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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.

The exchange of small molecules between the cell and the environment happens through transporter proteins. Besides nutrients and native metabolic products, xenobiotic molecules are also transported, however it is not well understood which transporters are involved. In this study, by combining exo-metabolome screening in yeast with transporter characterization in Xenopus oocytes, we mapped the activity of 30 yeast transporters toward six small non-toxic substrates. Firstly, using LC–MS, we determined 385 compounds from a chemical library that were imported and exported by S. cerevisiae. Of the 385 compounds transported by yeast, we selected six compounds (viz. sn-glycero-3-phosphocholine, 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, acrylic acid, 2-benzoxazolol) for characterization against 30 S. cerevisiae xenobiotic transport proteins expressed in Xenopus oocytes. The compounds were selected to represent a diverse set of chemicals with a broad interest in applied microbiology. Twenty transporters showed activity toward one or more of the compounds. The tested transporter proteins were mostly promiscuous in equilibrative transport (i.e., facilitated diffusion). The compounds 2,5-furandicarboxylic acid, 2-methylpyrazine, cefadroxil, and sn-glycero-3-phosphocholine were transported equilibratively by transporters that could transport up to three of the compounds. In contrast, the compounds acrylic acid and 2-benzoxazolol, were strictly transported by dedicated transporters. The prevalence of promiscuous equilibrative transporters of non-native substrates has significant implications for strain development in biotechnology and offers an explanation as to why transporter engineering has been a challenge in metabolic engineering. The method described here can be generally applied to study the transport of other small non-toxic molecules. The yeast transporter library is available at AddGene (ID 79999).

Tuberculosis (TB) claims nearly 1.5 million lives annually. Current TB treatment requires a combination of several drugs administered for at least 6 months. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can persist in infected humans and animals for decades. Moreover, during infection, Mtb produces differentially culturable bacteria (DCB) that do not grow in standard media but can be resuscitated in liquid media supplemented with sterile Mtb culture filtrates or recombinant resuscitation-promoting factors (Rpfs). Here, we demonstrate that, in an intranasal murine model of TB, Mtb DCB are detectable in the lungs after 4 weeks of infection, and their loads remain largely unchanged during a further 8 weeks. Treatment of the infected mice with dimethyl fumarate (DMF), a known drug with immunomodulatory properties, for 8 weeks eliminates Mtb DCB from the lungs and spleens. Standard TB treatment consisting of rifampicin, isoniazid, and pyrazinamide for 8 weeks reduces Mtb loads by nearly four orders of magnitude but does not eradicate DCB. Nevertheless, no DCB can be detected in the lungs and spleens after 8 weeks of treatment with DMF, rifampicin, isoniazid, and pyrazinamide. Our data suggest that addition of approved anti-inflammatory drugs to standard treatment regimens may improve TB treatment and reduce treatment duration.

Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli.

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.

The solute carrier (SLC) superfamily represents the biggest family of transporters with important roles in health and disease. Despite being attractive and druggable targets, the majority of SLCs remains understudied. One major hurdle in research on SLCs is the lack of tools, such as cell-based assays to investigate their biological role and for drug discovery. Another challenge is the disperse and anecdotal information on assay strategies that are suitable for SLCs. This review provides a comprehensive overview of state-of-the-art cellular assay technologies for SLC research and discusses relevant SLC characteristics enabling the choice of an optimal assay technology. The Innovative Medicines Initiative consortium RESOLUTE intends to accelerate research on SLCs by providing the scientific community with high-quality reagents, assay technologies and data sets, and to ultimately unlock SLCs for drug discovery.

AimsThe risk of cardiovascular events in patients with Rheumatoid Arthritis (RA) is disproportionately heightened as a result of systemic inflammation. The relative effect of autoimmune-associated citrullination on the structure and thrombotic potential of fibrin(ogen) remains unknown. We therefore compared indices of vascular function, inflammation, coagulation and fibrin clot composition in RA patients with healthy controls and evaluated parameter association with disease presence.MethodsBlood samples were collected from 30 RA patients and 30 age- and gender-matched healthy volunteers. Levels of serum amyloid A (SAA), c-reactive protein (CRP), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) was measured using a sandwich immunoassay. Whole blood coagulation was assessed using Thromboelastography (TEG®). Fibrin clot networks and fiber structure was investigated using Scanning Electron Microscopy. The detection and quantification of citrullination in formed fibrin clots was performed using a fluorescently labeled Citrulline monoclonal antibody with Fluorescence Wide Field Microscopy.ResultsConcentrations of SAA, CRP and ICAM-1 were significantly elevated in RA patients compared to controls. TEG parameters relating to coagulation initiation, rate of fibrin cross-linking, and time to reach maximum thrombus generation were attenuated in RA patients. Microscopic analysis revealed denser networks of thicker fibrin fibers in RA patients compared to controls and multiple citrullinated regions within fibrin clot structures in RA patients were present.ConclusionOur findings provide novel evidence for the citrullination of fibrin within vasculature is more prominent in RA plasma compared to control plasma and plasma is more accessible than synovial fluid. Citrullinated fibrinogen could play a role as a determinant of thrombotic risk in RA patients.

Background:Porphyromonas gingivalis and its inflammagens are associated with a number of systemic diseases, such as cardiovascular disease and type 2 diabetes (T2DM). The proteases, gingipains, have also recently been identified in the brains of Alzheimer's disease patients and in the blood of Parkinson's disease patients. Bacterial inflammagens, including lipopolysaccharides (LPSs) and various proteases in circulation, may drive systemic inflammation.Methods: Here, we investigate the effects of the bacterial products LPS from Escherichia coli and Porphyromonas gingivalis, and also the P. gingivalis gingipain [recombinant P. gingivalis gingipain R1 (RgpA)], on clot architecture and clot formation in whole blood and plasma from healthy individuals, as well as in purified fibrinogen models. Structural analysis of clots was performed using confocal microscopy, scanning electron microscopy, and AFM-Raman imaging. We use thromboelastography® (TEG®) and rheometry to compare the static and dynamic mechanical properties of clots.Results: We found that these inflammagens may interact with fibrin(ogen) and this interaction causes anomalous blood clotting.Conclusions: These techniques, in combination, provide insight into the effects of these bacterial products on cardiovascular health, and particularly clot structure and mechanics.

Lactoferrin is a nutrient classically found in mammalian milk. It binds iron and is transferred via a variety of receptors into and between cells, serum, bile, and cerebrospinal fluid. It has important immunological properties, and is both antibacterial and antiviral. In particular, there is evidence that it can bind to at least some of the receptors used by coronaviruses and thereby block their entry. Of importance are Heparan Sulfate Proteoglycans (HSPGs) and the host receptor angiotensin-converting enzyme 2 (ACE2), as based on other activities lactoferrin might prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from attaching to the host cells. Lactoferrin (and more specifically enteric-coated LF because of increased bioavailability) may consequently be of preventive and therapeutic value during the present COVID-19 pandemic.

Introduction: Migraine is a debilitating primary headache disorder with a poorly understood aetiology. An extensive body of literature supports the theory of migraine as a systemic vascular inflammatory disorder characterised by endothelial dysfunction. It is also well-known that chronic inflammation results in an excessive burden of oxidative stress and therefore cellular dysfunction. In this study the effects of excessive oxidative stress through the phases of female migraine-with-aura (FMA) were evaluated by examining the health of the systems of haemostasis.Methods: Blood was obtained from 11 FMA patients at baseline and during the headache phase of migraine, as well as from 8 healthy age-matched female controls. Samples were analysed using thromboelastography (TEG) to evaluate viscoelastic profiles, light microscopy for erythrocyte morphology, Scanning Electron Microscopy (SEM) for erythrocyte and fibrin clot structure, confocal microscopy for β-amyloid detection in fibrin clots.Results: Viscoelastic profiles from platelet poor plasma showed decreased clot reaction times in FMA at baseline (95% CI [5.56, 8.41]) vs. control (95% CI [7.22, 11.68]); as well as decreased time to maximum thrombus generation for the same comparison (95% CI [6.78, 10.20] vs. [8.90, 12.96]). Morphological analysis of erythrocytes indicated widespread macrocytosis, poikilocytosis and eryptosis in the migraineurs. Analysis of fibrin networks indicated that this hypercoagulability may be a result of aberrant fibrin polymerisation kinetics caused by the adoption of a β-amyloid conformation of fibrin(ogen).Conclusion: The results reaffirm the hypercoagulable state in migraine, and would suggest that this state is most likely a result of a systemic inflammatory state which induces oxidative damage to both erythrocytes and fibrin(ogen) in female episodic migraine-with-aura. Furthermore, if the amylodogenic changes to fibrin(ogen) were observed in a larger cohort, this would support theories of micro-embolisation in migraine-with-aura.

L-(+)-Ergothioneine (ERG) is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise ERG, but acquire it from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the baker's yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 ± 18 mg/L ergothioneine in fed-batch culture in 1 L bioreactors. Because S. cerevisiae is a GRAS (“generally recognized as safe”) organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

Parkinson’s disease (PD) is a well-known neurodegenerative disease with a strong association established with systemic inflammation. Recently, the role of the gingipain protease group from Porphyromonas gingivalis was implicated in Alzheimer’s disease and here we present evidence, using a fluorescent antibody to detect gingipain R1 (RgpA), of its presence in a PD population. To further elucidate the action of this gingipain, as well as the action of the lipopolysaccharide (LPS) from P. gingivalis, low concentrations of recombinant RgpA and LPS were added to purified fluorescent fibrinogen. We also substantiate previous findings regarding PD by emphasizing the presence of systemic inflammation via multiplex cytokine analysis, and demonstrate hypercoagulation using thromboelastography (TEG), confocal and electron microscopy. Biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. In our PD and control blood analysis, our results show increased hypercoagulation, the presence of amyloid formation in plasma, and profound ultrastructural changes to platelets. Our laboratory analysis of purified fibrinogen with added RgpA, and/or LPS, showed preliminary data with regards to the actions of the protease and the bacterial membrane inflammagen on plasma proteins, to better understand the nature of established PD.

Psoriasis vulgaris is a chronic inflammatory skin condition, associated with both a physical and a psychological burden. Our understanding of the etiology of this disease remains incomplete. Conventionally, psoriasis has been viewed as a condition that manifests solely in the skin. However, the systemic inflammatory nature of this disease has been confirmed by the presence of a wide array of dysregulated cytokines and inflammatory markers in the serum of these patients. Both dysregulated gut and skin microbiomes have been found in association with psoriasis. An evident association also exists between inflammatory bowel disease and this condition. Regarding the skin microbiome, changes have been observed in the relative abundance of Firmicutes, Actinobacteria, and Proteobacteria. Additionally, Staphylococcus and Streptococcus spp. were detected more frequently in lesional skin. Alterations in the gut microbiome have been characterized by a decrease in the Bacteroidetes phylum and an increase in the Faecalibacterium genus. We suggest that dysbiosis of the skin and gut microbiota may contribute to psoriasis, by promoting the translocation of microbes from these sites into the bloodstream. Consistent with the Iron Dysregulation and Dormant Microbes hypothesis, these microorganisms are in a physiologically dormant state, but may be awakened periodically and shed their cell wall components, such as lipopolysaccharide and lipoteichoic acid. Both of these inflammagens may contribute significantly to maintaining a chronic inflammatory state in the host, such as is seen in individuals diagnosed with psoriasis.

Alzheimer’s disease and other similar dementias are debilitating neurodegenerative disorders whose etiology and pathogenesis remain largely unknown, even after decades of research. With the anticipated increase in prevalence of Alzheimer’s type dementias among the more susceptible aging population, the need for disease-modifying treatments is urgent. While various hypotheses have been put forward over the last few decades, we suggest that Alzheimer’s type dementias are triggered by external environmental factors, co-expressing in individuals with specific genetic susceptibilities. These external stressors are defined in the Iron Dysregulation and Dormant Microbes (IDDM) hypothesis, previously put forward. This hypothesis is consistent with current literature in which serum ferritin levels of individuals diagnosed with Alzheimer’s disease are significantly higher compared those of age- and gender-matched controls. While iron dysregulation contributes to oxidative stress, it also causes microbial reactivation and virulence of the so-called dormant blood (and tissue) microbiome. Dysbiosis (changes in the microbiome) or previous infections can contribute to the dormant blood microbiome (atopobiosis1), and also directly promotes systemic inflammation via the amyloidogenic formation and shedding of potent inflammagens such as lipopolysaccharides. The simultaneous iron dysregulation and microbial aberrations affect the hematological system, promoting fibrin amylodiogenesis, and pathological clotting. Systemic inflammation and oxidative stress can contribute to blood brain barrier permeability and the ensuing neuro-inflammation, characteristic of Alzheimer’s type dementias. While large inter-individual variability exists, especially concerning disease pathogenesis, the IDDM hypothesis acknowledges primary causative factors which can be targeted for early diagnosis and/or for prevention of disease progression.

Many studies indicate that there is a (mainly dormant) microbial component in the progressive development of Alzheimer-type dementias (ADs); and that in the case of Gram-negative organisms, a chief culprit might be the shedding of the highly inflammagenic lipopolysaccharide (LPS) from their cell walls. We have recently shown that a highly sensitive assay for the presence of free LPS [added to platelet poor plasma (PPP)] lies in its ability (in healthy individuals) to induce blood to clot into an amyloid form. This may be observed in a SEM or in a confocal microscope when suitable amyloid stains (such as thioflavin T) are added. This process could be inhibited by human lipopolysaccharide-binding protein (LBP). In the current paper, we show using scanning electron microscopy and confocal microscopy with amyloid markers, that PPP taken from individuals with AD exhibits considerable amyloid structure when clotting is initiated with thrombin but without added LPS. Furthermore, we could show that this amyloid structure may be reversed by the addition of very small amounts of LBP. This provides further evidence for a role of microbes and their inflammagenic cell wall products and that these products may be involved in pathological clotting in individuals with AD.

Although it is widely considered, in many cases, to involve two separable stages (poor placentation followed by oxidative stress/inflammation), the precise originating causes of preeclampsia (PE) remain elusive. We have previously brought together some of the considerable evidence that a (dormant) microbial component is commonly a significant part of its etiology. However, apart from recognizing, consistent with this view, that the many inflammatory markers of PE are also increased in infection, we had little to say about immunity, whether innate or adaptive. In addition, we focused on the gut, oral and female urinary tract microbiomes as the main sources of the infection. We here marshall further evidence for an infectious component in PE, focusing on the immunological tolerance characteristic of pregnancy, and the well-established fact that increased exposure to the father’s semen assists this immunological tolerance. As well as these benefits, however, semen is not sterile, microbial tolerance mechanisms may exist, and we also review the evidence that semen may be responsible for inoculating the developing conceptus (and maybe the placenta) with microbes, not all of which are benign. It is suggested that when they are not, this may be a significant cause of PE. A variety of epidemiological and other evidence is entirely consistent with this, not least correlations between semen infection, infertility and PE. Our view also leads to a series of other, testable predictions. Overall, we argue for a significant paternal role in the development of PE through microbial infection of the mother via insemination.

Introduction: Unliganded iron both contributes to the pathology of Alzheimer’s disease (AD) and also changes the morphology of erythrocytes (RBCs). We tested the hypothesis that these two facts might be linked, i.e. that the RBCs of AD individuals have a variant morphology, that might have diagnostic or prognostic value. Methods: We included a literature survey of AD and its relationships to the vascular system, followed by a laboratory study. Four different microscopy techniques were used and results statistically compared to analyze trends between high and normal serum ferritin (SF) AD individuals. Results: Light and scanning electron microscopies showed little difference between the morphologies of RBCs taken from healthy individuals and from normal SF AD individuals. By contrast, there were substantial changes in the morphology of RBCs taken from high SF AD individuals. These differences were also observed using confocal microscopy and as a significantly greater membrane stiffness (measured using force-distance curves). Conclusion: We argue that high ferritin levels may contribute to an accelerated pathology in AD. Our findings reinforce the importance of (unliganded) iron in AD, and suggest the possibility both of an early diagnosis and some means of treating or slowing down the progress of this disease.