Biology subjects

Pretorius, E.

Publications and source records attributed to Pretorius, E..

11 recordsLinked to original sources

bioRxiv2026

Peer-Led Leadership, Mentoring and Promotion: Conversations Among Female Academics from South Africa, Ghana and the United Kingdom

Here, we report a one-day, cross-cultural, peer-led workshop at the University of Stellenbosch, facilitated by academics from South Africa, Ghana and the United Kingdom. The focus was on leadership, mentoring and promotion. Using short, structured activities alongside small-group discussion, the participants were encouraged to reflect on leadership, mentoring and the perceived gap between being "ready" and being "recognised" for promotion. Descriptive survey findings and free-text reflections highlight the demand for structured peer support, reciprocal mentoring opportunities, and clearer, more transparent promotion processes. Following the event, we performed a structured review of the impact. This highlighted that the workshop participants reported that the event allowed for greater self-awareness into their own leadership approaches, a stronger commitment to purposeful mentoring, and greater confidence and renewed motivation to take concrete steps towards promotion.

scientific communication and education↗

bioRxiv2026

An imaging flow cytometry method to study platelet-monocyte aggregates using Long COVID as a model

BackgroundLong COVID is characterised by persistent systemic inflammation and endothelial dysfunction, with increasing evidence implicating thromboinflammatory mechanisms. Platelet-monocyte aggregates (PMA) represent a sensitive marker of platelet activation and immune-vascular interactions, but their role in Long COVID remains incompletely defined. MethodsThis study quantified circulating PMA in 20 Long COVID patients and 20 healthy controls using a two-colour imaging flow cytometry assay targeting CD14 (a monocyte receptor for pathogen-associated molecular patterns, PAMPs) and CD62P (P-selectin). PMA were expressed as a percentage of total monocytes, and platelet attachment patterns were classified into single versus multiple platelet binding. Statistical analyses included Shapiro-Wilk normality testing, unpaired t-tests, Mann-Whitney U tests or two-way ANOVA as appropriate, and linear regression for correlation analysis. ResultsCirculating PMA were significantly elevated in Long COVID patients compared with controls (29.19 [20.02-37.26] vs 4.59 [2.67-7.16], p < 0.0001). Long COVID samples showed a reduced proportion of monocytes with single platelet attachment and a corresponding increase in multiple platelet binding (p < 0.0001). In controls, %PMA increased with age (p < 0.01), whereas no age association was observed in Long COVID, indicating an elevated baseline independent of age. ConclusionsLong COVID is associated with markedly increased platelet-monocyte aggregation and altered platelet attachment dynamics, consistent with sustained thromboinflammatory activity. PMA represent a sensitive cellular marker of platelet-driven immune activation and may have utility as an accessible biomarker for stratifying thromboinflammatory burden in Long COVID.

physiology↗

bioRxiv2025

Post-translational modifications within fibrinaloid microclot complexes distinguish Pre-COVID-19 Postural Orthostatic Tachycardia Syndrome, Long COVID, and Long COVID-POTS and reveal disease-specific molecular pathways

BackgroundPre-COVID-19 Postural orthostatic tachycardia syndrome (PC-POTS), Long COVID, and their overlap (LC-POTS) are chronic post-viral conditions marked by debilitating symptoms despite frequently normal routine laboratory results. We previously identified insoluble fibrinaloid microclot complexes (FMCs) in Long COVID. It is not known whether FMCs are also present in PC-POTS, or whether post-translational modifications (PTMs) within FMC-entrapped proteins contribute to disease mechanisms. MethodsPlatelet-poor plasma from healthy controls, PC-POTS patients (collected prior to the COVID-19 pandemic), Long COVID (without POTS) and LC-POTS patients underwent fluorescence imaging flow cytometry to quantify FMCs. Proteomic analyses were performed on insoluble protein fractions using a double trypsin digestion strategy and data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential protein abundance, PTMs, and amyloidogenicity were compared across groups. ResultsMeasured with imaging flowcytometry in objects/mL, higher levels of FMCs were present in disease groups compared to controls, although not statistically significant. Statistically significant differences potentially lay within FMC sizes and composition. Furthermore, despite only a few dysregulated proteins, FMC proteomics revealed extensive and disease-specific peptides with PTM dysregulation across coagulation, immune, and metabolic pathways. Long COVID displayed FMCs with PTMs of coagulation proteins including prominent advanced glycation end-products (AGE)- and oxidation-based modifications of fibrinogen subunits, particularly fibrinogen subunit A (FIBA), resembling diabetic glycation profiles. FMCs in PC-POTS showed fewer fibrinogen PTMs but markedly increased modifications in metabolic proteins, including oxidised apoA1 and apoB, and immune patterns with complement-related proteins (C3, C4A/B, IC1), immunoglobulin G1 (IGG1) and alpha 2 macroglobulin (A2MG). LC-POTS shared coagulation pathology with Long COVID and immune pathology with PC-POTS. Many dysregulated peptides were determined by in silco methods to be highly amyloidogenic, consistent with FMCs as {beta}-sheet-rich aggregates. Protein-level differences were minimal compared with PTM changes. ConclusionsThis study provides the first evidence that post-translational modifications (PTMs) within fibrinaloid microclots complexes (FMCs) uniquely distinguish pre-COVID-19 POTS, Long COVID, and Long COVID-POTS. Because PC-POTS samples were collected before SARS-CoV-2, their PTM patterns reflect intrinsic disease biology, allowing a clear separation from Long COVID-related changes. PTM profiling revealed pro-coagulant fibrinogen modifications in Long COVID and LC-POTS, metabolic-oxidative disruptions in Long COVID and PC-POTS, and immune dysregulation in PC-POTS and LC-POTS. None of these is detectable with routine assays, and all are independent of protein abundance. The consistent presence of amyloidogenic peptides suggests a contribution to microvascular dysfunction. These findings define disease-specific PTM landscapes and support new diagnostic and therapeutic avenues across autonomic and post-viral disorders.

physiology↗

bioRxiv2025

AmyloGram Reveals Amyloidogenic Potential in Stroke Thrombus Proteomes

BackgroundAmyloidogenic proteins play a central role in a range of pathological conditions, yet their presence in thrombi has only recently been recognized. Whether computational prediction tools can identify amyloid-forming potential in thrombus proteomes remains unclear. MethodsAmyloGram is a computational tool that estimates amyloid-forming potential based on n-gram sequence encoding and random forest classification. Using AmyloGram, we analyzed 204 proteins tagged by humans as amyloidogenic in UniProt. We then applied the same approach to proteins identified in thrombi retrieved using mechanical thrombectomy from patients with cardioembolic and atherothrombotic stroke. In addition we used AmyloGram to analyse the amyloidogenicity of 83,567 canonical human protein sequences. ResultsAmong the UniProt-annotated amyloid set, nearly all proteins received AmyloGram scores above 0.7, including 23 of the 24 human proteins. Even the lowest-scoring human protein, lysozyme (scoring 0.675), is known to form amyloid under certain conditions. In thrombi from both stroke subtypes, all detected proteins had AmyloGram scores above 0.7, suggesting a high likelihood of amyloid content. A majority of unannotated proteins also achieve AmyloGram scores exceeding 0.7. ConclusionsAmyloGram reliably identifies known amyloid-forming proteins and reveals that stroke thrombi are enriched for proteins with high amyloidogenic potential. These findings support the hypothesis that thrombus formation in stroke involves amyloid-related mechanisms and warrant further investigation using histological and functional validation.

bioinformatics↗

bioRxiv2024

The proteome content of blood clots observed under different conditions: successful role in predicting clot amyloid(ogenicity)

A recent analysis compared the proteome of (i) blood clots seen in two diseases - sepsis and long COVID - when blood was known to have clotted into an amyloid microclot form (as judged by staining with the fluorogenic amyloid stain thioflavin T) with (ii) that of those non-amy-loid clots considered to have formed normally. Such fibrinaloid microclots are also relatively resistant to fibrinolysis. The proteins that the amyloid microclots contained differed markedly both from the soluble proteome of typical plasma and that of normal clots, and also between the disease studies (an acute syndrome in the form of sepsis in an ITU and a chronic disease represented by Long COVID). Many proteins in the amyloid microclots were low in concentration in plasma and were effectively accumulated into the fibres, whereas many other abundant plasma proteins were excluded. The proteins found in the microclots associated with the diseases also tended to be themselves amyloidogenic. We here ask effectively the inverse question. This is: can the clot proteome tell us whether the clots associated with a particular disease contained proteins that are observed uniquely (or are highly over-represented) in known amyloid clots relative to normal clots, and thus were in fact amyloid in nature? The answer is in the affirmative in a variety of major coagulopathies, viz. venous thromboembolism, pulmonary embolism, deep vein thrombosis, various cardiac issues, and ischaemic stroke. Galectin-3-binding protein and thrombospondin-1 seem to be especially widely associated with amyloid-type clots, and the latter has indeed been shown to be incorporated into growing fibrin fibres. These may consequently provide useful biomarkers with a mechanistic basis.

bioinformatics↗

bioRxiv2024

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-{beta} motif of parallel or antiparallel {beta}-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena occur in blood from individuals with circulating inflammatory molecules (also those originating from viruses and bacteria). In the presence of inflammagens, pathological clotting can occur, that results 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 provide evidence against 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 overlap with the normal plasma proteome and no correlation between plasma protein abundance and presence in microclots. Notably, abundant plasma proteins like -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-{beta} 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.

biochemistry↗

bioRxiv2024

Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

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 quantification 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 degrading 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.

biochemistry↗

bioRxiv2020

The Atypical Fibrin Fibre Network in Rheumatoid Arthritis and its Relation to Autoimmunity, Inflammation and Thrombosis

ObjectiveThe risk of cardiovascular events in patients with 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 inter-parameter relationships. MethodsBlood samples were collected from 30 RA patients and 25 age- and gender-matched healthy volunteers. Levels of SAA, CRP, ICAM-1 and VCAM-1 was measured using a sandwich immunoassay. Whole blood coagulation was assessed using Thromboelastography. Fibrin clot networks and fiber structure was investigated using Scanning Electron Microscopy. The detection and quantification of citrullination in formed fibrin clots were performed using a fluorescently labeled Citrulline monoclonal antibody with Confocal Microscopy. ResultsConcentrations of SAA, CRP and ICAM-1 were significantly elevated in RA patients compared to controls. TEG parameters relating to coagulation initiation (R and K), rate of fibrin cross-linking (-Angle), and time to reach maximum thrombus generation (TMRTG) were attenuated in RA patients. Parameters relating to clot strength (MA, MRTG, TGG) did not statistically differ between RA and controls. Logistic regression modelling revealed stronger association between acute phase reactants (CRP, SAA) with TEG parameters than endothelial function markers. Microscopic analysis revealed denser networks of thicker fibrin fibers in RA patients compared to controls [median (interquartile range) 214 (170-285) vs 120 (100-144) nm respectively, p<0.0001, Odds ratio=22.7). Detection of multiple citrullinated regions within fibrin clot structures in RA patients, which was less prevalent in control samples (p<0.05, OR=2.2). ConclusionPatients with active RA display a coagulation profile that is dissimilar to general findings associated with other inflammatory conditions. The alteration of protein structures by autoimmune linked citrullination could play a role in determining the structure of fibrin and the potential of conferring a heightened thrombotic risk in RA patients.

biochemistry↗

bioRxiv2019

Parkinson’s disease: a systemic inflammatory disease accompanied by bacterial inflammagens

Parkinsons disease (PD) is a well-known neurodegenerative disease. Recently, the role of gingipains from Porphyromonas gingivalis was implicated in Alzheimers disease. Here we present evidence of systemic inflammation, accompanied by hypercoagulation; we also show that ginipains from P. gingivalis and its LPS may foster abnormal clotting, and that ginipains are present in PD blood, and thus that ginipains action on blood may be relevant to PD pathology. Bloods from both PD and healthy blood samples were analysed using thromboelastography (TEG), confocal and electron microscopies, and for cytokine and other circulating biomarkers. We also probed PD and healthy plasma clots with a polyclonal antibody for the bacterial protease, gingipain R1, from P. gingivalis. Low concentrations of recombinant gingipain R1 were also added to purified fluorescent fibrinogen. TEG, fibrin(ogen) amyloid formation and platelet ultrastructure analysis confirmed profound hypercoagulation, while the biomarker analysis confirmed significantly increased levels of circulating proinflammatory cytokines. We provide evidence for the presence of the protease, gingipain R1 in PD blood, implicating inflammatory microbial cell wall products in PD.

neuroscience↗

bioRxiv2017

Both Lipopolysaccharide And Lipoteichoic Acids Potently Induce Anomalous Fibrin Amyloid Formation: Assessment With Novel Amytracker™ Stains

In recent work, we discovered that the presence of highly substoichiometric amounts (10-8 molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name AmytrackerTM, also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thromboelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

biophysics↗

bioRxiv2017

Lipopolysaccharide-binding protein (LBP) can reverse the amyloid state of fibrin seen or induced in Parkinson’s disease: implications for its aetiology

The thrombin-induced polymerisation of fibrinogen to form fibrin is well established as a late stage of blood clotting. In recent work, we showed that the presence of tiny amounts of bacterial lipopolysaccharide (LPS) could cause these clots to adopt an amyloid form, that could be observed via scanning electron microscopy (SEM) or via the fluorescence of thioflavin-T. This could be prevented by the prior addition of lipopolysaccharide-binding protein (LBP). We had also observed by SEM this unusual clotting in the blood of patients with Parkinsons disease (PD). We here show that this too can be prevented by LBP, thereby implicating such inflammatory microbial cell wall products in the aetiology of the disease. This may lead to novel treatment strategies in PD designed to target microbes and their products.

neuroscience↗