Tag: methodology

Authors’ Response to “Comment on ‘SMPDL3B as a novel biomarker and therapeutic target in myalgic encephalomyelitis’”

Letter:

We thank Chen and Yan for their thoughtful and positive comments on our recent publication and for their interest in the translational implications of SMPDL3B biology in myalgic encephalomyelitis (ME) [, ]. Their letter provides a welcome opportunity to clarify methodological points related to biomarker validation, in vitro pharmacological assays, and mechanistic interpretation []. We appreciate this constructive dialogue and address each issue below in a collegial and scientifically grounded manner.

Read the rest of this letter HERE>>

Source: Rostami-Afshari B, Elremaly W, Franco A, Moreau A. Authors’ Response to “Comment on ‘SMPDL3B as a novel biomarker and therapeutic target in myalgic encephalomyelitis'”. J Transl Med. 2026 Jan 16;24(1):75. doi: 10.1186/s12967-025-07583-z. PMID: 41546078; PMCID: PMC12809929. https://pmc.ncbi.nlm.nih.gov/articles/PMC12809929/ (Full text)

Psychometric evaluation of the PROMIS® physical function short form 12a for use by adults with myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating, long-term illness that significantly impairs physical functioning. Despite its impact, the use of modern generic instruments to assess physical function in this population remains underexplored. This study aims to assess the psychometric properties of the Patient-Reported Outcome Measurement Information System® (PROMIS) Physical Function Short Form (PF-SF) 12a for use in adults with ME/CFS.

Methods: This study included 334 participants (173 with ME/CFS and 161 healthy controls) who took part in a Cognitive and Exercise sub-study of the Multi-Site Clinical Assessment of ME/CFS study from six clinics across the US. Data was used to examine the ceiling/floor effects, internal consistency reliability, known-groups validity, and convergent validity of the PROMIS PF-SF.

Results: The mean T-score of the PROMIS PF-SF was 40.5 for participants with ME/CFS, about one standard deviation below the national norm (T-score = 50). The PROMIS PF-SF showed no substantial floor/ceiling effects and high internal consistency (standardized Cronbach’s α = 0.88 and ω = 0.92). In addition, this instrument showed good known-groups validity with medium-to-large effect sizes (η2 = 0.08-0.35). A significant, monotonic increase of the physical function score was found across ME/CFS participant groups with low, medium, and high functional impairment as defined by four different measures. Participants with ME/CFS had significantly worse physical function scores than healthy controls (η2 = 0.70). The PROMIS PF-SF also demonstrated good convergent validity with high correlations (magnitude of r = 0.47-0.55) with other relevant measures.

Conclusions: The PROMIS PF-SF 12a demonstrated satisfactory reliability and validity for use in ME/CFS research and clinical practice.

Source: Yang M, Keller S, Rafiee P, Lin JS. Psychometric evaluation of the PROMIS® physical function short form 12a for use by adults with myalgic encephalomyelitis/chronic fatigue syndrome. Health Qual Life Outcomes. 2025 Oct 6;23(1):95. doi: 10.1186/s12955-025-02431-6. PMID: 41053836; PMCID: PMC12502361. https://pmc.ncbi.nlm.nih.gov/articles/PMC12502361/ (Full text)

AI-driven multi-omics modeling of myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic illness with a multifactorial etiology and heterogeneous symptomatology, posing major challenges for diagnosis and treatment. Here we present BioMapAI, a supervised deep neural network trained on a 4-year, longitudinal, multi-omics dataset from 249 participants, which integrates gut metagenomics, plasma metabolomics, immune cell profiling, blood laboratory data and detailed clinical symptoms.

By simultaneously modeling these diverse data types to predict clinical severity, BioMapAI identifies disease- and symptom-specific biomarkers and classifies ME/CFS in both held-out and independent external cohorts. Using an explainable AI approach, we construct a unique connectivity map spanning the microbiome, immune system and plasma metabolome in health and ME/CFS adjusted for age, gender and additional clinical factors.

This map uncovers altered associations between microbial metabolism (for example, short-chain fatty acids, branched-chain amino acids, tryptophan, benzoate), plasma lipids and bile acids, and heightened inflammatory responses in mucosal and inflammatory T cell subsets (MAIT, γδT) secreting IFN-γ and GzA.

Overall, BioMapAI provides unprecedented systems-level insights into ME/CFS, refining existing hypotheses and hypothesizing unique mechanisms—specifically, how multi-omics dynamics are associated to the disease’s heterogeneous symptoms.

Source: Xiong, R., Aiken, E., Caldwell, R. et al. AI-driven multi-omics modeling of myalgic encephalomyelitis/chronic fatigue syndrome. Nat Med (2025). https://doi.org/10.1038/s41591-025-03788-3  https://www.nature.com/articles/s41591-025-03788-3

Using the Ratio of Phosphorylated to Non-phosphorylated Forms of Stress Kinase PKR as a Potential Diagnostic Test for ME/CFS

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex illness characterized by a set of mainly neurological symptoms lasting for over 6 months. Currently, there is no definitive laboratory diagnostic test readily accessible to all clinicians and patients, and so clinical diagnosis occurs only after an exhaustive process of exclusion of all other possible causes of the varied symptoms experienced by the patient.

Here we present the development of a method that uses specific antibodies able to identify a changed ratio of phosphorylated and active protein kinase R in the peripheral blood monocyte cells (PBMCs) and neutrophil cells from a small group of ME/CFS sufferers, compared to age and sex-matched controls.

Protein kinase R (PKR) is an RNA-activated immune protein and stress kinase that has been observed to be present in its cleaved, auto-phosphorylated, and active form in past ME/CFS studies. After further validation, the activation status of PKR detected via specific antibodies in an ELISA format has potential for a simple readily accessible diagnostic tool for the early acute stage of ME/CFS illness, or as a long-term measure to evaluate the disease status.

Source: Sweetman E, Tate WP. Using the Ratio of Phosphorylated to Non-phosphorylated Forms of Stress Kinase PKR as a Potential Diagnostic Test for ME/CFS. Methods Mol Biol. 2025;2920:13-28. doi: 10.1007/978-1-0716-4498-0_2. PMID: 40372675. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_2

Using Single-Cell Raman Microspectroscopy to Profile Human Peripheral Blood Mononuclear Cells

Abstract:

A reliable, validated test would enhance our ability to treat and research chronic conditions. Early and accurate diagnosis would provide an entry point into clinical care, give access to benefits, remove the stigma associated with these conditions, and importantly, provide researchers with a fundamental tool they require to study these heterogeneous disorders.

In this chapter, we describe how Raman microspectroscopy can be utilised to study the biology of peripheral blood mononuclear cells (PBMCs) isolated from human blood samples. Using machine learning approaches, the data generated can be used to attempt to separate different patient and control groups, subgroups within a patient cohort, and identify differences in intracellular metabolites which may provide clues about disease mechanisms.

Source: Gan E, Stoker M, Guo E, Morten KJ, Xu J. Using Single-Cell Raman Microspectroscopy to Profile Human Peripheral Blood Mononuclear Cells. Methods Mol Biol. 2025;2920:29-37. doi: 10.1007/978-1-0716-4498-0_3. PMID: 40372676. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_3

MicroRNA Profiling of Blood Extracellular Vesicles in ME/CFS

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic debilitating neuroimmune disease affecting many organs and systems which, in the absence of validated biomarkers, remains diagnosed by clinical criteria. Extracellular vesicles (EV) in blood come from practically all cells in our body and therefore may carry the disease-specific biomarkers needed for the diagnosis of ME.

This chapter presents the methodology used on a single pilot study performed to evaluate this possibility to describe a workflow for EV isolation and the analysis of the miRNAs within, which could serve to interrogate additional cohorts of ME/CFS. Among the diverse nature of EV contents miRNAs may constitute a prominent regulatory layer in the development and progress of complex diseases such as ME/CFS, and therefore their study should be further pursued.

Source:Ljungström M, Nathanson L, Oltra E. MicroRNA Profiling of Blood Extracellular Vesicles in ME/CFS. Methods Mol Biol. 2025;2920:39-55. doi: 10.1007/978-1-0716-4498-0_4. PMID: 40372677. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_4

Deep Immunophenotyping in ME/CFS Using Spectral Flow Cytometry

Abstract:

Immune dysfunction is reported to play a significant role in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). To gain an understanding of the underlying immune abnormalities associated with this complex condition, a comprehensive approach for characterizing immune cell subsets and their inferred functional states is essential.

We developed a high-dimensional flow cytometry method that enables detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) from ME/CFS patients. By simultaneously measuring over 40 markers on individual cells within one sample, this approach provides a comprehensive assessment of immune cell subsets, incorporating effector or functional states, to enable assessment of their potential roles in disease pathogenesis.

Source: Gibson A, Chometon TQ, Damani T, Brooks AES. Deep Immunophenotyping in ME/CFS Using Spectral Flow Cytometry. Methods Mol Biol. 2025;2920:59-82. doi: 10.1007/978-1-0716-4498-0_5. PMID: 40372678. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_5

Application of DNA Methylome Analysis to Patients with ME/CFS

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome is a post-viral/stressor syndrome that has a complex pathophysiology reflecting multiple changes in many cell transcripts and proteins. These changes imply a change in the regulation of gene expression at the level of the DNA. A significant contributor to this is the modulation of the methylation at specific sites within regulatory regions throughout the genome that can either enhance or dampen expression depending on whether methylation is reduced or increased, respectively. DNA methylation can be analyzed by array technology or by reduced representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS).

This chapter describes RRBS, which has been very effective at analyzing the methylation states of ME/CFS patients both in single time point studies and in longitudinal studies with individual patients, for example, following a relapse recovery cycle. Here, we describe the step-by-step experimental methodology of how RRBS has been applied to DNA samples from ME/CFS patients and the analytical platforms used to detect the methylation changes that are statistically significant between patients and health controls. It has the potential to provide molecular biomarkers for a diagnostic test or to follow the progression of the condition in patients or through relapse/recovery fluctuations that occur frequently through the ongoing course of the disease. When effective therapies become available it has the potential to monitor the effectiveness on individual patients under treatment.

Source: Peppercorn K, Edgar CD, Al Momani S, Rodger EJ, Tate WP, Chatterjee A. Application of DNA Methylome Analysis to Patients with ME/CFS. Methods Mol Biol. 2025;2920:141-160. doi: 10.1007/978-1-0716-4498-0_9. PMID: 40372682. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_9

Repeated Cardiopulmonary Exercise Testing of ME/CFS Patients

Abstract:

Post-exertional malaise is a cardinal symptom present in 95% of individuals with myalgic encephalomyelitis (ME/CFS). Repeated cardiopulmonary exercise testing has been momentous in revealing that the physiological systems of those with ME/CFS are impaired or damaged and do not respond to exercise/physical activity like those without the condition. The 24-h repeated exercise test may demonstrate a reduction in peak oxygen consumption (VO2 peak), VO2 at ventilatory threshold, power output at both peak and ventilatory threshold, along with a reduction/diminished maximal heart rate commensurate with chronotropic intolerance. In this chapter, I describe the process and methods of repeated cardiopulmonary exercise testing, used to assess exercise tolerance in individuals with ME/CFS.

Source: Hodges L. Repeated Cardiopulmonary Exercise Testing of ME/CFS Patients. Methods Mol Biol. 2025;2920:163-172. doi: 10.1007/978-1-0716-4498-0_10. PMID: 40372683. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_10

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