Browsing by Department "Department of Integrative Biomedical Sciences"

Now showing 1 - 11 of 11
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    An electrocardiograph tutor using expert system technology
    (1989) Meyer, Derek Louis
    Computer systems for the interpretation of diagnostic ECGs are widely used, but currently provide no explanatory or teaching functions of value to the less experienced practitioner. The relevant literature is reviewed, and specifications are provided for an ECG analysis system which will function as a learning aid for undergraduate and postgraduate medical students. Key aspects of the specifications are implemented on an IBM-PC. Recommendations for further development are provided.
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    Elucidation of Early Evolution of HIV-1 Group M in the Congo Basin Using Computational Methods
    (2021-04-02) Tongo, Marcel; Martin, Darren P; Dorfman, Jeffrey R
    The Congo Basin region is believed to be the site of the cross-species transmission event that yielded HIV-1 group M (HIV-1M). It is thus likely that the virus has been present and evolving in the region since that cross-species transmission. As HIV-1M was only discovered in the early 1980s, our directly observed record of the epidemic is largely limited to the past four decades. Nevertheless, by exploiting the genetic relatedness of contemporary HIV-1M sequences, phylogenetic methods provide a powerful framework for investigating simultaneously the evolutionary and epidemiologic history of the virus. Such an approach has been taken to find that the currently classified HIV-1 M subtypes and Circulating Recombinant Forms (CRFs) do not give a complete view of HIV-1 diversity. In addition, the currently identified major HIV-1M subtypes were likely genetically predisposed to becoming a major component of the present epidemic, even before the events that resulted in the global epidemic. Further efforts have identified statistically significant hot- and cold-spots of HIV-1M subtypes sequence inheritance in genomic regions of recombinant forms. In this review we provide ours and others recent findings on the emergence and spread of HIV-1M variants in the region, which have provided insights into the early evolution of this virus.
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    Investigating expressed RNA variants that are related to disease severity in SARS-CoV-2-infected patients with mild-to-severe disease
    (2022-04-28) Okendo, Javan; Okanda, David
    Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a significant public health challenge globally. SARS-CoV-2 is a novel virus, and the understanding of what constitutes expressed RNAseq variants in healthy, convalescent, severe, moderate, and those admitted to the intensive care unit (ICU) is yet to be presented. We characterize the different expressed RNAseq variants in healthy, severe, moderate, ICU, and convalescent individuals. Materials and methods: The bulk RNA sequencing data with identifier PRJNA639275 were downloaded from Sequence Reads Archive (SRA). The individuals were divided into: (1) healthy, n = 34, moderate, n = 8, convalescent, n = 2, severe, n = 16, and ICU, n = 8. Fastqc version 0.11.9 and Cutadapt version 3.7 were used to assess the read quality and perform adapter trimming, respectively. STAR was used to align reads to the reference genome, and GATK best practice was followed to call variants using the rnavar pipeline, part of the nf-core pipelines. Results: Our analysis demonstrated that different sets of unique RNAseq variants characterize convalescent, moderate, severe, and those admitted to the ICU. The data show that the individuals who recover from SARS-CoV-2 infection have the same set of expressed variants as the healthy controls. We showed that the healthy and SARS-CoV-2-infected individuals display different sets of expressed variants characteristic of the patient phenotype. Conclusion: The individuals with severe, moderate, those admitted to the ICU, and convalescent display a unique set of variants. The findings in this study will inform the test kit development and SARS-CoV-2 patients classification to enhance the management and control of SARS-CoV-2 infection in our population.
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    Molecular and Kinetic Characterisation of wild type and mutant Porphobilinogen Deaminase
    (2016) Pienaar, Elaine; Meissner, Peter
    The purpose of this dissertation was to provide an overview of acute intermittent porphyria, focussing on the structure and function of the enzyme, porphobilinogen deaminase (PBGD), as well as experimentally demonstrating the use of kinetic, structural and thermodynamic approaches to shed light on the enzyme reaction. The key focus was to investigate the effect of three mutations of the active site lysine 98 residue (K98) on the enzyme’s stability and mechanism. Two clinically relevant PBGD mutants, the K98E and K98R were expressed. Both of these mutants have previously been described in patients. We engineered and expressed an additional mutant, K98A, in order to investigate the effect of charge at this residue. The K98E, K98R and K98A recombinant proteins were successfully engineered, expressed and purified. These mutations were kinetically characterised, and the low enzyme activity supports the fact that the K98E and the K98R are known-disease causing mutations. The negligible activity of the K98A and K98R mutants was predicted as a result of a loss of DPM co-factor binding, which was analysed and proved with a co-factor spectral shift assay. Further attempts to examine the interaction of co-factor binding involved removal of the bound cofactor from wild type enzyme, in order to investigate the possible interaction of the ‘apo’- enzyme with the DPM co-factor. However, no results were obtained to elucidate this interaction, largely due to the highly unstable nature of the generated ‘apo’-enzyme. Native polyacrylamide gel electrophoresis (PAGE) was performed in order to observe changes in enzyme-substrate complexes between the wild type and the different mutant proteins. The enzyme-substrate complexes for the wild type were clearly shown, however we could not do so in our mutant proteins. The secondary structure estimations as well as the conformational stability of the mutants were tested with the use of circular dichroism. Far- and near-UV analysis provided insight into the effect of each mutation on the enzyme’s secondary and tertiary structure respectively. Results indicate that the different mutations cause marginal alterations in secondary structure, and resulted in changes of aromatic ring conformations in the near-UV analysis. Finally, modelling of each mutation to known crystal structures of the human enzyme was done in order to provide a rationalisation of kinetic and conformational data. Although this provided only a static image and estimation of the structural effect of each mutation, it did allow for some speculation in order to rationalise the kinetic and conformational data obtained. Overall, this work illustrates how the characterisation of expressed, purified, AIP-associated mutant enzymes aids our understanding of the complex structure and mechanism of the PBGD enzyme.
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    Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding
    (2021-02-23) Makumire, Stanley; Dongola, Tendamudzimu Harmfree; Chakafana, Graham; Tshikonwane, Lufuno; Chauke, Cecilia Tshikani; Maharaj, Tarushai; Zininga, Tawanda; Shonhai, Addmore
    Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.
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    Proteomic insights into the modulation of foetal neurogenesis by the anti-retroviral efavirenz
    (2019) Albeldas, Claudia; Blackburn, Jonathan
    Background: 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.
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    Recent Zoonotic Spillover and Tropism Shift of a Canine Coronavirus Is Associated with Relaxed Selection and Putative Loss of Function in NTD Subdomain of Spike Protein
    (2022-04-21) Zehr, Jordan D; Pond, Sergei L Kosakovsky; Martin, Darren P; Ceres, Kristina; Whittaker, Gary R; Millet, Jean K; Goodman, Laura B; Stanhope, Michael J
    A canine coronavirus (CCoV) has now been reported from two independent human samples from Malaysia (respiratory, collected in 2017–2018; CCoV-HuPn-2018) and Haiti (urine, collected in 2017); these two viruses were nearly genetically identical. In an effort to identify any novel adaptations associated with this apparent shift in tropism we carried out detailed evolutionary analyses of the spike gene of this virus in the context of related Alphacoronavirus 1 species. The spike 0-domain retains homology to CCoV2b (enteric infections) and Transmissible Gastroenteritis Virus (TGEV; enteric and respiratory). This domain is subject to relaxed selection pressure and an increased rate of molecular evolution. It contains unique amino acid substitutions, including within a region important for sialic acid binding and pathogenesis in TGEV. Overall, the spike gene is extensively recombinant, with a feline coronavirus type II strain serving a prominent role in the recombinant history of the virus. Molecular divergence time for a segment of the gene where temporal signal could be determined, was estimated at around 60 years ago. We hypothesize that the virus had an enteric origin, but that it may be losing that particular tropism, possibly because of mutations in the sialic acid binding region of the spike 0-domain.
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    Ropporin-1 and 1B Are Widely Expressed in Human Melanoma and Evoke Strong Humoral Immune Responses
    (2021-04-09) Da Gama Duarte, Jessica; Woods, Katherine; Quigley, Luke T; Deceneux, Cyril; Tutuka, Candani; Witkowski, Tom; Ostrouska, Simone; Hudson, Chris; Tsao, Simon Chang-Hao; Pasam, Anupama; Dobrovic, Alexander; Blackburn, Jonathan M; Cebon, Jonathan; Behren, Andreas
    Antibodies that block immune regulatory checkpoints (programmed cell death 1, PD-1 and cytotoxic T-lymphocyte-associated antigen 4, CTLA-4) to mobilise immunity have shown unprecedented clinical efficacy against cancer, demonstrating the importance of antigen-specific tumour recognition. Despite this, many patients still fail to benefit from these treatments and additional approaches are being sought. These include mechanisms that boost antigen-specific immunity either by vaccination or adoptive transfer of effector cells. Other than neoantigens, epigenetically regulated and shared antigens such as NY-ESO-1 are attractive targets; however, tissue expression is often heterogeneous and weak. Therefore, peptide-specific therapies combining multiple antigens rationally selected to give additive anti-cancer benefits are necessary to achieve optimal outcomes. Here, we show that Ropporin-1 (ROPN1) and 1B (ROPN1B), cancer restricted antigens, are highly expressed and immunogenic, inducing humoral immunity in patients with advanced metastatic melanoma. By multispectral immunohistochemistry, 88.5% of melanoma patients tested (n = 54/61) showed ROPN1B expression in at least 1 of 2/3 tumour cores in tissue microarrays. Antibody responses against ROPN1A and ROPN1B were detected in 71.2% of melanoma patients tested (n = 74/104), with increased reactivity seen with more advanced disease stages. Thus, ROPN1A and ROPN1B may indeed be viable targets for cancer immunotherapy, alone or in combination with other cancer antigens, and could be combined with additional therapies such as immune checkpoint blockade.
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    Transcription analysis of virulent strains of Mycobacterium tuberculosis
    (2018) Ambler, Jon Mitchell; Mulder, Nicola
    Background: Despite the development of new drugs and success of social programs, tuberculosis remains a leading cause of mortality. This burden falls disproportionately on developing countries where the high burden of HIV has a potentiating e↵ect, but may soon return to areas where it was previously brought under control as resistant strains continue to emerge. In the Western Cape, two closely related strains of the Beijing family have been isolated that provide an opportunity to study virulence in a system with relatively little noise. The aim of this project was to identify the cause of the altered virulence displayed between the two strains, and describe how the di↵erences between the two genomes contributed to the phenotypic di↵erences. Results: GenGraph allows for the creation of graph genomes, and facilitated the creation of a pan-transcriptome that allowed for the mapping of gene annotations between isolates. This allowed for the mapping of reads to a more suitable Beijing family reference while interpreting the results with annotations from the H37Rv reference. We generated expression and target profiles for the known sRNA, and identified a large number of novel sRNA. Transcriptomic data from 4 di↵erent growth conditions was integrated with this sRNA data as well as variant data using the Cell pipeline. From this data we identified multiple sets of genes linked to copper sensing in MTB, including the di↵erentially expressed MoCo operon. Increasing evidence that macrophages use copper to poison bacteria trapped in their phagosomes provides the link to virulence and pathogenicity. Conclusions: Through the integration of data from multiple data types we were able to elucidate the most probable cause of the altered virulence found between the two isolates in this study. We developed reusable tools and pipelines, and noted a large number of undescribed sRNA expressed in these isolates. The identification of the copper response as a chief contributor to the phenotype increases both our understanding of the isolates, and the role of the element in infection. These results will be key in guiding further investigation of the variant linked genes to identify those linked to copper homeostasis or response.
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    The use of evaluation in the design and development of interactive medical record systems
    (1988) Herbst, Abraham J
    An explorative study was done to develop an evaluation methodology. This method can be applied during the development of interactive medical record systems in order to provide information which can be used to improve user interaction with the system. Th e evaluation methodology consists of a number of interactive sessions with potential users of the interactive medical record system. During the first two sessions the subjects are trained to use the system. During the third and last session the subjects are videotaped while they are doing a set of benchmark tasks on the system under evaluation. The video recordings are analysed to obtain performance data. This performance data consists of task timings and a list of problems experienced (errors made) by the subjects. The systems evaluated during the study were a problem-oriented manual medical record and an interactive computerized medical record. The computerized record system was specifically developed for this study. The design and subsequent improvements to this system are documented in the study.
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    VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses
    (2021-02-01) Guo, Jiarong; Bolduc, Ben; Zayed, Ahmed A; Varsani, Arvind; Dominguez-Huerta, Guillermo; Delmont, Tom O; Pratama, Akbar A; Gazitúa, M. C; Vik, Dean; Sullivan, Matthew B; Roux, Simon
    Background Viruses are a significant player in many biosphere and human ecosystems, but most signals remain “hidden” in metagenomic/metatranscriptomic sequence datasets due to the lack of universal gene markers, database representatives, and insufficiently advanced identification tools. Results Here, we introduce VirSorter2, a DNA and RNA virus identification tool that leverages genome-informed database advances across a collection of customized automatic classifiers to improve the accuracy and range of virus sequence detection. When benchmarked against genomes from both isolated and uncultivated viruses, VirSorter2 uniquely performed consistently with high accuracy (F1-score > 0.8) across viral diversity, while all other tools under-detected viruses outside of the group most represented in reference databases (i.e., those in the order Caudovirales). Among the tools evaluated, VirSorter2 was also uniquely able to minimize errors associated with atypical cellular sequences including eukaryotic genomes and plasmids. Finally, as the virosphere exploration unravels novel viral sequences, VirSorter2’s modular design makes it inherently able to expand to new types of viruses via the design of new classifiers to maintain maximal sensitivity and specificity. Conclusion With multi-classifier and modular design, VirSorter2 demonstrates higher overall accuracy across major viral groups and will advance our knowledge of virus evolution, diversity, and virus-microbe interaction in various ecosystems. Source code of VirSorter2 is freely available ( https://bitbucket.org/MAVERICLab/virsorter2 ), and VirSorter2 is also available both on bioconda and as an iVirus app on CyVerse ( https://de.cyverse.org/de ). Video abstract