School of Molecular & Cell Biology (ETDs)

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A Clot to Uncover: FOXP3 and SARS-CoV-2 Nucleocapsid Interactions and Their Effect on DNA Binding
(University of the Witwatersrand, Johannesburg, 2024) Mcinnes, Keiran; Fanucchi, Sylvia
During COVID-19, systemic coagulopathy, which can lead to strokes and embolisms, is often observed in COVID-19 patients and may also contribute to long COVID. This coagulopathy is the result of overactivated platelets in circulation that leads to inappropriate clot formation. FOXP3 is a transcription factor involved in platelet development and loss of FOXP3 function leads to platelets that resemble those seen during COVID-19. Thus, FOXP3 may be dysregulated in COVID-19. The SARS-CoV- 2 nucleocapsid (NC) is a multifunctional protein typically associated with viral genome packaging and virion assembly. However, it is also capable of binding DNA and is potentially able to alter regulation of host protein expression. Here, potential interactions between the DNA-binding forkhead domain (FHD) of FOXP3 and the SARS-CoV-2 NC were investigated. Identification of a novel interaction between FOXP3 and SARS CoV-2 NC may provide new clues as to the pathophysiology of COVID-19. To address this aim, both proteins were overexpressed in T7 E. coli, purified via immobilised metal affinity chromatography, and monitored for potential interactions in the absence and presence of DNA using pull-down assays and fluorescence anisotropy. A direct interaction was identified between the two proteins in the absence of DNA. Additionally, it was found that both proteins are capable of binding to DNA at the same time, but excess NC was found to cause FHD dissociation from the FHD- NC-DNA complex. This result implicates NC in FOXP3 dysfunction which may be associated with the coagulopathy and other symptoms seen during COVID-19. Additionally, NC DNA binding does not appear to be driven by the FOXP3 consensus sequence, indicating that FOXP3 may not be the only transcription factor potentially dysregulated by NC
A Phenotype Prediction Framework for Classifying Colorectal Cancer Patients’ Response to FOLFOX Treatment: An Integrated Approach
(University of the Witwatersrand, Johannesburg, 2024) Mashatola, Lebohang; Kaur, Mandeep
Colorectal cancer (CRC), characterised by its prevalence and heterogeneity, poses a significant challenge in understanding drug resistance, especially in the context of FOLFOX treatment. This study presents an innovative methodology that integrates diverse data analysis approaches to address the challenge of predicting the phenotype of CRC patients resistant or sensitive to FOLFOX. The initial analysis involved dierential and co-expression analyses, identifying pivotal hub genes crucial to drug resistance in CRC, regulating intricate molecular networks. Subsequent enrichment analysis revealed their significant roles in biological functions, particularly influencing DNA repair and nuclear division. To capture inherent topological characteristics within genetic expression data, a novel technique utilising topological data analysis (TDA) was employed. By applying persistence homology to generate persistence images, the Vietoris-Rips complex was constructed using the signed-topological overlap matrix, comprehensively capturing numerous topological features, including high-dimensional Betti-1 and Betti-2. This provided valuable insights into the structural patterns of gene expression between the hub genes. Furthermore, the integration of whole-slide images enhanced understanding of tissue anatomy, which is crucial for predicting cancer stages. Using a MobileNet architecture, a deep learning model classified cancer stages, contributing to a holistic understanding of colorectal tumor microenvironments. For predictive modelling of drug resistance, a multilayer perceptron applied topological summaries generated by TDA. The developed framework, GeTopology, exhibited remarkable performance metrics, achieving an overall 83% accuracy in predicting the FOLFOX response, demonstrating a 3% improvement over a previously published phenotype prediction framework (NSCLC ) that utilised similar data modes. Robust accuracies were consistently observed in independent datasets, classifying both cancer patients and healthy individuals. The results indicated an approximate 10% increase in model prediction accuracy compared to NSCLC, emphasising the potential clinical impact of this integrative approach. In conclusion, this study advances the understanding of drug resistance in CRC by proposing a novel approach that integrates topology with histopathological images, oering transformative insights into predictive modelling and precision medicine
Analysing RNA-sequence data for pancreatic ductal adenocarcinoma tissue samples to identify potential biomarkers
(University of the Witwatersrand, Johannesburg, 2023-09) Jamal, Khadija Sanober; Kaur, Mandeep
Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancer and is the fourth leading cause of death with a five-year survival rate of less than 10%. Patients are asymptomatic until detection is observed at a metastatic stage, hence contributing massively towards the high mortality rate. This study was conducted to explore PDAC and its two main subtypes, the classical and basal-like subtype, in an in-depth level via bioinformatic analysis. Bioinformatics is a computational approach to evaluate biological data by analysing omics data including genomic expression and proteomic sequences. A workflow consisting of programmes and web-tools was used to analyse PDAC RNA-sequence data. The sample sets were grouped according to tumour, stage, and subtype. The workflow began with quality control using FastQC and Trimmomatic. Alignment of sequencing files and counts were done through HISAT2 and HTSeq. The main component of this workflow was differential gene expression analysis to identify differentially expressed genes (DEGs), statistically significant genes, per compared conditions. WGCNA was used for co-expression analysis to identify the hub genes involved in regulating the biological network. Lastly, in-silico validation was done by using available web tools to support the findings of this workflow. The identified tumour genes included S100A11, PKM, GPRC5A, LAMC2 and ITGA2, which may represent as universal biomarkers as sample extraction was performed from data generated from individuals belonging to 8 different countries. KRT13 and IL6 were identified in the advanced stage and their role in cancer progression have been explored in this current study. The basal-like subtype had CAV1, DCVLD2 and TGFB2 genes that contribute to treatment resistance. The common dysregulated genes in the basal-like subtype and advanced stage were analysed to evaluate the link between subtype and stage which included WNT3A, TP63, KRT13 and IGF2BP. Coexpression analysis revealed hub genes for tumour (KIF4A, SPAG5, RRM2 and AURKA), basal-like subtype (BUB1, DEPDC1 and KIF14) and classical-subtype (PTPRN and CAMK2B). Through a machine learning model, recall, precision and accuracy scores per sample conditions for the DEGs were all above 94%. These potential biomarkers all have significant roles in promoting cancer progression, aggression and resistance. Hence, these may serve as a less invasive screening method for PDAC as DEGs were classified based on tissue or blood (extracellular vesicle) biomarkers. However, further wet laboratory validation is required for these biomarkers.
Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)
(University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.
Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.
Biochemical and biophysical analysis of a hinge region protease variant in HIV-1 subtype C
(University of the Witwatersrand, Johannesburg, 2023-10) Mokhantso, Tshele; Achilonu, Ikechukwu; Sayed, Yasien
HIV-1 protease plays a crucial role in the maturation of the virus by cleaving gag and gag-pol polyproteins. Understanding the structural and functional consequences of mutations in this enzyme is essential for developing effective anti-HIV drugs, especially in the face of emerging drug-resistant variants. This study focused on the N37T↑V+10•D25A mutant, a novel HIV-1 subtype C protease variant harbouring an insertion (↑V) and a substitution (N→T) at position 37, along with 10 naturally occurring polymorphisms. Mutations occurring distal to the active site have long been thought to contribute to drug resistance, with this in mind the study aimed to assess the impact these mutations have on the structure, stability, dynamics and drug binding of HIV-1 protease. The N37T↑V+10•D25A mutant and wild-type (WT•D25A) HIV-1 protease were overexpressed and purified from inclusion bodies formed in Escherichia coli cells using ion-exchange chromatography. Far-UV CD and SE-HPLC analysis showed that N37T↑V+10•D25A exhibited a predominantly β-sheet secondary structure (218 nm trough) and had a homodimeric size of ~23 kDa, respectively, both similar to WT•D25A. Assessment of the local tertiary structure by intrinsic tryptophan fluorescence indicated that the protease retained its tertiary structure in the presence of mutations, with partial exposure of Trp residues (Trp 6, Trp 6', Trp 42/43, and Trp 42'/43’). Overall, the insertion and substitution mutations did not significantly alter the overall structure of HIV-1 protease. However, the conformational stability of N37T↑V+10•D25A was found to be reduced compared to WT•D25A as determined by urea-induced equilibrium unfolding and thermal unfolding experiments. When denatured using urea and temperature, the mutant exhibited a two-state mechanism of unfolding without stable intermediates during the folding and unfolding process. Thermal unfolding experiment determined the melting temperature of N37T↑V+10•D25A as 58 ± 1.2 °C, which is lower than that of the wild type of 62 ± 0.9 °C. This suggests a potential decrease in dimer stability due to the mutations present in N37T↑V+10•D25A. Isothermal titration calorimetry with acetyl pepstatin as a model inhibitor was employed to examine the impact of mutations on drug binding. The enthalpy (ΔH) for WT•D25A and N37T↑V+10•D25A were 35.94 and 36.02 kJ/mol, respectively. The entropy (ΔS) for WT•D25A and N37T↑V+10•D25A was found to be 256.7 and 261.6 J.mol.K, respectively. These differences in thermodynamic parameters between the WT•D25A and N37T↑V+10•D25A proteases, may indicate altered drug-protease interactions. Induced fit molecular docking predicted the binding strengths of both WT•D25A and N37T↑V+10•D25A with nine protease inhibitors (atazanavir, darunavir, fosamprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir and tipranavir), revealing that specific background mutations, such as P40S present in N37T↑V+10•D25A, significantly decreased the binding energy of the HIV-1 protease to these inhibitors. Molecular dynamics simulations provided insights into the structural dynamics of N37T↑V+10•D25A, showing reduced stability and increased dynamics, particularly in the flaps and hinges of the protease. An increase in flap tip curling and involvement of the cantilever tips were observed to be related to the flap opening mechanism of HIV-1 protease. Interactions between the HIV-1 protease and inhibitors were examined and the radius of gyration and solvent accessible surface area were calculated to evaluate protein compactness and solvent accessibility of the bound inhibitors, respectively. In general, the N37T↑V+10•D25A mutant exhibited decreased compactness when bound to inhibitors, which correlated with the increased solvent exposure of PIs when bound to N37T↑V+10•D25A. This may contribute to the drug resistance mechanisms of the protease, as inhibitors would have difficulty binding the active site and exhibit weaker binding. During the N37T↑V+10•D25A and PI interactions, there was a decrease in hydrogen interactions, which form the basis for protease inhibitor drug-design and these were replaced by water-bridge interactions, which are weaker and can be easily broken. In conclusion, this study provides a comprehensive characterisation of the N37T↑V+10•D25A mutant of HIV-1 protease, shedding light on its structural alterations, conformational stability, drug binding properties, and dynamic behaviour. These findings contribute to our understanding of drug resistance mechanisms in HIV-1 protease and offer valuable insights for the design of more effective inhibitors to combat HIV-1.
Biophysical evaluation of the kinetics, thermodynamics, and structure-stability relationship of Wuchereria bancrofti glutathione transferase in comparison with human µ and π glutathione transferases
(University of the Witwatersrand, Johannesburg, 2024-06) Oyiogu, Blessing Oluebube; Achilonu, Ikechukwu Anthony
Lymphatic filariasis is an endemic disease caused mainly by the Wuchereria bancrofti parasite and has been classified as a major neglected tropical disease. The emergence of drug-resistant strains of W. bancrofti and the limited efficacy of the available drugs on adult worms threatens the eradication of the disease. W. bancrofti glutathione S-transferase (WbGST) is a homodimeric enzyme central to detoxifying electrophilic compounds in the parasite due to its lack of cytochrome P-450. Therefore, WbGST is a potential therapeutic target for lymphatic filariasis. Bromosulphophthalein (BSP) and epigallocatechin gallate (EGCG) were previously shown to inhibit glutathione S-transferase activity. In this study, the interaction of WbGST with BSP and EGCG in comparison with human glutathione S-transferase P1-1 (hGSTP1-1) and human glutathione S-transferase M1-1 (hGSTM1-1) isoforms was investigated. Soluble WbGST, hGSTP1-1 and hGSTM1-1 were recombinantly produced and purified successfully to homogeneity. Glutathione and 1-chloro-2,4-dinitrobenzene conjugation assay was employed to analyse the enzyme activity, kinetics and inhibitory potency of the compounds. Spectroscopic studies were employed to investigate the functional and structural impact of ligand binding to the enzymes. Both thermal and chemical stability studies were performed, and binding energetics were analysed using isothermal titration calorimetry. The activity of WbGST was predominantly inhibited, with IC50 values of 5 μM for BSP and 12 μM for EGCG. The EGCG displayed uncompetitive and mixed modes of inhibition towards WbGST with respect to glutathione and hydrophobic binding sites, respectively. Whereas BSP showed a mixed type of inhibition for both active sites of WbGST. Ligands reduced the turnover rates (kcat) and the catalytic efficiencies (kcat/KM) of the enzymes. Upon ligand binding, 8-anilino-1-napthalene sulphonate was displaced from WbGST and hGSTM1-1 by 67%(BSP), 24%(EGCG) and 72%(BSP), 5%(EGCG), respectively; suggesting that the ligands bind to the 8-anilino-1-napthalene sulphonate binding site. Stability studies indicate that WbGST is the least stable of the three enzymes and that glutathione increases its stability. Isothermal titration calorimetry showed that BSP binds to multiple sites in WbGST with binding at site-1 (S1) and site-2 (S2), which are entropically and enthalpically driven, respectively. S1 showed a higher affinity for BSP than S2. EGCG binding to WbGST was entropically driven. BSP had a higher affinity for the enzymes than EGCG. All the results indicated that the ligands significantly impact WbGST more than the human GSTs. Further investigations, such as crystallography and molecular dynamics simulations, will shed more light on the ligan-protein interactions on a molecular level. Overall, this study suggests that BSP and EGCG are efficient inhibitors of WbGST that probably bind to both L and H-sites of WbGST, altering catalytic activity of the enzyme. The unique properties of the L-site are particularly suitable for rational drug design. Therefore, both ligands can be repurposed as new-generation therapeutics against filariasis.
Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer
(University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, Mandeep
The cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasis
Comparison of different bioassay methods for the assessment of dose-response relationships of entomopathogens and toxins against Helicoverpa armigera (Hübner, 1809) (Lepidoptera: Noctuidae)
(University of the Witwatersrand, Johannesburg, 2024-11) Mogadingoane, Keitumetse Neo; Bouwer, Gustav
Bioassays are an important tool for developing bioinsecticides against agricultural pests. The aim of this study was to compare two bioassay methods – diet overlay and droplet feeding – to identify the most suitable method for assessing dose-response relationships of entomopathogens and toxins against second instar larvae of Helicoverpa armigera. The toxins used were purified Bacillus thuringiensis Cry1A.105 and Cry2Ab2.820 proteins, the spore-crystal complex (SCC) of B. thuringiensis subspecies kurstaki strain HD-73, and the entomopathogen Helicoverpa armigera nucleopolyhedrovirus (HearNPV). Based on the heterogeneity factor, coefficient of variance (CV) and relative precision, the diet overlay bioassay was determined to be the best fit for use with HD-73 SCC and HearNPV. Suitable bioassay methods could not be determined for the purified B. thuringiensis proteins due to a poor probit model fit and low precision of estimated LC50s and LD50s. Validation of CV and relative precision across bioassays will ensure the most suitable methods are used for sustainable integrated pest management.
Comparison of Saccharomyces cerevisiae and the novel wild yeast used in beer fermentation and their future in industrial biotechnology
(University of the Witwatersrand, Johannesburg, 2024) Zviuya, Patience; Moodley, Sanchia; Rumbold, Karl
The alcohol industry has grown over centuries due to the increase in alcohol demand by customers. Beer consumers now understand the brewing process and they well understand the role of yeast in fermentation. The alcohol industry now has a lot of customers because of the growth that this sector has made, and this has resulted in an increased demand for new beer styles and high flavor profile beers. Due to this demand, research has been conducted on unconventional wild yeasts that can be employed in making beer, demonstrating the variety of fermentation yeasts that are available and capable of enhancing beer quality and producing a wide range of new beer varieties. The commercial S. cerevisiae yeast and eight wild yeasts (Samson’s Saison, Ragnarok, Dark knight, B. brusc, Neipa, The Proletariat, La Trappist and B. clauss) were used to ferment pale ale in different fermentation vessels. The commercial yeast was the control of this research because this yeast has been used for generations in the brewing industry. The wild yeast strains used were identified using sanger sequencing and seven of these yeasts were S. cerevisiae wild species with similarity index of more than 80% and one was B. bruxellensis strain similarity index more than 95% before and after fermentation. The research outcomes demonstrated that most of the wildyeast performed the same as the commercial yeast in terms of physical and chemical parameters however most of the wild yeast produced more volatiles and esters as compared to the commercial brewing yeast. Commercial S. cerevisiae produced the highest alcohol content 4.5% and the average alcohol content for wild yeast was 3.4% because they are challenging to regulate during fermentation and have low alcohol tolerance unlike the commercial yeast that has been harvested and used for generations. Overall, the utilization of unconventional wild yeast to make beer was identified as a promising alternative to produce beers with exotic flavours and alcohol-free beers. Future work identifying specific yeast that suit different fermentation processes and beer types are recommended.
Computational modeling approaches to validate the druggability of the 26- and 28-kDa Schistosoma glutathione transferase enzymes using bromosulfophthalein as a benchmark ligand
(University of the Witwatersrand, Johannesburg, 2024) Valli, Akeel
PHARMACOPHORE MODELS are 3-D representations of the chemical and spatial features required for interaction with a drug target. These. models offer advantages in early-phase drug design by expediting screening experiments and enabling the sampling of highly specific chemical. space subsets, such as those containing quality drug-like candidates. The glutathione transferase enzyme of Schistosoma spp. (SGST) has been identified as an attractive drug target for the novel treatment of human schistosomiasis. We observed selective inhibition of SGST by bromosulfophthalein. Bromosulfophthalein was found to complex with SGST at a drug binding site in the target dimer interface, providing a suitable benchmark for the design of discriminative SGST pharmacophores. The aim of this research is to construct, deploy and evaluate pharmacophore models of the SGST drug binding site. The objectives are: to characterise the SGST drug binding site, to develop the pharmacophore models and finally to evaluate the drug-resolving ability of the models. We observed significant differences in the drug-binding character of SGST, compared to human glutathione transferase (hGST) counterparts, particularly that SGST supports binding of phenol and sulfonate moieties. Five- and four feature pharmacophores were developed for the respective 26- and 28 kDa SGST variants. Finally, the models demonstrated remarkable ability to retrieve candidates displaying drug-like qualities. In conclusion, we characterised and developed pharmacophore models of the drug binding domains from two major SGST variants. Assessment of drug-resolving power validates the capability of the models to sample drug-like chemicals. Altogether, these accomplishments enable efficient and reliable screening toward novel drug treatment for human schistosomiasis
Control of serine dehydratase activity in rat liver
(University of the Witwatersrand, Johannesburg, 2015-02-24) Abed, Suliman; Manchester, K.L.
Gluconeogenesis from amino acids in the liver is enhanced when the utilisation of glucose is limited under various hormonal and dietary conditions, such as diabetes, starvation or administration of a carbohydrate free diet. Pyruvate is of great importance as a carbon source for gluconeogenesis, since the sequence of gluconeogenic reaction is initiated by carboxylation of pyruvate to oxaloacetate. From this point of view, an important physiological role is suggested for serine dehydratase, which catalyses the degradation of serine to pyruvate and ammonia. The relationship of serine dehydratase levels to gluconeogenic activities, however, is poorly understood, A study of the hormonal and dietary control of serine dehydratase activity was carried out in vivo and in vitro in rat liver. Serine dehydratase was assayed by the colorimetric method of Suda and Nakagawa (1971) and the enzymatic method of Wimhurst and Manchester (1973). Both these methods have been found to be suitable since they are in agreement with each other and also give results which compare favourably with other published values. Activities of serine dehydratase from fresh liver and in slices of liver cultured for various periods have been compared. Also a study of the activity of another soluble enzyme, lactic dehydrogenase, was undertaken and the in vivo and in vitro levels were compared.
Differential expression analysis of PMA and 1,25(OH)2D3-induced monocyte-to-macrophage differentiation in THP-1 cells
(University of the Witwatersrand, Johannesburg, 2023-09) Perumal, Kelda Chloe; Meyer, Vanessa; Gentle, Nikki
The process of monocyte-to-macrophage differentiation is studied in vitro through the use of promonocytic model cell lines, such as the THP-1 cell line, where commonly used differentiation inducing agents include phorbol-12-myristate-13-acetate (PMA) and the active metabolite of vitamin D3, (1,25(OH)2D3; VD3). While both induce differentiation, differences in their mechanisms of action, as well as how the end states of the differentiation process differ, are not well understood. Therefore, this study used computational approaches to compare the effects of PMA and VD3 on the differentiation of monocytes into macrophages, using the promonocytic THP-1 cell line. Through the use of RNA-sequencing, gene expression was quantified in differentiated and undifferentiated THP-1 cells, treated with both PMA and VD3. Differential gene expression analysis was performed to determine genes that were differentially expressed as a result of either treatment relative to the untreated cells. This was followed by over-representation analysis to determine the pathways and processes in which the differentially expressed genes (DEGs) were involved. PMA treatment (3 989 DEGs) resulted in more changes in expression relative to VD3 treatment, where only 384 genes were found to be differentially expressed in response to treatment with VD3. Only TFE3, KIT and TRIB1 were observed to be crucial to the process of differentiation, irrespective of treatment. Apart from this, the treatments were observed to largely involve different biological pathways, resulting in cells that were phenotypically distinct from each other at the transcriptional level. This included changes observed in the expression of genes encoding transcription factors known to be involved in the differentiation process, such as CEBPA, GATA2, IRF8 and PU.1, as well as those encoding surface markers representative of monocytes and macrophages, such as CD14, CD64 and CD11b. The expression patterns observed here indicate that, at least at the concentrations and time points included in this study, PMA and VD3 induce macrophage-like cells that are at different stages of differentiation and are not comparable to either each other or primary macrophages. Furthermore, key differences observed in the expression of genes encoding pathogen recognition receptors and cytokines suggest that which differentiation inducing agent is used may have important implications for these cells’ capacity to recognise pathogens and produce cytokines. The findings of this study therefore emphasise that it is crucial to carefully consider the choice of differentiation-inducing agent when using THP-1 cells as an experimental system for studying monocyte-to-macrophage differentiation.
Differential Gene Expression Analysis of PMA Treated Pro-monocytic Cell Lines
(University of the Witwatersrand, Johannesburg, 2023) Kama, Asavela Olona; Meyer, Vanessa; Gentle, Nikki
HL-60, THP-1, and U937 are model cell lines that can undergo myeloid differentiation in vitro, allowing the study of myeloid cell function in drug metabolism, cytotoxicity, and the aetiology of infections. However, the differentiated end-state of these cells is not well characterised. Moreover, cell line-specific differences in the level of gene expression may confound results obtained from such studies. The aim of this study was thus to compare changes in gene expression between HL-60, THP-1, and U937 cells in response to the differentiation agent, phorbol 12-myristate 13-acetate (PMA), 48 hours after treatment. Gene expression profiles were compared across all three cell lines prior to and post-PMA treatment. Differential gene expression analysis between treated and untreated cells was performed using DESeq2 (v 4.2). Gene over-representation analysis was performed using cluster Profiler (v 4.0). HL-60, THP-1, and U937 cells had similar expression profiles prior to PMA treatment, but different sets of genes were significantly differentially expressed in these cell lines 48 h after treatment with PMA. A total of 475 genes were consistently differentially expressed across all cell lines. These genes were found to be involved in phagosome formation and cell cycle transition. HL-60, THP-1, and U937 cells had 944, 1231, and 624 uniquely differentially expressed genes, respectively. These genes were predominantly involved in energy metabolism and pathogen recognition. Overall, THP-1 cells showed greater potential to detect viruses, while U937 cells showed greater potential to detect bacteria. From this, it can be concluded that while all three cell lines did indeed undergo myeloid differentiation, the macrophage-like cell state produced in each case differed between cell lines.
Diversity and Abundance of Arthropods on Conventional Sugarcane under Field Conditions in South Africa
(University of the Witwatersrand, Johannesburg, 2024-09) Smith, Roshay; Malinga, Lawrence; Bouwer, Gustav
Insect diversity and abundance are often the base for formulating strategies that involve the appropriate application of pest control methods, considering the ecosystem services provided by insects. Therefore, the aim of this study was to provide recent baseline data on the diversity and abundance of insects in conventional sugarcane based on two sugarcane fields in KwaZulu-Natal. Three sampling methods, namely pitfall, sticky and water pan traps, were used to sample insects in rain-fed and irrigated sugarcane in Gingindlovu and Pongola from March to October 2022. This study collected 12 493 insects belonging to 14 insect orders and 88 families in rain-fed sugarcane and 22 309 insects belonging to 14 orders and 94 families in irrigated sugarcane. Significant differences in the diversity indices were found between the sampling methods and the sampling periods. This study provides recent baseline data on the diversity and abundance of insects in sugarcane.
Effects of acidification on the survival of pathogens in reconstituted infant formula
(University of the Witwatersrand, Johannesburg, 2023-10) Nemakonde, Mufunwa; De Maayer, Pieter
Infants are at a high risk of developing food-borne illnesses due to the consumption of powdered infant formulas (PIFs) contaminated by pathogenic microorganisms. These pathogens may be introduced during PIF production, transportation or during preparation and storage due to poor hygiene practices. Therefore, it is important to improve the microbiological safety of PIFs to reduce illness. Manufacturers have added further measures to ensure continued control of pathogenic microorganisms during the reconstitution and consumption, including the addition of prebiotics and probiotics, as well as acidification of the PIFs, which negatively affect the growth of pathogens. Different organic acids have been biologically or chemically added to commercial PIFs, however, little is known about the efficacy of the different acids in controlling pathogen growth post-reconstitution. The present study aimed to evaluate and compare the effects of two acids, namely citrate and lactate, integrated in commercial PIFs from two manufacturers on pathogen growth within the products. In Chapter 1, we present a review of the current literature pertaining to PIF production and consumption, its microbiological safety and key problem pathogens, as well as measures to control these pathogens (with specific emphasis on PIF acidification). In Chapter 2, the methodology of the present study is discussed. The effects of lactate and citrate incorporated in commercial PIFs against eight key PIF pathogens was evaluated using plate-based assays. Furthermore, the efficacy of PIF acidification at different storage temperatures and over prolonged storage periods was determined. Finally, to validate the effect of acidity on controlling pathogen growth, a spectrophotometric approach was used. Chapter 3 presents and discusses the key findings of the study. PIF acidification was found to negatively affect the growth of pathogens in comparison to the non-acidified infant formula. Acidification and storage at suboptimal temperatures resulted in little to no microbial growth. Lactate acidification of PIFs demonstrated greater inhibitory effects against most pathogens compared to citrate acidification. Further, lactate was found to have a bactericidal effect on the growth of some pathogens. Similarly, Luria Broth acidification resulted in the reduction of microbial growth. This validated that the acids and not other constituents of the infant formula were responsible for the inhibitory effect. Finally, Chapter 4 provides a summary of the key findings as well as recommendations and guidance on future studies which could be undertaken to enhance the effective control of pathogens in powdered infant formulas, thereby ensuring a safe and nutritious food source for infants.
Effects of Mg2+, Ni2+ and Ca2+ on ATP binding kinetics of nicotinate nucleotide adenylyltransferase from Klebsiella pneumoniae and Enterococcus faecium: insights from empirical and computational studies
(University of the Witwatersrand, Johannesburg, 2023-07) Van Deventer, Ruan; Achilonu, Ikechukwu Anthony
NNAT is an attractive target for drug development due to its crucial role in NAD+ synthesis. However, its characterisation in ESKAPE species, such as Klebsiella pneumoniae and Enterococcus faecium, remains limited. This study aimed to elucidate the binding mechanism of ATP, a pivotal substrate, to these NNAT species, focusing on the role of divalent metal ion cofactors. KpNNAT and EfNNAT enzymes were overexpressed and purified, yielding approximately 2 mg/ml for both. Various techniques were employed to investigate their properties, including far-UV CD, extrinsic ANS fluorescence, stopped-flow kinetic analyses, and MD simulations. The results revealed that KpNNAT could bind ATP independently of divalent metal ions, but catalytic activity required the presence of Mg2+. The kinetic analysis showed ka values of 5.99 μM-1 .sec-1 without divalent metal ions and 5.72 μM-1 .sec-1 in the presence of Mg2+. The "pseudo"-specific activity values were 0.005 μmol/min/mg without divalent metal ions and 0.374 μmol/min/mg in the presence of Mg2+. Conversely, recombinant EfNNAT exhibited limited ATP association, and the reasons for this remain unclear. Overall, this study shed light on the structural dynamics and functional kinetics of ATP association in both NNAT species. The findings contribute to our understanding of this druggable target and provide insights into the inactivity of EfNNAT, which warrants further investigation.
Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies
(University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.
Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.
Establishing and characterizing organoid cultures from colon tissue of South African individuals
(University of the Witwatersrand, Johannesburg, 2024) Du Plessis, Thea-Leonie; Kaur, Mandeep
Colorectal cancer (CRC) has been poorly studied in South Africa, with limited studies on disease progression and development. Studies that have investigated CRC in South Africa have indicated that there is racial disparity between different racial groups that may be attributed to alternative developmental pathways, differences in genetic compositions or CRC initiators that result in these different clinical presentations. Furthermore, the lack of population-based studies substantiates the need for more intensive CRC research. A particular model used to study cancer in general is the use of two-dimensional (2D) cell cultures, which have provided novel insight into many cancers and their development processes. However, these models lack the complex biology observed in vivo. One such model that is gaining research interest is the use of three-dimensional (3D) organoid cultures. Organoids are derived from stem cells and are able to self-organize and mimic the corresponding organ from which they were derived. Research has indicated that organoids are able to maintain cell-type heterogeneity as well as gene expression levels that resemble the organ of origin. Therefore, this project aimed at standardizing a protocol to establish and characterise colorectal organoid cultures from South African patient-derived tissues. Patient samples were obtained from individual patients with informed consent and were processed to generate organoids. The morphology of the organoids was monitored across several days and across passages. Once the organoids had reached maturity and were at passage 2, characterization was performed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence which indicated that the genetic composition and spatial localization of cell types of interest in non-cancerous tissue was recapitulated in the organoids. Based on these observations, it is proposed that organoids could be a promising model to investigate CRC disease development and progression and potentially search for novel therapeutics. This project has established the protocols for growing and characterizing organoids from African samples and provides baseline data, and outlines the complexities and issues involved in growing organoid cultures for the future studies
Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines
(University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne Jacqueline
Breast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.
Exploring temporal changes in the malting barley seed microbiome with meta-omics to understand nitrogen content effects
(University of the Witwatersrand, Johannesburg, 2024-10) Tshisekedi, Kalonji Abondance; De Maayer, Pieter; Botes, Angela
Barley (Hordeum vulgare L.) is a critical cereal crop, particularly in beer production, where it plays a significant role in the economy, especially in South Africa. Despite its importance, the barley seed microbiome, which affects seed storage and quality, is not well understood. This research addresses two key questions: (1) how microbial composition and function evolve during storage and (2) how the inherent nitrogen content of the grain affects these dynamics. Using metagenomic and metaproteomic approaches, eight barley samples from the Kadie cultivar, stored for various durations (harvest, three, six, and nine months) with high and low nitrogen content, were analysed. Metagenomic sequencing revealed a predominance of bacterial sequences and minimal fungal presence, with storage time having a greater impact on microbial diversity than nitrogen content. However, specific bacterial genera such as Erwinia, Pantoea, Pseudomonas, and Stenotrophomonas showed nitrogen-dependent prevalence. Metagenome-assembled genomes (MAGs) were reconstructed, representing 26 bacterial genera, with minimal shared orthologues, highlighting taxonomic diversity. Functional analysis identified key metabolic pathways and carbohydrate-active enzymes (CAZymes) essential for microbial adaptation during storage. Metaproteomic analysis further showed the active expression of proteins related to nutrient transport and stress response, indicating functional changes over storage time. Overall, this research enhances the understanding of the barley seed microbiome, providing valuable insights into storage practices that could improve brewing quality and agricultural sustainability.