Over-expression of FLO11 encoded mannoprotein in Saccharomyces cerevisiae.

Master of Science in Biochemistry. University of KwaZulu-Natal, Durban 2015.Abstract available in PDF file

Sub/supercritical fluid chromatography purification of biologics.

Doctoral Degree. University of KwaZulu-Natal, Durban.Peptide and protein drugs are highly versatile with numerous therapeutic properties such as anti-cancer, anti-diabetic, anti-hypertensive, and anti-microbial; which are therefore ideal candidates for the development of next-generation drugs. This is exemplified using these drugs for the treatment of diseases such as diabetes. Diabetes is one of the most prevalent non-communicable diseases worldwide. The rapid increase in the number of diabetic patients globally places a burden on current insulin manufacturers. The traditional reversed-phase high-performance liquid chromatography (RP-HPLC) purification methods of insulin and peptides are problematic, tedious with long run times of approximately 50 minutes, low yields employ harsh solvents such as acetonitrile, which has a negative impact on the environment. There is a need for a greener process for the purification of insulin and peptides. Sub/supercritical fluid chromatography (SFC) can provide the solution since it utilises greener mobile phases such as carbon dioxide (CO2) and methanol, which can be recycled. However, there is a paucity of knowledge regarding the SFC purification of human insulin and peptides. Therefore, this research study aimed to provide an efficient, innovative approach for the biosynthesis of human insulin and the SFC purification of biosynthesised human insulin, as well as the extension into the SFC purification of peptides. The background of these topics is presented in Chapter One. Chapter two (manuscript one) presents the development of a novel and more efficient method of human insulin biosynthesis in Escherichia coli (E. coli). Several of the conventional steps were eliminated. The crude biosynthesised protein sequence was verified using protein sequencing, which had a 100% similarity to the human insulin sequence. The biological activity of the biosynthesised human insulin was tested in vitro using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The biosynthesis of human insulin was conducted on a laboratory-scale basis; future studies should investigate scaling up of this method. Chapter three (manuscript two) was based on the SFC purification of the commercially available standard sample of human insulin and a crude biosynthesised sample of human insulin. The SFC purified standard the biosynthesised human insulin samples were detected and quantified using liquid chromatographymass spectrometry (LC-MS) and protein sequencing techniques. SFC columns, i.e., silica, 2’ ethyl pyridine, diol-HILIC, and the pentafluoro phenyl (PFP), were evaluated to determine the ideal column. The PFP column gave the best results since it displayed good peak shapes, resolution, retention factors, retention times, and the least relative standard deviation in comparison to the other columns. Therefore, the aforementioned column was selected for further analysis using the biosynthesised human insulin, whereby a column efficiency test was conducted on a semi-preparative scale, yielding 84% recovery. Subsequently, the biological activities of the SFC purified standard sample of human insulin and biosynthesised version were tested in vitro using a MTT assay. The results indicated that the biological activities of the standard and biosynthesised human insulin derivatives were retained subsequent to SFC purification. The biological activities were highly significant, with a p-value of < 0.0001. From chapter three, band broadening and phase separation peaks were experienced during SFC purification of the commercially available standard sample of human insulin and biosynthesised human insulin. Therefore, in Chapter four (manuscript three), a SFC purification method was developed to purify peptides at an analytical scale. A tetrapeptide [insulin β chain peptide (15-18)], octapeptide [angiotensin II], nonapeptide [insulin β chain peptide (15-23)] were purified using four SFC columns, i.e., PFP, diolHILIC, silica, and 2’ ethyl pyridine. Subsequently, the 2’ ethyl pyridine column was selected for further analysis based on the reproducibility, peak shapes, efficient separations and retention factors. The three peptides were monitored using LC-MS analysis. The successful peptide recoveries ranged from 80-102%. Chapter five pertains to the summary and conclusion drawn from the study and reflects on possible future endeavours. The present study was successful in providing a more affordable and innovative approach for the biosynthesis of human insulin. The work also successfully developed a rapid, greener, and more efficient method of SFC purifying biosynthesised human insulin and peptides as opposed to conventional HPLC purification methods. As far as we are aware, this study is the first of its kind to purify biosynthesised human insulin and this combination of peptides using SFC purification techniques. Future research studies can focus on the SFC purification of larger protein molecules and consider the use of custom columns and or other modifiers for the improvement of the isolation of other highly sought after biologics within the pharmaceutical industry

The analysis of alcohol content in hand sanitisers (in the Durban region) using gas chromatography-mass spectrometry during the COVID-19 pandemic

The COVID-19 pandemic has resulted in an unprecedented surge in the demand for alcohol-based hand sanitisers (ABHS). The Centre for Disease Control&nbsp; (CDC) and World Health Organisation (WHO) recommend alcohol, i.e., isopropanol or ethanol, at a 60-95% concentration in ABHS for sufficient antiviral&nbsp; protection. Consumers need to be vigilant of substandard hand sanitisers being marketed to the public. The frequent exposure of microorganisms to&nbsp; alcohol concentrations below the recommended range for infection prevention may lead to resistant mutations, and above the range may be ineffective.&nbsp; Therefore, this study aimed to verify the stated alcohol content in hand sanitisers from their respective labels. We analysed 50 hand sanitiser samples&nbsp; available to our region in Durban, KwaZulu-Natal, South Africa, using a Shimadzu GC-MS-QP2010 Ultra equipped with a Zebron ZB-wax capillary column.&nbsp; The hand sanitisers analysed had a range of 44–93% alcohol content. The data from our study also revealed that 32% (16) of hand sanitisers did not&nbsp; adhere to the stated alcohol indicated on their labels. 16% (8) contained &gt;80% and 12% (6) contained &lt;60%, while 6% (3) of the ABHS contained&nbsp; 1-propanol and ethyl acetate as contaminants, respectively. This study clearly emphasises manufacturers’ exploitation of the pandemic and the need for&nbsp; stricter guidelines and regulations for consistency amongst ABHS manufacturers. The public should also be more alert to the % alcohol stated (ideal&nbsp;&nbsp;&nbsp; range 60-80%) on the sanitizer bottle and note one needs to rub their hands together until it feels dry

Detection of insect-specific flaviviruses in mosquitoes (Diptera: Culicidae) in northeastern regions of South Africa

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.Gratama Fund; Uyttenboogaart-Eliasen foundation; RCN-IDEAS travel grant; National Research Foundation; The World Academy of Sciences; Global Health and Tropical Medicine; Professor Programme by the University of Pretoria, SA and Centres for Disease Control and Prevention.http://www.mdpi.com/journal/virusespm2022UP Centre for Sustainable Malaria Control (UP CSMC

Detection of insect-specific flaviviruses in mosquitoes (diptera: culicidae) in Northeastern regions of South Africa

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59) and Anopheles (5/17, 29.41). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species

An Overview of Repurposed Drugs for Potential COVID-19 Treatment

The COVID-19 pandemic caused by SARS-CoV-2 has placed severe constraints on healthcare systems around the globe. The SARS-CoV-2 virus has caused upheaval in the healthcare and economic sectors worldwide. On the 20th of May 2020, the World Health Organisation declared COVID-19 a global pandemic due to the unprecedented number of cases reported around the globe. As of the 4th of November 2022, there were 637,117,429 coronavirus cases reported globally by Worldometer stats, with 6,602,572 related deaths. In South Africa, there were approximately 4,029,496 coronavirus cases and 102,311 associated deaths. As such, there is a need for efficacious therapeutic regimes. There has been a paucity of knowledge encompassing the use of effective and specific antiviral drug therapies for treating COVID-19 since the outbreak. In this review, we provide valuable insights into the repurposing of current drugs for COVID-19. Drug repurposing provides a suitable option for the discovery of efficacious drugs for COVID-19, thereby decreasing the costs and turnaround times of drug development strategies. This review provides an overview of ten drugs, including antimalarial, antiparasitic, anti-inflammatory, nucleoside analogue, monoclonal-antibody drugs, that were repurposed for the potential treatment of COVID-19