Increased Levels of Inflammatory and Endothelial Biomarkers in Blood of Long COVID Patients Point to Thrombotic Endothelialitis.

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 the total burden of these inflammatory molecules has previously been shown to be entrapped inside fibrinolysis-resistant microclots (thus decreasing the apparent level of the soluble molecules). We conclude that presence of microclotting, together with relatively high levels of six biomarkers known to be key drivers of endothelial and clotting pathology, points to thrombotic endothelialitis as a key pathological process in Long COVID.

Vascular Pathogenesis in Acute and Long COVID: Current Insights and Therapeutic Outlook.

Long coronavirus disease 2019 (COVID-19)-a postacute consequence of severe acute respiratory syndrome coronavirus 2 infection-manifests with a broad spectrum of relapsing and remitting or persistent symptoms as well as varied levels of organ damage, which may be asymptomatic or present as acute events such as heart attacks or strokes and recurrent infections, hinting at complex underlying pathogenic mechanisms. Central to these symptoms is vascular dysfunction rooted in thrombotic endothelialitis. We review the scientific evidence that widespread endothelial dysfunction (ED) leads to chronic symptomatology. We briefly examine the molecular pathways contributing to endothelial pathology and provide a detailed analysis of how these cellular processes underpin the clinical picture. Noninvasive diagnostic techniques, such as flow-mediated dilation and peripheral arterial tonometry, are evaluated for their utility in identifying ED. We then explore mechanistic, cellular-targeted therapeutic interventions for their potential in treating ED. Overall, we emphasize the critical role of cellular health in managing Long COVID and highlight the need for early intervention to prevent long-term vascular and cellular dysfunction.

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.

Are fibrinaloid microclots a cause of autoimmunity in Long Covid and other post-infection diseases?

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, however, are likely to lead to the generation of autoantibodies. A convergent phenomenology, including distinct conformations and seeding of the anomalous form for initiation and propagation, is emerging to link knowledge in prions, prionoids, amyloids and now fibrinaloids. We here summarise the evidence for the above reasoning, which has substantial implications for our understanding of the genesis of autoimmunity (and the possible prevention thereof) based on the primary process of fibrinaloid formation.

Proteomic Evidence for Amyloidogenic Cross-Seeding in Fibrinaloid Microclots.

In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-ß motif of parallel or antiparallel ß-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena can occur in blood from individuals with circulating inflammatory molecules (and also some originating from viruses and bacteria). Such pathological clotting can result in an anomalous amyloid form termed fibrinaloid microclots. Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment. We thus provide evidence against such a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration. Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant quantitative overlap with the normal plasma proteome and no correlation between plasma protein abundance and their presence in fibrinaloid microclots. Notably, abundant plasma proteins like α-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand factor are well represented. Using bioinformatic tools, including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-ß elements into amyloid structures. This integration likely contributes to the microclots' resistance to proteolysis. Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders.

The Biology and Biochemistry of Kynurenic Acid, a Potential Nutraceutical with Multiple Biological Effects.

Kynurenic acid (KYNA) is an antioxidant degradation product of tryptophan that has been shown to have a variety of cytoprotective, neuroprotective and neuronal signalling properties. However, mammalian transporters and receptors display micromolar binding constants; these are consistent with its typically micromolar tissue concentrations but far above its serum/plasma concentration (normally tens of nanomolar), suggesting large gaps in our knowledge of its transport and mechanisms of action, in that the main influx transporters characterized to date are equilibrative, not concentrative. In addition, it is a substrate of a known anion efflux pump (ABCC4), whose in vivo activity is largely unknown. Exogeneous addition of L-tryptophan or L-kynurenine leads to the production of KYNA but also to that of many other co-metabolites (including some such as 3-hydroxy-L-kynurenine and quinolinic acid that may be toxic). With the exception of chestnut honey, KYNA exists at relatively low levels in natural foodstuffs. However, its bioavailability is reasonable, and as the terminal element of an irreversible reaction of most tryptophan degradation pathways, it might be added exogenously without disturbing upstream metabolism significantly. Many examples, which we review, show that it has valuable bioactivity. Given the above, we review its potential utility as a nutraceutical, finding it significantly worthy of further study and development.

A Perspective on How Fibrinaloid Microclots and Platelet Pathology May be Applied in Clinical Investigations.

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 small molecules including fluorogenic amyloid stains. Measuring these microclot parameters, together with microscopy imaging itself, along with methodologies like proteomics and imaging flow cytometry, as well as more conventional assays such as those for cytokines, might open up the possibility of a much finer use of these microclot properties in generative methods for a future where personalized medicine will be standard procedures in all clotting pathology disease diagnoses.

Quantitative LC-MS study of compounds found predictive of COVID-19 severity and outcome.

INTRODUCTION: Since 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. OBJECTIVES: In this study, we accurately quantitate a subset of compounds in patient serum that were found predictive of severity and outcome. METHODS: A 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 changes in those markers by disease severity and outcome relative to those of control patients' levels. RESULTS & CONCLUSION: Finally, we demonstrate the added value of the kynurenic acid/tryptophan ratio for severity and outcome prediction and highlight the viral detection potential of ddhC.

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.

A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications.

Post-acute sequelae of COVID (PASC), usually referred to as 'Long COVID' (a phenotype of COVID-19), is a relatively frequent consequence of SARS-CoV-2 infection, in which symptoms such as breathlessness, fatigue, 'brain fog', tissue damage, inflammation, and coagulopathies (dysfunctions of the blood coagulation system) persist long after the initial infection. It bears similarities to other post-viral syndromes, and to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Many regulatory health bodies still do not recognize this syndrome as a separate disease entity, and refer to it under the broad terminology of 'COVID', although its demographics are quite different from those of acute COVID-19. A few years ago, we discovered that fibrinogen in blood can clot into an anomalous 'amyloid' form of fibrin that (like other ß-rich amyloids and prions) is relatively resistant to proteolysis (fibrinolysis). The result, as is strongly manifested in platelet-poor plasma (PPP) of individuals with Long COVID, is extensive fibrin amyloid microclots that can persist, can entrap other proteins, and that may lead to the production of various autoantibodies. These microclots are more-or-less easily measured in PPP with the stain thioflavin T and a simple fluorescence microscope. Although the symptoms of Long COVID are multifarious, we here argue that the ability of these fibrin amyloid microclots (fibrinaloids) to block up capillaries, and thus to limit the passage of red blood cells and hence O2 exchange, can actually underpin the majority of these symptoms. Consistent with this, in a preliminary report, it has been shown that suitable and closely monitored 'triple' anticoagulant therapy that leads to the removal of the microclots also removes the other symptoms. Fibrin amyloid microclots represent a novel and potentially important target for both the understanding and treatment of Long COVID and related disorders.

Membrane transporter identification and modulation via adaptive laboratory evolution.

Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L-histidine, L-phenylalanine, L-threonine, and L-methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L-phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.

Relative Hypercoagulopathy of the SARS-CoV-2 Beta and Delta Variants when Compared to the Less Severe Omicron Variants Is Related to TEG Parameters, the Extent of Fibrin Amyloid Microclots, and the Severity of Clinical Illness.

Earlier variants of SARS-CoV-2 have been associated with hypercoagulability and an extensive formation of fibrin amyloid microclots, which are considered to contribute to the pathology of the coronavirus 2019 disease (COVID-19). The newer omicron variants appear to be far more transmissible, but less virulent, even when taking immunity acquired from previous infections or vaccination into account. We here show that while the clotting parameters associated with omicron variants are significantly raised over those of healthy, matched controls, they are raised to levels significantly lower than those seen with more severe variants such as beta and delta. We also observed that individuals infected with omicron variants manifested less extensive microclot formation in platelet-poor plasma compared with those harboring the more virulent variants. The measurement of clotting effects between the different variants acts as a kind of "internal control" that demonstrates the relationship between the extent of coagulopathies and the virulence of the variant of interest. This adds to the evidence that microclots may play an important role in reflecting the severity of symptoms observed in COVID-19.

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.

Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-Acute Sequelae of COVID-19 (PASC).

BACKGROUND: Fibrin(ogen) amyloid microclots and platelet hyperactivation previously reported as a novel finding in South African patients with the coronavirus 2019 disease (COVID-19) and Long COVID/Post-Acute Sequelae of COVID-19 (PASC), might form a suitable set of foci for the clinical treatment of the symptoms of Long COVID/PASC. A Long COVID/PASC Registry was subsequently established as an online platform where patients can report Long COVID/PASC symptoms and previous comorbidities. METHODS: In this study, we report on the comorbidities and persistent symptoms, using data obtained from 845 South African Long COVID/PASC patients. By using a previously published scoring system for fibrin amyloid microclots and platelet pathology, we also analysed blood samples from 80 patients, and report the presence of significant fibrin amyloid microclots and platelet pathology in all cases. RESULTS: Hypertension, high cholesterol levels (dyslipidaemia), cardiovascular disease and type 2 diabetes mellitus (T2DM) were found to be the most important comorbidities. The gender balance (70% female) and the most commonly reported Long COVID/PASC symptoms (fatigue, brain fog, loss of concentration and forgetfulness, shortness of breath, as well as joint and muscle pains) were comparable to those reported elsewhere. These findings confirmed that our sample was not atypical. Microclot and platelet pathologies were associated with Long COVID/PASC symptoms that persisted after the recovery from acute COVID-19. CONCLUSIONS: Fibrin amyloid microclots that block capillaries and inhibit the transport of O2 to tissues, accompanied by platelet hyperactivation, provide a ready explanation for the symptoms of Long COVID/PASC. Removal and reversal of these underlying endotheliopathies provide an important treatment option that urgently warrants controlled clinical studies to determine efficacy in patients with a diversity of comorbidities impacting on SARS-CoV-2 infection and COVID-19 severity. We suggest that our platelet and clotting grading system provides a simple and cost-effective diagnostic method for early detection of Long COVID/PASC as a major determinant of effective treatment, including those focusing on reducing clot burden and platelet hyperactivation.

Author Correction: Hitchhiking into the cell.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intelligent host engineering for metabolic flux optimisation in biotechnology.

Optimising the function of a protein of length N amino acids by directed evolution involves navigating a 'search space' of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is 'making such biology predictable'. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.

A palette of fluorophores that are differentially accumulated by wild-type and mutant strains of Escherichia coli: surrogate ligands for profiling bacterial membrane transporters.

Our previous work demonstrated that two commonly used fluorescent dyes that were accumulated by wild-type Escherichia coli MG1655 were differentially transported in single-gene knockout strains, and also that they might be used as surrogates in flow cytometric transporter assays. We summarize the desirable properties of such stains, and here survey 143 candidate dyes. We eventually triage them (on the basis of signal, accumulation levels and cost) to a palette of 39 commercially available and affordable fluorophores that are accumulated significantly by wild-type cells of the 'Keio' strain BW25113, as measured flow cytometrically. Cheminformatic analyses indicate both their similarities and their (much more considerable) structural differences. We describe the effects of pH and of the efflux pump inhibitor chlorpromazine on the accumulation of the dyes. Even the 'wild-type' MG1655 and BW25113 strains can differ significantly in their ability to take up such dyes. We illustrate the highly differential uptake of our dyes into strains with particular lesions in, or overexpressed levels of, three particular transporters or transporter components (yhjV, yihN and tolC). The relatively small collection of dyes described offers a rapid, inexpensive, convenient and informative approach to the assessment of microbial physiology and phenotyping of membrane transporter function.

Persistent clotting protein pathology in Long COVID/Post-Acute Sequelae of COVID-19 (PASC) is accompanied by increased levels of antiplasmin.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2)-induced infection, the cause of coronavirus disease 2019 (COVID-19), is characterized by acute clinical pathologies, including various coagulopathies that may be accompanied by hypercoagulation and platelet hyperactivation. Recently, a new COVID-19 phenotype has been noted in patients after they have ostensibly recovered from acute COVID-19 symptoms. This new syndrome is commonly termed Long COVID/Post-Acute Sequelae of COVID-19 (PASC). Here we refer to it as Long COVID/PASC. Lingering symptoms persist for as much as 6 months (or longer) after acute infection, where COVID-19 survivors complain of recurring fatigue or muscle weakness, being out of breath, sleep difficulties, and anxiety or depression. Given that blood clots can block microcapillaries and thereby inhibit oxygen exchange, we here investigate if the lingering symptoms that individuals with Long COVID/PASC manifest might be due to the presence of persistent circulating plasma microclots that are resistant to fibrinolysis. METHODS: We use techniques including proteomics and fluorescence microscopy to study plasma samples from healthy individuals, individuals with Type 2 Diabetes Mellitus (T2DM), with acute COVID-19, and those with Long COVID/PASC symptoms. RESULTS: We show that plasma samples from Long COVID/PASC still contain large anomalous (amyloid) deposits (microclots). We also show that these microclots in both acute COVID-19 and Long COVID/PASC plasma samples are resistant to fibrinolysis (compared to plasma from controls and T2DM), even after trypsinisation. After a second trypsinization, the persistent pellet deposits (microclots) were solubilized. We detected various inflammatory molecules that are substantially increased in both the supernatant and trapped in the solubilized pellet deposits of acute COVID-19 and Long COVID/PASC, versus the equivalent volume of fully digested fluid of the control samples and T2DM. Of particular interest was a substantial increase in α(2)-antiplasmin (α2AP), various fibrinogen chains, as well as Serum Amyloid A (SAA) that were trapped in the solubilized fibrinolytic-resistant pellet deposits. CONCLUSIONS: Clotting pathologies in both acute COVID-19 infection and in Long COVID/PASC might benefit from following a regime of continued anticlotting therapy to support the fibrinolytic system function.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome.

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.