Browsing by Author "Meyer, M."
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Item The assessment of the bactericidal effect of green synthesized silver nanoparticles against a panel of infectious microorganisms(University of the Western Cape, 2016) Mokone, Mmola; Beukes, D. R.; Antunes, E. M.; Meyer, M.The emergence of multiple drug resistant microorganisms poses a major threat to human life. These microorganisms have made the currently used antibiotics ineffective and therefore continue to thrive. Therefore, there is a need for development of new, broad-secptrum antibiotics which is effective against almost every infectious microorganism. These antibiotics should ensure high effectiveness against the infectious pathogens while it is less detrimental to human health. Thus the search is channelled in nanoscience and nanotechnology in order to develop antibiotics that can kill infectious microorganisms effectively and hindering the development of drug resistance by these microorganisms. Nanoscience is the study of properties of a material when reduced to it smallest size (below 100 nm). It is a newly developing field of science which includes chemistry, physics and biology and has made it easy to synthesise nanomaterials for applications in many sectors of life including in medicine. The synthesis of nanoparticles can be achieved by physical and chemical methods. However, these methods are energy and capital intensive. Additionally, chemical method of synthesis uses chemicals that may be toxic which restrict the use of resultant nanoparticles in medicine. Therefore, there is a need for the use of eco-friendly methods of nanoparticle synthesis. The synthesis of silver and gold nanoparticles in this study was carried out by a green synthesis method, at room temperature, using an aqueous extract from the endemic brown alga Sargassum incisifolium. For comparison, commercially available brown algal fucoidans were also used to synthesise these nanoparticle, in addition to conventional methods of synthesis. The formation of nanoparticles was followed by the use of UV-Vis spectrophotometry. The characterization of the nanoparticles was done by TEM, XRD, DLZ and FT-IR. The rate of nanoparticle formation varied with specific reducing agent used. The faster reaction rate was recorded with S. incisifolium aqueous extracts pretreated with organic solvents while extracts obtained without this pretreatment produced slightly slower reaction rates. However, the commercially available fucoidans were less effective and required elevated temperatures for nanoparticle formation. Sodium borohydride reduction of silver nitrate was faster than the biological methods while the reduction of auric chloride by the S. incisifolium extracts and sodium citrate proceeded at similar rates. The nanoparticles synthesised with the help of the untreated aqueous extract were bigger than those synthesised from pre-treated extracts with both giving irregular shaped of nanoparticles. Also the nanoparticles formed from commercially available fucoidans were not of the same size, with bigger sizes recorded with Macrocystis fucoidan and smaller sizes with Fucus fucoidan. The shapes of nanoparticles from these fucoidans were spherical. From the conventional method, the nanoparticle sizes were smaller compared to the green synthesised nanoparticles and were predominantly spherical. The silver nanoparticles synthesised from the Sargassum aqueous extracts showed excellent antimicrobial activity against five pathogenic microorganisms including A. baumannii, K. pneumoniae, E. faecalis, S. aureus, and C. albicans. The gold nanoparticles were much less effective. To adequately assess the antimicrobial activities of the nanoparticles, it is or paramount importance to also asses their cytotoxicity activity. Three cell lines were used in this study namely, MCF-7, HT-29 and MCF-12a. The silver nanoparticles were found to be toxic to HT-29 and MCF-7 cell lines, exhibiting sligtly less toxicity against MCF-12a cells. The gold nanoparticles showed lower toxicity but a similar trend was observed.Item Identification of miRNA's as specific biomarkers in prostate cancer diagnostics : a combined in silico and molecular approach(University of the Western Cape, 2015) Khan, Firdous; Pretorius, Ashley; Meyer, M.There are over 100 different types of cancer, and each of these cancers are classified by the type of cell that it initially affects. For the purpose of this research we will be focussing on prostate cancer (PC). Prostate cancer is the second most common form of cancer in men around the world and annually approximately 4500 men in South Africa are diagnosed making PC a global epidemic. Prostate cancer is a type of cancer which starts in the prostate it is normally a walnut-sized gland found right below the bladder. PC follows a natural course, starting as a tiny group of cancer cells that can grow into a tumour. In some men if PC is not treated it may spread to surrounding tissue by a process called direct invasion/ spread and could lead to death. Current diagnostic tests for prostate cancer have low specificity and poor sensitivity. Although many PC's are slow growing there is currently no test to distinguish between these and cancers that will become aggressive and life threatening. Therefore the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity of current available diagnostic test is required. Biomarkers have recently been identified as a viable option for early detection of disease for example biological indicators ie. DNA, RNA, proteins and microRNAs (miRNAs). Since first described in the 1990s, circulating miRNAs have provided an active and rapidly evolving area of research that has the potential to transform cancer diagnostics and prognostics. In particular, miRNAs could provide potentially new biomarkers for PC as diagnostic molecules. Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible bio-fluids, having the potential to be useful as diagnostic, prognostic and predictive biomarkers. In this study we aimed to identify miRNAs as potential biomarkers to detect and distinguish between various types of PC in its earliest stage. The major objectives of the study were to identify miRNAs and their gene targets that play a critical role in disease onset and progression to further understand their mechanism of action in PC using several in silico methods, and to validate the potential diagnostic miRNAs using qRT-PCR in several cell lines. The identification of specific miRNAs and their targets was done using an "in-house" designed pipeline. Bioinformatic analyses was done using a number of databases including STRING, DAVID, DIANA and mFold database, and these combined with programming and statistical analyses was used for the identification of potential miRNAs specific to PC. Our study identified 40 miRNAs associated with PC using our "in-house" parameters in comparison to the 20-30 miRNAs known to be involved in PC found in public databases e.g. miRBase. A comparison between our parameters and those used in public databases showed a higher degree of specificity for the identification PC-associated miRNAs. These selected miRNAs were analysed using different bioinformatics tools, and were confirmed to be novel miRNAs associated with PC. The identified miRNAs were experimentally validated using qRT-PCR to generate expression profiles for PC as well as various other cancers. Prostate lines utilised in this study included PNT2C2 (normal) which was compared to BPH1 (Benign) and LNCaP (Metastatic). In the study the expression profiles of eight potential miRNA biomarkers for the detection of PC was determined using qRT-PCR, and to distinguish PC from other cancers. QRT-PCR data showed that miRNA-3 and -5 were up-regulated in the BPH1 and LNCaP when compared to PNT2C2. In addition miRNA-8 was also shown to be up-regulated in LNCaP. Based on these results it was shown that a miRNA profile could be established to distinguish between BPH1 and the LNCaP prostate cell lines. The results suggest that one miRNA as a diagnostic marker may be sufficient to differentiate between different cancer cell lines. Furthermore by creating a unique profile for each cancer cell line by using a combination of miRNAs could be a suitable approach as well. Finally, it was shown that through the use of a single or combination of all eight miRNAs a unique profile for all the cancer cell lines tested in this study can be created. This is an important finding which could have potential diagnostic or prognostic implications in clinical practice.Item In-silico optimization and molecular validation of putative anti-HIV antimicrobial peptides for therapeutic purpose(University of the Western cape, 2016) Tincho, Marius Belmondo; Pretorius, A.; Meyer, M.; Morris, T.AIDS is considered a pandemic causing millions of deaths worldwide and a cure for this disease is still not available. Failure to implement early treatments due to the poor diagnostic methods and ineffective therapeutic regimens to treat HIV patients to achieve complete viral eradication from the human body has encouraged the escalation of this disease at an exponential rate. Though the current treatment regimens (High Active Antiretroviral Therapy) have aided in increasing the lifespan of HIV patients, it still suffers from some shortcomings such as adverse side effects and non-eradication of the virus. Thus, there is a need for a non-toxic therapeutic regimen to stop further infection of HIV-infected patients. Antimicrobial Peptides (AMPs) are naturally occurring peptides which are components of the first line of defence of many organisms against infections and have been proven to be promising therapeutic agents against HIV. The use of AMPs as anti-microbial agents is due to the fact that most AMPs have a net positive charge and are mostly hydrophobic molecules. These features allow AMPs to be site directed electro-statistically to the mostly negatively charged pathogens. In a previous study, a number of novel anti-HIV AMPs was identified using a predictive algorithm Profile Hidden Markov Models (HMMER). The AMP's threedimensional structures were predicted using an in-silico modelling tool I-TASSER and an insilico protein-peptide interaction study of the AMPs to HIV protein gp120 was performed using PatchDock. Five AMPs were identified to bind gp120, at the site where gp120 interacts with CD4 to prevent HIV invasion and HIV replication. Therefore, the aims of this research were to perform in-silico site-directed mutation on the parental anti-HIV AMPs to increase their binding affinity to the gp120 protein, validate the anti-HIV activity of these peptides and confirm the exclusivity of this activity by testing possible anti-bacterial and anti-cancer activities of the AMPs. Firstly, the five parental anti-HIV AMPs were used to generate mutated AMPs through insilico site-directed mutagenesis. The AMPs 3-D structures were determined using I-TASSER and the modelled AMPs were docked against the HIV protein gp120 using PatchDock. Secondly, an "in house" Lateral Flow Device (LFD) tool developed by our industrial partner, Medical Diagnostech (Pty) Ltd, was utilised to confirm the in-silico docking results. Furthermore, the ability of these AMPs to inhibit HIV-1 replication was demonstrated and additional biological activities of the peptides were shown on bacteria and cancer cell lines. In an effort to identify AMPs with increased binding affinity, the in-silico results showed that two mutated AMPs Molecule 1.1 and Molecule 8.1 bind gp120 with high affinity, at the point where gp120 bind with CD4. The molecular binding however showed that only Molecule 3 and Molecule 7 could prevent the interaction of gp120 protein and CD4 surface protein of human cells, in a competitive binding assay. Additionally, the testing of the anti-HIV activity of the AMPs showed that Molecule 7, Molecule 8 and Molecule 8.1 could inhibit HIV-1 NL4-3 with maximal effective concentration (EC₅₀) values of 37.5 μg/ml and 93.75 μg/ml respectively. The EC₅₀ of Molecule 8.1 was determined to be around 12.5 μg/ml. This result looks promising since 150 μg/ml of the AMPs could not achieve 80% toxicity of the human T cells, thus high Therapeutics Index (TI) might be obtained if 50% cytotoxic concentration (CC₅₀) is established. Further biological activity demonstrates that Molecule 3 and Molecule 7 inhibited P. aeruginosa completely after 24 hours treatment with peptide concentrations ranging from 0.5 mg/ml to 0.03125 mg/ml. Nevertheless, moderate inhibition was observed when CHO, HeLa, MCF-7 and HT-29 were treated with these peptides at peptides concentration of 100 μg/ml. The ability of these AMPs to block the entrance of HIV via the binding to CD4 of the host cells is a good concept since they pave the way for the design of anti-HIV peptide-based drugs Entry Inhibitors (FIs) or can be exploited in the production microbicide gels/films to suppress the propagation of the virus.Item Investigating the effect of particle size on the antibacterial activity of gold nanoparticles(University of the Western Cape, 2017) Maphasa, Retsepile Ephraim; Dube, A.; Meyer, M.The increase of antibiotic- and/or multidrug-resistant bacteria has become a major global challenge. Killing of antibiotic-resistant bacteria requires a high dose of multiple, expensive drugs, which possess unfavourable side effects to the infected individuals. As a result, treatment of antibiotic resistant-bacteria is costly and more time is required to complete treatment. Therefore, novel substitutes are required to combat drug resistant infections while preventing further microbial resistance. Spherical gold Nanoparticles (sAuNPs) prepared using the citrate reduction method have been found to exert antibacterial activity against a number of gram positive and gram negative bacteria. However, there is still uncertainty regarding the role of size on the antibacterial activity of sAuNPs. The effect of exposure time on the antibacterial activity of sAuNPs is also still not well understood. In this study, it was hypothesized that AuNPs will show a size- and concentration-dependentantibacterial activity against selected gram positive (+) and gram negative (-) bacteria.Item Investigating the effect of particle size on the antibacterial activity of gold nanoparticles(University of the Western Cape, 2017) Maphasa, Retsepile Ephraim; Dube, A.; Meyer, M.The increase of antibiotic- and/or multidrug-resistant bacteria has become a major global challenge. Killing of antibiotic-resistant bacteria requires a high dose of multiple, expensive drugs, which possess unfavourable side effects to the infected individuals. As a result, treatment of antibiotic resistant-bacteria is costly and more time is required to complete treatment. Therefore, novel substitutes are required to combat drug resistant infections while preventing further microbial resistance. Spherical gold Nanoparticles (sAuNPs) prepared using the citrate reduction method have been found to exert antibacterial activity against a number of gram positive and gram negative bacteria. However, there is still uncertainty regarding the role of size on the antibacterial activity of sAuNPs. The effect of exposure time on the antibacterial activity of sAuNPs is also still not well understood. In this study, it was hypothesized that AuNPs will show a size- and concentration-dependentantibacterial activity against selected gram positive (+) and gram negative (-) bacteria.Item Oral medicine case book 67: Oral manifestations of Evans syndrome: a presenting feature of HIV infection?(South African Dental Association, 2015) Ranchod, S.; Jeftha, Anthea; Meyer, M.; Dreyer, Wynand P.;A 19 year old female presented with spontaneous intra - oral bleeding of two days duration. The patient reported that she was, until recently, in good general health and also that she had an uncomplicated parturition three years ago. She recently started noticing blood in her stools and felt increasingly lethargic. There was no history of trauma or intra-oral intervention that may have initiated the bleeding. The clinical examination revealed marked pallor of the facial skin and multiple small petechiae were seen on both of her forearms. The intra-oral examination identified marked halitosis and multiple haemorrhagic lesions with a variable appearance, being plaque-like on the lip, nodular on the tongue and fungating and exophytic on the palate and in the retromolar regions. Even delicate manipulation of the tissues produced profuse bleeding.Item Synthesis and characterization of zinc-doped magnetic nanoparticles for diagnostic studies(University of the Western Cape, 2015) Allard, Garvin Richard Johan; Onani, Martin; Mushonga, Paul; Meyer, M.In the present study we report the synthesis and characterization of iron oxide magnetic nanoparticles doped with zinc in an attempt to enhance the magnetic properties. The nanoparticles were prepared via the co-precipitation route and capped with 3-phosphonopropionic acid (3-PPA). The amount of zinc dopant was varied to yield nanoparticles with the general formula ZnxFe3-xO4 (x=0, 0.1, 0.2, 0.3, 0.4). Characterization was carried out using high resolution transmission electron microscopy (HRTEM), X-ray diffraction spectroscopy (XRD), energy-dispersive X-ray spectroscopy (EDS), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and superconducting quantum interference device (SQUID) analysis. Results from HRTEM, XRD and SQUID confirm that doping took place and x=0.2 was found to be the doping limit for these nanoparticles with a maximum size of 10.73 nm and saturation magnetization of 73.37 emu/g. The EDS further confirmed successful doping with zinc, while FTIR and TGA confirmed successful capping with 3-PPA. Despite agglomeration at all doping levels, these nanoparticles show great potential for application in breast cancer diagnostic studies.