Browsing by Author "Blackburn, Jonathan"
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- ItemOpen AccessAn immunoproteomic approach to identifying cancer-associated autoantibody biomarkers(2018) Smith, Muneerah; Blackburn, JonathanCancer is a heterogenous disease capable of forming and spreading in most tissues of the human body. Cancer screening and diagnosis can be performed through medical procedures, which are highly invasive, requiring an intensive infrastructure. It is therefore important to create cost-effective, non-invasive cancer diagnostic tools that also gives an indication of disease prognosis. With this in mind, the Blackburn lab previously created a cancer-testis antigen microarray (CT100plus) functionalised with tumour-associated and tumour-specific antigens, capable of detecting plasma- or serum-derived autoantibodies in the picogram per millilitre (pg/ml) range. In this thesis, a newly established statistical pipeline was used to analyse colorectal cancer (CRC) patient-derived CT100plus data. Using the pipeline, the 10 antigens with the highest receiver operator characteristic (ROC)-derived area under the ROC curve (AUC)-values were identified as potential autoantibody-based biomarkers. The top 10 antigen biomarker candidates include CEACAM 1, COL6A1, GRWD1, MAGEA1, MAGEA5, MAGEA10, NY-CO-1, SGY-1, SPANXB1 and THEG. Using these biomarker candidates, distinct clusters of healthy controls (HCs) and CRC patients were observed using both unsupervised hierarchical clustering and principle component analysis (PCA) analysis. Combinatorial ROC analysis indicates that CEACAM1 and GRWD1 as the top autoantigen combination for CRC diagnosis, together producing sensitivity-, and specificity-, and AUC-values of 1.00, 0.77 and 0.94, respectively. Furthermore, other top autoantigens, including COL6A1, THEG and CEACAM7, a homologue of CEACAM1, were also identified in this thesis by affinity purification-mass spectrometry (AP-MS) for patients from the same cohort, providing supporting evidence that these antigens are associated with CRC. The CT100plus microarray content was enzymatically modified to include citrullinated proteins, with the subsequent assessment of CRC patient autoantibody response. Significantly (p-value ≤ 0.05; adjusted p-value ≤ 0.05) higher signal intensities were detected in CRC patients versus HCs for citrullinated CDK7, MAGEB1, MAGEB5, MAGEB6 and SYCP1, whereas no significant (adjusted p-value > 0.05) difference in autoantibody signal was detected for these autoantigens on the noncitrullinated microarray for the same patient cohort. ROC analyses of these antigens resulted in 2 an AUC-, sensitivity- and specificity-values of 0.91, 0.87 and 0.89, respectively. Together, this thesisshowsfor the first time that cancer patients elicit an autoantibody response to citrullinated proteins, resulting in potential novel CRC biomarkers. A novel AP-MS assay was developed to detect autoantibody responses to autologous native CRC tissue proteins. Using the optimised methodology, proteins or homologues of proteins that were significantly (> cut-off value) detected on the CT100plus microarray for the same 5 patients were re-identified by the orthogonal AP-MS method. Using the methodology, PAD2, an enzyme that catalyses the conversion of arginine to citrulline was also identified. In addition, citrullinated antigens associated with cancer were identified, including homologues of CDK7 and MAGEB supporting the conclusion that citrullinated homologues of these proteins induce an autoantibody response in CRC patients. Finally, serum and/or plasma samples of a cohort melanoma patients were analysed using the CT100plus microarray, and autoantibody signals were compared to those of healthy control (HC) samples. Using the established statistical pipeline, the 10 antigens with the highest ROC-derived AUC-values were identified as potential biomarkers. The top 10 biomarker autoantigen candidates for melanoma included CEACAM 1, DPPA2, FGFR2, ITGB1, MAGEA10, NANOG, PIM1, SPANXB1, THEG and XAGE1B. Using these biomarker candidates, distinct clusters of HCs and melanoma patients were identified in both unsupervised hierarchical clustering and PCA analysis. Combinatorial ROC analysis indicates that CEACAM1 and FGFR2 were identified as the top antigens for melanoma diagnosis, together producing sensitivity-, and specificity-, and AUCvalues of 0.96, 0.94 and 0.93, respectively. In conclusion, a statistical pipeline was established for microarray data, enabling the identification of potential antigens associated with cancer diagnosis, and the potential to determine disease prognosis. Using the established pipeline, cancer antigens associated with CRC and melanoma were identified. In addition, an AP-MS assay was developed for the identification of known and novel cancer antigen that can be added to the customisable CT100plus microarray.
- ItemOpen AccessAnalysis of urinary lipid biomarker candidates from tuberculosis patients by multiple reaction monitoring(2019) Waldron, Elizabeth Louise; Blackburn, JonathanBackground: Tuberculosis (TB) is an aggressive disease and is the leading cause of death by infectious disease in South Africa. With early diagnosis and correct treatment, almost all TB cases can be cured. The main diagnostic tests in South Africa are limited for people living in rural areas, require sputum which cannot be produced by very ill patients, and have low sensitivity in immune compromised individuals. There is an urgent need for a non-invasive and robust diagnostic test which uses an easily accessible biofluid and can be performed at the point-of-care. Urine has shown promise as a diagnostic biofluid for biomarker investigation. Full scan mass spectrometry is the gold standard for the unbiased discovery of biomarker candidates, and targeted multiple reaction monitoring (MRM) is the method of choice for subsequent validation of biomarker candidates. A list of candidate urinary biomarkers has previously been generated which can discriminate between latent and active TB infection using MS1 mass spectrometry, but these biomarkers have not yet been verified by targeted mass spectrometry. Aims and Objectives: The aim of this project is to verify a list of biomarker candidates using MRM assays by: 1) developing MRM assays for known fatty acid standards, and 2) developing MRM assays for unidentified urinary lipid biomarker candidates de novo, which can be applied to clinical cohorts for future validation. Methods: Fatty acid standards were initially assessed using direct infusion full-scan MS1 mass spectrometry on an orbitrap mass analyser. They were then optimised for fragmentation by compound optimisation on a triple-quadrupole mass analyser, the data from which was used to build MRM assays. Liquid chromatography was optimised for these lipids and the MRMs were validated by spiking the lipid standards into a complex mixture. For the second part of the project, lipid extract (containing unidentified biomarker candidates) from patient derived urine samples were analysed by data-dependent acquisition with inclusion lists on an orbitrap mass analyser. From this experiment MS/MS data was acquired for biomarker candidates which were then compiled into MRM assays and verified using a triple-quadrupole mass analyser. Results: From six fatty acid standards, reliable MRM assays were generated for five of them. The biomarker candidates formed a list of 70 molecules which were further refined to 10 molecules which were reproducibly measured by MRM assay. Discussion and Conclusions: From this work the fatty acid standards can be used as internal retention time predictors for future lipidomic work and quality checks, as they eluted across a wide retention time range. The biomarker candidates have been verified using MRM assays and can be validated in larger clinical cohorts in the future. The end-goal is to use these biomarker candidates as part of a panel which represents a unique biosignature according to the disease state of the patient.
- ItemOpen AccessA bioinformatic study on the feasibility of a cross-species proteomics analyses of mycobacteria(2013) Rajaonarifara, Elinambinina; Blackburn, Jonathan; Mulder, NicolaIncludes abstract. Includes bibliographical references.
- ItemOpen AccessCharacterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors(2019) Seele, Palesa Pamela; Sturrock, Edward; Blackburn, JonathanCavitation is a key facilitator in transmission of Mycobacterium tuberculosis (M. tb). Upregulation of matrix metalloproteases (MMPs) has been documented in patients with tuberculosis (TB), while their tissue inhibitor (TIMPs) levels remained the same. Animals which can develop cavities have well-conserved MMP-1 orthologs suggesting a pivotal role of MMP-1 in cavitation. The migration of immune cells to the site of infection and maturation of the granuloma is associated with MMP-9 expression. Our understanding of the phenotypic changes induced by Mycobacterium smegmatis (M. smeg) in THP-1 macrophages is central to understanding its avirulent nature especially its effects on MMPs. The aim of this study was to evaluate the role of MMP-1 and MMP-9, and their effectors in macrophages infected with M. smeg. Differentiated THP-1 monocytes were incubated in serum-free media with or without bacilli. Thereafter, the secretome and lysate were harvested at different time points. The activity of MMPs was analysed by zymography. The activity of MMP-1 and MMP-9 were specifically determined using an MMP-1 fluorogenic assay and a non-fluorogenic MMP-9 substrate monitored using the HPLC. Discovery proteomics was performed for the 18-hour time point with the use of mass spectrometry. The generated data was used to evaluate dysregulated proteins and those that act as upstream and downstream effectors of MMPs. The phenotypic changes induced by M. smeg were also analysed. In addition to that, the hosts’ response to lipoarabinomannan H37Rv (LAM) treatment was assessed by discovery proteomics and zymography. There was an increase in gelatinase activity of secreted MMP-9 which was maintained between the 1 and 18-hour time points. The fold difference in activity between uninfected and infected declined at 24 hours, and at the 72-hour time point the uninfected was slightly higher versus the infected. The data also suggests a switch in the proteolytic repertoire of the macrophages between the 6- and 18-hour time points to one that potentially generates the same degradation products as the uninfected macrophages. The intracellular gelatinase activity of MMP-9 (82 kDa) was not significantly altered by the M. smeg infection, in fact the activity was slightly higher in the uninfected in the 18 and 24-hour time points. In contrast to MMP-9, MMP-1 was secreted in the later time points and was significantly decreased by the infection. This supports the postulation that upregulation of MMP-1 is specific for M. tb infection. The proteomics data depict significant upregulation of MMP-9 in the lysate and secretome, while TIMP-1 was exclusively expressed and secreted by infected macrophages, validating the non-destructive ECM phenotype induced by M. smeg. The dysregulation of IL-1β and COX pathways were implicated in the overexpression of MMP-9, as well as tRNA aminoacylation in alternative splicing of MMPs. The GO enrichment of exosomes is postulated to play a role in the recycling of MMP-9. Intercellular communication is hypothesised to be delivered to neighbouring cells through exosomes carrying DNA, RNA, proteins and DNA/RNA binding proteins, and via signalling scaffolds formed by the 14-3-3 proteins amongst others. LAM treatment did not induce dysregulation in the activity of expressed and secreted MMP-9, however, TIMP-1 was upregulated explaining the lack of differential gelatinase activity between treated and non-treated macrophages. The C-type mannose receptor 2 (MRC2) and C-type lectin domain family 11 (CLEC11A) were only expressed by the LAM-treated macrophages and may partake in the recognition and uptake of M. tb. Interestingly, the data indicates the presence of chromatin in the secretome which may be responsible for the formation of extracellular traps (NETs) and facilitating the transport of LAM across the glomerular basal membrane (GBM) through exosomes. Inhibition of MMP activity by TIMPs could result in decreased aggregation of NETS (aggNETS) that trap the LAM from being transported by binding to the chromatin. This decreases the concentration of LAM in urine and means MMP inhibitors that chelate the active-site zinc could decrease the sensitivity of urine-LAM detection kits.
- ItemOpen AccessThe characterisation of the peanut agglutinin an evolved plant lectin, with improved specificity to the Thompson Freidenriech antigen(2013) Lagardien, Zaida; Blackburn, JonathanPeanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA).
- ItemOpen AccessComprehensive proteomic profiling of clinically relevant strains of Mycobacterium tuberculosis(2014) Peters, Julian S; Blackburn, JonathanTuberculosis is an airborne infectious disease caused by the bacillus known as Mycobacterium tuberculosis. Despite limited genetic variability, Mycobacterium tuberculosis strains exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. This study aims to use Mass Spectrometry (MS) tools to quantitatively and qualitatively investigate the total proteome expressed by various epidemiologically significant strains within the Mycobacterium tuberculosis complex (MTBC) as well as a clinically relevant non-tuberculous Mycobacteria (NTM) strain when cultured in vitro. We aim to use the experimental data obtained using discovery mass spectrometry to identify candidate proteins to use in the design of multiple reaction monitoring (MRM) MS experiments for targeted biomarker validation in patient derived biological samples such as sputum. Liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using the LTQ Orbitrap Velos. 1D LC was carried out on gel fractionated samples to increase proteome coverage. This allowed a significant increase in the number of protein identifications of up to 80% proteome coverage per strain. Comparative analysis of the datasets was carried out to identify and define the core-proteome expressed across all strains as well as to identify differentially expressed proteins amongst the strains.
- ItemOpen AccessDevelopment of computational methods for custom protein arrays analysis : a case study on a 100-protein ("CT100") cancer/testis antigen array(2010) Safari Serufuri, Jean-Michel; Blackburn, Jonathan; Mulder, Nicola; Kumuthini, JuditCustom antigen arrays offer a platform to assay the serological response of cancer patients to at set of selected cancer testis antigens in order to infer a diagnosis value or to assess the patient responses to particular treatments. However, the acquisition of the array data is subject to bias and noise. Therefore, array data processing and analysis is required to clear the data from bias, reduce noise and learn from the data. This study aims to address the issues of normalization and sample qualitative clustering for custom protein arrays.
- ItemOpen AccessDifferential Lipidomic and Proteomic Responses Induced by Sub-lethal Drug Challenge in Susceptible and Drug Resistant Mycobacterium smegmatis(2021) Giddey, Alexander D; Blackburn, JonathanTuberculosis remains a major global health challenge and the increasing strength and prevalence of drug resistance threaten to undo much of the good progress made. As one of the primary, frontline anti-tuberculous drugs, growing resistance to rifampicin in particular is concerning. Sub-lethal drug exposure and the development of adaptive phenotypic drug resistance, represent an important avenue by which genetic resistance and treatment failure or relapse may occur. Proteins and general metabolites are molecular classes that are highly dynamic, responsive and essential to understanding the state of an organism, while mass spectrometry-based proteomics and metabolomics are powerful tools by which these can be examined. For mycobacteria in particular, the lipidome and cell wall are compartments of major importance with respect to virulence, adaptation, host-pathogen interactions and persistence. As such, we sought to determine the effect of sub-lethal rifampicin exposure upon the model organism Mycobacterium smegmatis over time and determine what phenotypic adaptations might be observed and explained by alterations in the proteome and lipidome, with special focus on the cell wall sub-proteome. From these data we formed several new hypotheses with respect to virulence and mechanisms of both drug resistance and sensing, which were investigated further. Finally, we examined the effect of sub-lethal rifampicin exposure, and consequent proteomic alterations, upon the M. smegmatis lipidome and propose a model by which mycobacteria respond to sub-lethal challenge with rifampicin: Upon initial insult, drug-susceptible mycobacterial growth slows and stress response networks, including the SOS response, are temporarily activated. For both susceptible and resistant bacteria, cell wall remodelling begins early through dysregulation of cell wall and lipid synthesis enzymes — such as MtrAB, Mur proteins and PimB — resulting in ultimate accumulation of lipids with composition such as to impede passive diffusion of rifampicin into the cell. Some of this lipid accumulation, namely with PIMs, may take place rapidly and so ultimately reveal extremely large increases in abundance, which possibly necessitates downregulation of enzymes such as PimB by ~4 hours post treatment. In concert with ongoing lipid dysregulation, the cell wall proteome is altered as ABC transporter proteins are generally downregulated as an additional mechanism by which to control cell wall permeability through altered cell wall composition — through removal of cell wall penetrating transport proteins — and by limiting controlled influx of exogenous compounds. Bacterial efforts to resume normal growth and adapt to rifamipicin stress involves the dysregulation of numerous virulence factors, such as PknG, which results in impaired virulence. Transcriptional and translational machinery are also gradually upregulated so as to compensate for intracellular rifampicin's inhibition of RpoB, with transcriptional activity regulated separately to that of translational machinery. Ultimately, the combination of increased transcription, translation, and cell wall impermeability allows mycobacteria to overcome rifampicin challenge and resume normal growth. In M. smegmatis specifically, all this is accompanied by the gradual upregulation of the chromosomal resistance factor Arr which, at a later timepoint, modifies extracellular rifampicin to alleviate drug pressure.
- ItemOpen AccessDifferential proteomic profiling towards elucidation of TB-IRIS pathogenesis(2022) Peyper, Janique Michelle; Blackburn, Jonathan; Meintjes, GraemeBackground Up to 59% of tuberculosis (TB)/human immunodeficiency virus (HIV) co-treated patients develop paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) after addition of antiretroviral (ARV) therapy to anti-tuberculous therapy (ATT). The course can be prolonged and the average mortality rate is 2% (75% for TB-IRIS involving the central nervous system (CNS)). Immune elements – including neutrophils - involved in the anti-Mycobacterium tuberculosis (Mtb) response are implicated in pathogenesis, which remains incompletely understood. Diagnosis is one of exclusion, no reliable laboratory markers exist, corticosteroid-mediated prophylaxis and therapy are only partially effective, and no treatment targets tissue damage. Disentangling cause and effect in complex disorders such as TB-IRIS requires techniques capable of interrogating complex biological systems. Neutrophils are the major circulating leukocyte population, the earliest innate system responders, and exhibit various unusual immunometabolic functional specialisations. Proteins represent the most functionally-proximal and commonly pharmacologically-targeted cellular biomolecules. Label-free high-performance liquid chromatography-coupled tandem mass spectrometry (HPLCMS/MS) is well-suited to differentially profiling the ex vivo neutrophil proteome in an unbiased manner, in order to investigate TB-IRIS predisposition and pathogenesis. Methods Applying first principles to existing human literature, the most parsimonious holistic hypothetical model regarding paradoxical TB-IRIS predisposition and pathogenesis was inferred. A clinical cohort of control (CTRL) and matched TB-IRIS case (IRIS) study participants was assembled. Demographic, clinical, and biochemical characteristics were analysed for statistically-significant differences and to identify potential risk/protective factors (relative risk (RR) with 95% confidence interval (CI)). A small group (n = 9) of TB-HIV- healthy volunteers (HVs) was also assembled. Phlebotomy occurred at two timepoints: just prior to ARV initiation (week 0) and at the typical time of IRIS manifestation (week 2). Neutrophils were isolated and lysed, proteins underwent on-filter protein trypsinisation, peptide salts and detergents were removed, and neutrophil-optimised HPLC-MS/MS was conducted. Spectra were submitted to MaxQuant for parent protein identification and quantitation. Comparisons of (a) CTRL0 and IRIS0 to HV (and resultant differences) identified class-differential impacts of partial ATT-treated coinfection (IRIS predisposition) and of (b) CTRL2 to CTRL0 and IRIS2 to IRIS0 (and resultant differences) identified class-differential impacts of ARV therapy (IRIS pathogenesis). Class-discriminating proteomic differences were visualised using principal components analysis (PCA), protein differential expression analysis was performed (including for detectable/undetectable and significantly differentiallyexpressed (SDE) proteins), and results informed differential functional profiling via gene ontology overrepresentation analysis (GO-ORA) and pathway activation state prediction. To address shortcomings of current knowledgebases and automated tools, a novel deep manual analysis approach focused on key inference-friendly proteins, convergent findings, and neutrophil-specific functional modules. Integrated findings extended the literature-derived TB-IRIS model, generating testable novel hypotheses, one of which was partially validated using live-cell fluorescence microscopy. Proteomic data were additionally analysed to detect Mtb proteins, preliminarily analyse variable post-translational modifications (PTMs) of interest, and identify candidate prognostic and diagnostic biomarkers. Finally, mechanistic hypotheses facilitated identification of novel potential prophylactic and therapeutic targets. Results (1) Literature suggests advanced TB/HIV-coinfection (including a higher Mtb/antigen load) as the major TB-IRIS risk factor. Attendant significant immunometabolic state perturbations include myeloid overactivation, metabolic stress (possibly including adaptogen depletion), a lack of regulatory receptors, impaired pro-inflammatory signal transduction, and impaired antigen clearance. These likely predispose to lytic cell death - including release of host- and pathogen-derived inflammatory/cytotoxic molecules and proteolytic enzymes - and less restrained/more abnormal inflammation as well as tissue damage, on restoration of HIV-suppressed inflammatory signalling pathways by ARV therapy. (2) Regarding the clinical cohort, a sample size of > 42 participants per characteristic-matched comparison class provides > 95% power to detect a two-fold change with 99% confidence. Cases exhibited known TB-IRIS risk factors, and largely expected white cell count (WCC), body mass index (BMI), and C-reactive protein (CRP) level changes in response to ARV therapy (e.g. WCC increase and BMI decrease). None of the few participants on alternate (efavirenz (EFV)- or tenofovir (TDF)-lacking) regimens developed TB-IRIS. Pre-ARV prednisone (or incidental antihistamine/anti-fungal) use was associated with a non-significantly decreased TB-IRIS risk. Interestingly, smoking is associated with a significant decrease in TB-IRIS risk by 60%. (3) Regarding sample processing and analysis, the average sample collection to neutrophil isolation interval was within recommended limits. Isolation yield exceeded 15 x 106 and 25 x 106 per sample in the CTRL and IRIS groups, respectively. Isolated neutrophil purity exceeded 80% in both groups; the few low-purity samples were excluded from subsequent proteomic analysis. Lysates from 5 x 106 neutrophils routinely yielded over 100μg total protein, tryptic digestion was efficient (on average < 96% missed cleavages), and equivalent peptide injection volumes yielded comparable total ion chromatogram (TIC) profiles and intensities. An average 23% spectral identification rate resulted in a total of 2532 protein group identifications, the deepest neutrophil proteome coverage achieved to date without pre-fractionation, representing ~12% of human protein coding genes, and ~25% of the detectable human proteome. Samples were analysed in two (randomised) batches, producing independent datasets A (N = 37) and B (N =74). Withindataset technical replicates exhibit excellent agreement in protein identities and quantities; betweendataset protein identities and functional inferences also exhibit excellent agreement. We identify a number of proteins apparently not known to be expressed by human neutrophils, as well as one predicted human protein never before observed empirically. Overall, parent pathways of level-altered proteins suggest perturbation of nine major neutrophil function modules at both time-points: (a) signal transduction, (b) pattern recognition receptor (PRR) and cytokine signalling, (c) the eicosanoid cascade, (d) neutrophil antimicrobial functions, (e) carbon-energy metabolism, (f) protein homeostasis, (g) integrated nitrogen-sulfur-B-vitamin and redox/xenobiotic/glyoxal metabolism, (h) gene expression, and (i) cytoskeletal dynamics. (4) Regarding impacts of partially ATT-treated co-infection (week 0), neutrophil proteomic profiles successfully distinguish between HV and IRIS0 or CTRL0. Many differences from HV are shared between IRIS0 and CTRL0 (i.e. driven by partially ATT-treated co-infection), but some are class-unique (i.e. driven by factors predisposing to or protecting from TB-IRIS). Findings are supported by a head-to-head comparison of the CTRL0 and IRIS0 proteomes, including changes suggesting: more prevalent type I IFN, TGFβ, and Th2-type cytokine signalling; poorer capacity for restraint of alternate complement activation; mitochondrial and oxidative stress (including proneness to necrosis); impaired function (e.g. microbicidality, TLR/IL-1R-MyD88-NFκB signalling, and caspase 1- mediated IL-1β and IL-18 maturation) of activated neutrophils; and enhanced lipid and upstream (but inhibited downstream) isoprenoid synthesis (including decreased steroidogenesis). Candidate biomarkers distinguish CTRL- and IRIS-class partially ATT-treated neutrophils from HV neutrophils and from each other. (5) Regarding impacts of ARV therapy (week 2), both IRIS and CTRL neutrophil proteomes exhibit significant changes in response to ARV therapy. Many changes are shared between IRIS and CTRL (i.e. driven by ARV therapy and declining viral load (VL)), but some are class-unique (i.e. driven by factors preventing/contributing to TB-IRIS pathogenesis). Findings are supported by a head-to-head comparison of the CTRL2 and IRIS2 proteomes, including changes suggesting: a slower decline in type I IFN signalling; increased inflammatory cytokine (e.g. IL-6, TNFα, and IFNγ) signalling and protease (e.g. MMP-8) activity; decreased sensitivity to immunoregulatory glucocorticoids and vitamin A; and increased mitochondrial, endoplasmic reticulum (ER), and oxidative stress. Candidate biomarkers distinguish CTRL- and IRIS-group ARV-exposed neutrophils from baseline and from each other. (6) Livecell fluorescence microscopy of HV neutrophils suggests that in vivo-equivalent levels of EFV rapidly alter mitochondrial, lysosomal, and aggresomal architecture in a manner consistent with organelle and protein folding stress, and suggesting cell death commitment. (7) Integrated neutrophil immunometabolic changes suggested by proteomic findings support and extend the biologically compelling literature-derived model. Model highlights include more advanced baseline TB/HIV (including higher type I IFN, TGFβ, and possibly Th2-type cytokine levels), with consequent impaired myeloid-mediated Mtb antigen clearance and depletion of cellular adaptogens. The resultant abnormal immunometabolic state produces myeloid cells less able to counteract metabolic stress and primed for less-restrained inflammation. Introduction of mitotoxic ARV drugs and rapid lifting of HIVmediated immune embargoes escalates myeloid metabolic (including oxidative) stress and overactivation (including via NLRC4, CASP4/5, TLR/IL-1R-MyD88-NFκB, and MAPK-AP1 signalling), producing - instead of Mtb clearance - inflammatory cell death with release of immune-activating and tissue-damaging host- and Mtb-derived molecules. Reactivation of Mtb lymphocyte memory responses likely only produces clinically-apparent inflammation (TB-IRIS) when multiple simultaneous but incompatible immune programmes (e.g. overzealous myeloid activity, Th1, Th2, Th17, and Treg) coexist. Based on this model, existing compounds with the potential for rational, safe, effective TB-IRIS prophylaxis/therapy are identified (e.g. glutathione, vitamins B-complex and A/D/E, rapamycin, and metformin) which may assist in restoring system homeostasis.
- ItemOpen AccessExploring the effects of polymorphic variation on the stability and function of human cytochrome P450 enzymes in silico and in vitro(2014) Arendse, Lauren Beth; Blackburn, JonathanCytochrome P450s are highly polymorphic enzymes responsible for the Phase I metabolism of over 80% of pharmaceutical drugs. Polymorphic variation can result in altered drug efficacy as well as adverse drug reactions so the lack of understanding of the effects of single amino acid substitutions on cytochrome P450 drug metabolism is a major problem for drug development. In order to begin to address this problem, this thesis describes an in silico analysis of over 300 nonsynonymous single nucleotide polymorphisms found across nine of the major human drug metabolising cytochrome P450 isoforms. Information from functional studies - in which regions of the cytochrome P450 structure important for substrate recognition, substrate and product access and egress and interaction with the cytochrome P450 reductase were delineated - was combined with in silico calculations on the effect of mutations on protein stability in order to establish the likely causes of altered drug metabolism observed for cytochrome P450 variants in functional assays carried out to date. This study revealed that 75% of all cytochrome P450 mutations showing altered activity in vitro are either predicted to be damaging to protein structure or are found within regions predicted to be important for catalytic activity. Furthermore, this study showed that 70% of the mutations that showed similar activity to the wild-type enzyme in in vitro studies lie outside of functional regions important for catalytic activity and are predicted to have no effect on protein stability. Based on these results, a cytochrome P450 polymorphic variant map was created that should find utility in predicting the functional effect of uncharacterised variants on drug metabolism. To further test the accuracy of the in silico predictions, in vitro assays were performed on a panel of CYP3A4 and CYP2C9 variants heterogeneously expressed in E.coli. All mutations predicted to alter protein function by stabilising or destabilising the apo-protein structure in silico were found to significantly alter the thermostability of the holo-protein in solution. Thermostability assays also suggest that other mutations may affect stability by disrupting haem binding, changing protein conformation or altering oligomer formation. The utility of a fluorescence-based functional P450 protein microarray platform, previously developed in our laboratory, for generating kinetic data for multiple CYP450 variants in parallel was also examined. Since the microarray platform in its current stage of development was found to be unsuitable for this purpose, kinetic data for the full panel of CYP3A4 and CYP2C9 variants was generated using solution phase assays, revealing several variants with altered catalytic turnover and/or binding affinity for fluorescent substrates.
- ItemOpen AccessFunctional effects of cytochrome P450 variants on drug metabolism and adverse drug reactions: developing and extending high throughput P450 protein technology platforms(2014) Nair, Omesan; Blackburn, JonathanCytochrome P450 (CYPs) are a superfamily of heme containing enzymes that catalyse a diverse range of biological reactions. They are responsible for over 80% of primary metabolism of currently available drugs and are therefore central to its medical importance. Investigating the effects of these enzymes on drugs by metabolite detection and kinetic studies is a step forward to the vision of personalised medicine. The enzyme family is known to be associated with the development of adverse drug reactions which are usually only discovered in late stages of drug development, therefore screening for potential adverse drug reactions earlier on would aid minimising such adverse events occurring. There is therefore a need to analyse the interaction profile of new drugs with CYPs in a cost effective and high throughput manner for early stage screening, since drug discovery efforts tend to utilise large compound libraries. Recently, a novel functional CYP microarray has been developed in the Blackburn laboratory at UCT to enable label-dependent analysis of metabolism of substrates by the major CYP3A4 isoform in a high throughput manner. This thesis describes efforts involved in expanding the functional CYP microarray format to the other major CYP isoforms namely, CYP2C9 and CYP2D6 and developing a new immobilisation-free technology with label-free mass spectrometric identification and quantitation of metabolites formed. The goals of expansion of functional CYP microarrays were achieved by using microarray or confocal fluorescence scanning in conjunction with atomic force microscopy to more accurately quantitate active CYP3A4, CYP2C9 and CYP2D6 protein levels for catalytic substrate-dependent turnover rates. Finally the label- and immobilisation-free CYP technology was evaluated using probe substrates and a complex drug, rifampicin. These two platforms are primed to be a useful tool in pre-clinical drug screening for use in the drug discovery field by the academic, pharmaceutical and biotechnology industries.
- ItemOpen AccessGenerating a proteomic profile of neurogenesis, through the use of human foetal neural stem cells(2019) Garnett, Shaun; Blackburn, Jonathan; Kidson, SusanIntroduction Neurogenesis, the development of new neurons, starts soon after the formation of the neural tube and is largely completed by birth. Development of the brain after birth is mainly reliant on the formation of new connections between surviving neurons. However, adult neurogenesis does continue in the subgranular zone of the hippocampus from quiescent adult neural stem cells. Traditionally neural stem cells were cultured as neurospheres, a heterogeneous agglomeration of neural cells at various stages of differentiation. This heterogeneity prevented accurate quantitative analysis. In 2008 Sun et al produced the first non-immortalised human foetal neural stem (NS) cell line from nine week old human foetal cortex. These cells are cultured as monolayers, have a radial glia like appearance, self renew and form all three neural cell types, neurons, astrocytes and oligodendrocytes upon differentiation. More recently human foetal neuroepithelial like (NES) stem cells have been produced from five week old human foetal hind-brain, they resemble neuroepithelial cells, with characteristic rosettes, upon differentiation they appear to form a pure population of neurons. These homogeneous monolayer cultures enable quantitative proteomic analysis, to increase our understanding of early brain development Methods Three NES and two NS cell lines were available for analysis. They proliferate by stimulation from FGF and EGF, removal of these growth factors results in spontaneous differentiation. Proliferating NES and NS cells were compared using SILAC labelling. In addition, each cell line was differentiated for 12 days, 6 timepoints were taken and compared using label free quantitation. Results 4677 proteins were quantitated with 473 differentially expressed, revealing fundamental differences between NES and NS cells. NES cells are less differentiated, expressing SOX2 and LIN28, have active cell cycle processes, DNA elongation, histone modification and miRNA mediated gene silencing. Whereas NS cells are more developmentally defined, express multiple membrane proteins, have activated focal adhesion, thereby increasing their binding and interaction with their environment. NS metabolism is more oxidative, utilises lipid metabolism, the pentose phosphate pathway and produces creatine phosphate. Upon differentiation the cell cycle processes are downregulated and neurogenic and gliogenic processes increased. Conclusion This work represent a detailed in vitro characterisation of non immortalised human foetal neural stem cells, it describes the regulatory, metabolic and structural changes occurring within neural stem cells in early brain development. The information herein points towards de-differentiation potentially through LIN28-let7, as a means to produce more neurogenic neural stem cells in vitro thus aiding regenerative therapies, as well as provides a wealth of information for better understanding neurological developmental disorders.
- ItemOpen AccessHigh throughput proteomic analysis of Mycobacterium tuberculosis associated Immune Reconstitution Inflammatory Syndrome (TB-IRIS)(2012) Bell, Liam Templeton; Blackburn, JonathanIncludes abstract. Includes bibliographical references.
- ItemOpen AccessIdentification of a novel HIV-1C protease from a microbial source(2023) Sonday, Zarinah; Blackburn, JonathanHIV-1 subtype C is currently the most prevalent in the epidemiology of HIV/AIDS cases in subSaharan Africa. Most clinical protease inhibitors (PIs) were designed against subtype B and are reported to have reduced activity against subtype C proteases. Our initial hypothesis was to create an Escherichia coli based life-or-death selection system for the screening of potential PIs against HIV-1 subtype C protease (PR). This system was engineered by inserting an HIV PR cleavage sequence between the export signal peptide of the commonly used TEM-1 β-lactamase, which upon co-expression of the HIV PR in vivo, would cleave the modified β-lactamase thus preventing its translocation to the periplasmic space. This would result in the host cells' sensitivity to β-lactam antibiotics supplemented in the growth media. The presence of an inhibitor would restore resistance and therefore ‘life'. Despite validation of the E. coliscreening system using the Tobacco Etch Virus (TEV) protease, co-expression of HIV protease subtype C did not inhibit cell growth. Further investigations revealed PR C activity was inhibited by an endogenous E. coli protein. The inhibitor was isolated from E. coli crude cell lysates using ammonium sulphate precipitation, gel filtration and anion exchange chromatography fractionation. It was identified using peptide fingerprinting mass spectrometry (PMF), as alkyl hydroperoxide reductase C22 subunit (AhpC22). Mass-Assisted Laser Desorption-Ionisation-time of flight (MALDI-TOF) analysis of the precursor pre-incubated with AhpC22 revealed reduced autocatalytic cleavage occurring at the N-terminus of PR C. Inhibition kinetics using a recombinant source of AhpC22 characterized the enzyme as a non-competitive inhibitor of PR C activity with an inhibition constant (Ki ) of 0.88 µM. We also describe a protocol to express, purify and refold the HIV-1C protease which is well known for aggregation into inclusion bodies.
- ItemOpen AccessIntegrative genomic analyses of bacterially-associated colorectal cancer(2015) Viljoen, Katie S; Blackburn, JonathanSporadic colorectal cancer (CRC) has been linked to various lifestyle factors, including the consumption of alcohol and red meat, smoking, and obesity. CRC is one of most extensively characterised cancers, both at a molecular and 'omic' level; nevertheless, the precise mechanism driving CRC initiation remains unknown. To date, numerous studies have identified changes in the microbial profiles of CRCs compared to adjacent normal mucosa and compared to healthy controls; however, CRC-associated bacteria have not been concurrently quantified across a single cohort; nor have the relationships between CRC-associated bacteria, clinicopathological features of CRC and genomic subtypes of CRC been investigated. The main aim of this thesis was therefore to gain insight into the potential contribution of CRC-associated bacteria in the aetiopathogenesis of CRC by leveraging both host genomic and clinicopathological data as well as to investigate patterns of tissue colonisation between different CRC-associated bacteria. The objectives were 1) to quantify, using quantitative-PCR, CRC-associated bacteria in a cohort of 55 paired tumour and adjacent histologically normal samples collected during surgical resection as well as in an additional 18 formalin-fixed paraffin-embedded (FFPE) samples; 2) to determine their relationships to patient age, gender, ethnicity, stage of disease, site of disease and MSI status (Chapter 4); 3) to evaluate the relationship between each bacterium and host gene expression (Chapter 8) and methylation changes (Chapter 6); and 4) to determine genomic subtypes of CRC using unsupervised clustering of gene expression data in the context of patient clinicopathological features and bacterial quantitation data; and 5) to gain a deeper biological understanding of the results from the objectives 1–4 using pathway analyses of the genomic subtypes obtained (Chapter 7). The main finding of this thesis is that a transcriptomic subtype of colorectal cancer, characterised by an increase in CpG island methylation, displays an increased frequency of colonisation by Enterococcus faecalis and by high levels of Fusobacterium. At the pathway-level, this subtype is enriched for pathways related to damage response, infection, inflammation and cellular proliferation; notably, these findings were confirmed in a well-defined publically available CRC gene expression dataset of colorectal adenocarcinomas (N=155). These findings suggest that specific bacterial colonisation underlie s a distinct genomic subtype of colorectal cancer that is characterise d by inflammatory-related gene expression changes ; these findings however require validation in a larger cohort. In addition, novel associations between colonisation by specific bacteria and host clinicopathological, transcriptomic and DNA methylation features were identified.
- ItemOpen AccessIsolation and characterisation of novel DNA aptamers against Mycobacterium tuberculosis biomarkers: new tools for tuberculosis diagnostics(2018) Amos-Brown, Bianca; Blackburn, JonathanTuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements.
- ItemOpen AccessModulation of protein expression in Mycobacterium tuberculosis-stimulated TB- IRIS patient-derived PBMCs by omega n-3 polyunsaturated fatty acids(2018) Barnabe, Marine; Blackburn, JonathanTuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory disorder which affects up to 54% of TB-HIV co-infected patients. Its pathogenesis remains unclear, and although treatment with the corticosteroid prednisone has shown some benefits, there is no specific treatment currently available for TB-IRIS. N-3 polyunsaturated fatty acids (PUFA) have safely and successfully been used in the treatment of other inflammatory diseases, and we hypothesized that they will have beneficial anti-inflammatory effects on TB-IRIS patient-derived immune cells. To investigate this hypothesis, we used mass spectrometry (MS)-based proteomic methods to probe the secretome of peripheral blood mononuclear cells (PBMCs) re-stimulated ex vivo with Mycobacterium tuberculosis (Mtb) whole cell lysate (WCL), and treated with n-3 PUFA. Optimization experiments were performed on cells obtained from eight healthy donors to assess the secretome changes induced by re-stimulation with Mtb WCL and treatment with n-3 PUFA. In addition, experiments were repeated using PBMCs obtained from five non-IRIS and five TB-IRIS patients. The secretome was prepared via chloroform/methanol precipitation and overnight Trypsin digestion, and investigated via MS-based shotgun proteomics. MaxQuant was used for protein identification and Perseus for statistical analysis. Stimulation with Mtb WCL shifted the secretome of healthy PBMCs towards an inflammatory state, and this was altered by treatment with EPA/DHA via changes in the regulation of several proteins. Preliminary results from TB-IRIS patient-derived PBMCs show the same trend. These promising early results suggest the potential benefits of n-3 PUFA dietary supplementation for patients with TB-IRIS, and warrant further studies with increased sample size to confirm findings, and the possible inclusion of n-3 PUFA in a future clinical trial on patients with TBIRIS.
- ItemOpen AccessP450 biochips : development of a protein microarray platform for investigating cytochrome P450 clinical drug metabolism(2010) Beeton-Kempen, Natasha; Blackburn, JonathanThis thesis describes the development of a novel cytochrome P450 array format, the P450 Biochip that allows quantitative and truly high-throughput measurement of cytochrome P450-mediated turnover reactions in sub-nanolitre volumes.
- ItemOpen AccessPreparation and evaluation of polymer microspheres for enhanced lateral flow immunoassay: the case study for malaria(2015) Hobbs, Henriëtte Renée; Blackburn, Jonathan; Jordaan, JustinWe proposed that the development of a new high capacity polymer microsphere technology, termed ReSyn, could be developed as viable detection reagents for lateral flow technology. This body of work outlines the development of this new high capacity polymer microsphere technology for suitability to flow on lateral flow membranes, and highly specific biomarker detection for immunoassay development. Proof-of-concept was achieved using hCG (pregnancy biomarker) and validated for detection of pLDH and HRP2 as biomarkers of malaria. The sensitivity, stability and multiplex capability of these microspheres were further explored and compared against the current ‘gold' standard detection agent for lateral flow, colloidal gold. Malaria was selected as a suitable target for evaluation of the microsphere technology since it is considered to be a global epidemic that can benefit greatly from improved point-of-care diagnostics. Malaria affects almost half of the world's population and is responsible for causing approximately 655 000 deaths per annum in 2010, with 90% of these deaths occurring in Africa and 85% of these deaths occurring in children under 5 years of age (del Prado et al., 2014; Kokwaro, 2009; White et al., 2014; WHO, 2009). Febrile disease diagnosis at point-of-care is often based on symptomatic diagnosis rather than on the use of validated diagnostic technologies, and is considered one of the major contributing factors for the high morbidity and mortality rate of malaria (Chandler et al., 2008; Kain et al., 1998; Kokwaro, 2009). Improved diagnostic technologies, allowing for sensitive and accurate diagnosis at the point-of-care, could assist alleviating these problems through the improved management of disease (Bell et al., 2006). Lateral flow rapid diagnostic tests are the preferred method for point-of-care diagnostics in resource constrained areas but have several limitations including sensitivity and stability in resource constrained settings (Bell et al., 2006). Improvements in detection agents are seen as a viable approach to improving these features of diagnostic assays. The results of this study show that the polymer microspheres provide improved stability to immobilised antibodies, with potential for translation into improved stability for diagnostic assays in tropical malaria endemic regions. The polymer microspheres offered high specificity and comparable visual sensitivity to the market leader colloidal gold and is therefore considered as alternate detector agents in lateral flow assays. Additionally, the microspheres can be dyed various colours (red and blue in this study), allowing for specific and sensitive multiplex detection of multiple analytes in a single sample. This increases the versatility of the microspheres for lateral flow diagnostic application, and improves the interpretation of lateral flow diagnostic technology at the point-of-care.
- ItemOpen AccessProteomic insights into the modulation of foetal neurogenesis by the anti-retroviral efavirenz(2019) Albeldas, Claudia; Blackburn, JonathanBackground: South African guidelines recommend that HIV-positive pregnant women immediately initiate antiretroviral therapy (efavirenz, emtricitabine, and tenofovir), regardless of trimester. Efavirenz causes central nervous system neuropathy and has been linked to birth defects such as encephalocoele. Cohort studies of HIV-uninfected children exposed to antiretroviral treatment in utero report minor learning delays but are inconclusive. Non-transformed human derived neuroepithelial stem (NES) represent a unique pre-clinical model in which to investigate the effects of efavirenz on the developing neural system. Efavirenz-induced global cellular molecular changes may be characterised using mass spectrometry (MS). Aims: To optimise an MS-based efavirenz extraction and detection assay, and to investigate efavirenzinduced NES proteomic responses. Methods: A TSQ Vantage triple quadrupole mass spectrometer was employed to optimise targeted detection of efavirenz extracted from cultured cells and supernatant. Cells were cultured for 72 hours, incorporating a 24-hourly efavirenz treatment. Efavirenz concentration dynamics were assessed over this period, and cells were harvested every 24 hours for discovery proteomic analysis using a Q-Exactive quadrupole-Orbitrap mass spectrometer. Results: Drug extraction with acetonitrile was selected as the optimal extraction and detection technique. In cell culture, efavirenz concentration increased after 24 hours and decreased after 48 hours. A total of 1663 protein groups were identified, with 26, 39, and 80 protein groups differentially expressed 24, 48, and 72 hours respectively post EFV treatment. The most significantly enriched deregulated pathways included cholesterol biosynthesis, mRNA splicing, and JAK/STAT and Wnt signalling. Conclusions: Efavirenz-altered protein expression reflects functional pathway perturbations, which may contribute to clinically-observed neurological effects. Orthoganal and in vivo confirmation is required.