic phenomenology methods. The approach prioritized respecting and engaging patients as they share individual experiences of living with ME/CFS.

Methods: We developed a 5-step process that could be replicated for interviewing patients in their own words. The process prioritized respecting patients while developing, documenting, and sharing individual accounts of living with ME/CFS. The standardized process for gathering each VOP story enabled individuals to share and participate on their own terms.

Results: Over four years, eight VOP stories were completed and posted on CDC’s ME/CFS website. The stories received over 196,000 page views. Each story was completed in approximately six months. Participants expressed gratitude for the opportunity to share experiences and were appreciative of the process that involved them in the development of stories.

Conclusions: Qualitative methods guided the process for participants taking a central role in sharing stories, which in turn may help educate about patient experiences with ME/CFS. Standardization of steps enabled consistency and transparency. Building flexibility into the process allowed interviewing a range of people with ME/CFS (i.e. bed bound to working) and enabled patients to give narratives in their voice. This process may help to share experiences of people with other chronic diseases or infection associated chronic conditions.

Source: Brimmer DJ, Lin JS, Unger ER. Voice of the patient: people with myalgic encephalomyelitis/ chronic fatigue syndrome (ME/CFS) share in their own words. Fatigue. 2025;13(2):1-11. doi: 10.1080/21641846.2024.2444826. PMID: 40123856; PMCID: PMC11926923. https://pmc.ncbi.nlm.nih.gov/articles/PMC11926923/ (Full text)

ME patient experiences: Sampling bias limits the external validity of findings

Abstract:

In a recent paper in Journal of Health Psychology, Kielland et al. present a study with the first objective of documenting how helpful or unhelpful persons with ME perceive common services and interventions. The authors recruited participants by respondent driven sampling, a method that aims to produce estimates that correct for sampling bias. However, we argue that the main assumptions of the method are not met, and that the results of the study thus cannot be generalised to the intended target population.

Source: Selvakumar J, Wyller VBB. ME patient experiences: Sampling bias limits the external validity of findings. J Health Psychol. 2025 Jan 21:13591053241310320. doi: 10.1177/13591053241310320. Epub ahead of print. PMID: 39838600. https://pubmed.ncbi.nlm.nih.gov/39838600/

Classification Accuracy and Description of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in an Integrated Health Care System, 2006-2017

Abstract:

Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic illness characterized by marked functional limitations and fatigue. Electronic health records can be used to estimate incidence of ME/CFS but may have limitations.

Methods: The authors used International Classification of Diseases (ICD) diagnosis codes to identify all presumptive cases of ME/CFS among 9- to 39-year-olds from 2006 to 2017. The authors randomly selected 200 cases for medical record review to classify cases as confirmed, probable, or possible, based on which and how many current clinical criteria they met, and to further characterize their illness. The authors calculated crude annual rates of ME/CFS coding stratified by age and sex using only those ICD codes that had identified confirmed, probable, or possible ME/CFS cases in the medical record review.

Results: The authors identified 522 individuals with presumptive ME/CFS based on having ≥ 1 ICD codes for ME/CFS in their electronic medical record. Of the 200 cases selected, records were available and reviewed for 188. Thirty (15%) were confirmed or probable ME/CFS cases, 39 (19%) were possible cases, 119 (60%) were not cases, and 12 (6%) had no medical record available. Confirmed/probable cases commonly had chronic pain (80%) or anxiety/depression (70%), and only 13 (43%) had completed a sleep study. Overall, 37 per 100,000 had ICD codes that identified confirmed, probable, or possible ME/CFS. Rates increased between 2006 and 2017, with the largest absolute increase among those 30-39 years old.

Conclusions: Using ICD diagnosis codes alone inaccurately estimates ME/CFS incidence.

Source: Liles EG, Irving SA, Koppolu P, Crane B, Naleway AL, Brooks NB, Gee J, Unger ER, Henninger ML. Classification Accuracy and Description of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in an Integrated Health Care System, 2006-2017. Perm J. 2024 Jun 19:1-12. doi: 10.7812/TPP/23.170. Epub ahead of print. PMID: 38980763. https://www.thepermanentejournal.org/doi/10.7812/TPP/23.170 (Full text)

BioMapAI: Artificial Intelligence Multi-Omics Modeling of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome

Abstract:

Chronic diseases like ME/CFS and long COVID exhibit high heterogeneity with multifactorial etiology and progression, complicating diagnosis and treatment. To address this, we developed BioMapAI, an explainable Deep Learning framework using the richest longitudinal multi-‘omics dataset for ME/CFS to date.

This dataset includes gut metagenomics, plasma metabolome, immune profiling, blood labs, and clinical symptoms. By connecting multi-‘omics to asymptom matrix, BioMapAI identified both disease- and symptom-specific biomarkers, reconstructed symptoms, and achieved state-of-the-art precision in disease classification. We also created the first connectivity map of these ‘omics in both healthy and disease states and revealed how microbiome-immune-metabolome crosstalk shifted from healthy to ME/CFS.

Thus, we proposed several innovative mechanistic hypotheses for ME/CFS: Disrupted microbial functions – SCFA (butyrate), BCAA (amino acid), tryptophan, benzoate – lost connection with plasma lipids and bile acids, and activated inflammatory and mucosal immune cells (MAIT, γδT cells) with INFγ and GzA secretion. These abnormal dynamics are linked to key disease symptoms, including gastrointestinal issues, fatigue, and sleep problems.

Source: Xiong R, Fleming E, Caldwell R, Vernon SD, Kozhaya L, Gunter C, Bateman L, Unutmaz D, Oh J. BioMapAI: Artificial Intelligence Multi-Omics Modeling of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome. bioRxiv [Preprint]. 2024 Jun 28:2024.06.24.600378. doi: 10.1101/2024.06.24.600378. PMID: 38979186; PMCID: PMC11230215. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11230215/ (Full text available as PDF file)

Assessing Functional Capacity in Myalgic Encephalopathy/Chronic Fatigue Syndrome: A Patient-Informed Questionnaire

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an acquired disease with significant morbidity that affects both children and adults. Post-exertional malaise is a cardinal symptom of ME/CFS and impacts a patient’s functional capacity (FC). The absence of effective tools to assess FC has significant consequences for timely diagnosis, clinical follow-up, assessments for patient disability benefits, and research studies. In interventional studies, the inability to assess FC can result in an incomplete assessment of the potential benefit of the intervention, leading to beneficial treatment outcomes being missed.
Methods: Using extensive, repeated patient feedback, we have developed a new questionnaire, FUNCAP, to accurately assess FC in ME/CFS patients. The questionnaire consists of eight domains divided by activity types: A. personal hygiene/basic functions, B. walking/movement, C. being upright, D. activities in the home, E. communication, F. activities outside the home, G. reactions to light and sound, and H. concentration.
Results: Through five rounds of anonymous web-based surveys and a further test–retest validation round, two versions of the questionnaire were developed: a longer version comprising 55 questions (FUNCAP55), developed for improved diagnostic and disability benefit/insurance FC assessments; and a shorter version (FUNCAP27) for clinical patient follow-up and potential use in research. Good reliability and validity and negligible floor and ceiling effects were found, with comparable findings in all aspects in both a large Norwegian (n = 1263) and a separate English-language international sample (n = 1387) demonstrating the validity and reliability of FUNCAP.
Conclusions: Our findings support the utility of FUNCAP as an effective, reliable and valid tool for assessing FC in ME/CFS patients.
Source: Sommerfelt K, Schei T, Seton KA, Carding SR. Assessing Functional Capacity in Myalgic Encephalopathy/Chronic Fatigue Syndrome: A Patient-Informed Questionnaire. Journal of Clinical Medicine. 2024; 13(12):3486. https://doi.org/10.3390/jcm13123486 https://www.mdpi.com/2077-0383/13/12/3486 (Full text)