Philosophiae Doctor - PhD (Medical BioScience)

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Potamanautes warreni biomarker assays to monitor silver nanomaterial contaminants in aquatic environments
(University of the Western Cape, 2016) Walters, Chavon Rene
There has been extensive growth in nanoscale technology in the last few decades to such a degree that nanomaterials (NMs) have become a constituent in a wide range of manufactured commercial and domestic products. This surge has resulted in uncertainties regarding their environmental impact, due to the significant increases in the amount of NMs released into the environment (Dowling et al., 2004) through intentional and unintentional releases. Like many other toxins, the aquatic environment is particularly vulnerable as it acts as a sink for nanoparticles (NPs) (Scown et al., 2010). The escalating growth of NMs has not advanced without efforts to understand its properties. Despite the dramatic advances in both the production and application of NMs, very little is known regarding their interaction with and effects on environmental and human health. Given the lack in scientific knowledge, particularly under various environmental conditions, it is often difficult to accurately assess the potential exposure pathways to ecological receptors. Of all NMs, silver nanoparticles (AgNPs) are the most widely used NPs, present in several consumer products mainly because of their anti-bacterial properties. It is estimated that the annual production exceeds 1000 tons/year (Piccinno et al., 2012). The increase uses of AgNPs in consumer products (e.g. textiles, cosmetics and personal hygiene), household appliances (e.g. washing machines and vacuum cleaners) and medical equipment have led to their increase release into the environment, thereby posing an environmental risk and human health concern. Silver NPs are known to induce the production of Reactive Oxygen Species (ROS) (Ahamed et al., 2010; Levard et al., 2012; Piao et al., 2011). Also since AgNPs are oxidized to ionic Ag (Ag+), it is still unclear whether the effects of ROS can be attributed to Ag+ release or to the AgNP itself (Fabrega et al., 2009; Miao et al., 2009). The behaviour of AgNPs is collectively influenced by inherent (nanoparticle size, shape, surface area, surface charge, crystal structure, coating, solubility/dissolution) and environmental factors (temperature, pH, ionic strength, salinity, organic matter). Climate change predictions indicate that the frequency, intensity and duration of extreme natural events (such as temperature elevations) will increase in the future (IPCC, 2001; IPCC, 2007). Global warming and climate change could increase atmospheric temperatures by 2.4 – 6.4 °C (IPCC, 2001; IPCC, 2007). The main feature associated with global climate change is the anticipation of wetter winters (i.e. increased flood events) and drier, warmer summers (i.e. extreme temperatures). These changes are likely to affect the inputs of contaminants into the environment as well as affect their behaviour, fate and transport, and toxicity in aquatic environments. It is known that the current temperature predictions in climate change scenarios could directly affect aquatic ecosystem communities (Carpenter et al., 1992), since temperature is also regarded as an important abiotic factor influencing growth and production of primary producers (i.e. algae, macrophytes etc.), and may also affect species distribution. For example, Liu et al. (2010) reported higher dissolution rates of AgNPs with increased temperature. Similarly, sudden hydrographic activity like high flood conditions may cause resuspension and redistribution of sediments. Few studies have linked the foreseeable climate change with contaminant release and ecosystem impacts. Similarly, few studies have analyzed the behaviour of NMs in the environment considering these predicted changes in mean temperatures. This thesis focuses on the effects of AgNPs on oxidative stress responses in the Cape River crab Potamonautes perlatus. The present work was undertaken to interpret the biological effects of AgNPs (<100 nm) on P. perlatus, as well as to assess its effects under different environmental conditions. To understand the uptake, accumulation and biological effects of AgNPs, freshwater microcosms were produced to mimic a typical aquatic environment and temperature manipulated microcosms to which a commercially-available AgNP powder was added. Nanoparticles were characterized in the dry state and in suspension under different environmental conditions. Dissolution of total Ag was measured by inductively coupled plasma mass spectrometry (ICP-OES). Nanoparticle toxicity was assessed by measuring mortality and biomarkers of oxidative stress (CYP450, SOD, CAT, GST) evaluated in crab tissues. The overall results demonstrated that: (1) AgNPs may be transformed in both size and state under variable environmental conditions. The formation of smaller aggregates at higher temperatures suggests higher toxicity, (2) the release of free metal ions from NPs and NPs aggregates contribute to a higher toxicity towards aquatic organisms, (3) oxidative stress is a significant mechanism of AgNP toxicity and consequently enzymatic activation/ inhibition with increasing AgNP concentration and temperatures, (4) oxidative stress responses to AgNPs particles were significantly modulated by temperature stress in P. perlatus, (5) mortality was observed from day 2 with maximum mortality achieved at day 7, (6) enzymes involved in detoxification, i.e. CYP450, has functional significance in the haemocytes, (7) P. perlatus has proved to be a significant target for AgNP exposure and, furthermore, has proved to be a suitable species to assess the ecotoxicity of AgNP in the aquatic environment, (8) antioxidant enzymes activities (are valuable tools to assess the oxidative status of crab tissues co-exposed to AgNPs and temperature. Furthermore, the results obtained in this study contributed to the understanding of the behaviour, bioavailability, uptake and toxicity of AgNPs under variable temperatures. Recommendations for future work are given in Chapter 6.
Holarrhena floribunda leaves as a potential source of bioactive anticancer compounds
(University of the Western Cape, 2014) Abiodun, Badmus Jelili
Cancer is one of the leading causes of morbidity and mortality in developed and developing nations. It is estimated that 86% of new cases and 64% of death due to cancer are from Africa and 13.1 million deaths are estimated to occur worldwide by the year 2030. Cancer death rates have not subsided despite recent advances in cancer drug development and treatment. Present cancer drug regimens are limited due to unpredictable efficiency, severe side effects, resistance and high cost. Plants provide a vast array of natural compounds such as terpenoids, phenolics and alkaloids with antiproliferative pro-apoptotic and antioxidant effects. Plants are principal sources of compounds for drug discovery and development of several clinically proven useful anticancer drugs. The present study focused on the isolation of compounds from the Holarrhena floribunda (H. floribunda) leaves for their potential anticancer activities. Standard methods were employed to assess the antiproliferative potential, apoptosis, cell cycle analysis and reactive oxygen species of the methanolic leaf extract (MLE) of H. floribunda. The standard methods of isolation such as column chromatography, thin layer chromatography, high performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) were used to isolate and purify bioactive compounds from the leaves. To elucidate the mechanism of cytotoxicity of the isolated compounds, apoptosis effect was studied by flow cytometry analysis using the ApopercentageTM dye, Annexin-V/PI stain, induction of caspase-3 using the Caspase-3/7 Glo assay kit and PARP-1 deactivation using Western blot analysis. The mode of action was further assessed by evaluating reactive oxygen species (ROS), mitochondrial toxicity, light and fluorescent microscopic morphological evaluations of F-actin and topoisomerase-I relaxation assay. In addition, potential cancer prevention of the plant was also evaluated by assessing the antioxidant activity of the flavonoids compounds isolated from the MLE. The results of the present study show that the MLE of H. floribunda inhibited the proliferation of the cancer cell lines (HeLa, HT-29 and MCF-7) in a dose- and timedependent manner. The anti-proliferative activity of the methanolic extract is selective towards cancer cells more than normal KMST-6 fibroblast cells used in the study. The extract showed cytotoxicity, cell cycle arrest, induced generation of ROS and apoptosis. The methanolic extract of the leaves led to the isolation of two pure steroidal alkaloid and four flavonoid compounds. The two steroidal alkaloids - holamine and funtumine were found to be selectively cytotoxic to human cancer cells more than the normal fibroblasts. The cytotoxicity of the two steroidal alkaloids was mediated through the induction of apoptosis. The apoptosis induction was found to be triggered by the activation of caspase-3, deactivation of PARP-1, increased ROS, cell cycle arrest at G0/G1 and G2/GM phases, mitochondria toxicity, F-actin disorganization and topoisomerase-I inhibition. However, four flavonoids (kaempferol-3-O-rutinoside, quercetin-3-galactoside/glucoside, quercetin-3-O-glucoside and kaempferol-3-O-glucoside) isolated were subjected to antioxidant activity assay using oxygen radical absorbance capacity (ORAC), ferric reducing/antioxidant power (FRAP), trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation inhibition. Two flavonoids with a quercetin nucleus were found to be active in the entire antioxidant spectrum, except pro-oxidant activity while the remaining two compounds with a kaempferol nucleus were selective in their activity. Structure-activity relationship analysis shows that the activity of the flavonoids depends on the ortho dihydroxyl group on the B-ring of the flavonoids. The present study on H. floribunda has shown that the leaves contain bioactive compounds with potential as anticancer agents and cancer protective and preventive activity. More work is still needed to elucidate other possible anticancer mechanisms that might be relevant to the cytotoxic effects of the two isolated alkaloids.
The effect of maternal oral health on pregnancy outcomes
(University of the Western Cape, 2014) Turton, Mervyn Sydney
Adverse pregnancy outcomes such as preterm birth and low birth weight are major causes of maternal and neonatal morbidity and mortality. Increasing evidence points to an association between periodontal disease and adverse pregnancy outcomes and thus a better understanding of the nature of this association will assist in treatment planning to reduce adverse pregnancy outcomes. Among the Gram-negative anaerobic bacteria frequently associated with periodontal disease are Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis which may be detected in plaque using the BANA test (N-benzoyl-DL-arginine-2- naphthylamide). The aim of this study was to investigate the effect of periodontal disease on pregnancy outcomes and evaluate the use of BANA as a screening test for the risk of adverse pregnancy outcomes. This study complied with the Declaration of Helsinki (2013) and included 443 pregnant women attending ante-natal clinics in KwaZulu Natal. At first visit, maternal oral health status was assessed by the measurement of periodontal indices and BANA testing of dental plaque from the same teeth. Patient demography and medical history were obtained by means of a questionnaire and all data compared with pregnancy outcomes. While controlling for other factors, significant differences were found between the distributions of periodontal disease at BANA-negative and BANA-positive sites and between infant birth weight and maternal periodontal index scores such as plaque index and gingival index. The birth weight and gestational age at delivery of infants born of BANA-positive periodontally diseased mothers were significantly lower than those born of BANA-negative mothers with no periodontal disease. We may conclude that the presence of periodontal disease during pregnancy has a significant association with negative pregnancy outcomes and suggest that the risk for adverse pregnancy outcomes may be reduced by monitoring the oral health status of women during pregnancy.
Investigations on the effects of Typha capensis on male reproductive functions
(University of the Western Cape, 2016) Ilfergane, Abdulkarem
Introduction Typha capensis, commonly referred to as bulrush also called „love reed‟ growing in Southern Africa‟s wetlands, is one of South Africa indigenous medicinal plants that are traditionally used to treat male fertility problems and various other ailments. Previous studies revealed that T. capensis has indeed a beneficial effect on male reproductive functions and aging male symptoms. The T. capensis rhizomes are used in traditional medicine during pregnancy to ensure easy delivery, for venereal diseases, dysmenorrhea, diarrhoea, dysentery, and to enhance the male potency and libido. Typha genuses contain flavones and other phenolic compounds, which exhibit anti-oxidative capacity. Materials and Methods This study encompasses three parts (part 1: Exposure of different cell lines to crude aqueous extracts of T. capensis rhizomes; part 2: HPLC analysis of Typha capensis crude rhizome extract and exposure of different cell lines to the F1 fraction of the summer season; part 3: Compound identification by means of NMR spectrometric analysis and exposure of different cell lines to bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes. Part 1: TM3-Leydig cells and LNCaP cells incubated with different concentrations of crude aqueous extract of T. capensis rhizomes (0.01, 0.02, 0.1, 1, 10 and 100 µg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology and viability (determined by means of MTT assay). Part 2: The crude extract HPLC profiles were obtained by preparing the extracts for different seasons (Autumn, Winter, Spring, Summer). TM3-Leydig cells, LNCaP cells and PWR-1E cells incubated with different concentrations T. capensis rhizomes extract F1 fraction of the summer season (0.01, 0.02, 0.1, 1, 10 and 100 µg/ml) and control (without extract) for 24 and 96 hours, after incubation. The following parameters were evaluated: cell morphology was observed and recorded, viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding), DNA fragmentation (determined by means of the TUNEL assay). Part 3: NMR spectrometric analysis was performed on a 13C spectra were recorded at 400 MHz. TM3-Leydig cells and LNCaP cells incubated with different concentrations of bioactive compounds (Quercetin and Naringenin) isolated from T. capensis rhizomes, for acute exposure (24, 96 hours) and chronic exposure (96 hours), after incubation, the following parameters were evaluated: cell morphology and viability (determined by means of MTT assay), testosterone production (testosterone ELISA test), cell early apoptosis (determined by means of Annexin V-Cy3 binding) and DNA fragmentation (determined by means of the TUNEL assay). Results Part 1: for TM3-Leydig cells the results reveal no observable morphological changes and no significant influence on cell viability except at highest concentration indicating cellular stress. However, LNCaP cells showed a decline in cell viability at the incubation period 96 hours (-82.4%) more than 24 hours (-64.7%) indicating more cell death. Part 2: HPLC data showed that the most effective fraction was the F1 fraction from the summer harvest. Results revealed that the T. capensis rhizome extract F1 fraction of the summer season significantly enhanced testosterone production in TM3 cells and was more toxic towards cancer cells (LNCaP cells ) compared to the normal cell lines (TM3-Leydig, PWR-1E cells). Part 3: NMR data showed 2 bioactive compounds which were identified as Quercetin and Naringenin. The assays showed that LNCaP cells are more sensitive to the cytotoxic effects and apoptosis induction of both compounds, whereas, the assays resulted in weak effects toward TM3- Leydig cells. However, testosterone production in TM3-Leydig cells was significantly enhanced at low concentrations of Quercetin and Naringenin at all exposure types (acute and chronic) testosterone beak significantly at around 0.100 and 0.125 μg/ml (P<0.0001), stimulatory activity in a dose-dependent manner.
Synergistic effects of mixtures of fungicides and medicinal plant extracts against Botrytis cinerea
(University of the Western Cape, 2008) Vries, Filicity Ann
We hypothesize that South African medicinal plants contain compounds that can act in synergism with synthetic antifungal compounds. Four fungicides - Sporekill™, Rovral™, Terminator™ and Teldor™ at doses 0.1, 0.2, 0.4 and 0.8 mL L-1 and plant species Galenia africana, Elytropappus rhinocerotis and Tulbaghia violacea were tested alone and in different combinations for their potency (efficacy) on radial growth inhibition of Botrytis cinerea strains on potato dextrose plates. Four doses of plant extract for each of the respective plant species were used. A total of 48 combinations were tested for each strain. Mixtures of plant extracts were far more effective in controlling strains compared to the individual components alone, representing significant levels of in vitro synergistic interactions. Combinations of these components represent an attractive future prospect for the development of new management strategies for controlling B. cinerea. Since the in vitro tests of these mixtures showed inhibitory activity, the mixtures were tested for activity in assays on Granny Smith apples. In vitro tests can be used to screen mixtures to obtain information on their inhibitory activity on a pathogen, however, the environmental conditions of the fruit and the ability of the pathogen to grow into the fruit cannot be simulated in vivo. A series of two-fold doses of medicinal plant extracts were combined with fungicides to conduct decay inhibition studies. The incidence of gray mold was significantly reduced by mixtures of plant extracts and fungicides. Under conditions similar to those in commercial storage, a drench treatment with G. africana and Rovral™ significantly (p=0.05) inhibit gray mold on the apples and was more effective than the plant extract and fungicide alone. The treatments exerted synergistic effects and were markedly better than the components applied alone. The wound colonization assay was used for optimal decay control. In a drench, much higher volumes of the treatments are used to ensure that the components of the suspension are deposited evenly over the entire fruit surface. Drenching of fruit to apply other chemicals is an established practise in the pome (fleshy) fruit industry, and simplifies the commercial application of the mixtures, as no additional infrastructure at commercial packing houses will be required. This approach not only makes it possible to reduce fungicide concentrations while maintaining adequate decay control, but also ensures a reduction of the chemical residue on the fruit.
The effect of maternal exposure to alcohol and nicotine on pancreas and kidney size, aorta and carotid intima thickness and visceral fat in their children.
(University of the Western Cape, 2019) De Smidt, Juléy Janice Abigail
Background: In utero exposure to teratogens, increasing urbanization, rapid nutritional transition from poverty to affluence, adoption of a Western-style diet and physical inactivity have contributed to the growing obesity epidemic in the low income countries. Aim: To investigate the associations between in utero exposure to alcohol and nicotine on the growth and development of children aged five years from a low income setting. These effects will be observed in children aged five years as a reduced pancreas and kidney size, higher aorta and carotid intima thickness as well as higher visceral abdominal adiposity measurements. Methods: A prospective cohort study of children aged five years from a low-income setting. This is a further follow-up study of children born in the Safe Passage Study. Data was collected from 500 mother-child pairs at antenatal clinic visits, at birth and at the age five years. Maternal data were collected at antenatal clinics in the residential area of Bishop Lavis, Western Cape, when women enrolled for their first antenatal visit. All other assessments were done at follow-up study visits at Tygerberg Academic Hospital in Bellville, South Africa. Dependent variables included: anthropometric measurements at birth, weight (BW), length (BL), mid-upper-arm circumference (MUAC). And, body mass index (BMI), skinfold thickness (SFT) and waist circumference (WC) at age five years. Also, clinical assessments at five years, blood pressure (BP), mean arterial pressure (MAP) and heart rate (HR). And, ultrasound assessments consisted of the aorta and carotid intima media thickness (aIMT and cIMT) and visceral adipose tissue (VAT), kidney and pancreas size. With independent variables: maternal in utero exposure to nicotine and alcohol, maternal BMI and MUAC. Results: We observed higher cIMT and lower visceral adipose tissue values as a result of dual in utero exposure to alcohol and nicotine in males; maternal adiposity influenced the adiposity measures of their children. Females had higher SBP, DBP and HR values compared to males. Also, females born to overweight mothers had higher SFT values compared to those born to normal weight mothers (OR 1.62, 95 % confidence interval 1.24 – 2.13). Pancreas body and kidney length were associated with in utero nicotine exposure [F (3, 485) = 2.86 at p = 0.04] and [F (3, 493) = 2.99 at p = 0.03]. Conclusions: In utero exposure to alcohol and nicotine had both individual as well as compounding negative effects on the growth and development. Ultimately, higher cIMT and visceral adipose tissue values translate to cardiometabolic risk factors which are present in these five-year-old children from a low-income setting in South Africa.
Bionomics of vector-borne diseases in sites adjacent to lakes Victoria and Baringo in Kenya
(University of the Western Cape, 2016) Ouma, David Omondi
Bionomics of vector-borne pathogens (VBPs) is a complex phenomenon that involves understanding the ecology of arthropod borne pathogens and vertebrate hosts potentially involved in their transmission cycles. Investigations into the bionomics of viral and bacterial VBPs circulating in Baringo and Homa Bay Counties of Kenya were carried out. Specifically, vertebrate hosts represented in mosquito bloodmeals, presence of arboviruses in blood fed mosquitoes and patients presenting with acute undiagnosed febrile illnesses in rural health facilities, and tick borne pathogens (TBPs) diversity in ticks of animals were identified. Mosquitoes were trapped by BG sentinel and CDC light traps, while ticks were sampled directly from domestic animals and tortoises close to human habitation along the shores and adjacent islands of Lakes Victoria and Baringo in Kenya. Blood and sera were also sampled from patients presenting with acute febrile illnesses visiting four rural health facilities in Homa Bay County. Mosquitoes and ticks were sorted and identified to species using standard morphological taxonomic keys. All the biological samples (blood-fed mosquitoes, ticks and blood/sera) were processed using molecular and culture procedures for detection of VBPs (arboviruses, Ehrlichia, Anaplasma, Rickettsia and protozoa). Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes, 33 bloodmeal hosts were identified including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. Further detection of Sindbis and Bunyamwera viruses was done on blood-fed mosquito homogenates by Vero cell culture and RTPCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. In TBPs assay, 585 tick pools were analysed consisting of 4,126 ticks collected in both study areas. More ticks were sampled in Baringo (80.5%), compared to Homa Bay (19.5%). In Baringo, agents of ehrlichiosis were detected from Amblyomma and Rhipicephalus ticks including Ehrlichia ruminantium (12.3%), Ehrichia canis (10.5%) and Paracoccus sp. (4.4%). Agents of anaplasmosis included Anaplasma ovis (7.2%), Anaplasma platys (4.4%) and Anaplasma bovis (4.0%), all from Hyalomma, Amblyomma and Rhipicephalus ticks, as well as agents of rickettsiosis, including Rickettsia africae, Rickettsia aeschlimannii, Rickettsia rhipicephali, Rickettsia montanensis and a Rickettsia sp. that was not conclusively characterized. Babesia caballi, Theileria sp. and Hepatozoon fitzsimonsi were also detected from both Rhipicephalus ticks and Amblyomma ticks. In Homa Bay, Ehrichia ruminantium (17.5%) and Ehrichia canis (9.3%) were isolated from Amblyomma latum and Rhipicephalus pulchellus, as well as Anaplasma platys (14.4%) and Anaplasma ovis (14.4%) from Amblyomma and Rhipicephalus species. In determination of the occurrence of arboviruses among patients presenting with acute febrile illnesses, acute Bunyamwera 3 (0.9%) and Sindbis 2 (0.6%) infections were detected by RT-PCR and cell culture and Sindbis seroprevalence was determined by plaque assay. Though a significant proportion of these patients tested positive for low Plasmodium parasitemia, none were co-infected with Plasmodium parasites and arboviruses. This study highlights the presence and relative importance of zoonotic VBPs in both study areas.
Structural and functional characterization of human DDX5 and its interaction with NS5B of hepatitis C virus
(University of the Western Cape, 2011) Choi, Yook-Wah
Hepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the Cterminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional - helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 autoinhibit its interaction with NS5B. Interestingly, the -helix in NTR is essential for this autoinhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.
Effects of Libyan traditional plants on the reproductive system of male and female rats
(University of the Western Cape, 2016) Elgenaidi, Abdalla Ramadan
In different parts of the world, medicinal plants have demonstrated a lot of health benefits to mankind and remains an important source for the discovery of new bio-active compounds. Libya is a typical example of a country where medicinal plants are widely used. Plant extracts of five Libyan medicinal plants were used in this study to investigate their in vivo effects on spermatogenesis and steroidogenesis in male rats and on ovulation and fertility in female rats. The In vitro effects of these plant extracts were also investigated on TM3 Leydig cells and MCF 7 breast cancer cells. A phyto-chemical analysis of the five Libyan medicinal plants (flaxseed, black seeds, radish seed, date palm pollen and nutmeg) was done. The results showed that date palm pollen had a higher antioxidant activity than all of the above mentioned plants. In addition to this, Nigella sativa was observed to possess high flavonol content as well as high antioxidant activity. Male rats exposed to flaxseed, radish seeds and date palm pollen showed no significant alterations in body weight gain, whereas date palm pollen (240 mg/kg, p < 0.05) promoted an increase in body gain. This study also revealed a significant increase in the relative testicular weight of animals exposed to either flaxseed (300mg/kg) or date palm pollen (120mg/kg). In addition, the relative weights of the seminal vesicles of all treated groups showed significant increased values. The level of serum testosterone showed a significant increase after exposure to radish seed (80mg/kg) and a significant dose- dependent increase for date palm pollen when compared to control (P< 0.05). In contrast, flaxseed caused a dose-dependent significant (p <0.01) decrease in testosterone level at radish seed (300mg/Kg). All plant extracts caused a significant increase in sperm concentration. Sperm vitality significantly (p < 0.05) increased by radish seed (80mg/kg), flaxseed (300mg/kg) and date palm pollen (120, 240mg/kg) respectively. Total progressive motility improved significantly at flaxseed (300 mg/kg) (p < 0.001) as well as date palm pollen (p < 0.01). Histological examination of the cross sections of the testis showed clear presence of all stages of spermatogenesis in all the treated groups. Rat epididymides showed normal morphological appearance and their lumen were filled with spermatozoa. The diameter of seminiferous tubules in male rats exposed to date palm pollen (120 and 240 mg/kg) was significantly higher (p < 0.001). The heights of the germ cell epithelia within the seminiferous tubules were also significantly increased in all treated groups. Liver and renal functions tests showed a significant decrease in Alanine transaminase (ALT) and creatinine in all treated groups (p < 0.05), and this demonstrates the lack of cytotoxic effects of date palm pollen, radish seed and flaxseed on the rats. However, these plant extracts produced a non-significant (p > 0.05) increase in Aspartate transaminase (AST) levels. Besides this, superoxide dismutase activity (SOD) in testis was increased significantly by radish seed (160 mg/kg), flaxseed (200 mg/kg) and date palm pollen (120 mg/kg). There was also improved catalase activity in testis of male rats exposed to radish seed and date palm pollen. Regarding male sexual behavior, the time to reach the female and the mount frequency decreased significantly in male rats exposed to flaxseed (300 mg/kg) and date palm pollen (120 and 240 mg/kg; p > 0.05) thus, these plant extracts exhibit aphrodisiac properties. In addition, exposure of male rats to date palm pollen (120 mg/kg) produced a significant (p < 0.01) increase in the number of embryos in untreated female rats. In the female rats, the body weight gain was not affected (p > 0.05). However, the relative uterus weights exposed to nutmeg (200 mg/kg) and date palm pollen (120 and 240 mg/kg) were significantly decreased (p < 0.05). In addition, the relative weights of ovaries after treatment with nutmeg (400 mg/kg) and black seed (400 mg/kg) showed significantly increased values (p < 0.01). Serum FSH was significantly increased (p > 0.05 or 0.01) when the female rats have been exposed to black seed (200 mg/kg), nutmeg (200 mg/kg) or date palm pollen (120 mg/kg). The LH level significantly (p < 0.01) decreased following exposure to black seed (200 mg/kg), date palm pollen (120 mg/kg). On the other hand, serum LH concentration was significantly increased in female rats exposed nutmeg (400 mg/kg; p > 0.05). The creatinine activity in female rat serum in all treated groups was significantly decreased (p < 0.05). Whereas the higher dose of date palm pollen (240 mg/kg) caused only a non-significant decrease. ALT activity in serum of female rat exposed to either black seed (400 mg/kg) or date palm pollen (120 and 240 mg/kg) was shown to decrease significantly (p < 0.05). Histology of the reproductive organs, kidney and liver in the female rats showed no obvious alterations in any of the treated groups. In addition, the number of embryos in female rats significantly increased (p < 0.01; p < 0.001) following exposure of female rats to black seeds 400 and date palm pollen 240 mg/kg, respectively. Incubation of TM3 Leydig cells with radish seeds for 24, 48 or 72 hours caused a significant (p < 0.01) decrease in mitochondrial dehydrogenase activity. Besides that, date palm pollen and flaxseed increased the mitochondrial dehydrogenases activity of TM3 Leydig cells. In addition, higher concentration of date palm pollen, nutmeg and black seed were cytotoxic to MCF7 breast cells. In testis slices testosterone secretion in vitro was significantly increased by flaxseed (500 µg/ml; p > 0·05) and date palm pollen (500 µg/ml; p > 0·01). MCf-7 cells treated with BS 10-50 µg/ml black seed and nutmeg 10-50µg/ml significantly increased cell proliferation. However, the treatment with date palm pollen produced only a weak estrogenic effect, which resulted in a concentration dependent significant increase as observed between 50-1000 µg/ml date palm pollen. In conclusion, in this study, we observed that date palm pollen, radish seed and flaxseed increased libido as well as steroidogenesis and spermatogenesis, improved hepato and nephron-protective effects. In female rats, the plant extracts NM, BS and date palm pollen potentiated the production of gonadotropic hormones. In addition to this, at lower concentrations these medicinal plants promoted cell growth, whereas at higher concentrations they inhibited cell proliferation of MCF7 breast cancer cells. The anti-oxidant effects of these plant extracts have been implicated for the above mention effects.
Modulation of colon carcinogenesis by dietary ω-6/ω-3 fatty acid ratios : a chemopreventive strategy
(University of the Western Cape, 2015) Abrahams, Celeste H
The aim of this study was to determine whether dietary fats constituting specific ω-6/ω-3 fatty acids (FA) ratio has chemopreventive modulating effects on the development of colon cancer. Western diets intake of saturated FA (SATS) and ω-6 polyunsaturated FA (PUFA) are very high relative to low ω-3 PUFA consumption. This high ω-6 and low ω-3 FA intake, resulting in a high ω-6/ω-3 FA ratio, appears to have a promoting effect on disease outcome, whilst increased ω-3 FA intake exhibiting anti-cancer effects. An animal cancer model was employed to evaluate the effects of dietary fat ratios on chemically induced carcinogenesis during cancer promotion. This was to determine whether the FA diets have a promoting or inhibitory effect on early neoplastic lesions by quantifying aberrant crypt foci (ACF) development and monitoring the crypt cells proliferative and apoptotic indices. The expressions of genes associated with changes in cells redox balance were also assessed. Common dietary fats were combined to produce the dietary fat ratios: sunflower oil (S), borage oil (B) and fish oil (F). Combinations of these oils generated the different ω-6/ω-3 FA ratios: SB (ω-6/ω-3: 38:1), SF (ω-6/ω-3: 13:1) and SBF (10:1). To represent the Western diet’s high ω-6/ω-3 FA ratio profile, S (ω-6/ω-3: 501: 1) was used as a control, and canola oil and olive oil as additional reference. The dietary fats had no toxic effects on the liver and kidney based on serum clinical biochemical measurements. Diets containing borage oil (SB and SBF diets), canola and olive oil decreased (p<0.05) the crypt multiplicity of large (7 crypts/focus) ACF, exhibiting anti-cancer effects by decreasing (p<0.05) the proliferative activity of the rat colon crypts. Borage oil’s protective effect resulted from the enhanced supply of C18:3ω-6 that has anti-inflammatory and anti-proliferative properties. The observed decrease (p<0.05) in apoptosis in the ACF was also facilitated by the up- and downregulation of DNA repair and DNA replication associated genes, Xpa and Ercc2 by borage oil, respectively. Canola oil and olive had the largest inhibitory effect on suppressing crypt multiplicity by reducing (p<0.05) proliferation in the colon. Both oils effected the up-regulation (p<0.05) of the expression of several oxidative stress and anti-oxidant defence genes mediating the regulation of cell proliferation. The increased supply of C18:1ω-9 (canola and olive) and total polyphenolic content (olive) protected cells against oxidative stress induced apoptosis, which provided interesting interactive effects between FA and polyphenolic oil constituents that should be further elucidated. In contrast, the fish oil containing (SF diet) and the control sunflower (S diet) increased (p<0.05) the total ACF and colon crypt multiplicity (7 crypts/focus) when compared to the SB, SBF, olive oil and canola oil diets. An increased resistance to oxidative stress induced apoptosis appears to facilitate fish oil’s enhancing effect on crypt multiplicity despite the increased supply of LC ω-3 FA, which are prone to oxidation and leads to increased oxidative stress. This protective effect on crypt multiplicity and ACF development was mainly due to enhanced cellular antioxidant and DNA repair responses through the up-regulation (p<0.05) of Gpx4 and Nudt1, which favoured the increase (p<0.05) of crypt cells proliferation. The in vitro study demonstrated that oil ratio emulsions (S: ω-6/ω-3 = 249:1; SB: ω-6/ω-3 = 28:1; SF: ω-6/ω-3 = 12:1 and SBF: ω-6/ω-3 = 12:1) had differential effects on the survival indices of HT-29 and Caco-2 colon cancer cells. Contrary to the in vivo model, fish oil (SF and SBF emulsions) significantly (p<0.05) reduced the viability and proliferation of both cell lines, with the HT-29 cells showing greater sensitivity to the oil’s anti-proliferative effect. The HT-29 cells exposure to increased levels of C20:5ω-3 and C22:6ω-3 predisposes it to lipid peroxidation that increases the potential for cell removal via apoptosis. However, apoptotic effects were absent due to the HT-29 cells removal via necrosis as the cells energy status (ATP production) was significantly (p<0.05) depleted. Similar to the animal cancer model, borage oil (SB and SBF emulsions) had a reducing (p<0.05) effect on cell proliferation in both cell lines. However, as ATP was decreased (p<0.05), the S, SF and SBF emulsions resulted in an increased (p<0.05) apoptotic response in the Caco-2 cells in a dose dependent manner. This response resulted from the altered FA and lipid composition effected by the oil emulsions. Increased (p<0.05) incorporation of C20:5ω-3 and C22:6ω-3 in membrane phospholipid, phosphatidylethanolamine (PE), resulted in a significant decrease (p<0.05) in total SATS and MUFA content. A decrease (p<0.05) in membranes ω-6/ω-3 FA ratio was noted as well. This effect seems to selectively favour the induction of apoptosis by borage oil (SB and SBF). Similarly, an increase (p<0.05) in the PC/PE ratio by all oil emulsions, and a decrease (S and SB) and increase (SF and SBF) (p<0.05) in the chol/PL ratio appears to facilitate apoptosis too. A different threshold of the FA and lipid composition parameters elicits the inhibition of cell proliferation utilising lower oil emulsion concentrations. Therefore, the dietary supply of fats characterised by a defined low ω-6/ω-3 FA ratio can selectively modulate the growth indices of colon cancer. Specific oil ratio combinations by incorporating borage oil and fish oil hereby provide a selective strategy for chemoprevention in the colon, although underlying interactions and threshold effects of specific FA seems to prevail that should be further unravelled.
The frequency and characterization of streptococci in aerobic vaginitis (AV) and its association with pregnancy outcomes
(University of the Western Cape, 2014) Kaambo, Eveline
Aerobic vaginitis has been implicated in preterm delivery of low birth weight infants. The presence of aerobic vaginitis (AV) -associated bacteria such as group B streptococci (GBS) and E. faecalis in pregnant women are regarded to be the leading cause of neonatal mortality and morbidity world-wide. The aim of the study was to detect the prevalence of AV and its associated bacteria with preterm delivery in the Western Cape, South Africa. Furthermore, it sought particularly to examine and investigate the predictive value of GBS and E. faecalis for preterm delivery (PTD). It also aimed to establish other factors which may predict adverse pregnancy outcomes. Three hundred and one pregnant women were recruited from four different antenatal in the Western Cape, South Africa. The study conformed with the Declaration of Helsinki (2013). Maternal data was collected from a questionnaire and maternal medical records. Vaginal and rectal swabs were collected and microscopically examined for AV, followed by culture characterization of GBS and E. faecalis. Antimicrobial susceptibility testing was also performed. In this study, AV was detected in 79 (26.2%) of the 301 pregnant women, and GBS and E. faecalis isolated from 50 (16.6%) and 21 (7.0%) respectively. GBS serotype V was the predominant serotype, followed by serotype III. Pulse field gel electrophoresis (PFGE) profile analysis for both GBS and E. faecalis yielded a total of 24 restrictions profiles for GBS and 16 for E. faecalis. Multivariable analysis revealed that parity, gravidity, vaginal discharge, urinary tract infection, and smoking were significantly associated with PTD. The results from the study provides improved guidelines maternal screening of pregnant women. The early detection of AV-related bacteria may significantly reduce maternal and neonatal morbidity.
Ursolic acid and oleanolic acid as novel therapeutic agents in breast cancer
(University of the Western Cape, 2015) Abukhattala, Emhemed Mohamed
Breast cancer is one of the most common cancers among women in South Africa and the second leading cause of cancer death after lung cancer. According to the American Cancer Society 2015, women have a 12% chance of developing invasive breast cancer and a 3% chance of dying from it. Despite the wide variety of breast cancers e.g. lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS), many share the same etiology and target tissue. Estrogen related carcinogenesis with regard to breast cancer typically results from the activation of distinct signalling pathways. These pathways are not mutually exclusive and are often constituted by receptor mediated stimulation of cell proliferation caused by specific transcriptional gene activation, reactive oxygen species (ROS) formation causing DNA damage and consequently mutations. The molecular pathways that cause drug resistance are not fully understood and the search continues to find novel targets for treatment. The effects of non-toxic triterpenes, oleanolic acid and ursolic acid and the role of autophagy and apoptosis as mechanisms to overcome drug resistance in breast cancer were studied in vitro in MCF-7 breast cancer cells and MCF10A breast cells. In this study the first aim was to establish the influence of OA and UA on cell growth and to see if opposing proliferation patterns could observed between the presumably ERɑ negative (ERɑ/ß - /+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells. This was followed by morphology studies to establish the possible presence of cytotoxicity and examination of molecular pathways contributing to the anti-cancerous properties of UA and OA and their validity as therapeutic agents. The MCF-7 breast cancer cell line and the immortalized normal mammary cell line, MCF-10A were treated with different concentrations of UA and OA for 6hrs, 12hrs, 24hrs, 48hrs, and 72hrs respectively. Cell morphology was studied in hematoxylin and eosin as well as Hoechst and acridine orange stained cells and viability was measured using crystal violet staining. Molecular techniques employed included the Tali® Apoptosis - and the cellROX assays, flow cytometry and western blotting. Morphological, viability and apoptotic studies have shown that at their lowest concentration, both UA and OA have anti-proliferative and apoptotic effects on MCF-7 and to a lesser extent on MCF-10A. Flow cytometric analysis of treated cells has demonstrated cell arrest in the S- and G2/M phase. The MCF-7 and MCF-10A cells growth inhibition effect may be due to increased autophagy and apoptosis as an alternative to decreased proliferation in MCF-7 cells. This possibility should be evaluated in further studies. The results showed that UA was more effective OA in decreasing cell numbers and it may be applied as treatment for breast cancer. Our observation has shown the treatment with OA and UA increased cell death in MCF-7 cells. The opposing proliferation patterns observed between the presumably ERɑ negative (ERɑ/ß -/+) MCF-10A and ERɑ positive (ERɑ/ß +/+) MCF-7 cells could possibly be ascribed to ERß forming homodimers that may facilitate proliferation, whereas ERɑ/ß heterodimers (expressed in 59% of breast cancers) are frequently associated with the ERɑ antagonising actions of ERß. The results indicate a trend towards biphasic and anti- proliferative effects of the reactants in breast cancer cells which may contribute towards the development of anticancer therapies. However, further work is must be done to identify the OA and UA mechanism(s) responsible for anticancer activity.
An evaluation of cancer biomarkers in normal ovarian epithelial cells and ovarian cancer cell lines
(University of the Western Cape, 2019) Fruka, Tayra
Introduction: Globally, there are over 190,000 new reported cases of ovarian cancers per annum. This comprises 3% to 4% of all cancers in women. Ovarian cancer is one of the leading causes of deaths in women. Ovarian cancer is the second most diagnosed gynaecological malignancy and over all the fifth cause leading to death among all types of cancer in the UK in 2004. More than 70% of epithelial ovarian cancers are diagnosed at an advanced stage. Consequently, the prognosis is poor and the mortality rate high. Thus, the survival rate is affected by how far the disease has progressed or spread. A dire need exists to identify ovarian cancer biomarkers, which could be used as good indicators of expression in ovarian cancer cells in vitro. Aim: The aim of this study was to analyse selected cancer biomarkers, which are currently under intense investigation for their suitability to diagnose epithelial ovarian cancer at an early stage. These biomarkers were analysed in terms of their in vitro expression in normal epithelial cells and ovarian cancer cell lines, which allows for their genomic and proteomic classification. The expression analysis of each biomarker is related to the malignancy of a tumour and, therefore, advocates its use for potential future improvement of sensitive tumour markers. Methods: The primary human ovarian surface epithelial cell line (HOSEpiC), SKOV-3 cells and the OAW42 human epithelial ovarian tumour cell lines were used to evaluate the selected cancer biomarkers. Cells were cultured using appropriate media and supplements, and real-time quantitative polymerase chain reaction (RT-PCR) utilized to validate expression levels of the following genes: HDAC1, HDAC2, HDCA3, HDAC5, HDAC6, HDAC7, HDAC8, LPAR1, LPAR2, MUC16 and FOSL1, against normal housekeeping genes GAPDH and HPRT. In addition, immunocytochemistry was also used in the validation process of the aforementioned genes. Significance: ovarian cancer cells express gene signatures, which pose significant challenges for cancer drug development, therapeutics, prevention and management. The present study is an effort to explore ovarian cancer biomarkers to provide a better diagnostic method that may offer translational therapeutic possibilities to increase five- year survival rate. Results: HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 expressed distinctively in ovarian cancers matched to other tissues or cancer types have already been identified by RT-QPCR and confirmed by immunocytochemistry and efforts to generate monoclonal antibodies to the other six genes (HDAC1, HDAC2, HDAC3, HDAC7, HDAC8 and FOSL1) encoded proteins are underway. Conclusions: here we provide strong evidence suggesting that HDAC5, HDAC6, LPAR1, LPAR2, except MUC16 are up regulated in ovarian cancer. These data were confirmed by examining Human Protein Atlas (HPA) databases, in addition to protein expression of HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 in cells cytoplasm. For future prospective, using other techniques that assess the variant expression that could explain the release of these gene candidates into the circulation with serum tumour markers, and protein expression will be strengthened.
Phenotypic and molecular characteristics of Methicillin-resistant Staphylococcus Aureus isolates from stored patient samples in Misurata hospitals and poultry from commercial markets, Libya
(University of the Western Cape, 2019) Elakrout, Alhussien Ali
The emergence of virulent and drug-resistant bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA) is a global public health burden. The World Health Organization (WHO) has placed MRSA and vancomycin-intermediate-sensitive S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) on a high global priority pathogens list of antibiotic-resistant bacteria to promote the research and development of novel and effective antibiotic therapeutic rationales. Uncomplicated S. aureus bacteraemia (e.g., mild skin infections) may be treatable with the conventional regimens of antibiotics, but resistance strains of the bacteria (e.g., invasive infections), often persist as a high load of bacterial DNA in blood, and has been linked to increased mortality in world populations, irrespective of country or location. Several lines of evidence imply that combinations of vancomycin (a glycopeptide antibiotic that targets cell wall synthesis) and ß-lactam antibiotics that target the penicillin-binding proteins (PBPs) improve clearance of MRSA bloodstream infections (BSIs). However, acquired resistance to virtually the entire spectrum of ß-lactams hinders the therapeutic benefit of such antibiotic combinations. Hospital/health care-associated resistant S. aureus infections (HA-MRSA) and community-associated resistant S. aureus infections (CAMRSA), continue to be among the most common and challenging life-threatening infections worldwide. Furthermore, the increased rate of resistance among MRSA and vancomycin resistant Enterococcus (VRE) underscore the dire need for the discovery of novel anti-MRSA and anti-VRE compounds. The degree of ß-lactam resistance varies among clinical MRSA isolates, particularly with regard to those mediated by chromosomal mutations and the novel exogenous resistance gene which encodes PBP2a, i.e., mecA. PBP2a is the key resistance factor of ß-lactams, but the evolution of this mecA gene product and its mechanisms remain elusive. Nonetheless, it is widely accepted that in MRSA, PBP2a reduces the binding affinity to ßlactam antibiotics, rendering them ineffective. Besides HA-MRSA and CA-MRSA, colonization by livestock-associated MRSA (LA-MRSA) presents a major threat to both animal and human health, e.g., MRSA clonal complex (CC) 398 has spread from pigs to humans, but rarely from person to person. Even though LA-MRSA CC398 has been deemed less virulent than other MRSA strains, it particularly colonizes pig farmers. Recent studies indicate that an increasing number of people are being infected with LA-MRSA CC398. LAMRSA in mink is considered a human health hazard to farmers and farm workers, who handle the animals and are at risk of bites and scratches from colonized sites. Likewise, MRSA is also present in rabbits, cattle (e.g., dairy cows) and poultry, and, as such, has become an emerging threat to public health because of the spread from animals to humans via animal husbandry, and the health care and food processing industry. Generally, the irrational use of antibiotics is the main cause of the emergence of antibiotic resistant S. aureus strains. Globally, in both developed and developing countries, MRSA represents a serious public health concern because of the rapid spread of this bacterium around the world coupled with the evolution of new genetically distinct HA-MRSA, CA-MRSA and LA-MRSA strains. Additionally, the development of cross-resistance to other non-ß-lactams, including vancomycin, has only exacerbated the burden of MRSA infections. In many countries such as the USA, UK, Europe and Iran, the MRSA epidemic has been well documented. However, in South America and Africa, there seems to be a paucity on available data concerning MRSA strains. Equally, in Libya, detailed information is lacking regarding MRSA contagiousness risk, but conscious efforts are currently being directed at understanding the molecular epidemiology of S. aureus isolates, not only in terms of the persistence and spread of CA-MRSA, HA-MRSA and LA-MRSA, but also to focus awareness on prudent antibiotic prescription policy and use, as well as prevention and control of MRSA transmission in hospital, community and food production settings. The aim of this study was to compare antibiotic sensitivities of Staphylococcus aureus isolates in human patient samples from Misurata hospitals and laboratories as well as poultry samples from commercial markets in Libya. The objectives of the study were to (1) analyze laboratory and hospital samples from patients as well as poultry samples for bacterial and fungal growth, using standard bacterial isolate identification and antibiotic susceptibility tests; (2) compare traditional bacterial culture methods that are used to measure MRSA strains with modern molecular methods to isolate the mecA1 and mecA2 genes, using PCR; (3) determine the diagnostic profile of the bacterial and fungal species in patient and poultry specimens; (4) evaluate the Staphylococcus aureus antibiotic sensitivity and resistance profiles for patient and poultry samples; (5) compare Staphylococcus aureus antibiotic sensitivity and resistance profiles in patient specimens according to gender, age group and location (site collected); (6) compare Staphylococcus aureus antibiotic sensitivity and resistance profiles in poultry samples according to location and different parts of the chicken after slaughter; (7) identify and detect the MRSA contamination in chicken samples.
Human coronavirus NL63 envelope protein-protein interactions: role in pathogenesis
(University of the Western Cape, 2024) Schoeman, Dewald
Human coronaviruses (HCoVs) have garnered intense interest due to the large outbreaks caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which have resulted in severe respiratory disease and significant global morbidity and mortality. In contrast, four other HCoVs—HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1—are associated with milder, more frequent, and seasonal respiratory tract infections. Of the four canonical coronavirus structural proteins, the envelope (E) protein remains the least well-characterised, despite its critical functions in viral assembly and release, as well as its role as a major contributor to viral pathogenesis. Its contribution to viral pathogenesis has been strongly linked to a C-terminal PDZ-binding motif (PBM) that interacts with host post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (ZO-1) (PDZ) domain-containing proteins, facilitating immune dysregulation and hyperinflammation. Early work on SARS-CoV-1 revealed that the PBM of its E protein serves as a pathogenic determinant, which was later supported by SARS-CoV-2 studies that demonstrated similar immunopathological outcomes. This raised a central question: if all HCoVs possess an E protein containing a PBM, why do some induce severe disease while others cause only mild infections?
Antibody production with synthetic peptides against human Coronavirus-NL63 ORF3 protein
(University of the Western Cape, 2025) Mnyamana, Yanga Eddie
Human Coronavirus (HCoV) NL63 was detected in an infant in the Netherlands in 2004 and will hereafter be referred to as CoV-NL63. Four years later, it was classified into Group 1 coronaviruses by the International Committee for Taxonomy of Viruses (ICTV), which was modified into alphacoronavirus in 2009. It appears that CoV‐NL63 originates from bats and its hosts are bats and palm civets. A 27,553‐bp CoV‐NL63 genome structure and the spike glycoprotein (S1 domain) use angiotensin‐converting enzyme 2 (ACE2) for infectious entry. Furthermore, CoV‐NL63 simplifies binding to ACE2 by attaching to heparan sulphate proteoglycans on the cell surface. ACE2 has a significant expression in airway differentiated epithelial cells and CoV‐NL63 enter these cells from their apical surface. It is imperative to note that CoV‐NL63, CoV‐229E, CoVHKU1 and CoV‐OC43 infect polarized epithelial cells, and the entry and release of these viruses are related to apical cells in addition to respiratory signs, there are two non-respiratory symptoms including gastroenteritis and Kawasaki disease, both of which are associated with CoV‐NL63. Moreover, neurological manifestations of CoV-NL63 have been reported, especially in children. The main aim of this research study was to generate monoclonal antibodies (mAbs) against the human coronavirus NL63 Open Reading Frame 3 (ORF3) protein. Firstly, bioinformatic tools were used to assist in identifying antigenic determinants and antigenic epitopes were used to generate peptides of ORF3 protein. Thereafter, the ORF3 protein was characterized using various molecular techniques across the bacterial and mammalian expression systems. A CoV-NL63 full-length ORF3 protein (Protein Identity Number: ABE97138.1) was utilized for the synthesis of the peptide from the N-terminus and peptides conjugated with a MAP; KLH and MAP+KLH linkage on the carboxyl end. The mAbs were generated in vivo by immunizing BALB/c mice. Antigen-specific ELISA assessed the antibody specificity and sensitivity. The mAbs that exhibited the highest specificity and sensitivity for antigenic peptide (s) were used to characterize the ORF3 protein by Western blot and immunofluorescence analysis (IFA).
Assessment of the biological quality of raw and treated effluents from three sewage treatment plants in the Western Cape, South Africa
(University of the Western Cape, 2012-03-06) Hendricks, Rahzia; Pool, Edmund J.
The aim of this study was to compare the water quality of raw wastewater and treated sewage effluents from three different sewage treatment plants in the Western Cape, South Africa. The treatment plants investigated are on the same river system. Sewage treatment plant 1 and 2 use older technologies, while sewage treatment plant 3 has been upgraded and new technologies (membrane bioreactor) were incorporated in the treatment processes. The first objective was to determine the occurrence of total coliforms, Escherichia coli (E. coli) and fluoroquinolone and sulfamethoxazole antibiotic residues in raw wastewater and treated sewage effluents. Bacteria in treated sewage effluents can result in diseases such as dysentery, gastroenteritis, and typhoid upon exposure. A chromogenic test was used to screen for coliforms and E. coli. Enzyme linked Immunosorbent Assays (ELISA) were used to quantitate antibiotic residues (fluoroquinolones and sulfamethoxazole) in raw wastewater and treated sewage effluents. This study showed that bacteria are present in raw wastewater and residual bacteria are released with treated sewage effluents from sewage treatment plants.
Apoptotic markers in ejaculated human spermatozoa
(University of the Western Cape, 2005-12-01) Brooks, Nicole Lisa; Van der Horst, Gerhard; Dyer, Silke
The role of male germ cell death in spermatogenesis is an important one as it removes dysfunctional or genetically damaged germ cells and is necessary to maintain an optimal germ cell to Sertoli cell ratio. The formation of the bloodtestis barrier requires the elimination of excessive germ cells and a surge of germ cell apoptosis occurs prior to puberty regulating the ratio of germ cells to Sertoli cells. The aim of this study was to evaluate the presence of four apoptotic markers on sperm from patients with various grades of fertility using flow cytometry. Furthermore, any correlations between the apoptotic marker assays and the standard semen analysis results were identified. This study compares early and late parameters of apoptosis with morphological features in spermatozoa in the same samples. The three sample groups were identified as: teratozoospermic [G-pattern] (n=26), teratozoospermic [P-pattern] (n=98) and oligoteratozoospermic [Ppattern] (n=36). Standard semen analysis was conducted on the semen samples according to the WHO guidelines. Four apoptotic marker assays using flow cytometry was applied in this study to examine the apoptotic alterations in ejaculate sperm. These assays included the Annexin-V staining for the determination of phosphatidylserine exposure, APO-Direct to identify DNA fragmentation, caspase-3 to detect expression of this active protease during early apoptosis and Fas expression. For the Annexin-V and caspase-3 assays, statistically significant differences (P<0.05) were evident between the three groups. No significant differences (P>0.05) were found between the groups with respect to the APO-Direct assay. A significant difference (P<0.05) was found when comparing the teratozoospermic [G-pattern] group and the oligoteratozoospermic [P-pattern] group for the Fas assay. A strong positive correlation was evident between the Fas and the caspase-3 assays in the teratozoospermic [G-pattern] group. For the teratozoospermic [P-pattern group] the following positive correlations existed between the APO-Direct and the Fas assays, APO-Direct and caspase-3 assays and between caspase-3 and Fas assays. The only strong positive correlation was between the caspase-3 and APO-Direct assays in the oligoteratozoospermic [P-pattern] group. The presence of spermatozoa showing microscopic features resembling apoptosis has been identified in ten human ejaculate samples per sample group. Electron microscopy was used to identify morphological features of apoptosis in these human sperm samples. Classical apoptosis as observed in diploid cells could be identified in sperm and these included: loose fibrillarmicrogranular chromatin network, presence of vacuoles in the nuclear chromatin, membranous bodies within the vacuoles of the chromatin, partially disrupted nuclear membranes, plasma membrane protuberances and apoptotic bodies containing cytoplasmic vacuoles and dense masses. This study has confirmed that semen samples with abnormal semen parameters exhibit the presence of apoptotic markers in sperm. The identification of apoptotic markers on the sperm suggests that abnormalities occur during their developmental process, however, the exact mechanism thereof remains unclear. These findings may suggest that certain apoptotic markers may be an indicator of abnormal sperm function and possibly indicative of male infertility.

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