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

Cerebrospinal fluid immune phenotyping reveals distinct immunotypes of myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex heterogeneous multiorgan disease that can have severe impact on individuals’ quality of life. Diagnosis of ME/CFS is based on symptom presentation, and a significant goal for the field is to establish meaningful subtypes. The heterogeneity in the literature suggests that individuals living with ME/CFS may suffer from overlapping but different underlying pathophysiological mechanisms.

We enrolled 40 participants with ME/CFS and 41 matched healthy control subjects at the Bragée Clinic in Sweden. We assessed plasma samples from both ME/CFS cases and control groups and cerebrospinal fluid (CSF) samples from individuals with ME/CFS.

We investigated dysregulated pathways and disease profiles through clinical questionnaires; multiplex analyses of cytokines, hormones, and matrix metalloproteinases; pathogen seroreactivity through peptide display bacteria libraries; and high-throughput microarray for autoantibodies. All samples used were from humans.

We show altered interaction patterns between circulating biological factors in plasma of ME/CFS participants. Our analysis of CSF from individuals with ME/CFS revealed different immunotypes of disease. We found 2 patient clusters based on matrix metalloproteinases profiles. The subgroups had similar clinical presentation but distinct pathogen exposure and CSF inflammatory profiles.

Our findings shed light on ME/CFS immune phenotypes and generate hypotheses for future research in disease pathogenesis and treatment development by exploring disease subgroups.

Source: Bastos VC, Greene KA, Tabachnikova A, Bhattacharjee B, Sjögren P, Bertilson B, Reifert J, Zhang M, Kamath K, Shon J, Gehlhausen JR, Guan L, VanElzakker M, Proal A, Bragée B, Iwasaki A. Cerebrospinal fluid immune phenotyping reveals distinct immunotypes of myalgic encephalomyelitis/chronic fatigue syndrome. J Immunol. 2025 May 15:vkaf087. doi: 10.1093/jimmun/vkaf087. Epub ahead of print. PMID: 40373264. https://academic.oup.com/jimmunol/advance-article/doi/10.1093/jimmun/vkaf087/8133211 (Full text)

Therapeutic Approaches to the Neurologic Manifestations of COVID-19

Abstract:

As of May 2022, there have been more than 527 million infections with severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) and over 6.2 million deaths from Coronavirus Disease 2019 (COVID-19) worldwide. COVID-19 is a multisystem illness with important neurologic consequences that impact long-term morbidity and mortality.

In the acutely ill, the neurologic manifestations of COVID-19 can include distressing but relatively benign symptoms such as headache, myalgias, and anosmia; however, entities such as encephalopathy, stroke, seizures, encephalitis, and Guillain-Barre Syndrome can cause neurologic injury and resulting disability that persists long after the acute pulmonary illness. Furthermore, as many as one-third of patients may experience persistent neurologic symptoms as part of a Post-Acute Sequelae of SARS-CoV-2 infection (Neuro-PASC) syndrome.

This Neuro-PASC syndrome can affect patients who required hospitalization for COVID-19 or patients who did not require hospitalization and who may have had minor or no pulmonary symptoms. Given the large number of individuals affected and the ability of neurologic complications to impair quality of life and productivity, the neurologic manifestations of COVID-19 are likely to have major and long-lasting personal, public health, and economic consequences.

While knowledge of disease mechanisms and therapies acquired prior to the pandemic can inform us on how to manage patients with the neurologic manifestations of COVID-19, there is a critical need for improved understanding of specific COVID-19 disease mechanisms and development of therapies that target the neurologic morbidities of COVID-19. This current perspective reviews evidence for proposed disease mechanisms as they inform the neurologic management of COVID-19 in adult patients while also identifying areas in need of further research.

Source: Graham EL, Koralnik IJ, Liotta EM. Therapeutic Approaches to the Neurologic Manifestations of COVID-19. Neurotherapeutics. 2022 Sep;19(5):1435-1466. doi: 10.1007/s13311-022-01267-y. Epub 2022 Jul 21. PMID: 35861926; PMCID: PMC9302225. https://pmc.ncbi.nlm.nih.gov/articles/PMC9302225/ (Full text)

Untargeted analysis in post-COVID-19 patients reveals dysregulated lipid pathways two years after recovery

Abstract:

Introduction: Similar to what it has been reported with preceding viral epidemics (such as MERS, SARS, or influenza), SARS-CoV-2 infection is also affecting the human immunometabolism with long-term consequences. Even with underreporting, an accumulated of almost 650 million people have been infected and 620 million recovered since the start of the pandemic; therefore, the impact of these long-term consequences in the world population could be significant. Recently, the World Health Organization recognized the post-COVID syndrome as a new entity, and guidelines are being established to manage and treat this new condition. However, there is still uncertainty about the molecular mechanisms behind the large number of symptoms reported worldwide.

Aims and Methods: In this study we aimed to evaluate the clinical and lipidomic profiles (using non-targeted lipidomics) of recovered patients who had a mild and severe COVID-19 infection (acute phase, first epidemic wave); the assessment was made two years after the initial infection.

Results: Fatigue (59%) and musculoskeletal (50%) symptoms as the most relevant and persistent. Functional analyses revealed that sterols, bile acids, isoprenoids, and fatty esters were the predicted metabolic pathways affected in both COVID-19 and post-COVID-19 patients. Principal Component Analysis showed differences between study groups. Several species of phosphatidylcholines and sphingomyelins were identified and expressed in higher levels in post-COVID-19 patients compared to controls. The paired analysis (comparing patients with an active infection and 2 years after recovery) show 170 dysregulated features. The relationship of such metabolic dysregulations with the clinical symptoms, point to the importance of developing diagnostic and therapeuthic markers based on cell signaling pathways.

Source: López-Hernández Y, Oropeza-Valdez JJ, García Lopez DA, Borrego JC, Murgu M, Valdez J, López JA, Monárrez-Espino J. Untargeted analysis in post-COVID-19 patients reveals dysregulated lipid pathways two years after recovery. Front Mol Biosci. 2023 Mar 3;10:1100486. doi: 10.3389/fmolb.2023.1100486. PMID: 36936993; PMCID: PMC10022496. https://pmc.ncbi.nlm.nih.gov/articles/PMC10022496/ (Full text)

Long-COVID in children and their parents: A prospective cohort study

Abstract:

Background: Long-COVID is a significant global health concern, regardless of age. However, few reports have longitudinally evaluated the characteristics, prevalence, and risk factors of long-COVID in children.

Methods: Participants were Japanese children younger than 18 years hospitalized for COVID-19 between November 2021 and October 2022, along with their COVID-19 affected parents. During hospitalization and at 1-, 3-, and 6-month follow-ups, participants completed age-appropriate questionnaires on long-COVID symptoms. The quality of life (QOL) score was assessed in children older than 2 years. The prevalence of long-COVID symptoms by age group was compared. Multivariable logistic regression analysis was conducted to investigate risk factors affecting long-COVID. Analysis of covariance adjusted for potential confounders was conducted to determine which symptoms affect QOL score.

Results: Of 108 children enrolled, the prevalence of long-COVID was 44.9%, 37.8%, and 22.8% at 1, 3, and 6 months, respectively, after SARS-CoV-2 infection. There were no specific risk factors for long-COVID. Cough, fatigue, and sleep disturbance were the most common long-COVID symptoms, with sleep disturbance associated with a change in lower QOL score from admission at all three follow-ups (mean difference 9.25, 20.15, and 19.81; 95% CI, 1.58-16.91, 3.38-36.92, and 5.51-34.11). The prevalence of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) symptoms among 0-6 years was significantly lower than among 7-17 years and parents; there was no significant difference between 7 and 17 years and parents.

Conclusion: Even 6 months after SARS-CoV-2 infection, 22.8% of pediatric patients still had long-COVID symptoms. Some of these symptoms were similar to those of ME/CFS, potentially affecting children’s QOL.

Source: Iijima H, Funaki T, Kubota M. Long-COVID in children and their parents: A prospective cohort study. Pediatr Int. 2025 Jan-Dec;67(1):e70042. doi: 10.1111/ped.70042. PMID: 40351239. https://onlinelibrary.wiley.com/doi/full/10.1111/ped.70042 (Full text)

The potential therapeutic approaches targeting gut health in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): a narrative review

Abstract:

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex disorder characterized by persistent fatigue and cognitive impairments, with emerging evidence highlighting the role of gut health in its pathophysiology. The main objective of this review was to synthesize qualitative and quantitative data from research examining the gut microbiota composition, inflammatory markers, and therapeutic outcomes of interventions targeting the microbiome in the context of ME/CFS.

Methods: The data collection involved a detailed search of peer-reviewed English literature from January 1995 to January 2025, focusing on studies related to the microbiome and ME/CFS. This comprehensive search utilized databases such as PubMed, Scopus, and Web of Science, with keywords including “ME/CFS,” “Gut-Brain Axis,” “Gut Health,” “Intestinal Dysbiosis,” “Microbiome Dysbiosis,” “Pathophysiology,” and “Therapeutic Approaches.” Where possible, insights from clinical trials and observational studies were included to enrich the findings. A narrative synthesis method was also employed to effectively organize and present these findings.

Results: The study found notable changes in the gut microbiota diversity and composition in ME/CFS patients, contributing to systemic inflammation and worsening cognitive and physical impairments. As a result, various microbiome interventions like probiotics, prebiotics, specific diets, supplements, fecal microbiota transplantation, pharmacological interventions, improved sleep, and moderate exercise training are potential therapeutic strategies that merit further exploration.

Conclusions: Interventions focusing on the gut-brain axis may help reduce neuropsychiatric symptoms in ME/CFS by utilizing the benefits of the microbiome. Therefore, identifying beneficial microbiome elements and incorporating their assessments into clinical practice can enhance patient care through personalized treatments. Due to the complexity of ME/CFS, which involves genetic, environmental, and microbial factors, a multidisciplinary approach is also necessary. Since current research lacks comprehensive insights into how gut health might aid ME/CFS treatment, standardized diagnostics and longitudinal studies could foster innovative therapies, potentially improving quality of life and symptom management for those affected.

Source: Hsu CY, Ahmad I, Maya RW, Abass MA, Gupta J, Singh A, Joshi KK, Premkumar J, Sahoo S, Khosravi M. The potential therapeutic approaches targeting gut health in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): a narrative review. J Transl Med. 2025 May 11;23(1):530. doi: 10.1186/s12967-025-06527-x. PMID: 40350437. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-025-06527-x (Full text)