College of Agriculture and Environmental Sciences
https://hdl.handle.net/10500/130
2024-03-28T16:59:01ZSupramolecular modification of isoniazid : an in vitro and in silico analysis of combination drug efficacy against mycobacteria species
https://hdl.handle.net/10500/30938
Supramolecular modification of isoniazid : an in vitro and in silico analysis of combination drug efficacy against mycobacteria species
Setshedi, Itumeleng Bridgette
Tuberculosis was for the longest of time a leading infectious killer instigated by a single pathogen until the emergence of SARS-CoV-2. The disease continues to be a major global health problem causing high rates of morbidity and mortality. Unfortunately, both the estimated disease incidence and mortality related to tuberculosis infections have been reported to be on the rise as treatment of the disease has been neglected due to a shift in attention to address the COVID-19 global pandemic. The remnants thereof still pose a direct negative impact on tuberculosis diagnosis and treatment, resulting in the partial reversal of any progress that had been made in the quest to end tuberculosis prior to the global COVID-19 pandemic. It is presumed that the challenges around tuberculosis treatment and misdiagnosis are as a result of the continuous ability of the pathogen to evade the host’s immune system and development of resistance against tuberculosis drugs. However, diagnosis seems to be the weakest link contributing to delayed treatment thereof.
If detected early, tuberculosis can be cured. However, if tuberculosis is not detected, it definitely cannot be treated; and, if it cannot be treated, it certainly cannot be eradicated. In addition, there is also an emergence of nontuberculous mycobacteria which are caused by mycobacteria species other than those belonging to the Mycobacterium tuberculosis complex. Some of these organisms cause pulmonary infections indistinguishable to those caused by M. tuberculosis infections, presenting with pulmonary and extrapulmonary tuberculosis-like diseases. The prevalence of these diseases has steadily gained traction in recent years, reported and isolated from clinical isolates around the world. Yet, these mycobacteria are not targeted when screening for tuberculosis in clinical isolates. This is a public health problem as nontuberculous mycobacteria are generally resistant to the tuberculosis treatment regimen used in screening assays and if not screened, they can easily be misdiagnosed as Mycobacterium tuberculosis. If there are no obvious ways in which these organisms can be distinguished during diagnosis, it would be beneficial if drugs that are designed could target both Mycobacterium tuberculosis and nontuberculous mycobacteria as nontuberculous mycobacteria are not susceptible to first- and second-line tuberculosis treatment regimens.
The aim of this study was to design and synthesise isoniazid derivatives that are effective against clinically infectious Mycobacterium tuberculosis as well as nontuberculous mycobacteria isolates. To achieve this, methods such as the one-pot reflux method were
employed for the synthesis of the isoniazid derivative. Crystallographic data were collected using the Bruker APEX II CCD area detector diffractometer. Data were reduced and corrected using the SAINT-Plus software and the SADABS software, respectively. Diagrams were generated using WinGX and PLATON software. To test for the efficacy of the derivatives against mycobacteria of choice (M. tuberculosis H37Ra, M. bovis BCG, M. smegmatis, M. avium and M. fortuitum) in comparison to the efficacy of isoniazid and that of rifampicin, a number of disciplines with varying techniques were employed and these include microbiology, where minimum inhibitory concentration experiments were conducted, and cell biology where cell viability was assessed using real-time cell analysis techniques and flow cytometry analysis conducted using the xCELLigence RTCA system and the BD FACSArai™ III cell sorter, respectively. The potential safety of these compounds was also assessed on RAW 264.7 murine macrophage cells where methods such as the MTT assay and real-time cell analysis assay as well as flow cytometry assay (Muse® Cell Analyzer) were employed to conduct cell viability assessment. Lastly, molecular docking and modelling experiments were conducted to determine the target protein and binding of the ligands thereof (protein-ligand binding).
Unit cells were collected for all nineteen synthesised derivatives, where nine have been previously synthesised and ten were found to be novel. Of the ten, six were refined successfully and five of those published in peer-reviewed journals. All derivatives were screened against mycobacteria of choice and only a few were found to have inhibitory effects against some of the mycobacteria with some noteworthy concentrations varying between 0.25 μg/mL and 11.36 μg/mL as single compounds, and 0.09 μg/mL and 0.59μg/mL for combination drug treatments. Isoniazid was comparable against single and combination derivatives with concentrations ranging between 3.9 μg/mL and 22.73 μg/mL, and rifampicin gave competitive results at concentrations ranging between 0.16 μg/mL and 15.65 μg/mL against single and derivatives. In most cases, the derivatives were observed to cause direct apoptotic effects on mycobacterial cells at competitive percentages of between 59% and 90%, thus better than the apoptosis percentage induced by isoniazid and rifampicin of less than 10%. Nonetheless, the related apoptosis results were in accordance with RTCA and flow cytometry results that saw most derivatives posing less toxicity against murine macrophage cells and the ability of the said cells to recover as compared to when exposed to isoniazid and rifampicin. The molecular modelling results also saw a few molecules and combinations possessing exceptional binding abilities with the catalase-peroxidase enzyme with no degree of displacement and good drug residence time, thus suggestive of some derivatives being effective mycobacteria inhibitors targeting the catalase-peroxidase enzyme with docking scores ranging from -4.879 kcal/mol, -5.390 kcal/mol, to -6.065 kcal/mol.
2023-10-12T00:00:00ZThe ecological assessment of the influence of anthropogenic activities on Palala River, Limpopo, South Africa
https://hdl.handle.net/10500/30835
The ecological assessment of the influence of anthropogenic activities on Palala River, Limpopo, South Africa
Sibiya, Sifundile
Water quality integrity deterioration is a severe global issue due to urbanisation, population growth, pollution and other anthropogenic activities threatening freshwater integrity. Anthropogenic activities alter freshwater integrity which leads to negative impacts on general ecological functioning of rivers. The current study was conducted along the Palala River in the Waterberg district in Limpopo, South Africa. The study was designed to employ multivariate and multimeric methods to determine relationships between environmental variables and macroinvertebrate communities. Macroinvertebrates were used to assess spatial and temporal changes in water quality using SASS5 (South African Scoring System version 5). The ecological status of the Palala River was clearly revealed through measuring of nutrient concentrations and environmental variables that influence water quality, and the macroinvertebrate communities that are found within the river. The results indicated the water quality was significantly impacted by changes in chloride concentrations at the site which had the most human interactions. The multivariate analysis revealed that macroinvertebrate communities were impacted by changes in the concentrations of chlorides (Cl-), total dissolved solids (TDS), as well as electrical conductivity (EC). Additionally, simple linear regression indicated that the abovementioned environmental variables had an impact on Taxa richness, total abundance and taxa diversity. The river was revealed to be in a natural state as it drains through upstream (P1 and P2) and midstream sampling sites (P3 and P4). Interestingly, there was a massive improvement in water quality as the river drains within Lapalala Wilderness Reserve (sites P3 and P4). Unfortunately, there was a dramatic decrease in water quality as the river exited the reserve draining through downstream sites P5 and P6. The sampling site P5 was highly impacted by human settlements and domesticated livestock increasing nutrient concentrations in the river. The results revealed that an increase in chloride concentrations affected macroinvertebrate abundances at sampling site P5. Sampling site P5 was dominated by highly tolerant taxa at 75%, intermediate 25% and 0% sensitive taxa. The tolerant animals such as Chironomidae, Hydracarina and Ceratopogonidae were found in abundance at this site. The highest recorded SASS5 score was 165 at sampling site P4 and lowest was 80 at sampling site P5. The highest ASPT score was 8,5 at sampling site P3 and lowest was 3.8 at sampling site P6. The scores indicated that the river was severely impaired at sites P5 and P6.
2023-08-01T00:00:00ZExpression, purification, and characterisation of the Alpha-helical and Beta-sheet domains of Rotavirus VP6
https://hdl.handle.net/10500/30822
Expression, purification, and characterisation of the Alpha-helical and Beta-sheet domains of Rotavirus VP6
Strachan, Milaan
The capsid protein VP6 is of paramount importance to the stability and infectivity of Rotaviruses. Through interactions of VP6s’ beta-sheet (VP6) and alpha-helical (VP6) domains with the viral particle's outer- and innermost layers, respectively, VP6 stabilises matured Rotaviruses and activates transcription of the viral genome upon cell entry. This study focused on the individual domains of Rotavirus VP6. The aim of the study was to probe the structure and stability of VP6 and VP6 when expressed independently of each other. The objectives of the study were: (1) optimise the bacterial expression of VP6 and VP6 through modulation of the expression conditions (2) solubilise and then purify the domains by immobilised metal chromatography (IMAC), (3) characterise by means of spectroscopy (mass spectroscopy, far-UV circular dichroism (CD), and intrinsic tryptophan fluorescence spectroscopy) and gel electrophoresis (native-PAGE), the primary, secondary, tertiary, and quaternary structures of the domains, (4) characterise the conformational stability by means of spectroscopy (far-UV CD and intrinsic tryptophan fluorescence) of VP6 and VP6 when thermally and chemically challenged, and (5) determine the melting temperature by differential scanning calorimetry (DSC). To this end, two Escherichia coli strains BL21(DE3) and NiCo21 (DE3) were transformed with pET15a plasmids containing the codon-optimised DNA consensus sequences of VP6 and VP6. The expression of VP6 and VP6 was done at different temperatures (37°C and 20°C), inducer concentrations (1 × and 10 × IPTG), and post-induction incubation times (2 h – 7 h, and 16 h) in both E. coli strains and the outcomes were visualised by SDS-PAGE. All conditions tested produced the domains in an insoluble form and though expression levels appeared to be comparable between strains, the NiCo21 (DE3) was ultimately selected for further expression of the domains as expression could be induced with the lowest concentration of IPTG. The insoluble domains were subjected to a solubilisation study where the domains were frozen in various Tris-HCl buffers differing in pH (7 – 10) and urea concentration (0 M, 2 M, and 5 M) and thawed. The results of the solubilisation study showed that both domains could effectively be solubilized in 2 M urea, provided that the pH of the freezing buffer was at least one unit higher than the pI of the domain. The solubilised VP6 and VP6 were purified by nickel affinity chromatography in yields of 13.32 mg and 25 mg from 1 L of NiCo21 (DE3) culture, respectively and were confirmed by mass spectroscopy, far-UV CD, and intrinsic tryptophan fluorescence spectroscopy, to have native-like sequences and structural features. The quaternary analysis revealed that VP6 existed as a single monomeric species in solution while VP6 formed different-sized structures in solution. The conformational stability of VP6 and VP6 was demonstrated as the domains had resisted structural changes up to 46°C and 50°C, respectively and the DSC analysis revealed melting points of 67.94°C for VP6 and 68.55°C for VP6. The domains were noted to aggregate extensively which prevented the recovery of their native structures upon cooling. The chemical unfolding study was done in 1 M – 5 M guanidine hydrochloride (GdCl) and 1 M – 8 M urea, and revealed the chemical stability of the domains and their respective unfolding pathways. Approximately 1.5 M and 2.25 M GdCl were needed to denature 50% of VP6 and VP6, respectively. Urea concentrations of 4.5 M (VP6) and 4 M (VP6) also resulted in a 50% loss of native structures. The observation of non-cooperative unfolding pathways that differed between the spectroscopic probes suggested a complex unfolding process involving the formation of one or more intermediates. Though native-like structures could be recovered upon denaturant removal, the refolding and unfolding pathways differed, which was indicative of irreversibility. Overall, VP6 and VP6 were easily producible and purifiable in quantities suitable for further studies. Further investigations could highlight the potential applications the domains could have in vaccine development and drug-delivery. Non-cooperative folding indicated the necessity of interactions between VP6 and VP6 in the full-length protein for cooperative folding.
2023-08-20T00:00:00ZMetabolomic analysis of GM and non-GM maize and its preference by cattle
https://hdl.handle.net/10500/30693
Metabolomic analysis of GM and non-GM maize and its preference by cattle
Payne, Joseph
GM organisms have started to infiltrate South Africa's agricultural landscape, altering how
farmers deal with weeds and insects. Adopted from the United States of America, it is a
technology that the commercial farming sector cannot do without anymore and the uptake
was enormous in the last 10 years. The technology involves altering the DNA of an
organism to provide a specific benefit using genetic engineering techniques.
But all new technologies and inventions bring the responsibility of stewardship and
accountability with it, and it is impossible to predict all possible outcomes of such an
invention to every possible scenario in terms of environmental stewardship. The question
and occurrence of cattle and wild animals seemingly choosing non-GM containing maize
plants have raised the question whether this is really the case of the presence of the GM or
maybe it is just due to the differences in plant genetic makeup. Quicker deterioration of
short, seasoned cultivars opposed to the longer seasoned cultivar that is known to be more
resistant to diseases and plant pathogens, might therefore not be as easily affected by mainly
pathogens and stay much tastier for longer.
This study used beef cattle of the Drakensberger indigenous breed to determine the taste
preference with the use of a trial that is planted with the different types of maize cultivars
that includes GM and non-GM maize and short and longer seasoned cultivars. The trial was
performed on a commercial farm near Standerton, Mpumalanga in the high rainfall area of
South Africa. The results clearly indicated that cattle could not differentiate the GM maize
from the non-GM maize, but that they preferred the longer season cultivar.
The chemical profiles of different materials, including those made from GM and non-GM,
were analyzed using NMR spectroscopy and OPLS-DA and pre-processing techniques. The
results of the metabolomic study revealed that the former had slightly different
metabolomics profiles than the latter. The differences were mainly concentrated in the
aliphatic and sugar regions. On the other hand, the long and short-season GM had similar
metabolomic profiles.
The study therefore clearly indicates that cattle could not differentiate GM from non-GM
material on taste and that any material can be provided to cattle. The slight difference in the
metabolomics profiles however raise a concern for differential metabolomic pathways and
should be investigated further.
2023-01-01T00:00:00Z