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Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1.
Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles. Their trafficking from lymphoid tissues into the circulation is dependent on sphingosine-1-phosphate (S1P) gradients, sensed by S1P receptor 5 (S1P5). We hypothesized that NK-cell function and trafficking could be affected in NPC disease due to the combined effects of the lysosomal calcium defect and sphingosine storage. In an NPC1 mouse model, we found the frequency of NK cells was altered and phenocopied S1P5-deficient mice, consistent with defects in S1P levels. NK cells from NPC1 mice also had a defect in cytotoxicity due to a failure in degranulation of cytotoxic granules, which was associated with reduced lysosomal calcium levels. Affected NPC1 patients and NPC1 heterozygote carriers had reduced NK-cell numbers in their blood and showed similar phenotypic and developmental changes to those observed in the NPC1 mouse. These findings highlight the effects of lysosomal storage on the peripheral immune system.
A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.
Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.
NMR analysis reveals significant differences in the plasma metabolic profiles of Niemann Pick C1 patients, heterozygous carriers, and healthy controls.
Niemann-Pick type C1 (NPC1) disease is a rare autosomal recessive, neurodegenerative lysosomal storage disorder, which presents with a range of clinical phenotypes and hence diagnosis remains a challenge. In view of these difficulties, the search for a novel, NPC1-specific biomarker (or set of biomarkers) is a topic of much interest. Here we employed high-resolution 1H nuclear magnetic resonance spectroscopy coupled with advanced multivariate analysis techniques in order to explore and seek differences between blood plasma samples acquired from NPC1 (untreated and miglustat treated), heterozygote, and healthy control subjects. Using this approach, we were able to identify NPC1 disease with 91% accuracy confirming that there are significant differences in the NMR plasma metabolic profiles of NPC1 patients when compared to healthy controls. The discrimination between NPC1 (both miglustat treated and untreated) and healthy controls was dominated by lipoprotein triacylglycerol 1H NMR resonances and isoleucine. Heterozygote plasma samples displayed also increases in the intensities of selected lipoprotein triacylglycerol 1H NMR signals over those of healthy controls. The metabolites identified could represent useful biomarkers in the future and provide valuable insight in to the underlying pathology of NPC1 disease.
Machine learning-based prediction of anxiety disorders using blood metabolite and social trait data from the UK Biobank
Anxiety disorders are the most prevalent type of mental health disorders and are characterised by excessive fear and worry. Despite affecting one in four individuals within their lifetime, there remains a gap in our understanding regarding the underlying pathophysiology of anxiety disorders, which limits the development of novel treatment options. Exploring blood-based biomarkers of anxiety disorder offers the potential to predict the risk of clinically significant anxiety in the general population, increase our understanding of anxiety pathophysiology, and to reveal options for preventative treatment. Here, using psychosocial variables in combination with blood and urine biomarkers, reported in the UK Biobank, we sought to predict future anxiety onset. Machine learning accurately predicted (ROC AUC: ∼0.83) ICD-10-coded anxiety diagnoses up to 5 years (mean 3.5 years) after blood sampling, against lifetime anxiety-free controls. Analysis of the blood biochemistry measures indicated that anxious individuals were more anaemic and exhibited higher levels of markers of systemic inflammation than controls. However, blood biomarkers alone were not predictive of resilience or susceptibility to anxiety disorders in a subset of individuals rigorously matched for a wide range of psychosocial covariates (ROC AUC: ∼0.50). Overall, we demonstrate that the integration of biological and psychosocial risk factors is an effective tool to screen for and predict anxiety disorder onset in the general population.
Small-molecule dissolution of stress granules by redox modulation benefits ALS models.
Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.
Integrating TSPO-PET imaging with metabolomics for enhanced prognostic accuracy in multiple sclerosis.
BACKGROUND: Predicting disease progression in multiple sclerosis (MS) remains challenging. PET imaging with 18 kDa translocator protein (TSPO) radioligands can detect microglial and astrocyte activation beyond MRI-visible lesions, which has been shown to be highly predictive of disease progression. We previously demonstrated that nuclear magnetic resonance (NMR)-based metabolomics could accurately distinguish between relapsing-remitting (RRMS) and secondary progressive MS (SPMS). This study investigates whether combining TSPO imaging with metabolomics enhances predictive accuracy in a similar setting. METHODS: Blood samples were collected from 87 MS patients undergoing PET imaging with the TSPO-binding radioligand 11C-PK11195 in Finland. Patient disability was assessed using the expanded disability status scale (EDSS) at baseline and 1 year later. Serum metabolomics was performed to identify biomarkers associated with TSPO binding and disease progression. RESULTS: Greater TSPO availability in the normal-appearing white matter and perilesional regions correlated with higher EDSS. Serum metabolites glutamate (p=0.02), glutamine (p=0.006), and glucose (p=0.008), detected by NMR, effectively distinguished future progressors. These three metabolites alone predicted progression with the same accuracy as TSPO-PET imaging (AUC 0.78; p=0.0001), validated in an independent cohort. Combining serum metabolite data with PET imaging significantly improved predictive power, achieving an AUC of 0.98 (p<0.0001). CONCLUSION: Measuring three specific serum metabolites is as effective as TSPO imaging in predicting MS progression. However, integrating TSPO imaging with serum metabolite analysis substantially enhances predictive accuracy. Given the simplicity and affordability of NMR analysis, this approach could lead to more personalised, accessible treatment strategies and serve as a valuable tool for clinical trial stratification.
Early plasma ceramide and sphingomyelin levels reflect APOE genotype but not familial Alzheimer's disease gene mutations in female 5xFAD mice, with brain-region specific sphingolipid alterations.
Pathophysiological changes associated with Alzheimer's disease (AD) begin decades before dementia onset, with age and APOE ε4 genotype as major risk factors [1-4]. Primary risk factors for developing AD include aging and number of copies of the apolipoprotein E (APOE) ε4 allele. Altered sphingolipid metabolism is increasingly implicated in early AD. However, the relationship between early plasma and brain sphingolipid changes-particularly in the context of APOE genotype-remains poorly defined. In this study, we analyzed plasma and brain sphingolipid profiles in transgenic AD mice carrying human APOE3 or APOE4 variants, with or without familial AD mutations (E3FAD and E4FAD). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we assessed 110 sphingolipid species across four major classes (ceramides (Cers), hexosylceramides (HexCers), lactosylceramides (LacCers), and sphingomyelins (SMs)) at 2, 4, and 6 months in plasma and at 6 months in brain tissue in the cortex, hippocampus, striatum, and cerebellum. Our results demonstrate that early plasma sphingolipid alterations are largely driven by APOE genotype rather than AD pathology. Specifically, APOE4 carriers showed significant increases in SM species and reductions in Cer species compared to APOE3 carriers, independent of age or AD genotype. Brain lipid profiles showed minimal changes across genotypes after region correction. However, combined p-value analyses revealed APOE- and EFAD-dependent differences in the composition of primarily cortical sphingolipids. ROC analyses demonstrated high discriminative power of plasma sphingolipids for APOE, but not for AD genotype. These findings suggest that early plasma lipid profiles in female 5xFAD mice are more strongly influenced by APOE genotype than by overt AD pathology, potentially reflecting systemic pathways linked to APOE4-associated AD risk, while early disease-associated changes in the brain appear to be subtle and region-specific. These results underscore the importance of accounting for APOE genotype in early-stage AD lipidomic studies and in the interpretation of peripheral lipid biomarkers.
Increased TMEM106B levels lead to lysosomal dysfunction which affects synaptic signaling and neuronal health
Background: Genetic variation in Transmembrane protein 106B (TMEM106B) is known to influence the risk and presentation in several neurodegenerative diseases and modifies healthy aging. While evidence from human studies suggests that the risk allele is associated with higher levels of TMEM106B, the contribution of elevated levels of TMEM106B to neurodegeneration and aging has not been assessed and it remains unclear how TMEM106B modulates disease risk. Methods: To study the effect of increased TMEM106B levels, we generated Cre-inducible transgenic mice expressing human wild-type TMEM106B. We evaluated lysosomal and neuronal health using in vitro and in vivo assays including transmission electron microscopy, immunostainings, behavioral testing, electrophysiology, and bulk RNA sequencing. Results: We created the first transgenic mouse model that successfully overexpresses TMEM106B, with a 4- to 8-fold increase in TMEM106B protein levels in heterozygous (hTMEM106B(+)) and homozygous (hTMEM106B(++)) animals, respectively. We showed that the increase in TMEM106B protein levels induced lysosomal dysfunction and age-related downregulation of genes associated with neuronal plasticity, learning, and memory. Increased TMEM106B levels led to altered synaptic signaling in 12-month-old animals which further exhibited an anxiety-like phenotype. Finally, we observed mild neuronal loss in the hippocampus of 21-month-old animals. Conclusion: Characterization of the first transgenic mouse model that overexpresses TMEM106B suggests that higher levels of TMEM106B negatively impacts brain health by modifying brain aging and impairing the resilience of the brain to the pathomechanisms of neurodegenerative disorders. This novel model will be a valuable tool to study the involvement and contribution of increased TMEM106B levels to aging and will be essential to study the many age-related diseases in which TMEM106B was genetically shown to be a disease- and risk-modifier.
Membrane-permeable trehalose 6-phosphate precursor spray increases wheat yields in field trials.
Trehalose 6-phosphate (T6P) is an endogenous sugar signal in plants that promotes growth, yet it cannot be introduced directly into crops or fully genetically controlled. Here we show that wheat yields were improved using a timed microdose of a plant-permeable, sunlight-activated T6P signaling precursor, DMNB-T6P, under a variety of agricultural conditions. Under both well-watered and water-stressed conditions over 4 years, DMNB-T6P stimulated yield of three elite varieties. Yield increases were an order of magnitude larger than average annual genetic gains of breeding programs and occurred without additional water or fertilizer. Mechanistic analyses reveal that these benefits arise from increased CO2 fixation and linear electron flow ('source') as well as from increased starchy endosperm volume, enhanced grain sieve tube development and upregulation of genes for starch, amino acid and protein synthesis ('sink'). These data demonstrate a step-change, scalable technology with net benefit to the environment that could provide sustainable yield improvements of diverse staple cereal crops.
Repeated administration of L-alanine to mice reduces behavioural despair and increases hippocampal mammalian target of rapamycin signalling: Analysis of gender and metabolic effects.
BACKGROUND: The amino acid L-alanine, has been shown to be elevated in biofluids during major depression but its relevance remains unexplored. AIM: We have investigated the effects of repeated L-alanine administration on emotional behaviours and central gene expression in mice. METHODS: Mice received a daily, 2-week intraperitoneal injection of either saline or L-alanine at 100 or 200 mg/kg and were exposed to the open field, light-dark box and forced swim test. The expression of L-alanine transporters (asc-1, ASCT2), glycine receptor subunits (GlyRs), NMDA receptor subunits (GluNs) mRNAs were measured, together with western blots of the signalling protein mammalian target of rapamycin (mTOR). Since L-alanine modulates glucose homeostasis, peripheral and central metabolomes were evaluated with 1H-NMR. RESULTS: L-alanine administration at 100 mg/kg, but not at 200 mg/kg, to both male and female mice increased latency to float and reduced floating time in the forced swim test, but had no effect on anxious behaviour in the open field and light-dark box tests. There was a significant reduction in mRNAs encoding asc-1 and ASCT2 and GluN2B in the hippocampus of mice following 100 mg/kg L-alanine only. On western blots, hippocampal GluN2B immunoreactivity was reduced, but mTOR signalling was increased in the 100 mg/kg L-alanine group. 1H-NMR revealed gender-specific changes in the forebrain, plasma and liver metabolomes only at 200 mg/kg of L-alanine. CONCLUSIONS: Our data suggest that L-alanine may have antidepressant-like effect that may involve the modulation of glutamate neurotransmission independently of metabolism. In major depression, therefore, elevated L-alanine may be a homeostatic response to pathophysiological processes, though this will require further investigation.
Myelopoiesis is temporally dynamic and is regulated by lifestyle to modify multiple sclerosis.
Monocytes and neutrophils from the myeloid lineage contribute to multiple sclerosis (MS), but the dynamics of myelopoiesis during MS are unclear. Here we uncover a disease stage-specific relationship between lifestyle, myelopoiesis and neuroinflammation. In mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), myelopoiesis in the femur, vertebrae and spleen is elevated prior to disease onset and during remission, preceding the peaks of clinical disability and neuroinflammation. In progressive EAE (P-EAE), vertebral myelopoiesis rises steadily throughout disease, while femur and splenic myelopoiesis is elevated early before waning later during disease height. In parallel, sleep disruption or hyperlipidemia and cardiometabolic syndrome augment M-CSF generation and multi-organ myelopoiesis to worsen P-EAE clinical symptoms, neuroinflammation, and spinal cord demyelination, with M-CSF blockade abrogating these symptoms. Lastly, results from a previous trial show that Mediterranean diet restrains myelopoietic activity and myeloid lineage progenitor skewing and improves clinical symptomology of MS. Together, our data suggest that myelopoiesis in MS is dynamic and dependent on disease stage and location, and that lifestyle factors modulate disease by influencing M-CSF-mediated myelopoiesis.
Permissive central tolerance plus defective peripheral checkpoints license pathogenic memory B cells in CASPR2-antibody encephalitis.
Autoantibody-mediated diseases targeting one autoantigen provide a unique opportunity to comprehensively understand the development of disease-causing B cells and autoantibodies. Convention suggests that such autoreactivities are generated during germinal center reactions. Here, we explore earlier immune checkpoints, focusing on patients with contactin-associated protein-like 2 (CASPR2)-autoantibody encephalitis. In both disease and health, high (~0.5%) frequencies of unmutated CASPR2-reactive naïve B cells were identified. By contrast, CASPR2-reactive memory B cells were exclusive to patients, and their B cell receptors demonstrated affinity-enhancing somatic mutations with pathogenic effects in neuronal cultures and mice. The unmutated, precursor memory B cell receptors showed a distinctive balance between strong CASPR2 reactivity and very limited binding across the remaining human proteome. Our results identify permissive central tolerance, defective peripheral tolerance, and autoantigen-specific tolerance thresholds in humans as sequential steps that license CASPR2-directed pathology. By leveraging the basic immunobiology, we rationally direct tolerance-restoring approaches, with an experimental paradigm applicable across autoimmunity.
The modernized classification of cardiac anti-arrhythmic drugs: its application to clinical practice.
Cardiac arrhythmias pose a major public health problem and pharmacological intervention remains key to their therapy. The landmark Vaughan Williams (VW, 1970) classification utilizing known actions of then available anti-arrhythmic drugs (AADs) became and remains central to management, but requires revision in response to extensive subsequent advances. Our modernized antiarrhythmic drug (AAD) classification reflected and sought to facilitate such fundamental physiological and clinical development. We here respond to requests for an adaptation of our scheme specifically focussed at clinical practice. This adaptation: (1) improves accessibility of our original scheme to clinical practice, focussing on key AADs in clinical use rather than investigational new drugs (INDs) whilst still conserving and encompassing the classic VW scheme. We nevertheless (2) preserve a rational conceptual framework based on current understanding of the relevant electrophysiological events, their underlying cellular or molecular cardiomyocyte targets and the functional mechanisms they mediate. Additionally, (3) the adopted subclasses within each AAD class parallel clinical practice in including only subclasses containing established AADs, or approved potential off-label drugs, as opposed to those only including INDs. Finally, (4) the simplified scheme remains flexible, permitting drugs to be placed in multiple classes where required, and the future addition of classes and subclasses in the light of future investigations and clinical approvals. We thus derive from our comprehensive modernized AAD classification a more focussed and simpler scheme, for clinical use. This both modernizes but preserves the classic Vaughan Williams classification, and remains flexible accommodating for future developments.
Physiological Function of Cyclic Nucleotide Phosphodiesterases in Atrial Myocytes and their Potential as Targets in Atrial Fibrillation.
Cyclic nucleotide hy drolysing phosphodiesterases (PDEs) are key regulators of cyclic nucleotide (e.g. cAMP and cGMP) signalling. Here we examine the role of PDEs in the physiology of atrial myocytes (AMs), the pathogenesis of atrial fibrillation (AF) and the potential of PDEs as therapeutic targets for AF. PDE1-5 and 8 are present and functional in AMs. PDE2-4 are important regulators of AM contraction but their role beyond atrial contractility is unclear. The role of PDE1,5 and 8 in healthy AMs is unknown but of interest because of their roles in ventricular myocytes. We propose that PDE2-5 and PDE8 are potential targets to prevent the triggering of AF considering their effects on Ca2+ handling and /or electrical activity. PDE1-5 are possible targets to treat patients with paroxysmal or persistent AF caused by pulmonary vein automaticity. PDE8B2 is a possible target for patients with persistent AF due to its altered expression. Research should aim to identify the presence, localisation, and function of specific PDE isoforms in human atria. Ultimately, the paucity of PDE isoform-specific small molecule modulators and the difficulty of delivering PDE-targeted medications or therapies to particular cell types limit current research and its application.
Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.
Development of a fluorescence-based assay for RecBCD activity using Functional Data Analysis and Design of Experiments
Biochemical assays are essential tools in biological research and drug discovery, but optimisation of these assays is often a challenging and lengthy process due to the wide range of input...
Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide.
The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.
Protocol: A metabolomic analysis of convalescent inflammatory conditions
Background ‘The term ‘long covid’ describes persistent symptoms following infection with SARS-CoV-2 that are not explained by an alternative diagnosis. It embraces a number of globally used terms and reported prevalence is highly variable. In the United Kingdom (UK) in 2023, approximately 2.9% of the population were thought to be affected. The condition manifests in a constellation of fluctuant symptoms, which persist beyond the acute infection and frequently profoundly impact an individual’s functional and relational capacity. The underlying mechanisms remain imperfectly understood and there is great demand for diagnostic tools that distinguish long covid from other chronic conditions. This study aims to utilise metabolomics to develop such a test and identify potential pathophysiological mechanisms. Methods Blood and urine samples will be collected at two timepoints at least 9 months apart from non-hospitalised individuals with a previous confirmed COVID-19 infection. This population will be divided into those who recovered completely within six weeks and those who continue to experience persistent symptoms. Samples will be analysed using 1H NMR spectroscopy and the resultant metabolomic profiles will be subject to multivariate pattern recognition techniques. This will produce mathematical models capable of distinguishing these long covid and control groups. Symptoms, potential confounders, and qualitative narrative data will be collected alongside this process to add deeper richness to the subsequent analysis. Primary Outcome The creation of a diagnostic test for long covid using 1H NMR metabolomics. Secondary Outcomes The development of algorithms that predict the severity and chronicity of long covid, identification of subgroup differences in metabolomic and immune profiles, and triangulation with symptom and narrative data to produce a deeper understanding of the patient experience. Conclusion This study seeks to advance the understanding of long covid using advanced multi-omic and narrative techniques, which may offer potential diagnostic and therapeutic avenues.