Douglas Kell

Transport systems play a pivotal role in bacterial physiology and represent potential targets for medical and biotechnological applications. However, even in well-studied organisms like Escherichia coli, a notable proportion of transporters, exceeding as many as 30%, remain classified as orphans due to their lack of known substrates. This study leveraged high-resolution LC-MS-based untargeted metabolomics to identify candidate substrates for these orphan transporters. Human serum, including a diverse array of biologically relevant molecules, served as an unbiased source for substrate exposure. The analysis encompassed 26 paired transporter mutant contrasts (i.e., knockout vs. overexpression), compared with the wild type, revealing distinct patterns of substrate uptake and excretion across various mutants. The convergence of candidate substrates across mutant scenarios provided robust validation, shedding light on novel transporter-substrate relationships, including those involving yeaV, hsrA, ydjE, and yddA. Furthermore, several substrates were contingent upon the specific mutants employed. This investigation underscores the utility of untargeted metabolomics for substrate identification in the absence of prior knowledge and lays the groundwork for subsequent validation experiments, holding significant implications for both medical and biotechnological advancements.

We very much welcome open scientific debate, which is a cornerstone of academic development and progress. Thus, we are delighted to provide the following reply to the Letter to the Editor written by Kell et al.[

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 …

Postural orthostatic tachycardia syndrome (POTS) is a common accompaniment of a variety of chronic, inflammatory diseases, including long COVID, as are small, insoluble, ‘fibrinaloid’ microclots. We here develop the argument, with accompanying evidence, that fibrinaloid microclots, through their ability to block the flow of blood through microcapillaries and thus cause tissue hypoxia, are not simply correlated with but in fact, by preceding it, may be a chief intermediary cause of POTS, in which tachycardia is simply the body’s exaggerated ‘physiological’ response to hypoxia. Similar reasoning accounts for the symptoms bundled under the term ‘fatigue’. Amyloids are known to be membrane disruptors, and when their targets are nerve membranes, this can explain neurotoxicity and hence the autonomic nervous system dysfunction that contributes to POTS. Taken together as a system view, we indicate that fibrinaloid microclots can serve to link POTS and fatigue in long COVID in a manner that is at once both mechanistic and explanatory. This has clear implications for the treatment of such diseases.

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.

Understanding the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that herpesviruses' 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. The 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 ECs of ME/CFS patients. This review offers a conceptual advance by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research towards potentially unexplored avenues in understanding and treating this complex syndrome.

Aromatic compounds are used in pharmaceutical, food, textile and other industries. Increased demand has sparked interest in exploring biotechnological approaches for their sustainable production as an alternative to chemical synthesis from petrochemicals or plant extraction. These aromatic products may be toxic to microorganisms, which complicates their production in cell factories. In this study, we analysed the toxicity of multiple aromatic compounds in common production hosts. Next, we screened a subset of toxic aromatics, namely 2‐phenylethanol, 4‐tyrosol, benzyl alcohol, berberine and vanillin, against transporter deletion libraries in Escherichia coli and Saccharomyces cerevisiae. We identified multiple transporter deletions that modulate the tolerance of the cells towards these compounds. Lastly, we engineered transporters responsible for 2‐phenylethanol tolerance in yeast and showed improved 2 …

Nattokinase, from the Japanese fermented food natto, is a protease with fibrinolytic activity that can thus degrade conventional blood clots. In some cases, however, including in Long COVID, fibrinogen can polymerise into an anomalous amyloid form to create clots that are resistant to normal fibrinolysis and that we refer to as fibrinaloid microclots. These can be detected with the fluorogenic stain thioflavin T. We describe an automated microscopic technique for the quanti-fication of fibrinaloid microclot formation, which also allows the kinetics of their formation and aggregation to be recorded. We also here show that recombinant nattokinase is effective at de-grading the fibrinaloid microclots in vitro. This adds to the otherwise largely anecdotal evidence, that we review, that nattokinase might be anticipated to have value as part of therapeutic treatments for individuals with Long COVID and related disorders that involve fibrinaloid microclots.

Atrial fibrillation (AF) is a comorbidity of a variety of other chronic, inflammatory diseases for which fibrinaloid microclots are a known accompaniment (and in some cases, a cause, with a mechanistic basis). Clots are, of course, a well-known consequence of atrial fibrillation. We here ask the question whether the fibrinaloid microclots seen in plasma or serum may in fact also be a cause of (or contributor to) the development of AF. We consider known ‘risk factors’ for AF, and in particular, exogenous stimuli such as infection and air pollution by particulates, both of which are known to cause AF. The external accompaniments of both bacterial (lipopolysaccharide and lipoteichoic acids) and viral (SARS-CoV-2 spike protein) infections are known to stimulate fibrinaloid microclots when added in vitro, and fibrinaloid microclots, as with other amyloid proteins, can be cytotoxic, both by inducing hypoxia/reperfusion and by other means. Strokes and thromboembolisms are also common consequences of AF. Consequently, taking a systems approach, we review the considerable evidence in detail, which leads us to suggest that it is likely that microclots may well have an aetiological role in the development of AF. This has significant mechanistic and therapeutic implications.

IntroductionSince the beginning of the SARS-CoV-2 pandemic in December 2019 multiple metabolomics studies have proposed predictive biomarkers of infection severity and outcome. Whilst some trends have emerged, the findings remain intangible and uninformative when it comes to new patients.ObjectivesIn this study, we accurately quantitate a subset of compounds in patient serum that were found predictive of severity and outcome.MethodsA targeted LC–MS method was used in 46 control and 95 acute COVID-19 patient samples to quantitate the selected metabolites. These compounds included tryptophan and its degradation products kynurenine and kynurenic acid (reflective of immune response), butyrylcarnitine and its isomer (reflective of energy metabolism) and finally 3′,4′-didehydro-3′-deoxycytidine, a deoxycytidine analogue, (reflective of host viral defence response). We subsequently examine …

Acute COVID-19 infection is followed by prolonged symptoms in approximately one in ten cases: known as Long COVID. The disease affects ~65 million individuals worldwide. Many pathophysiological processes appear to underlie Long COVID, including viral factors (persistence, reactivation, and bacteriophagic action of SARS CoV-2); host factors (chronic inflammation, metabolic and endocrine dysregulation, immune dysregulation, and autoimmunity); and downstream impacts (tissue damage from the initial infection, tissue hypoxia, host dysbiosis, and autonomic nervous system dysfunction). These mechanisms culminate in the long-term persistence of the disorder characterized by a thrombotic endothelialitis, endothelial inflammation, hyperactivated platelets, and fibrinaloid microclots. These abnormalities of blood vessels and coagulation affect every organ system and represent a unifying pathway for the …

Microscopy imaging has enabled us to establish the presence of fibrin(ogen) amyloid (fibrinaloid) microclots in a range of chronic, inflammatory diseases. Microclots may also be induced by a variety of purified substances, often at very low concentrations. These molecules include bacterial inflammagens, serum amyloid A, and the S1 spike protein of severe acute respiratory syndrome coronavirus 2. Here, we explore which of the properties of these microclots might be used to contribute to differential clinical diagnoses and prognoses of the various diseases with which they may be associated. Such properties include distributions in their size and number before and after the addition of exogenous thrombin, their spectral properties, the diameter of the fibers of which they are made, their resistance to proteolysis by various proteases, their cross-seeding ability, and the concentration dependence of their ability to bind …

Background:Fibrin (ogen) amyloid microclots and platelet hyperactivation are key pathological findings in patients with acute COVID-19 infection and also in those with Long COVID/Post-Acute Sequelae of COVID-19 (PASC). These pathologies may represent a suitable target for pharmacological treatment of Long COVID.Methods:Here we report on the symptoms displayed by a cohort of 91 South African Long COVID patients at baseline and after a clinician-initiated anticoagulant regime was completed. For laboratory analysis, patients provided a blood sample before and after treatment. Fibrinaloid microclot presence was studied by adding thioflavin T to platelet poor plasma (PPP), whilst platelet hyperactivation was studied using two platelet markers-PAC1 and CD62P (P-selectin). The anticoagulant regime included dual antiplatelet therapy (DAPT-Clopidogrel 75mg+ Aspirin 75mg) once a day, and a direct oral anticoagulant (DOAC-Apixaban) 5mg twice a day. A proton pump inhibitor (PPI) pantoprazole 40 mg/day was also prescribed for gastric protection. Each of the treated cases reported their main Long COVID symptoms, and whether their symptoms resolved following treatment or not.Results:In our cohort a most participants did not report any comorbidities before acute COVID-19 infection. Hypertension and dyslipidaemia were the commonest underlying illnesses, whilst the most commonly reported Long COVID symptoms included fatigue, cognitive dysfunction, shortness of breath, and joint and muscle pains. Following completion of treatment, each of the different symptoms resolved in the majority of patients. This was also reflected in the …

The prevailing hypotheses for the persistent symptoms of Long COVID have been narrowed down to immune dysregulation and autoantibodies, widespread organ damage, viral persistence, and fibrinaloid microclots (entrapping numerous inflammatory molecules) together with platelet hyperactivation. Here we demonstrate significantly increased concentrations of von Willebrand factor (VWF), platelet factor 4 (PF4), serum amyloid A (SAA), α-2 antiplasmin (α-2AP), endothelial-leukocyte adhesion molecule 1 (E-selectin), and platelet endothelial cell adhesion molecule (PECAM-1) in the soluble part of the blood. It was noteworthy that the mean level of α-2 antiplasmin exceeded the upper limit of the laboratory reference range in Long COVID patients, and the other 5 were significantly elevated in Long COVID patients as compared to the controls. This is alarming if we take into consideration that a significant amount of …

Long COVID has become a significant global health and economic burden, yet there are currently no established methods or diagnostic tools to identify which patients might benefit from specific treatments. One of the major pathophysiological factors contributing to Long COVID is the presence of hypercoagulability; this results in insoluble amyloid microclots that are resistant to fibrinolysis. Our previous research using fluorescence microscopy has demonstrated a significant amyloid microclot load in Long COVID patients. However, this approach lacked the elements of statistical robustness, objectivity, and rapid throughput. In the current study, we have used imaging flow cytometry for the first time to show a significantly increased concentration and size of these microclots. We identified notable variations in size and fluorescence between microclots in Long COVID and those of controls even using a 20× objective. By …

Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder affecting 50 million people globally. It is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles, consisting of amyloidand hyperphosphorylated tau proteins, respectively. Despite global research efforts, there is currently no cure available, due in part to an incomplete understanding of the disease pathogenesis. Numerous possible mechanisms, or hypotheses, explaining the origins of sporadic or late-onset AD have been proposed, including the amyloid-, inflammatory, vascular, and infectious hypotheses. However, despite ample evidence, the failure of multiple trial drugs at the clinical stage illuminates the possible pitfalls of these hypotheses. Systems biology is a strategy which aims to elucidate the interactions between parts of a whole. Using this approach, the current paper shows how the four …

Long COVID has become a significant global health and economic burden, yet there are currently no established diagnostic tools to identify which patients might benefit from specific treatments. One of the major pathophysiological factors contributing to Long COVID is the presence of hypercoagulability; this results in insoluble amyloid microclots that are resistant to fibrinolysis. Our previous research using fluorescence microscopy has demonstrated a significant amyloid microclot load in Long COVID patients. However, this approach lacked statistical robustness, objectivity, and rapid throughput. In the current study, we have used imaging flow cytometry for the first time to show significantly increased concentration and size of these microclots. We identified notable variations in size and fluorescence between microclots in Long COVID and those of controls even using a 20x objective. By combining cell imaging and the high-event-rate nature of a conventional flow cytometer, imaging flow cytometry can eliminate erroneous results and increase accuracy in gating and analysis beyond what pure quantitative measurements from conventional flow cytometry can provide. Although imaging flow cytometry was used in our study, our results suggest that the signals indicating the presence of microclots should be easily detectable using a conventional flow cytometer. Flow cytometry is a more widely available technique which has been used in pathology laboratories for decades, rendering it a potentially more suitable and accessible method for detecting microclots in individuals suffering from both Long COVID and other conditions with similar pathology …

It is now well established that the blood-clotting protein fibrinogen can polymerise into an anomalous form of fibrin that is amyloid in character; the resultant clots and microclots entrap many other molecules, stain with fluorogenic amyloid stains, are rather resistant to fibrinolysis, can block up microcapillaries, are implicated in a variety of diseases including Long COVID, and have been referred to as fibrinaloids. A necessary corollary of this anomalous polymerisation is the generation of novel epitopes in proteins that would normally be seen as ‘self’, and otherwise immunologically silent. The precise conformation of the resulting fibrinaloid clots (that, as with prions and classical amyloid proteins, can adopt multiple, stable conformations) must depend on the existing small molecules and metal ions that the fibrinogen may (and is some cases is known to) have bound before polymerisation. Any such novel epitopes …

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 …

Ergothioneine, an antioxidant nutraceutical mainly at present derived from the dietary intake of mushrooms, has been suggested as a preventive for pre-eclampsia (PE). We analysed early pregnancy samples from a cohort of 432 first time mothers as part of the Screening for Endpoints in Pregnancy (SCOPE, European branch) project to determine the concentration of ergothioneine in their plasma. There was a weak association between the ergothioneine levels and maternal age but none for BMI. Of these 432 women, 97 went on to develop pre-term (23) or term (74) PE. If a threshold was set at the 90th percentile of the reference range in the control population (≥462 ng/ml), only one of these 97 women (1%) developed PE, versus 96/397 (24.2%) whose ergothioneine level was below this threshold. One possible interpretation of these findings, consistent with previous experiments in a reduced uterine …