Data-independent LC-MS/MS analysis of ME/CFS plasma reveals a dysregulated coagulation system, endothelial dysfunction, downregulation of complement machinery.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic condition that is characterized by unresolved fatigue, post-exertion symptom exacerbation (PESE), cognitive dysfunction, orthostatic intolerance, and other symptoms. ME/CFS lacks established clinical biomarkers and requires further elucidation of disease mechanisms. A growing number of studies demonstrate signs of hematological and cardiovascular pathology in ME/CFS cohorts, including hyperactivated platelets, endothelial dysfunction, vascular dysregulation, and anomalous clotting processes. To build on these findings, and to identify potential biomarkers that can be related to pathophysiology, we measured differences in protein expression in platelet-poor plasma (PPP) samples from 15 ME/CFS study participants and 10 controls not previously infected with SARS-CoV-2, using DIA LC-MS/MS. We identified 24 proteins that are significantly increased in the ME/CFS group compared to the controls, and 21 proteins that are significantly downregulated. Proteins related to clotting processes - thrombospondin-1 (important in platelet activation), platelet factor 4, and protein S - were differentially expressed in the ME/CFS group, suggestive of a dysregulated coagulation system and abnormal endothelial function. Complement machinery was also significantly downregulated, including C9 which forms part of the membrane attack complex. Additionally, we identified a significant upregulation of lactotransferrin, protein S100-A9, and an immunoglobulin variant. The findings from this experiment further implicate the coagulation and immune system in ME/CFS, and bring to attention the pathology of or imposed on the endothelium. This study highlights potential systems and proteins that require further research with regards to their contribution to the pathogenesis of ME/CFS, symptom manifestation, and biomarker potential, and also gives insight into the hematological and cardiovascular risk for ME/CFS individuals affected by diabetes mellitus.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: the biology of a neglected disease.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, debilitating disease characterised by a wide range of symptoms that severely impact all aspects of life. Despite its significant prevalence, ME/CFS remains one of the most understudied and misunderstood conditions in modern medicine. ME/CFS lacks standardised diagnostic criteria owing to variations in both inclusion and exclusion criteria across different diagnostic guidelines, and furthermore, there are currently no effective treatments available. Moving beyond the traditional fragmented perspectives that have limited our understanding and management of the disease, our analysis of current information on ME/CFS represents a significant paradigm shift by synthesising the disease's multifactorial origins into a cohesive model. We discuss how ME/CFS emerges from an intricate web of genetic vulnerabilities and environmental triggers, notably viral infections, leading to a complex series of pathological responses including immune dysregulation, chronic inflammation, gut dysbiosis, and metabolic disturbances. This comprehensive model not only advances our understanding of ME/CFS's pathophysiology but also opens new avenues for research and potential therapeutic strategies. By integrating these disparate elements, our work emphasises the necessity of a holistic approach to diagnosing, researching, and treating ME/CFS, urging the scientific community to reconsider the disease's complexity and the multifaceted approach required for its study and management.

Herpesvirus Infection of Endothelial Cells as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Understanding the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that a herpesvirus infection of endothelial cells (ECs) may underlie ME/CFS symptomatology. We review evidence linking herpesviruses to persistent EC infection and the implications for endothelial dysfunction, encompassing blood flow regulation, coagulation, and cognitive impairment-symptoms consistent with ME/CFS and Long COVID. This paper provides a synthesis of current research on herpesvirus latency and reactivation, detailing the impact on ECs and subsequent systemic complications, including latent modulation and long-term maladaptation. We suggest that the chronicity of ME/CFS symptoms and the multisystemic nature of the disease may be partly attributable to herpesvirus-induced endothelial maladaptation. Our conclusions underscore the necessity for further investigation into the prevalence and load of herpesvirus infection within the ECs of ME/CFS patients. This review offers conceptual advances by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research toward potentially unexplored avenues in understanding and treating this complex syndrome.

Cardiovascular and haematological pathology in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): A role for viruses.

ME/CFS is a debilitating chronic condition that often develops after viral or bacterial infection. Insight from the study of Long COVID/Post Acute Sequelae of COVID-19 (PASC), the post-viral syndrome associated with SARS-CoV-2 infection, might prove to be useful for understanding pathophysiological mechanisms of ME/CFS. Disease presentation is similar between the two conditions, and a subset of Long COVID patients meet the diagnostic criteria for ME/CFS. Since Long COVID is characterized by significant vascular pathology - including endothelial dysfunction, coagulopathy, and vascular dysregulation - the question of whether or not the same biological abnormalities are of significance in ME/CFS arises. Cardiac abnormalities have for a while now been documented in ME/CFS cohorts, with recent studies demonstrating major deficits in cerebral blood flow, and hence vascular dysregulation. A growing body of research is demonstrating that ME/CFS is accompanied by platelet hyperactivation, anomalous clotting, a procoagulant phenotype, and endothelial dysfunction. Endothelial damage and dysregulated clotting can impair substance exchange between blood and tissues, and result in hypoperfusion, which may contribute to the manifestation of certain ME/CFS symptoms. Here we review the ME/CFS literature to summarize cardiovascular and haematological findings documented in patients with the condition, and, in this context, briefly discuss the potential role of previously-implicated pathogens. Overall, cardiac and haematological abnormalities are present within ME/CFS cohorts. While atherosclerotic heart disease is not significantly associated with ME/CFS, suboptimal cardiovascular function defined by reduced cardiac output, impaired cerebral blood flow, and vascular dysregulation are, and these abnormalities do not appear to be influenced by deconditioning. Rather, these cardiac abnormalities may result from dysfunction in the (autonomic) nervous system. Plenty of recently published studies are demonstrating significant platelet hyperactivity and endothelial dysfunction in ME/CFS, as well as anomalous clotting processes. It is of particular importance to determine to what extent these cardiovascular and haematological abnormalities contribute to symptom severity, and if these two systems can be targeted for therapeutic purposes. Viral reservoirs of herpesviruses exist in ME/CFS, and most likely contribute to cardiovascular and haematological dysfunction directly or indirectly. This review highlights the potential of studying cardiac functioning, the vasculature, and coagulation system in ME/CFS.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system.

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

The potential role of ischaemia-reperfusion injury in chronic, relapsing diseases such as rheumatoid arthritis, Long COVID, and ME/CFS: evidence, mechanisms, and therapeutic implications.

Ischaemia-reperfusion (I-R) injury, initiated via bursts of reactive oxygen species produced during the reoxygenation phase following hypoxia, is well known in a variety of acute circumstances. We argue here that I-R injury also underpins elements of the pathology of a variety of chronic, inflammatory diseases, including rheumatoid arthritis, ME/CFS and, our chief focus and most proximally, Long COVID. Ischaemia may be initiated via fibrin amyloid microclot blockage of capillaries, for instance as exercise is started; reperfusion is a necessary corollary when it finishes. We rehearse the mechanistic evidence for these occurrences here, in terms of their manifestation as oxidative stress, hyperinflammation, mast cell activation, the production of marker metabolites and related activities. Such microclot-based phenomena can explain both the breathlessness/fatigue and the post-exertional malaise that may be observed in these conditions, as well as many other observables. The recognition of these processes implies, mechanistically, that therapeutic benefit is potentially to be had from antioxidants, from anti-inflammatories, from iron chelators, and via suitable, safe fibrinolytics, and/or anti-clotting agents. We review the considerable existing evidence that is consistent with this, and with the biochemical mechanisms involved.

Immuno-Thrombotic Complications of COVID-19: Implications for Timing of Surgery and Anticoagulation.

Early in the coronavirus disease 2019 (COVID-19) pandemic, global governing bodies prioritized transmissibility-based precautions and hospital capacity as the foundation for delay of elective procedures. As elective surgical volumes increased, convalescent COVID-19 patients faced increased postoperative morbidity and mortality and clinicians had limited evidence for stratifying individual risk in this population. Clear evidence now demonstrates that those recovering from COVID-19 have increased postoperative morbidity and mortality. These data-in conjunction with the recent American Society of Anesthesiologists guidelines-offer the evidence necessary to expand the early pandemic guidelines and guide the surgeon's preoperative risk assessment. Here, we argue elective surgeries should still be delayed on a personalized basis to maximize postoperative outcomes. We outline a framework for stratifying the individual COVID-19 patient's fitness for surgery based on the symptoms and severity of acute or convalescent COVID-19 illness, coagulopathy assessment, and acuity of the surgical procedure. Although the most common manifestation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is COVID-19 pneumonitis, every system in the body is potentially afflicted by an endotheliitis. This endothelial derangement most often manifests as a hypercoagulable state on admission with associated occult and symptomatic venous and arterial thromboembolisms. The delicate balance between hyper and hypocoagulable states is defined by the local immune-thrombotic crosstalk that results commonly in a hemostatic derangement known as fibrinolytic shutdown. In tandem, the hemostatic derangements that occur during acute COVID-19 infection affect not only the timing of surgical procedures, but also the incidence of postoperative hemostatic complications related to COVID-19-associated coagulopathy (CAC). Traditional methods of thromboprophylaxis and treatment of thromboses after surgery require a tailored approach guided by an understanding of the pathophysiologic underpinnings of the COVID-19 patient. Likewise, a prolonged period of risk for developing hemostatic complications following hospitalization due to COVID-19 has resulted in guidelines from differing societies that recommend varying periods of delay following SARS-CoV-2 infection. In conclusion, we propose the perioperative, personalized assessment of COVID-19 patients' CAC using viscoelastic hemostatic assays and fluorescent microclot analysis.

Membrane Transporters Involved in the Antimicrobial Activities of Pyrithione in Escherichia coli.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal-drug compounds.

A protet-based, protonic charge transfer model of energy coupling in oxidative and photosynthetic phosphorylation.

Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150-170mV; to achieve in vivo rates the imposed pmf must reach 200mV. The key question then is 'does the pmf generated by electron transport exceed 200mV, or even 170mV?' The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines.

INTRODUCTION: It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. OBJECTIVES: Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. METHODS: We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the 'exometabolome' or metabolic footprint). RESULTS: Our method measures some 4000-5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. CONCLUSION: Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.