College of Agriculture, Engineering and Science

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Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Comparative antidiabetic effects and mechanisms of actions of five Chinese and South African indigenous teas.
(2020) Xiao, Xin.; Islam, Shahidul.
The present thesis assessed the in vitro, ex vivo and in vivo anti-oxidative and antidiabetic activities of five teas which are widely consumed in China or South Africa. Three of the selected five teas are from South Africa, namely red rooibos (Aspalathus linearis), green rooibos (Aspalathus linearis) and red honeybush (Cyclopia genistoides) tea. The remaining two from China are jasmine green (Camellia sinensis) and zhengshanxiaozhong (ZSXZ) black tea (Camellia sinensis). The different sequential solvent extracts following increasing polarity index (dichloromethane, ethyl acetate, ethanol, and water) and hot water extract of different teas were evaluated at in vitro and ex vivo conditions for their antioxidant properties, inhibitory potentials on α-glucosidase, α-amylase and pancreatic lipase, effects on ameliorating Fe2+- induced oxidative pancreatic or hepatic injury, as well as the glucose absorption inhibition in small intestine and the glucose uptake stimulation in isolated psoas muscle of rats. Possible bioactive components responsible for the activities of the extracts were identified by using Gas Chromatography-Mass Spectrometry (GC-MS) analysis or liquid chromatograph-mass spectrometry (LC-MS) analysis. In vitro and ex vivo tests presented promising antioxidant and antidiabetic activities of these five teas. The red honeybush, jasmine green and green rooibos teas, were further subjected to an in vivo intervention trial in a fructose-streptozotocin (STZ) induced T2D model of Sprague-Dawley rats. Assays were carried out to reveal the effects of these teas on lowering blood glucose level, improving oral glucose tolerance ability, stimulating insulin secretion and hepatic glycogen synthesis and ameliorating some diabetes related parameters such as serum lipid profile, hepatic and renal function tests and calculated insulin resistance (HOMA-IR), β-cell function (HOMA-β) from the blood glucose and serum insulin data. Furthermore, in vivo oxidative stress markers such as reduced glutathione, superoxide dismutase and catalase activity and lipid peroxidation were analysed in harvested organs (liver, kidney, heart and pancreas). The results of in vivo tests demonstrated that high dose of jasmine green tea showing the best activity followed by the high dose of red honeybush tea, low dose of jasmine green tea, high dose of green rooibos tea, low dose of red honeybush tea, when lowest activity was observed for the low dose green rooibos tea. The results of this study indicated promising anti-T2D properties of the above-mentioned teas. However, further clinical trials are needed to ascertain the results of these in vitro, ex vivo and in vivo studies.
Cytotoxic and mechanistic studies of novel phenanthroline-derived oxovanadium(IV) complexes.
(2022) Barry, Kristy-Lyn.; Munro, Orde Quentin.
In this work, a new series of ternary oxovanadium(IV) complexes of the type [VO(ONO)(DPQ/DPPZ)], [VO(ONN)(PHEN/DPQ)](PF6) and [VO(ONN)(DPPZ)](Cl) have been synthesised and characterised for the purpose of developing novel anticancer agents. ONO represents a tridentate Schiff base ligand derived from salicylaldehyde and various 4-substituted-2-aminophenols. ONN represents a tridentate Schiff base ligand derived from 1-methyl-2-imidazolecarboxaldehyde and various 4-substituted-2-aminophenols. PHEN represents 1,10-phenanthroline, DPQ represents dipyrido[3,2-d:2′,3′-f]quinoxaline and DPPZ represents dipyrido[3,2-a:2′,3′-c]phenazine. The bidentate N,N-donor polypyridyl co-ligands were incorporated to enhance DNA binding and to stabilise the vanadium(IV) metal centre. The known N,N-bidentate ligands were synthesised and characterised by ESI-mass, 1H and 13C NMR spectroscopy. The tridentate O,N,O′ and O,N,N′ ligands were synthesised and characterised by ESI-mass, NMR, IR, and UV/visible spectroscopic techniques, elemental analysis and single crystal X-ray crystallography. Single crystal X-ray diffraction, 1H NMR and DFT simulations confirmed the O,N,N′ ligands in this work form dimeric hydrogen-bonded supramolecular structures that are stable in solution. The oxovanadium(IV) complexes were characterised by elemental analysis, UV/visible, FT-IR and EPR spectroscopy as well as mass spectrometry. Elemental analysis and mass spectrometry confirmed the identity and purity of the complexes. EPR spectroscopy confirmed the monomeric VIVO-bound species. IR and UV/visible spectroscopy confirmed coordination of the ligands to the metal centre. Highresolution solid-state structures were elucidated for the cationic complexes with PHEN coligands and the 4-chloro and 4-tert-butyl derivatives of the [VO(ONO)(DPQ)] complexes. The crystal structures of the complexes showed a monomeric vanadium(IV) species with the bidentate N,N-donor co-ligand and respective dianionic O,N,O′- or monoanionic O,N,N′-tridentate Schiff base ligand coordinated to the oxovanadium centre in a distorted octahedral geometry. The respective neutral VIVO3N3 and monocationic VIVO2N4+ species were formed. A least-squares fit of the solid-state and DFT-simulated (B3LYP-/6-311G(dp)) structures of the oxovanadium chelates indicate the experimental and simulated structures are in good agreement. DFT-simulated structures were determined for those complexes where X-ray data are not available. The geometry-optimised structures for the neutral and cationic complexes all indicate that the respective bidentate polypyridyl ligands are free from steric hindrance by the tridentate ligand and should be available to bind DNA, which is their proposed cellular target. DFT simulations indicated the neutral complexes have larger HOMO-LUMO energy gaps than the corresponding cationic complexes, suggesting that the neutral complexes are more stable with respect to ligand substitution than the cationic complexes. Experimental mass spectrometry and UV/visible spectroscopy confirmed slower solvolysis processes for the neutral complexes versus the cationic analogues. 51V NMR studies indicate partial oxidation of the vanadium(IV) species in DMSO to VVO2(ONO/ONN)(DMSO) analogues. The cationic complexes with PHEN and DPQ co-ligands were deemed suitable to proceed with absorption DNA binding studies. The cytotoxicity screening of the oxovanadium complexes in this work revealed that, in general, the neutral complexes with DPQ co-ligands are cytotoxic against the triple-negative breast cancer MDA-MB and neuroblastoma SH-SY5Y tumour cell lines and non-toxic towards the cervical cancer HeLa cell line. The charge of the complexes was found to influence the cytotoxic properties. The cationic complexes with PHEN and DPQ co-ligands are cytotoxic towards the HeLa cell line as well as the MDA-MB and SH-SY5Y cell lines. The neutral DPQ and cationic complexes with PHEN and DPQ co-ligands were found to be more cytotoxic towards MDA-MB cell lines than cisplatin and the cationic complexes were found to be more cytotoxic towards the HeLa cell line than cisplatin. Steric bulk of the Schiff base functional group influences cytotoxicity with larger functional groups, such as tert-butyl and sulfonyl, leading to lower cytotoxicity. The N,N-donor co-ligand and steric bulk of the Schiff base functional group also influenced the selectivity index of the cationic complexes. The cationic oxovanadium-DPQ complex with a methyl substituent on the tridentate ligand is significantly more toxic to the carcinoma cell lines than the healthy renal cell line HEK293. In comparison, the cationic oxovanadium-PHEN analogue with a methyl substituent and the cationic oxovanadium-DPQ analogue with a bulky tert-butyl substituent are less selective in their cytotoxicity. The DNA binding studies show that the neutral and cationic DPQ compounds do have an affinity for DNA. A positive correlation between antitumour activity and DNA binding affinity was found. The [VO(ONN)(DPQ)](PF6) analogue with a bulky tert-butyl substituent has a lower intrinsic ct-DNA binding constant than the [VO(ONN)(DPQ)](PF6) analogue with a methyl substituent (1.3 × 104 M–1 and 2.8 × 104 M–1 respectively). The cationic DPQ derivatives also bind more strongly to DNA than the cationic complexes with PHEN co-ligands. The steric effect is also evident in the neutral complexes. The [VO(ONO)(DPQ)] complex with a tert-butyl substituent has a lower apparent binding constant than the [VO(ONO)(DPQ)] complex with no substituents on the Schiff base ligand. The cationic charge also led to a higher apparent binding constant for the [VO(ONO)(DPQ)](PF6) complex with a tert-butyl functional group than for the corresponding neutral [VO(ONO)(DPQ)] analogue with a tert-butyl functional group. Absorption and fluorescence spectroscopic and DNA viscosity studies indicate at least a partial DNA intercalative ability for the cationic oxovanadium-DPQ derivatives and the neutral oxovanadium-DPQ complexes with less bulky substituents. Molecular docking studies further highlighted the affinity of the metal chelates towards DNA, including interactions between DNA and the tridentate ligand. The lowest energy molecular docking poses range from ca. -48 to -67 kJ mol–1. Gel electrophoresis studies showed the cationic vanadium complexes with DPQ co-ligands (unlike the neutral DPQ and cationic PHEN analogues) were able to cleave plasmid DNA without adding external oxidising or reducing reagents. Experimental data suggest a singlet oxygen pathway is the most likely. It was also shown that the combination of metal ion and ligand is needed to induce DNA cleavage. The neutral [VO(ONO)(DPQ)] derivative with a methyl functional group on the Schiff base was shown to oxidatively cleave supercoiled plasmid DNA in the presence of H2O2 through the generation of hydroxyl radicals. EPR spintrapping studies with DMPO further support the idea that hydroxyl radicals are formed from reaction of the oxovanadium complex and H2O2. In summary the charge of the complex, type of substituent on the tridentate ligand and the identity of the N,N-donor heterocyclic ligand affected the stability, cytotoxic properties, selectivity, DNA binding, DNA cleavage abilities and DNA cleavage mechanism of the oxovanadium compounds in this study.
Evaluation of the larvicidal potential of Bacillus velezensis strain PHP1601 as a viable biological control agent against selected fly species.
(2024) Ramesar, Danvir Rajesh.; Hunter, Charles Haig.
Flies are one of the most abundant and prevalent insect pests posing a growing threat to various sectors of the economy. In response to this, a study was undertaken to evaluate Bacillus spp. strain PHP1601 as a candidate biocontrol agent against Lucilia cuprina larvae as a proxy for fly species of biocontrol significance. The identity of PHP1601 was confirmed as B. velezensis using MLSA and species-specific PCR. Bioassays demonstrated a larvicidal effect of cell, endospore (102 – 1010 cells/endospores g -1 ) and cell-free supernatant (1 – 30% v w -1 ) treatments on second instar larvae of L. cuprina. Studies were directed to the larvicidal effect of extracellular compounds, namely lipopeptides. Crude lipopeptide extract (CLP) was acquired using organic extraction from Landy broth. Bioassays with CLP extract (5 – 1000 μg g -1 ) resulted in a dose-dependent larvicidal response. Lipopeptides in the CLP extract were purified by TLC and characterised using UPLC ESI-TOF MS. This indicated the presence of iturin, fengycin and surfactin homologues of which, the purified surfactin fraction (Rf 0.91) was the most larvicidal. Bioassays were repeated with commercial surfactin, confirming its larvicidal potency, exhibiting an LC50 of 9.87 μg g -1 at 240 h. Larvae scent choice tests using TSB and MG bioassay medium fermented by PHP1601 showed that resulting VOCs were attractive to fly larvae, which was considered a viable trait of a fly biocontrol agent. CG-MS of the VOCs produced indicated that ketones were the dominant VOC class and, presumably, the major contributor to this larvae attraction effect. Field performance evaluation using pig manure trials demonstrated successful inhibition of several fly species of agricultural and veterinary importance using endospore treatments (105 and 1010 endospores g -1 ) of PHP1601. qPCR and REP-PCR fingerprinting confirmed that PHP1601 could grow in the manure slurries and was amiable to recovery and monitoring. Zebrafish embryo toxicity bioassays of the CLP produced by PHP1601 indicated that they achieved an LC50 of 22.77 µg ml-1, which characterised these metabolites as slightly toxic. Genome mining detected no genes associated with pathogenicity or virulence and presented no apparent pathogenic threat. The investigation demonstrated that B. velezensis PHP1601 is a viable fly biocontrol candidate and constitutes the first report of a B. velezensis antagonist of Brachycera flies.
Flexible Bayesian hierarchical spatial modelling in disease mapping.
(2022) Ayalew, Kassahun Abere.; Manda, Samuel.
The Gaussian Intrinsic Conditional Autoregressive (ICAR) spatial model, which usually has two components, namely an ICAR for spatial smoothing and standard random effects for non-spatial heterogeneity, is used to estimate spatial distributions of disease risks. The normality assumption in this model may not always be correct and misspecification of the distribution of random effects could result in biased estimation of the spatial distribution of disease risk, which could lead to misleading conclusions and policy recommendations. Limited research studies have been done where the estimation of the spatial distributions of diseases under the ICAR-normal model were compared to those obtained from fitting ICAR-nonnormal model. The results from these studies indicated that the ICAR-nonnormal models performed better than the ICAR-normal in terms of accuracy, efficiency and predictive capacity. However, these efforts have not fully addressed the effect on the estimation of spatial distributions under flexible specification of ICAR models in disease mapping. The overall aim of this PhD thesis is to develop approaches that relax the normality assumption that is often used in modeling and fitting of ICAR models in the estimation of spatial patterns of diseases. In particular, the thesis considers the skew-normal and skew-Laplace distributions under the univariate, and skew-normal for the multivariate specifications to estimate the spatial distributions of either univariable or multivariable areal data. The thesis also considers non-parametric specification of the multivariate spatial effects in the ICAR model, which is a novel extension of an earlier work. The estimation of the models was done using Bayesian statistical approaches. The performances of our suggested alternatives to the ICAR-normal model are evaluated by simulating studies as well as with practical application to the estimation of district-level distribution of HIV prevalence and treatment coverage using health survey data in South Africa. Results from the simulation studies and analysis of real data demonstrated that our approaches performed better in the prediction of spatial distributions for univariable and multivariable areal data in disease mapping approaches. This PhD work shows the limitations of relying on the ICAR-normal model for the estimations of spatial distributions for all spatial analyses, even when the data could be asymmetric and non-normal. In such scenarios, skewed-ICAR and nonparametric ICAR approaches could provide better and unbiased estimation of the spatial pattern of diseases.
Genetic analyses of antimicrobial resistance and virulence genes in Enterococcus species isolated from livestock production systems in South Africa.
(2021) Mnguni, Anele Buhle.; Zishiri, Oliver.
Enterococcus species are widely dispersed in the environment this includes soil, water, plants, food and animals. Although Enterococcus constitute mostly as a commensal bacterium; over the past years the bacterium has evolved to cause nosocomial infections. The proliferation of this pathogen is attributed to its ability in successfully transferring antimicrobial and virulence genes using several channels such as mobile genetic elements. This study investigated the prevalence of Enterococcus spp. in small-scale commercial farms in rural South Africa. The dissemination of virulent E. faecium and E. faecalis isolates allied with livestock production in the Eastern Cape and KwaZulu-Natal provinces was investigated. A total of 276 samples randomly sampled from livestock and their associated environments (feed, soil and water) were screened for Enterococcus spp. using selective media and using DNA molecular methods. E. faecalis and E. faecium prevalence was confirmed by the amplification of the tuf and sodA genes. Sixty-one percent of total presumptive isolates were E. faecalis (n=61) and only 8% (n=8) were identified as E. faecium. The presence of virulence determining factors such as asa1, ccf, cylA, esp, gelE and hyl was screened in all samples that tested positive for Enterococcus species. Presumptive E.faecalis and E. faecium isolates were mostly recovered from Amandawe (KZN). E. faecalis isolates harboured the most virulence genes asa1 (25%; n=), ccf (84%; n=), esp(4%;n= ), gelE (69%; n=) and hyl (12%; n= ). Whilst E. faecium isolates only harboured of asa1(12.5%; n=1), ccf (100%; n=8), gelE (75%;n=6 ) and hyl (25%;n=2). The current study also evaluated the antibiotic resistance profiles and their associated genes in these two species. Antibiotic susceptibility profiles of E. faecium and E. faecalis were assessed using Kirby-Bauer disk-diffusion assay as per the CSLI guidelines. Erythromycin had the highest occurrence of resistant isolates in both species with 75% (n=6) and 54.1% (n=33) respectively. Isolates were least resistant to ampicillin, with 0.03% resistance in E. faecalis and 0% in E. faecium. E. faecalis had the highest prevalence of Multi Drug Resistance (MDR), exhibiting phenotypic resistance to macrolides, aminoglycoside, tetracyclines and fluoroquinolones. TET-CIP-ERY was the most observed antibiotic resistance pattern. Furthermore, the isolates were screened for vanA, vanB, vanC1, vanC2/3, aac(6”)-aph(2”) ,ermA and ermB. The resistance genes that amplified in E. faecalis included vanB (8%;n=5), vanC1 (37%;n=23), vanC2/3 (37%; n=23), ermB (96%;n=58), ermA (8%;n=5) and aac(6”)-aph(2”) (1.6%;n=1). The immense dissemination of E. faecalis that has potentially pathogenic virulent determinants is a cause for concern in livestock production systems. In addition, faecal contamination from livestock poses a threat to the dissemination of virulent strains. The study demonstrated that E. faecium and E. Faecalis isolated from livestock and their associated environment were predominantly resistant to macrolides, glycopeptides, tetracyclines and fluroquinolones. In addition to be the first study in South Africa to document the emergence of inducible vanC determinants in Vancomycin Resistant Enterococci isolates.
Identification of arthropods of forensic importance during cold and warm seasons in KwaZulu-Natal Province of South Africa.
(2021) Tembe, Danisile.; Mukaratirwa, Samson.
Forensic entomology is the study and use of insects and other arthropods in forensic investigations associated with death, abuse and neglect of both humans and animals. Although there has been an increased interest in forensic entomology and its application in predicting post-mortem interval (PMI) amongst other issues in many developed countries, the results cannot be extrapolated beyond the countries/regions of study since the arthropods species spectra may vary with region and geographical conditions. The present study aimed to determine the arthropod species of forensic importance found during different stages of decomposition of sheep (Ovis aries) and pig (Sus scrofa domesticus) carrion during the warm and cold season in KwaZulu-Natal province of South Africa. A scoping review was conducted to determine the state of knowledge of forensic entomology research and application in southern Africa. To determine the arthropod species associated with sheep and pig carcass during different stages of decomposition, two medium sized Large-White pigs and two medium sized Merino sheep were humanely killed and used for the cold and warm season. Adult arthropods found on and around the carcasses during different stages of decomposition were collected and identified using combined morphological identification keys and molecular technique based on the mitochondrial gene. The review showed that arthropod species that were found on a decomposing carcass could be useful in the estimation of PMI and provided clues in cases of criminal investigations. The review also confirmed the scarcity of forensic entomology research, and its application in southern Africa. Experimental results from this study showed that dipteran flies from the families Calliphoridae, Muscidae and Sarcophagidae were the first to colonize the sheep and pig carcasses during both warm and cold seasons. These include species of Chrysomya marginalis, Ch. putoria, Ch. albiceps, Ch. chloropyga, Lucilia cuprina, Musca domestica and Sarcophaga calcifera. On the sheep carcasses, Ch. marginalis, Ch. albiceps and M. domestica were the most dominant fly species, contributing 63.2 % of the collected flies in the warm season, and 68.9 % in the cold season. Colonization by coleopterans during the warm season started as early as the fresh stage with Dermestes maculatus, Thanatophilus micans and Onthophagus crassicollis. In the cold season these same beetle species were collected from the bloated stage of the sheep carcass. On the pig carcasses, Ch. marginalis (n = 111), Ch. albiceps (n = 99) and M. domestica (n = 131) were the most abundant species during the warm season. The same species were the most abundant species in the cold season (n = 55), (n = 34) and (n = 81) respectively, although in lower numbers than the warm season. Among the collected Coleoptera species, D. maculatus (n = 112) and N. rufipes (n=62) were the most abundant species found on the carcass during the warm season and the same species were the most abundant species in the cold season (n = 66) and (n = 48) respectively. In the warm season Dermestes maculatus was recorded on the pig carcass during the fresh stage and persisted on the carcass until the last of decomposition. However, in the cold season Dermestes maculatus was first recorded on the carcass during the active stage of decomposition. Molecular analyses confirmed the identification of twelve (12) arthropod taxa collected from both sheep and pig carcasses during the cold season. Results showed that 11/12 arthropod species were common in both sheep and pig carcasses, with exception to Onthophagus sp. and Atherigona soccata species which were unique to sheep and pig carcasses respectively. However, during the warm season, the sheep carcass attracted more (n=13) taxa as compared to the pig carcass. The variation in the arthropod was due to the presence of Onthophagus sp. which was also unique to the sheep carcass during this season. Furthermore, there was an addition of a beetle species Hycleus lunatus, which was collected from both sheep and pig carcasses but unique to the warm season. This study generated important information on the endemic arthropod species that are of forensic importance KwaZulu-Natal province. The arrival time and association of arthropods species with different stages of decomposition during the warm and cold season highlighted their value in estimating the PMI in forensic investigations in the locality of KwaZulu-Natal province. The studied arthropods can potentially be useful in the estimation of PMI and other cases of criminal investigations. The seasonal variations in abundance of both Diptera and Coleoptera in the two seasons seemed to indicate influence of seasons which subsequently influenced temperature. It is recommended that similar studies be conducted at other geographical locations of South Africa with a different ecological system to build a database of dipteran and coleopteran species of forensic importance which are endemic in these areas.
Influence of human-associated tsetse habitat degradation on tsetse fly (Diptera: Glossinidae) populations and prevalence of infection with trypanosomes in North-Eastern Zambia.
(2021) Chilongo, Kallinga.; Mukaratirwa, Samson.
African trypanosomiasis is among the most important parasitic diseases of livestock and humans caused by several species of trypanosomes, and the disease occurs in 36 countries in sub-Saharan Africa. Human African Trypanosomiasis (HAT) causes a considerable public health burden on rural populations, and Animal African Trypanosomiasis (AAT) is an important constraint to livestock production and full utilization of land for agricultural production, such that if not controlled the disease can induce important losses through limiting crop production and access to land, and diminishing income from meat, milk and other livestock products, consequently resulting in poverty. In Zambia, approximately 40% of the country’s land is tsetse-infested and the infestation in the Luangwa valley is among the most important with respect to occurrence of both human and animal trypanosomiais. In affected areas, occurrence of trypanosomiasis in humans and in livestock normally correlates with the prevalence of trypanosome infection in tsetse flies. Laboratory studies have shown that among the major factors that affect such trypanosome infection in tsetse flies, is occurrence of stress in tsetse flies. Occurrence of stress in wild tsetse fly populations is associated with unfavourable environmental conditions for the flies, and this is usually a consequence of tsetse habitat degradation. In many parts of Zambia’s eastern tsetse belt, human-associated degradation of the tsetse habitat has been on the increase over the last decades. This suggests that research to determine the effects of such human-associated tsetse habitat degradation, on tsetse populations and prevalence of trypanosome infection in the tsetse population in the area, could provide some insights into the epidemiology of trypanosomiasis in the area. In this study undertaken in three sites, Mpika, Lundazi and Rufunsa sites, in north-eastern Zambia (in parts of the eastern tsetse belt), the objectives were, to determine and measure (i) variation in size, age and hunger stages in tsetse flies and (ii) variation in prevalence of trypanosome infection in the tsetse flies, with increase in distance away from the edge into the inner parts of tsetse belt, and in relation to the distribution of human settlements; and (iii) to detect, assess and evaluate the contribution and importance of existing agricultural and other forms of ecosystem utilization, to tsetse-habitat degradation in the three sites. Three study sites were selected based on level and pattern of human settlement, i.e. Mpika and Rufunsa sites with human settlement concentrated at or close to the edge of the tsetse belt, and Lundazi site with human settlement evenly distributed from the edge into the innermost parts of the tsetse belt. Samples of two species of tsetse flies found in the sample sites, i.e. Glossina morsitans morsitans and G. pallidipes, were collected and (i) size, age and hunger stage in the tsetse flies were recorded and assessed with reference to distance away from the edge of the tsetse belt; (ii) variation in prevalence of trypanosome infection in the tsetse population in the study sites, with reference to distance away from the edge of the tsetse belt, and in relation to distribution of human settlements; and (ii) key land-use and socioeconomic factors in the human settlements, with reference to human-associated tsetse habitat degradation in the study sites. Trapping of the tsetse flies was done in defined sample points, identified with use of a Global Position System (GPS) unit, in the transect line, with use of the Black-screen fly round (BFR) and Epsilon traps. From the sampled flies, the following were recorded; species of fly, sex, body size, age and hunger stage (as indicators of levels of occurrence of stress), and screening for trypanosome infection using microscopy and the loop-mediated isothermal amplification (LAMP). A semi-structured questionnaire was administered in each of the settlements within our study location, and national land cover maps for the years 2000 and 2010, produced by the country’s Forest Department, were used to estimate vegetation cover change during the period 2000 to 2010 in each of the sites. Regression models were applied to determine and measure the level of association of the distance from the edge into the inner parts of the tsetse belt with; size, age and hunger stages of tsetse samples, and prevalence of trypanosome infection in the tsetse flies. In each settlement, data were collected on key land-use and socio-economic factors that may be linked to human-associated habitat degradation and changes in the vegetation cover during the period 2000 and 2010, was calculated in QGIS. The results showed that in the Mpika and Rufunsa sites, the number of Glossina morsitans morsitans tsetse flies caught increased along with the increase in distance from the edge into the inner parts of the tsetse belt. This was also associated with increase in the body size (p < 0.0001 in both sites), and reduction in the age (p < 0.001 in each site) and the hunger stages (p < 0.0001 in both sites), and reduction in the prevalence of trypanosome infection (p = 0.024 and p = 0.012 in the case of all sub-species of trypanosomes tested for in the Mpika and Rufunsa sites respectively; and p = 0.013 and p = 0.025 in the case of only nannomonas sub-species in the two sites, respectively). The level of vegetation cover change was insignificant in each of the sites, such that it was unlikely to have had any significant impact on the quality of the tsetse habitat in each of the sites. In the Mpika and Rufunsa sites, human activities associated with access to resources might have had significant effect on the distribution of wild mammals that served as tsetse hosts in the area, such that numbers (of wild mammals) were low in locations that were close to the settlements and high in locations that were furthest from the settlements – giving rise to a gradient of increasing levels of availability of tsetse hosts with increase in distance away from the human settlements. This same trend was observed with regard to the distribution of body size of the flies, age, hunger stages, and prevalence of nannomonas and trypanozoon trypanosome infection, in G. m. morsitans in the Mpika and Rufunsa sites. This was indication that (in the Mpika and Rufunsa sites) increase in the levels of availability of tsetse hosts was associated with increase in levels of tsetse wellbeing – in turn associated with increase in levels of tsetse habitat quality. With regard to the findings in the Lundazi site (where human settlement was evenly distributed in transect line), the absence of any such variation (in each of the respective attributes in G. m. morsitans) with distance from the edge of the belt, could be taken as supportive to the reason indicated above as the likely basis for the existence of a gradient of reducing levels of tsetse habitat degradation in the Mpika and Rufunsa sites. In the case of G. pallidipes, the results showed no variation in the respective features in the tsetse flies, with increase in distance from the edge of the tsetse belt, and factors such as the relatively fewer numbers of the species caught, and a large proportion of the transect length not having registered any catch of the species, in each site in the study, likely contributed to this outcome.
Investigating the role of small RNAS in transcriptome regulation of genetically diverse clinical strains of mycobacterium tuberculosis.
(2021) Govender, Divenita.; Mvubu, Nontobeko Eunice.
Tuberculosis (TB), caused by the human adapted members of the Mycobacterium tuberculosis complex (MTBC), is a threat to global health. Understanding the regulatory network of the MTBC members may reveal novel vaccine candidates and drug targets. The small RNAs (sRNAs) have only recently been investigated for their role in Mycobacterium tuberculosis (M. tb) transcriptome regulation with none being explored in clinical strains or within the MTBC lineages. The present study aimed to investigate the regulatory role of sRNAs on the M. tb transcriptome in a lineage-specific manner, with emphasis on the clinical strains most prevalent in South Africa. In silico whole genome sequence alignment of strains belonging to the eight MTBC lineages was performed to identify sRNAs containing lineage-specific mutations and their respective potential targets. To elucidate transcriptome regulation in clinical strains of M. tb belonging to the Beijing and F15/LAM4/KZN lineages, mRNA and sRNA sequencing were performed followed by Hisat-Ballgown Bioinformatics analysis to identify novel sRNAs and their respective targets. The sRNAs discovered from sRNA sequencing were confirmed through real time qPCR. The in silico data revealed several sRNAs that may play a role in transcriptome regulation at a lineage-specific level, such as those involved in macrophage entry, lipid biosynthesis pathway, adaptation mechanisms during antibiotic exposure, and environmental stress. They may also be able to disrupt genes that are detrimental and restore functions to those that are beneficial. The mutated and consensus sRNAs were identified to target the same function, but one pathway may be more efficient than the other. Novel sRNAs were discovered from sRNA sequencing of the Beijing and F15/LAM4/KZN clinical strains, with their predicted targets absent from the mRNA sequencing results, indicating these sRNAs may elicit an inhibitory function. Real time-PCR analysis revealed significant fold change differences between the clinical strains belonging to the Beijing, F15/LAM4/KZN, F11 and Unique families suggesting an underlying regulation of these transcripts at a family level. This data could explain the underlying phenotypic differences observed within the MTBC and understanding of the regulatory function of these sRNAs, may identify novel alternative strategies in the fight against M. tb.
Micromorphology and biological activities of leaves and stem bark of Diospyros villosa (L.) De Winter.
(2021) Adu, Oluwatosin Temilade.; Naidoo, Yougashree.; Lin, Johnson.
Review Article: The genus Diospyros consists of many species which are distributed throughout the world. There is no elaborate information about the holistic importance of the plants. This review aims to delve for more information about the usefulness of the plants for humans. A detailed study of the genus Diospyros with a targeted focus on Diospyros villosa (L.) De Winter plant was carried out information on the plants’ morphology was taken as observed during harvest. Other information with respect to geographical distribution, secretory structures, chemical composition, embedded bioactive constituents and the associated mechanism of action of the bioactive constituents were acquired. The genus Diospyros yields products of medicinal importance throughout the world. This study gives some detailed information about the genus Diospyros as well as the potential use of the species as functional medicinal plants with bioactive compounds. Manuscript 1: This study evaluated the phytochemical constituents, antioxidant and antimicrobial potentials of Diospyros villosa (L.) De Winter leaves and stem bark. The extracts were obtained using different media (methanol, chloroform and hexane). DPPH and FRAP methods were used to investigate the antioxidant potentials of the crude extracts. The antimicrobial potency of Diospyros villosa extracts against five pathogenic bacteria was determined using MIC, MBC and agar well diffusion methods. Flavonoids, alkaloids and phenols were identified in D. villosa extracts. The mean concentrations of methanol extracts Diospyros villosa leaves and stem against DPPH providing 50% inhibition were 9.53 ± 0.25 μg ml-1 and 9.52 ± 0.30 μg ml-1 respectively. The methanolic leaves extracts further showed promising antimicrobial activity against Klebsiella pneumonia, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and Staphylococcus aureus with inhibition zones which are almost as high as the control. The antimicrobial methods also demonstrated that the leaves and stem bark extracts had wide antimicrobial abilities. The results revealed that D. xxvii villosa leaves and stem bark contain reasonable amount of bioactive compounds. Hence, these compounds may serve as natural antioxidants and antibacterial agents for the treatment of bacterial infections and diseases. Manuscript 2: Nanoparticles are synthesized through wet chemical techniques. Meanwhile, the chemicals used often are toxic and flammable. While considering safe substances, Diospyros villosa leaves and stem bark are both observed to be suitable for the biosynthesis of silver nanoparticles. In this research study, we described a less expensive and environmental-friendly technique for the biosynthesis of silver nanoparticles from silver nitrate (AgNO3) solution and Diospyros villosa extracts. The obtained silver nanoparticles were characterized using the UV-vis absorption spectroscopy, FT-IR, EDX, SEM and TEM, DPPH scavenging ability, ferric reducing antioxidant potential, antimicrobial susceptibility and quorum sensing inhibition tests. The biosynthesized silver nanoparticles showed good antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis and Enterococcus faecalis. The D. villosa nanoparticles demonstrated potential antioxidant and quorum sensing inhibition and may thus represent a source of antioxidant as well as quorum sensing inhibition candidate for infection and disease control. Manuscript 3: The structural adaptation, histological compatibility and ecological functions of trichomes in Diospyros villosa, however remain largely unclear. This study aimed to interpret the structure and histochemical analysis of the leaves and stem bark of Diospyros villosa using light and electron microscopy and different histochemical tests as well as to elucidate the composition of the secretory products. Trichome density and length were also determined in three developmental stages of the leaves. The results of histochemical tests also revealed the presence of alkaloids and phenolic compounds which are medicinally important and used to treat multiple ailments. xxviii Manuscript 4: Diospyros villosa is traditionally used for processing anti-bacterial properties. Its cytotoxic effects have not been studied before. Therefore, this study aimed to examine the nutritional properties as well the cytotoxic effects of D. villosa. The leaves and stem barks were subjected to three different extraction methods (methanol, chloroform and hexane) and their nanoparticles were synthesized at two different temperatures (room temperature and at 80 ºC). Thereafter, extracts were assessed using the associated AOCC protocols, for their nutritional content (moisture, fibre, proteins, lipid, ash and hydrolysable carbohydrates). Diospyros villosa extracts and their corresponding nanoparticles were then incubated overnight with cancerous and noncancerous cell lines to evaluate their cytotoxic potential.
Morphological and molecular characterization of Fasciola hepatica and Fasciola gigantica phenotypes from Mpumalanga and KwaZulu-Natal provinces of South Africa.
(2020) Haridwal, Sayurika.; Mukaratirwa, Samson.
Fascioliasis is a food- and waterborne disease. It is one of the most common helminthic infections in domesticated ruminants. The disease is caused by liver flukes, Fasciola hepatica and Fasciola gigantica. Increased attention has been geared toward studying these flukes due to their ever-expanding geographical distribution, enormous economic impact, increased human infections, increased resistance to treatment and the existence of hybrid forms. Both these species are co-endemic in the Mpumalanga and KwaZulu-Natal provinces of South Africa and even though, hybrids have been reported in other areas where both species exist it has not been attempted in South Africa. Therefore, this study was conducted to determine the existence of Fasciola hybrids in South Africa using morphological and molecular characterization. A total of 71 flukes were collected from naturally infected cattle slaughtered at abattoirs located in Enhlazeni and Nelspruit in Mpumalanga province and Pietermaritzburg in KwaZulu-Natal province of South Africa as well as control samples from Zimbabwe (Bulawayo abattoir) where only F. gigantica exist. The samples were categorized morphologically as either F.hepatica, F.gigantica, or Fasciola sp. The morphometrics (body length, body width, and length/width) were analyzed through a PCA and produced three distinct groups. A one-way ANOVA indicated that the length and length/width could be used to differentiate the species (P < 0.05) and the width was not useful in differentiating the species (P > 0.05). Molecular analysis based on ITS-1/5.8S/ITS2 marker showed that specimens morphologically identified as Fasciola sp were F.gigantica, with one sample morphologically identified as F.gigantica was molecularly identified as Fasciola sp. Similar results were observed with the CO1 marker, however, one sample came up as unknown, this sample however, formed a well-supported sister clade to F. gigantica. . It was also observed that aspermatic specimens are not only limited to hybrids, as some individuals that were molecularly identified as F. hepatica lacked sperm in their seminal vesicles. This study confirms species identification of F. hepatica and F.gigantica cannot be solely based on morphological characters where both these species are co-endemic. This was also the first study to report the existence of hybrid Fasciola spp. in South Africa.
The morphological characterization, chemical composition and biological activity of barleria albostellata (Acanthaceae).
(2021) Gangaram, Serisha.; Naidoo, Yashini.; Dewir, Yaser Hassan.
Herbal preparations of plants continue to present mankind with novel remedies as many of these plants contain important secondary metabolites. Medicines manufactured by pharmaceutical companies are largely synthetic. The fear for ineffectiveness, adverse side-effects and toxicity, has brought about further scientific investigations on the potential usage of medicinal plants. Plants of the family Acanthaceae are rich in bioactive phytochemicals. Several plant species are being utilized for their ethnomedicinal properties based on their phytocompounds they acquire, with Barleria (Acanthaceae) being one of such genera. Barleria albostellata C.B. Clarke (Acanthaceae) is a shrub indigenous to South Africa. Herbal use of this plant has not been fully documented, however, several species of Barleria are used in traditional medicine. Little or no investigations have been undertaken to evaluate the micromorphology secretory mechanisms, through which such phytochemicals are synthesized and secreted; floral biology; phytochemical profile; antibacterial and antioxidant activity, cytotoxicity and synthesis of nanoparticles using the leaf and stem extracts of B. albostellata. Therefore, this study aimed at bridging these gaps by first characterizing the morphology, chemical composition and biological activity of leaves and stems of B. albostellata, using various microscopy techniques and biological assays. Floral biology of the plant was conducted using stereo- and scanning electron microscopic (SEM) techniques. ImageJ was used to measure the length and diameter of the different trichome types and pollen structures. Histo-phytochemical, thin-layer chromatography (TLC), fluorescence microscopy and gas-chromatography mass spectrometry (GC-MS) analysis were performed on crude extracts (hexane, chloroform and methanol) to determine the composition of the compounds that may be of medicinal importance. Biological (antibacterial and antioxidant) analyses were also conducted on the crude extracts. Cytotoxicity of the crude extracts were evaluated established using 3-[(4,5- dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay in the human embryonic kidney (HEK293), cervical cancer (HeLa), and breast adenocarcinoma (MCF-7) cell lines. Silver nanoparticles (AgNPs) were synthesized using methanolic, aqueous- powdered and -fresh leaf and stem extracts. These AgNPs were characterized using UV–visible spectroscopy, high resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FTIR) spectral analysis and nanoparticle tracking analysis (NTA). Antibacterial analysis of the synthesized AgNPs was assessed using the disk diffusion method. Cytotoxicity of the synthesized nanoparticles were investigated using the MTT assay on the three cell lines. Histochemically stained sections, stereo, SEM and TEM micrographs revealed a dense indumentum with the presence of five morphologically distinct glandular capitate trichome types, multangulate-dendritic branched (MDB) non-glandular trichomes and a glandular head attached to a branched non-glandular trichome. Both glandular and non-glandular trichomes in the leaves and stems of B. albostellata are important diagnostic features of this species. The secretory head of glandular typed trichomes varied from 20.72 ± 1.36 µm to 54.23 ± 2.08 µm. Additionally, the length of these stalks varied from 15.11 ± 3.89 µm to 101.92 ± 1.78 µm, and the width ii from 11.62 ± 1.03 µm to 35.32 ± 3.87 µm. Multangulate-dendritic branched trichomes had a stalk length of 554.10 ± 92.27 µm and width of 28.55 ± 2.48 µm. Stained sections and crude extracts indicated the presence of important medicinal compounds such as alkaloids and phenolics. The histochemical tests indicated the presence of various phytocompounds that have medicinal importance and aid in defense against pathogens and herbivores. Pollen grains of B. albostellata are complex, intricate and display reticulate sculpturing, with a diameter of 77.53 ± 5.63 μm and aperture of 14.31 ± 0.59 µm. This will contribute significantly to our growing understanding on the floral and pollen biology of this species. Qualitative phytochemical screening, GC-MS and florescence microscopy of the leaf and stem extracts revealed various biologically active compounds and the presence of different colours in the leaf and stem powder. Different colour intensities obtained from TLC suggested concentrations of the separated compounds were varied. Additionally, the phyto-constituents found in the leaf and stem crude extracts of could inhibit the growth of various pathogenic strains. Results from this study revealed the medicinal potential of B. albostellata in the treatment of various bacterial diseases Ultraviolet spectra, NTA and EDX, revealed varying absorption peaks, size distribution, and elemental Ag in all extracts. Crude extracts and synthesized AgNPs displayed varying degrees of antioxidant, antibacterial and cytotoxic activities. Significance was established at P 63 µg/mL and >9 µg/mL, respectively) may be due to the presence of flavonoids, phenols, and antioxidant activity in the different parts of this species. Synthesized AgNPs showed possible bacteriostatic effects against Gram-positive and -negative human pathogenic bacteria. Their broad spectrum of bioactivity suggested that they may be as promising agents in fighting infections. This study ultimately proved that leaf and stem extracts of B. albostellata contained numerous biologically active compounds such as alkaloids and phenolics. These results are suggestive that the leaves and stems of B. albostellata are rich in bioactive compounds which, could be a possible source of antibacterial agents in treating several diseases .There is a great potential for B. albostellata as this plant displays valuable biological activities. Future studies on this plant are recommended, as this could advance the use of indigenous herbal medicine or product novel drug leads.
Morphology, phytochemistry, and medicinal properties of South African Mangifera indica L. leaves for summer and winter seasons.
(2021) Maharaj, Arvish.; Naidoo, Yougasphree.; Dewir, Yaser Hassan.
Herbal preparations of plants continue to present mankind with novel remedies as many of these plants contain important secondary metabolites. Plant species of the family Anacardiaceae are rich in bioactive phytochemicals. Mangifera indica (Anacardiaceae) is an introduced and naturalised species to South Africa. Herbal use of this plant has not been fully documented; however, it is used in traditional medicine. This study aimed at characterizing the morphology, phytochemistry, and biological activity of Mangifera indica leaves harvested in winter and summer. The foliar biology of the plant was conducted by various microscopy techniques such as stereo- and Scanning electron microscopy. The length and diameter of the different trichome types were measured using ImageJ. The non-glandular trichome lengths range between 70 - 200 μm. The peltate gland trichomes consist of 2 rows of 8 oblong cells each with a size ranging from 32- 48 μm. Morphological observations using stereo- and SEM revealed the presence of non-glandular trichomes with cuticular warts and glandular peltate trichomes on the leaves of Mangifera indica. Transmission electron micrographs showed the presence of numerous mitochondria, starch grains, plastoglobuli, and plastids. The results for summer and winter leaves resembled somewhat similar-to-identical morphological characteristics on all fronts. For the phytochemical and biological assays, this study aimed to investigate some of the phytochemical and biological properties using different solvents (hexane, chloroform, and methanol) for extraction of the leaves of Mangifera indica for the summer and winter seasons. Preliminary phytochemical screening for the hexane, chloroform and methanolic extracts was done using a reflux extraction apparatus to uncover the presence of different metabolites and the anti-oxidant screening was done by the radical scavenging activity, which was established using the 2,2-diphenyl-1-picrylhydrazyl assay. Potent radical scavenging activity was exhibited for both summer and winter seasons with hexane and methanolic extracts for summer (IC50 of 19.53 μg/mL and 12.71 μg/mL respectively) and winter (22.32 μg/mL and 14.35 μg/mL respectively) in comparison to the control ascorbic acid which produced an IC50 of 3.20 μg/mL. The summer extracts had better radical scavenging IC50 capacity than winter extracts. The antibacterial activity of the methanolic leaf extracts for summer and winter of Mangifera indica were evaluated against the bacterial species: Gram-negative Escherichia coli (ATCC 25922) and Gram-positive: Staphylococcus aureus (ATCC ATCC 43300). For S. aureus (ATTC 43300), the summer crude extract displayed lower antibacterial activity than the control streptomycin, the summer extracts had a zone of inhibition of 14.17 mm while streptomycin had a 16.67 mm zone of inhibition. winter extracts had a zone of inhibition of 12 mm while streptomycin had a 13.67 mm zone of inhibition. For E. coli (ATCC 25922), the summer crude extract displayed higher antibacterial activity than the control gentamycin; the summer extract had a zone of inhibition of 18.05 mm while gentamycin had a 17.5 mm zone of inhibition. The winter extracts had a zone of inhibition of 8.5 mm. while gentamycin had a 14.5 mm zone of inhibition. Between seasons, summer had better antibacterial activity compared to winter for both Gram-positive and Gram-negative bacteria. Phytochemical screening showed the presence of phenols, flavonoids, tannins, and terpenoids, alkaloids, phytosterols, saponins, steroids, and carbohydrates. Potent radical scavenging activity was exhibited for the hexane and methanolic extracts for summer and winter, indicating that Mangifera indica is a potential source of medicinally important compounds. Antibacterial screening showed positive results with antibacterial properties for both summer and winter samples revealing its valuable biological activities. Summer overall performed better than the winter season. Future studies on this plant species are recommended to advance the use of indigenous herbal medicine or produce novel drug leads. To our knowledge, this study represents the first recent investigation in South Africa describing key foliar micromorphological features, phytochemicals, and biological activities of Mangifera indica L.
Nutritional management of adult patients hospitalised with covid-19 by dietitians in KwaZulu-Natal.
(2023) Ebrahim, Naseera.; Pillay, Kirthee.
Background: The outbreak of a novel coronavirus disease (COVID-19) in December 2019, led to a worldwide pandemic. Over the past three years, over 4 million people in South Africa (SA) have been infected with COVID-19, which mainly affects the respiratory system. The presence of existing co-morbidities influences the severity of the illness, and the long-term prognosis. Patients who require hospitalisation for respiratory support are often critically ill and, in most cases, cannot consume enough nutrients. Although dietitians have been involved in the nutritional management of patients hospitalised with COVID-19, there were no guidelines on the nutritional management of COVID-19 available for them to use and they were not trained on its management. There is no consensus on how dietitians managed COVID-19 and which nutritional management guidelines they consulted. Aim: This study aimed to determine how dietitians managed adult patients hospitalised with COVID-19 in KwaZulu-Natal (KZN). Objectives: (i) to determine which nutritional guidelines dietitians used in the management of adult COVID-19 patients; (ii) to identify the challenges that dietitians faced when nutritionally managing adult COVID-19 patients; (iii) to determine whether dietitians took or estimated anthropometric measurements in bed-bound and mobile adult COVID-19 patients; (iv) to determine whether dietitians assessed malnutrition risk in adult COVID-19 patients, and if so, which nutrition screening tools were used; (v) to determine if dietitians recommended unconventional mega-doses of micro- and immunonutrient supplements for the management of adult COVID-19 patients and the reasons for use. Method: A cross-sectional descriptive study, which included dietitians employed in the public and private sectors in KZN was conducted. An electronic self-administered questionnaire was developed and used to collect data via the online platform Google Forms. Initially, the KZN Department of Health (DOH) and the Association for Dietetics in South Africa (ADSA) assisted with distributing the link to the study to dietitians in KZN. However, after an initial poor response, the data collection period was extended and the researcher was granted permission to directly contact and invite dietitians to participate, using publicly-available contact details. Data were collected between 14 August 2022 and 31 March 2023 and analysed using the Statistical Package for Social Sciences (SPSS) version 25. Results: Of the forty-two dietitians who participated in this study, 52.4% (n=22) were KZN DOH-employed dietitians and 31% (n=13) were ADSA members. Seven-percent (n=3) of the dietitians were both KZN DOH-employed dietitians and ADSA members and approximately 10% (n=4) of the dietitians were neither KZN DOH-employed dietitians nor ADSA members. An equal number of dietitians worked in private hospitals (n=16; 38.1%) and in public districtlevel hospitals (n=16; 38.1%). A significant number of dietitians began treating COVID-19 patients from the start of the pandemic or during and/or after the first wave of infection (p=0.001). Just over half of the dietitians were no longer treating any COVID-19 patients at the time of data collection (n=22; 52.4%) (p<0.001). Sixty-nine percent (n=29) of dietitians consulted a nutrition society for recommendations on the nutritional management of COVID-19 patients. The European Society for Parenteral and Enteral Nutrition (ESPEN) expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection were most used in the current study. The most frequently used values for calculating macronutrient requirements were: 25-30 kCal/kg/day for energy, 1.2-1.5 g/kg/day for protein, 30% of the total energy requirement (TER) for fat and 50-60% of TER for carbohydrates. Dietitians also reported using actual body weight (ABW) (n=13; 31.0%) or estimated body weight (n=19; 45.2%) to calculate nutritional requirements (p=0.004). Individual challenges faced by the dietitians were similarly grouped. There was significant disagreement among the dietitians that a lack of support and resources (p<0.001) and nutrition-related external factors were challenges they experienced (p<0.001). Anthropometry was assessed in all patient groups, with the main methods used being estimated weight, height and body mass index (BMI) for patients who were bed-bound and unconscious (n=31; 73.8%) (p=0.003). Actual weight, height and BMI were assessed in patients who were fully mobile (n=35; 83.3%) (p<0.001). There was also a significant agreement amongst the dietitians that COVID-19 patients did not feel well enough to have their anthropometric measurements taken. Half of the dietitians reported using a nutrition screening tool to screen for malnutrition (n=21; 50%) and a significant number of dietitians (n=13; 61.9%) used the Nutrition Risk Screening 2002 (NRS-2002) tool (p<0.001). Only 12 dietitians (28.6%) recommended the use of mega-doses of micro- and immunonutrient supplements in their COVID-19 patients, with an overall significant agreement that mega-doses could benefit the patient (p=0.012). All 12 dietitians who recommended mega-doses of micro and immunonutrient supplements did not report any adverse side-effects in their patients (p<0.001), and most (n=10; 83.3%) noticed an improvement in the condition of their COVID-19 patients (p=0.039). Conclusion: This was one of the first studies in SA to investigate the nutritional management of adult patients hospitalised with COVID-19. Although dietitians were involved in treating COVID-19 patients from the onset of the pandemic, most were not treating any COVID-19 patients at the time of data collection. Most dietitians consulted the ESPEN practical recommendations on the nutritional management of critically ill patients to manage their COVID-19 patients. There was no significant consensus amongst the dietitians that medical conditions and complications were challenges faced by the dietitians. Depending on the degree of mobility of the COVID-19 patient, anthropometry was assessed in COVID-19 patients, either by estimation or actual measurements. The most common malnutrition screening tools used by dietitians were the NRS-2002 and the Malnutrition Universal Screening Tool (MUST). Megadosing of micro- and immunonutrient supplements was not popular among the dietitians in the current study and further studies are needed in this area.
One-pot, multicomponent oxidative synthesis of 2,4,5-trisubstituted imidazoles from internal alkenes using an I2/DMSO system.
(2023) Majola, Nonhlelo.; Jeena, Vineet.
Imidazoles are vital heterocyclic compounds usually incorporated in natural products such as biotin, vitamin B12, histamine, and histidine. 2,4,5-trisubstituted imidazoles, in particular, possess versatile biological and pharmaceutical activities such as antidiabetic, antimalarial, and analgesic properties. A traditional procedure for the synthesis of these elegant compounds involves the cyclocondensation reaction between a 1,2-diketone, an aldehyde, and ammonia in the presence of an acid or metal catalyst. However, this methodology suffers from various shortcomings such as the use of acid or metal catalysts, tedious work-up procedures, use of toxic reagents, and substrate scope limitations. Hence, the development of new methods to synthesize 2,4,5-trisubstituted imidazoles is of vital importance. This study describes the preparation of 2,4,5-trisubstituted imidazoles from alkenes using an environmentally benign iodine/DMSO system. This novel methodology was applied to a broad substrate scope such as substituted benzaldehydes, heterocyclic aldehydes, bulkier aldehydes, and substituted stilbenes, and afforded the target compounds in moderate to high yields under mild reaction conditions. Preliminary mechanistic studies revealed that 1,2-diketone is a key intermediate and that the mechanism is not radical-mediated. It also revealed that the oxygen source is DMSO and that the coupling step is catalyzed by iodine coordination and hydrogen bonding from the solvent. Based on the results obtained from the preliminary mechanistic investigations, a reasonable mechanism is proposed.
Recombinant expression and enzymatic characterisation of Trypanosoma vivax cathepsin L-like protease (TviCATL) for single chain variable fragment antibody production.
(2022) Ramjeawon, Bhavana Roshenlal.; Coetzer, Theresa Helen Taillefer.
Humans and animals in sub-Saharan Africa are at risk of African trypanosomiasis (AT), caused by tsetse fly-transmitted protozoan blood parasites of the Trypanosoma genus. Animal African trypanosomiasis (AAT), or nagana, is caused by T. brucei, T. congolense and T. vivax and negatively impacts livestock farming and consequently the economy of the continent. Since AAT occurs in rural areas, affordable rapid diagnostic tests (RDTs) and drugs are required. Diagnostic tests focus on antibody detection; however, antigen detection is more favorable since anti-trypanosome antibodies persist in blood for years following recovery. Due to the parasite’s defense by antigenic variation, development of a vaccine is unlikely. Molecules that are essential for parasite survival, such as peptidases, are currently being targeted for diagnosis and chemotherapy. A cathepsin-L-like cysteine protease from T. vivax, TviCATL, is released by dying parasites in the host bloodstream and was shown to be a diagnostic target for detecting host antibodies. To achieve diagnosis of current infections, detection of TviCATL is being explored. The overall aim of this study was to enzymatically characterise TviCATL; and to study the interaction of antibodies against the TviCATL antigen which could be used as a chemotherapeutic drug for the diagnosis of T. vivax infections. The protease, TviCATL, was recombinantly expressed in E. coli using the pET-28a expression vector and purified using a nickel chelate affinity column. The resulting 47 kDa protein was identified using western blot and was shown to hydrolyse H-D-Ala-Leu-Lys-AMC and was inhibited by bestatin and E-64 and had optimal activity between pH 6.5 and 7.5. The crossreactivity between TviCATL and antibodies produced against other Trypanosoma spp cysteine proteases was evaluated in western blots, and results confirmed cross-reactivity. In addition, chicken anti-TviCATL antibodies were able to detect TviCATL in TviCATL-spiked bovine serum. The production of antibodies using the Nkuku® phage library was employed as an alternative to the animal-based antibody production and single-chain variable fragment (scFvs) antibodies were selected by panning against the TviCATL antigen. After four rounds of panning, TviCATL-scFvs binders were enriched and four clones gave the highest signal when evaluated using a monospecific ELISA. Due to the low values obtained, optimisation of panning is necessary for improved results. Optimisation of recombinant expression and purification of the identified scFvs for use in a sandwich ELISA were explored to this end. This study showed that TviCATL is a promising chemotherapeutic and diagnostic target for African animal trypanosomiasis.
Statistical modelling on childhood anaemia, malaria and stunting in Malawi, Lesotho, and Burundi.
(2023) Gaston, Rugiranka Tony.; Ramroop, Shaun.; Habyarimana, Faustin.
The current research aimed to produce and expand statistical models in the discipline of biostatistics with a focus on childhood anaemia, malaria, and stunting. Malaria, anaemia, and stunting together continue to be public health issues worldwide in both industrialised and underdeveloped countries, particularly in children younger than 5 years (Osazuwa and Ayo, 2010; Kanchana et al., 2018). Malaria, anaemia, and stunting are dangerous, mostly in children from underdeveloped nations and they still remain the biggest contributor to morbidity and mortality. In addition, anaemia, malaria, and stunting are associated, and if not treated on time can damage children’s emotional, physical, mental status and poor performance at school (Gaston et al., 2022). The current study evaluates the link between anaemia, stunting, and malaria simultaneously. Furthermore, the study assessed whether socioeconomic, geographical, environmental, and child demographic variables have a significant effect on childhood malaria, anaemia, and stunting. This study used a national secondary cross-sectional data from Malawi Malaria Indicator Survey (MMIS); Lesotho Demographic Health Survey (LDHS); and Burundi Demographic Health Survey (BDHS). The data was collected based on multi-stage sampling, stratified, and cluster sampling with an unequal chance of sampling. It is for this reason we first used the survey logistic regression model in Chapter 3, which accounted for the complexity of sampling design and heterogeneity between observations from the same cluster. However, this model includes only the fixed effect and does not have the option of adding the random effect to model the correlation between observations. We extend the model in Chapter 4, to a generalised mixed additive model (GAMM) to include the random effect. The GAMM is also an extension of the generalised linear mixed model (GLMM) and enables the parametric fixed effects from GLMM to be modelled as a non-parametric model using the additive smooth function. These models were applied to single response variables, and we wanted to evaluate the relationship which might exist between anaemia, stunting, and malaria. We then explore the multivariate joint model under GLMM in Chapter 5 to simultaneously joint either malaria and anaemia or anaemia and stunting. Finally, we introduce a structural equation model (SEM) in Chapter 6, to evaluate the complex interrelationships between socioeconomics, demographics, and environmental factors, as well as their direct or indirect relationship with childhood malaria, anaemia and stunting co-morbidity. The previous chapters could not address these interrelationships among the variables of interest. Each model used in this study has its weaknesses and strengths which can depend on the goal of the xii researcher. However, the multivariate model under GLMM and the structural equation model were found to be more adaptive and attractive to researchers interested in innovative scientific research. The findings from this study revealed that the child’s nutrition status, age, the child with fever, diarrhoea, altitude, place of residence, toilet facility, access to electricity, children who slept under a mosquito bed net the night before the survey, mother's education level, and mother’s body mass index have a significant effect on both childhood anaemia and malaria. The age of a child, the mother’s educational status, place of residence, wealth index, and child weight at birth were the determinants of stunting or malnutrition. The findings also indicated that the geographical, geophysical, environmental, household and child demographic factors were statistically significant and have either a direct or an indirect effect on childhood co-morbidity factors. The geographical factors were statistically significant and had a positive direct effect on childhood malaria, anaemia, and stunting. The estimated indirect path for the impact of geophysical factors on childhood co-morbidity factors, as mediated by household factors was statistically significant and positive. However, the estimated indirect paths for the effect of geophysical factors on childhood co-morbidity factors, as mediated by environmental factors were statistically significant but negative. The child demographic factors revealed a direct statistically significant impact on childhood co-morbidity factors. Furthermore, the estimated indirect path effect on childhood comorbidity as mediated effect on household factors was statistically significant and negative. Moreover, household and environmental factors indicate a positive direct effect on childhood co-morbidity anaemia, malaria, and stunting. Finally, the results of this study revealed a positive relationship between stunting, anaemia, and malaria. This means that malaria, anaemia, and stunting increase or decrease in the same direction. Hence, controlling one or two between malaria, anaemia, and stunting can reduce the effect of other(s), which can assist the policymakers and government in the allocation of financial resources to fight against childhood comorbidity anaemia, malaria, and stunting. Furthermore, understanding the link between anaemia, malaria, and stunting other factors associated with them will assist in focusing on those areas and go a long way toward achieving the United Nations Sustainable Development Goals (SDGs3), known as the complete elimination of under-5 mortality by 2030.
Status of research on two parasitic zoonoses (Toxoplasmosis and toxocariasis) in Sub-Saharan Africa and their prevalence in selected rural communities of Kwazulu-Natal Province of South Africa using free-range chickens as a case study.
(2022) Omonijo, Adejumoke Oluwatosin.; Mukaratirwa, Samson.
Free-range chickens are among the popular livestock that are owned by most households in rural communities. They constitute a major source of food security, financial income, and are used in socio-cultural practices. However, due to their habit of scavenging for food they are exposed to parasitic agents thereby making them host for several disease pathogens such as Toxoplasma gondii and Toxocara spp. Toxoplasma gondii and Toxocara spp are the etiological agents of human toxoplasmosis and human toxocariasis respectively. Humans become directly infected via accidental ingestion of sporulated oocysts of T. gondii from felids and tachizoites/bradyzoites of T. gondii from raw/undercooked meat or embryonated eggs with second stage larva of Toxocara spp via contact with contaminated faeces of definitive hosts (dogs and cats), or indirectly via ingestion of contaminated water or consumption of raw or undercooked chickens. Following infection, the parasites migrate through the human body causing varying degree of diseases known as toxoplasmosis and toxocariosis respectively. Consumption of poultry meat viscera is an increasing dietary habit common in different communities worldwide and depending on socio-cultural preferences it can either be eaten raw, undercooked, or well cooked. However, the habit of eating raw/under cooked meat or viscera poses the risk of transmitting T. gondii and Toxocara spp from animals to humans. Limited information exist on the epidemiology of T. gondii and Toxocara spp in sub-Saharan Africa and let alone the role of free-range chickens in the transmission of these zoonotic parasites, hence, this study was designed to: ➢ review the status of research on these two parasitic zoonoses in sub-Saharan Africa. ➢ determine prevalence of the parasites in free-range chickens from selected rural communities in KwaZulu-Natal province through molecular approach. determine the level of awareness of the zoonotic transmission of these parasites when the viscera or meat of Free-range chicken are consumed raw or undercooked. A sytematic review and meta-analysis was conducted following the Preferred Reporting items for systematic Reviews and Meta-Analysis (PRISMA) guidelines on the epidemiology of T. gondii in animals and humans in southern Africa and epidemiology of Toxocara spp in canine, feline, and humans in sub-Saharan Africa respectively. The reviews showed that there is paucity of information on T. gondii and Toxocara spp in food animals including free-range chickens. Furthermore, to determine the prevalence of T. gondii and Toxocara spp in free-range chickens, free-range chickens were randomly purchased from selected rural communities namely, Gingindlovu (GI), Ozwathini (O), uMzinto (MZ), and Shonwgweni (SH) in KwaZulu-Natal (KZN). The chickens were euthanized according to ethical guidelines. The brain tissue of each chicken was divided into two equal halves. One half was examined for the presence of parasites while the remaining half was preserved in 70% alcohol for molecular analysis. To detect the presence of T. gondii, the preserved brain tissues were subjected to molecular analysis based on analysis of DNA sequences of the nuclear ribosomal internal transcribed spacer (ITS-1 and ITS-2) region using TOX4 and TOX5 primers. To detect the presence of Toxocara spp, various parts of chickens such as brain, heart, liver, spleen, kidney, duodenum, pectoral, thigh, and breast were digested using the acid/pepsin; 1:1 method and the larvae were recovered with 20-μm sieve. Three (3) larvae were recovered from the right pectoral from a chicken collected in GI; two (2) from the lungs of a chicken from MZ; three (3) each in the liver and left thigh of two separate chickens from SH. The recovered larvae were subjected to molecular analysis using Nem_18S primers. Toxoplasma gondii was not detected in the tissue samples which were subjected to molecular analysis, however, Toxocara canis was identified in Gingindlovu (n=1), uMzinto (n=1), and Shongweni (n=2). The identified T. canis showed 100% homology with Genbank isolates from China, the United Kingdom, and the United State of America. The occurrence of T. canis in free-range chickens from KZN province reveals the possibility of human toxocariasis transmission in the province. Moreover, we conducted a questionnaire survey to determine the knowledge and practices relating to consumption of free-range chicken viscera in selected rural communities of KwaZulu-Natal with respect to zoonotic transmission of T. gondii and Toxocara spp. There was low level of awareness of risk of zoonotic transmission of the parasites via ingestion of raw/undercooked free-range chicken meat/viscera and the majority of respondents consumed free-range chicken viscera. They preferred the viscera well cooked which reduces the risk of transmission of the the two parasites. The study contributes new knowledge on the prevalence of zoonotic parasites in free-range chickens as well as the level of knowledge and awareness on zoonosis transmission via consumption raw/undercooked free-range chicken viscera or meat.
Synthesis of non-natural amino acids as covalent inhibitors for protein-protein interactions.
(2023) Dladla, Siphamandla Austen.; Sithebe, Siphamandla.; Veale, Clinton Gareth Lancaster.
There is still a need to develop new cancer therapies for troubling cancers. Hence, a resurging interest in compounds that engage their target through covalent interactions. Lysine’s amine can be engaged covalently with a weak electrophile (SO2F) extending the potential of covalent inhibitors. Herein, we were prompted to investigate the synthesis of non-natural amino acids, modified to include weakly electrophilic warheads, which could potentially target specific lysine residues. Three new non-natural amino acids were successfully synthesized, methyl (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((fluorosulfonyl)oxy)phenyl)propanoate, 3.5, methyl (S)-2-((tert-butoxycarbonyl)amino)-2-(4-((fluorosulfonyl)oxy)phenyl)acetate, 3.9, and methyl (S)-2-((tert-butoxycarbonyl)phenyl)propanoate, 3.35, in 85%, 89%, and 63.7% yield, respectively. Our study explored the synthetic pathway of a three-step procedure toward the target compounds, with the initial esterification of the carboxylic acid group, followed by the N-Boc protection of the amine group. Finally, the key sulfonation of the N-Boc protected amino methyl ester, where for 3.5 and 3.9, was performed through ex-situ generation of sulfuryl fluoride, which was installed following the substitution of the hydrogen on the hydroxyl group by SO2F. For 3.35, it was achieved through a palladium-catalyzed system and an in-situ fluorine introduction, where para iodine was substituted by the SO2 generated from DABSO. Under physiological conditions, compound 3.5 was assessed for possible interaction through its electrophilic warhead, with nucleophilic N-Boc-lysine side chain. The LCMS and NMR buffered assays were conducted, and in both these studies, the characteristics of a possible binding happening can be observed, hence an adduct N2-(tert-butoxycarbonyl)-N6-((4-((S)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)phenoxy)sulfonyl)-L-lysine 3.5a formation.

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