School of Laboratory Medicine & Medical Sciences

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1,4,7,10,13,16-Hexaazacyclooctadecane (Hexacyclen) Induced Nitrosative Stress and Downregulated NF-κB Cell Survival Pathway in Human Embryonic Kidney (Hek293) and Colorectal Adenocarcinoma (Caco2) Cells.
(2022) Nxumalo, Mthokozisi Bongani.; Khan, Rene Bernadette.; Khumalo, H.
Colorectal cancer (CRC) is the third most common malignancy detected and the second leading cause of cancer-related mortality. Mammalian cells require metals for the physiological process as they are part of the structure or co-factor of many proteins. However, excessive accumulation may manifest in toxicity. In addition, the promotion of oncogenesis and tumour growth has been associated with an increased presence of metals. Promising anticancer compounds that disrupt the onset and progression of carcinogenesis are currently being intensely investigated by the scientific community. Hexacyclen, a nitrogen electron donor and a potent metal ion chelator that binds various metal and transition metal cations, is one such anticancer drug. The cytotoxic effects of Hexacyclen on human colorectal adenocarcinoma cells (Caco2) and normal embryonic kidney cells (Hek293) were investigated in this work after acute exposure (48 hours). The toxicity of Hexacyclen was studied in Hek293 and Caco2 cells at different concentration ranges [(0-500 μM) and (0-50 μM), respectively]. The MTT (to determine IC20 and IC50), ATP and mitochondrial membrane potential (ΔΨM) assays were used to assess metabolic activity, while TBARS, NOS and GSH assays were used to assess oxidative activity. Caspase activity (-8, -9, -3/7), phosphatidylserine externalisation and LDH leakage were used to assess cell death by apoptosis. In addition, western blotting was used to examine the expression of antioxidant (SOD2, GPx, catalase), pro-and anti-apoptotic (p-p53, Bcl-2, HSP70, PARP, cPARP) and inflammatory (NF-κB, STAT3 and p-STAT3) proteins. From the dose-dependent MTT curve, an IC20 and IC50 of 6μM and 38μM (Hek293) and 1.2μM and 5μM (Caco2 cells) were determined. The decreased ATP concentration in Hek293 (p<0.05) and Caco2 (p>0.05) cells for both treatments was consistent with altered ΔΨM in both cell lines, indicating reduced metabolic activity. Elevated RNS was implied by increased iNOS particularly at the Caco2 IC50 (p<0.05) that promoted nitric oxide production at the IC20 (p>0.05) and IC50 (p<0.05) for Hek293 and Caco2 cells respectively. The decreased MDA in Hek293 cells (p>0.05) was associated with increased SOD2 (p<0.05) and GPx (p<0.05), while slightly increased MDA in Caco2 cells (p>0.05) accompanied increased SOD2 (p>0.05) and GPx (p<0.05 at the IC50 only). Furthermore, GSH levels were increased significantly in IC50-treated Hek293 and Caco2 cells (p<0.05), but downregulation of catalase in Hek293 and Caco2 cells was not significant. In this study, apoptosis was initiated by an increase in caspase-9 (IC50, p<0.05) but not caspase 8, which was decreased for both treatments in Hek293 cells (p<0.05). In Caco2 cells, caspase-8 (p<0.05) and caspase 9 (p>0.05) were increased. Anti-apoptotic Bcl-2 (p<0.05) and HSP70 (p<0.05 for Caco2 cells) were downregulated in both cell lines. The activity of p-p53 was not affected in IC20, whereas it was significantly reduced in IC50-treated (p<0.05) in Hek293 and Caco2 cells. Apoptosis was executed as caspase 3/7 was increased in all treatments (p<0.05), albeit non-significantly for IC20-treated Hek293 cells. Moreover, phosphatidylserine externalisation, an early apoptosis marker, was increased in both cell lines (p<0.05 for IC50-treated Hek293 cells), while LDH (a late marker) was increased for Hek293 cells (p<0.05) but not Caco2 cells (p>0.05). Interestingly, decreased cPARP/PARP activity was observed for IC50-treated cells (p<0.05) in both cell lines. Finally, the inflammatory markers NF-κB (p>0.05 for IC20-treated Hek293 cells) and p-STAT3/STAT3 (p>0.05 for IC20-treated Caco2 cells) were downregulated in this study. Hexacyclen induced apoptosis in Hek293 and Caco2 cells via an RNS-mediated mechanism. Intrinsic apoptosis was noted in Hek293 cells, while both pathways facilitated apoptosis in Caco2 cells. Interestingly, apoptosis proceeded concurrently with a reduction in the NF-κB cell survival pathway.
Acquired and transmitted drug resistance in HIV-1 subtype C : implications of novel mutations on replication capacity, cleavage and drug susceptibility.
(2015) Singh, Urisha.; Gordon, Michelle Lucille.
Introduction Large scale roll-out of combination antiretroviral therapy (cART) has been successful in improving the quality of life of HIV-1 infected individuals in South Africa (SA). However the development and transmission of drug resistance threatens the future success and longevity of cART in the country. Studies have shown that resistance to Protease inhibitors (PI’s), in the absence of mutations in Protease (PR), is increasing in SA. Whilst some studies attribute this to poor treatment adherence, others have shown that mutations in Gag contribute to PI resistance. The majority of these studies however have been conducted on HIV-1 subtype B, despite HIV-1 subtype C being the most prevalent subtype globally. Given that Gag is highly polymorphic between subtypes, studies focusing on HIV-1 subtype C are required. Despite the high rate of virologic failure of patients on PI inclusive treatment regimens, no transmitted drug resistance (TDR) studies have identified PI associated TDR mutations. This could be due to the high fitness cost associated with PR mutations which would result in rapid reversion or low frequency of mutations within the viral quasispecies. Most TDR studies in SA, as in other resource limited settings, have used recently infected cohorts to measure TDR. It is however unlikely that rapidly reverting mutations would be detected in recent infection. Furthermore, these studies have all used Sanger sequencing which only detects mutations at frequencies >15-20%. With recent studies showing that low frequency mutations present at frequencies as low as 1% impact treatment outcomes, the elucidation of these mutations using deep-sequencing techniques is necessary. For a true measure of TDR, studies employing acute infection cohorts and deep-sequencing techniques are required. The current study aimed to identify mutations in Gag-Protease associated with PI resistance/exposure, and to determine their impact on replication capacity and drug susceptibility. The prevalence of low frequency TDR mutations in an HIV-1 subtype C acute infection cohort was also investigated. Methods A cohort of 80 HIV-1 subtype C infected participants failing a PI inclusive treatment regimen (i.e. PCS cohort) from 2009–2013 in Durban, South Africa was used to assess the role of Gag in PI resistance. Gag mutations were divided into three groups: PI exposure associated Gag mutations; resistance associated Gag mutations (rGag) and novel Gag mutations (nGag). Frequencies of each of these mutations were compared amongst: 80 PCS cohort sequences, 2,481 HIV-1 subtype B treatment naïve sequences, 954 HIV-1 subtype C treatment naïve sequences and 54 HIV-1 subtype C sequences from acutely infected individuals, in order to identify PI associated mutations and natural polymorphisms. Next, recombinant viruses for all 80 participants were generated by co-transfection of a CEM derived T-cell line (i.e. GXR cells) with an NL43-deleted-gag-protease (NL43Δgag-protease) backbone and patient derived Gag-Protease amplicons. Thereafter, the replication capacity of each virus was assessed using a replication assay that employed a green fluorescent protein reporter cell line and flow cytometry. Associations between replication capacity and Gag-Protease mutations were established. Eighteen viruses with mutations of interest were then selected for use in drug susceptibility assays, where the impact of mutations on susceptibility to lopinavir (LPV) and darunavir (DRV) was assessed in a luciferase based assay. Lastly, the impact of novel Gag mutations on replication capacity and drug susceptibility was validated by generating site-directed mutant viruses with mutations of interest and using these mutant viruses in replication capacity and drug susceptibility assays. Furthermore the cleavage profile of each site-directed mutant virus was established by western blotting. Samples available from 47 HIV-1 subtype C acutely infected individuals collected from 2007-2014 in Durban, South Africa, was used to assess low frequency TDR mutations in HIV-1 subtype C acute infection. Firstly the RT and PR region of each virus was genotyped using the Viroseq HIV-1 genotyping system in order to identify the prevalence of TDR in the cohort. Thereafter 14 participant samples were selected, based on the availability of plasma at one week after onset of plasma viremia (OPV), for sequencing by ultra-deep pyrosequencing (UDPS). This served to identify low frequency mutations. Comparisons in TDR prevalence was made between Sanger sequencing and UDPS. Thereafter, the impact of low frequency TDR mutations on treatment outcomes was assessed by comparing time to virologic suppression for two participants with low frequency mutations to that of four participants without low frequency mutations. Results Protease resistance associated mutations (RAMs) occurred in 34/80 (42.5%) participants, whilst Gag mutations associated with PI resistance in subtype B were detected in 67/80 (84%) participants. Overall, 12 Gag mutations associated with PI exposure (i.e. E12K, V35I, G62R, V370A/M, S373P/Q/T, A374P, T375N, I376V, G381S, I389T, I401T and H219Q), eight rGag mutations (i.e. R76K, Y79F, V128I, A431V, K436R, L449F, R452K and P453L) and four nGag mutations (i.e. Q69K, S111C/I, T239A/S and I256V) were identified in the PCS cohort. The E12K, V370A/M, T375N, G381S, R76K and Y79F mutations all occurred as natural polymorphism in HIV-1 subtype C. The A431V, K436R, L449F, R452K, P453L, Q69K, S111C/I, T239A/S and I256V mutations were all associated with PI resistance/exposure. Interestingly all viruses with PR RAMs harboured rGag and nGag mutations, however rGag and nGag mutations were also found to occur without PR RAMs. Protease RAMs were associated with significantly reduced replication capacity. The K335R and A431V mutations were the only Gag mutations associated with significantly reduced replication capacity. Viruses with PR RAMs were associated with significantly reduced susceptibility to LPV (>15 FC in IC50) and DRV (>6 FC in IC50). Furthermore, the following combinations of rGag and nGag mutations were found to confer reduced susceptibility to LPV and DRV in the absence of PR RAMs: R76K+Y79F+K436R+L449P+I256V (5.2 fold increase in IC50 for DRV), R76K+R453L (23.88 fold increase in IC50 for LPV and a 6.73 fold increase in IC50 for DRV) and R76K+K436R+Q69K+S111C (7.40 fold increase in IC50 for LPV). Analysis of recombinant viruses showed that the Q69K nGag mutation rescued replication capacity of all viruses harbouring A431V+PR RAMs. This was validated by SDM, where Q69K rescued the replication capacity of site-directed mutant viruses harbouring A431V+V82A. The Q69K mutation was also associated with increasing polyprotein cleavage when found in conjunction with A431V+V82A. With regards to TDR, we demonstrated a prevalence of 57% of TDR mutations with UDPS and 2.2% with Sanger sequencing. Sanger sequencing identified the K103N non-nucleoside reverse transcriptase inhibitor (NNRTI)-associated TDR mutation. In addition to K103N (frequency: >99%), the following low frequency mutations were detected by UDPS: the K65R (1-1.5%) and D67N (3.88%) nucleotide reverse transcriptase inhibitor (NRTI)-associated TDR mutations, the F53L (17.6%) and M46L (6.3%) Protease inhibitor (PI)-associated TDR mutations, and the T97A (2.90%) integrase strand transfer inhibitor (InSTI)-associated TDR mutations. Participants with low frequency TDR mutations took 40 days longer to achieve viral suppression than participants without low frequency TDR mutations, when placed on fixed dose combination antiretroviral therapy.
Activation of silent biosynthetic gene clusters and profiling of secondary metabolites secreted by Endophytic Fungi for use as potential anti-HIV agents.
(2022) Makhwitine, John Phuti.; Ndlovu, Sizwe Innocent.; Mkhwanazi, Nompumelelo Prudence.
The continuous burden of Human Immunodeficiency Virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated five endophytic fungi isolated from Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Single-round fractionated extracts of treated P.chrysogenum showed potent anti-HIV activity with an IC₅₀ of 5.90 μg/mL and a 5-fold increase compared to the untreated fraction. The active fractionated extracts were subjected to gas chromatography-mass spectrometry (GCMS), and more bioactive compounds were detected in treated P.chrysogenum fractions than in untreated fractions. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites as therapeutic compounds
The activity of nybomycin against mycobacterium tuberculosis.
(2018) Niehaus, Abraham Johannes.; Moodley, Prashini.; Sturm, Adriaan Willem.
Nybomycin was discovered in 1955, but was never developed for clinical use. The compound was noticed again in recent years when it displayed bactericidal activity against certain fluoroquinolone-resistant bacterial species. The work presented here aims chiefly at describing the effect of nybomycin on Mycobacterium tuberculosis. The study is made up of three parts. In the first part, in vitro nybomycin susceptibility testing was conducted with various fluoroquinolone-susceptible and fluoroquinolone-resistant bacterialspecies. All M. tuberculosis isolates displayed low nybomycin inhibitory concentrations regardless of fluoroquinolone resistance. Similar susceptibility results were obtained for N. gonorrhoeae isolates, but results obtained with other bacterial species were less promising. In the second part, in silico investigations were conducted to elucidate the mechanism of action of nybomycin in M. tuberculosis. Results show that nybomycin binds to M. tuberculosis gyrase enzyme with an affinity at least similar to that of fluoroquinolones. No clear differences in binding affinity were observed when gyrA mutations, commonly associated with fluoroquinolone resistance, were considered. The results suggest that the mechanism of action of nybomycin against M. tuberculosis involves inhibition of gyrase enzyme. In the third part, M. tuberculosis mutants with increased nybomycin minimum inhibitory concentrations were selected and compared with the wild type organism through whole genome sequencing. None of the isolates harbored any mutations commonly linked to known drug resistance mechanisms. This indicates that M. tuberculosis likely employs a novel mechanism of resistance against nybomycin. This may further signify that nybomycin has an additional mechanism of action against M. tuberculosis, besides the action on gyrase enzyme, as suggested by the in silico results from this study. Twenty-two genes were identified through whole genome sequencing that may potentially be linked to the mechanism of resistance and possibly an additional mechanism of action.
The acute effects of dioxidovanadium on blood glucose concentration and oxidative stress in the hippocampus of non-diabetic male Sprague Dawley rats and the chronic effects of dioxidovanadium on selected markers associated with hippocampal dysfunction in male streptozotocin-induced diabetic rats.
(2022) Dayanand, Yalka.; Ngubane, Phikelelani Siphosethu.; Khathi, Andile.; Sibiya, Ntethelelo Hopewell.
Diabetes mellitus is a disease associated with derangements in glucose metabolism and chronic hyperglycaemia. Chronic hyperglycaemia induces oxidative stress and inflammation that affect glucose sensitive hippocampal neurons resulting in generation of amyloid plaques and tau tangles. These are the primary markers used in the detection of neurodegenerative diseases such as Alzheimer’s and dementia. Hence, there is a strong correlation between diabetes and memory impairment. Current therapeutic options such as bolus insulin have been successful in the management of the disease. Despite the efficacy of these therapies, they however have been shown to possess undesirable effects that exacerbate the secondary pathological effects of diabetes on the hippocampus thereby contributing to the detriment of cognitive tasks such as learning and memory. Therefore, there is a need to explore alternative treatments. Transition metals have been shown to possess therapeutic effects with vanadium possessing the greatest potency in lowering blood glucose concentrations. However, studies have demonstrated toxic accumulation of vanadium in the hippocampus which result in the generation of oxidative stress and neurodegeneration. In our laboratory, we have synthesised dioxidovanadium (V) complex by attaching organic ligands to reduce the toxicity and improve potency of the metal. This complex has been shown to efficiently reduce blood glucose and elicit cardio and reno-protective properties. Despite these advancements the effects of this complex on the hippocampus and learning and memory are yet to be established. Therefore, in this study the aim was to evaluate the effect of dioxidovanadium complex on selected learning and memory parameters. Methodology The effect of vanadium on the brain was studied acutely and chronically. In the acute study, animals were separated into 2 groups, non-diabetic control group and a non-diabetic animal group which was were treated with vanadium complex (40 mg.kg-1 p.o). The treatment was administered at time 0. Subsequently an n=3 from each group was sacrificed at regular time intervals (1 hour, 2 hours, 6 hours, 24 hours, 5 days, 10 days) in each group. Blood glucose concentration was monitored before sacrificing and hippocampal tissue was harvested for malonaldehyde (MDA) analysis and glutathione peroxidase (GPx1) and tumour necrosis alpha (TNF-α). The second study was conducted over 5 weeks and consisted of an untreated non-diabetic control, a diabetic control, a positive insulin treated group (0.175 mg.kg-1 s.c) and two dioxidovanadium (V) treated groups (40 mg.kg-1 p.o), a non-diabetic and a diabetic group. Blood glucose was monitored weekly and the Morris water maze was conducted on the last week of the study. After 5 weeks the animals were sacrificed and hippocampal tissue was harvested for malonaldehyde (MDA) analysis, glutathione peroxidase (GPx1) tumour necrosis alpha (TNF-α), amyloid beta (Aβ) and hyperphosphorylated tau (pTau) ELISA’s. Results Acutely, dioxidovandium (V) did not lower blood glucose significantly in comparison to the control group. Interestingly, MDA, GPx1 and (TNF-α) were also not significantly different from the control group over all time periods in the study. Chronically, the glucose concentration of the dioxidovandium (V) treated diabetic group was significantly lowered when compared to the untreated group which displayed significantly increased glucose concentration in comparison to the non-diabetic control. The non-diabetic dioxidovanadium (V) treated group did not show a significant difference in glycaemic level. Increased MDA concentration in the diabetic group was significantly lowered by dioxidovanadium(V) treatment. GPx1 concentration in the dioxidovanadium (V) treated group significantly improved in comparison to the diabetic untreated control. The non-diabetic dioxidovandium (V) treated group showed no significant change in MDA and Gpx1 after the 5-week period. There was no significant difference in TNF-α in dioxidovanadium (V) treated groups, diabetic and non-diabetic. The concentration of Amyloid β was significantly lower in the diabetic control when compared to the non-diabetic control. The dioxidovanadium (V) treated groups, both diabetic and non-diabetic did not have a significant difference in comparison to the diabetic control. pTau concentrations in all groups did not significantly differ. Latency times for the last day of training the Morris water maze followed the same trend. The probe test results, which measured spatial memory, for the diabetic untreated and dioxidovanadium (V) treated groups were significantly reduced in comparison to the non-diabetic control group. The non-diabetic untreated and non-diabetic dioxodivanadium (V) treated were not significantly different. Conclusion Dioxidovanadium (V) treatment in non-diabetic animals did not induce hypoglycaemia acutely however reduced blood glucose concentration in diabetic animals when administered chronically. Dioxidovanadium (V) did not induce oxidative stress and may protect against neurodegeneration by enhancing antioxidant status and therefore was considered as a pro-oxidant in the hippocampus.
Aflatoxin B1 modulates oxidative stress and apoptosis in human embryonic kidney cells.
(2019) Dlamini, Nomali Zanele.; Khan, Rene Bernadette.
Introduction: Aflatoxin B1 (AFB1) is produced by filamentous fungal strains of Aspergillus flavus and Aspergillus parasiticus that infect field crops, therefore AFB1 is a frequent contaminant of dietary staples such as rice, maize and peanuts. Humans and animals are exposed to AFB1 through consumption of contaminated foods, predisposing them to various diseases. AFB1 is a potent hepatotoxin that has been classified by the International Agency of Research on cancer (IARC) as a group1 carcinogen. The carcinogenic effects of AFB1 have been attributed to the metabolism of this toxin to an epoxide that promotes the production of free radicals, mitochondrial toxicity and induction of cell death. With the increasing prevalence of kidney associated diseases in humans, and the AFB1-associated kidney toxicity observed in animals, this study investigated the cytotoxic effects/mechanism of AFB1 in human embryonic kidney (Hek293) cells. Methods: Hek293 cells were exposed to AFB1 (0-100μM) for 24hrs. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. Results and discussion: The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1-treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalisation of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated Hek293 cells. Conclusion: The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in Hek293 cells. Keywords : Aflatoxin B1 (AFB1), oxidative stress, apoptosis, Hek293 cells
Allele-specific polymerase chain reaction (ASPCR) to detect resistance mutations in minor variants of HIV-1 subtype C in patients failing highly active antiretroviral therapy (HAART).
(2014) Maharaj, Shevani.; Gordon, Michelle Lucille.
The World Health Organization (WHO) has recommended Tenofovir disoproxil fumarate (TDF) as one of the preferred first-line antiretrovirals (ARVs). TDF and Abacavir (ABC) were introduced into the South African National Antiretroviral Treatment Guidelines in 2010. However, exposure to TDF and ABC can result in the development of the K65R and L74V resistance mutations, respectively. The K65R mutation occurs preferably in subtype C viruses, due to the unique polymorphisms found at codons 64 and 65 (which are not present in subtype B). This is a cause for concern in South Africa, where subtype C is the most common HIV-1 subtype. In addition, these mutations may be present in the minor viral population (i.e <20% of the viral population) and it has been shown that the presence of a resistance mutation in a frequency as low as <0.5% may be associated with an increase in the risk of virological failure. This study investigated the prevalence of K65R and L74V in the minor viral population, using Allele-specific PCR (ASPCR), in a cohort of subtype C infected patients that failed their first-line treatment regimen that did not include TDF or ABC. RNA was extracted from stored plasma samples from a subset of the South African Resistance Cohort Study (SARCS) and the pol region was reverse transcribed and amplified using a one-step RT-PCR kit (Invitrogen; California, USA). For both the K65R and L74V mutations, ASPCR was performed using specific and non-specific primers. A specific and non-specific standard curve was optimised for each mutation (using a mutant plasmid control) and these standard curves were used to perform an absolute quantification. Subsequently, the percentage of each mutation (in each sample) was calculated by dividing the quantity of mutant sequences in the sample by the quantity of total viral sequences in the sample and multiplying this ratio by 100. The Limit of Detection (LOD) of the K65R ASPCR was 0.72%. Of the 84 patients that were assayed, the K65R mutation was detected in 7 (8.33%) of the patients. Five of the 7 samples were detected above 1% (i.e 3 were approximately 2%, 1 was 9.48% and 1 was 100%) and 2 were detected below 1% (i.e 1 was 0.88% and the other was 0.93%). The limit of detection for the L74V ASPCR was 0.013%.We found the L74V mutation to be prevalent in 9 (10.7%) of 84 patients. In 4 of the 9 patients, the L74V mutation was found in ≥1% of the viral population (viz. 2.82%, 10.10%, 12.02% and 18.22%) and in the other 5 patients, the L74V mutation was detected in <1% of the viral population (2 were between 0.5% and1%, while 3 were detected between 0.013% and0.5%). In this study, ASPCR detected additional K65R and L74V mutations in the minor viral population of TDF and ABC-inexperienced patients that were missed by standard genotyping. These minorityK65R mutations could contribute to treatment failure in these patients when switched to TDF or ABC-containing ARV regimens. ASPCR is a useful tool for screening for minority mutations before starting or switching regimens.
Allicin ameliorates some deoxynivalenol-induced cytotoxic effects in human embryonic kidney (Hek293) cells, but also elicits synergistic and potentiating adverse effects.
(2020) Mamane, Yandisa Zintle.; Khan, Rene Bernadette.
Introduction: Deoxynivalenol (DON), a type B trichothecene produced by plant pathogenic fungi, especially Fusarium graminearum and F. culmorum, is a highly toxic mycotoxin found throughout South Africa. DON is consumed unintentionally through maize derived products and is rapidly becoming a potential health risk to humans and animals. It is a known immunosuppressant that induces apoptosis and oxidative stress and may cause liver lesions and kidney problems. Recently, dietary therapeutics have demonstrated a role against mycotoxin-induced cytotoxicity. Garlic (Allium sativum) is part of the Alliaceae family. The garlic bulb is used for medicine and as food consumption. The aqueous extract has recently demonstrated the potential to protect against mycotoxin-induced cell death and decrease reactive oxygen species (ROS). Aim: This study investigated the induction of apoptosis and oxidative stress by DON in Hek293 cells, and the ability of allicin to ameliorate these effects. Methods: Hek293 cells were treated with a range of allicin concentrations (0-150mM) over 24hrs. An EC50 of 1.7mM was obtained from the MTT assay and used in all subsequent assays. Hek293 cells were treated with 5μM DON, 1.7mM allicin (A), or a combination (DON+A) for 24hrs; untreated cells served as the control. Lipid peroxidation [malondialdehyde (MDA) and lactate dehydrogenase (LDH) assays] were used to indirectly quantify reactive oxygen species (ROS) and oxidative stress; reactive nitrogen species (RNS) were quantified using the nitrates assay. Apoptotic induction was determined by the detection of phosphatidylserine (annexin V) and DNA fragmentation. Necrotic cells were distinguished by propidium iodide uptake. Luminometric quantification of ATP, reduced glutathione (GSH), and caspase 9, 3/7, were used to verify these events. In addition, antioxidant enzymes protein expression of superoxide dismutase (SOD2), catalase and glutathione peroxidase (GPx1); as well as nuclear factor erythroid 2-related factor 2 (Nrf2) and heat shock protein (Hsp70), and apoptotic markers associated protein expression of p53, Bax, and poly (ADP-ribose) polymerase (PARP) were detected by western blotting. Results: DON-induced ROS production was suggested by the depletion of antioxidants including SOD2 (p < 0.0001), catalase (p < 0.0001) and GSH (p = 0.0886). Decreased lipid peroxidation indicated by the decreased MDA concentration (p < 0.0001) and reduced LDH (p = 0.0342) imply that the Hek293 cells were spared from the membrane-damaging effect of oxidative stress. A reduction in Hsp70 (p = 0.0056) and Nrf2 (p < 0.0001), and upregulation of GPx1 (p = 0.0362) protein expression was noted. In addition, increased nitrate concentration in all treatments compared to the control (p < 0.0001) suggested a shift to RNS production. Notably, allicin maintained Nrf2 protein expression similar to the control. The decrease in MDA concentration (p = 0.0109) by allicin was concurrent with depleted GSH (p = 0.0504)and increased SOD2, catalase and GPx1 (p < 0.0001), and suggests allicin induced an oxidative stress response. Allicin also protected DON-treated cells from oxidative stress by upregulating Hsp70 (p < 0.0001), catalase (p = 0.0006) and GPx1 (p = 0.0018), with concurrent decreased GSH (p = 0.0342) and ATP (p = 0.2028) concentration, which were also decreased by DON. In addition, allicin increased MDA (p < 0.0001) and LDH (p = 0.1267) towards control levels in the combined treatment. Apoptosis was reduced in the DON (p = 0.4631) and DON+A (p < 0.0488) treated cells in comparison to the control, necrosis was not evident in any treatment. The slight induction of p53 (p = 0.0008) and PARP-1 (p = 0.4036) by DON implies an attempt at DNA repair, but the Hek293 cells experienced reduced levels of apoptosis. Indeed, Bax expression was slightly reduced (p = 0.1071), caspases 9 (p = 0.0705) and 3/7 (p = 0.4431) activities were diminished, phosphatidylserine was not externalized, and PARP-1 was not cleaved. A non-fragmented DNA profile in allicin-treated and DON+A-treated Hek293 cells may be explained by increased expression of DNA repair proteins, PARP-1 (p = 0.0048 and p = 0.0004 respectively) and p53 (p < 0.0001). The upregulation of p53 is associated with an increase in Bax expression (p < 0.0001 and p = 0.0026 respectively). However, caspases 9 (p = 0.0596) and 3/7 (p = 0.0311) were not activated and apoptosis did not occur. Conclusion: DON treatment induced oxidative stress but not apoptosis in Hek293 cells at the concentration tested. In addition, its mechanism of toxicity in Hek293 cells appears to be more related to nitrosative stress and induction of DNA damage. Oxidative stress and not apoptosis is the possible mechanism of allicin-induced effects in Hek293 cells. Although allicin ameliorated some of the effects of DON in Hek293 cells, it also elicited synergistically or potentiating adverse effects that require further investigation.
The alteration of dopamine receptors in L-DOPA (L-3,4-dihydroxyphenylalanine) induced dyskinesias.
(2021) Mokgokong, Makwena.; Mabandla, Musa Vuyisile.; Msibi, Zama Ndlondlo Princess.
L-3,4-dihydroxyphenylalanine (L-DOPA) can ease symptoms of Parkinson’s disease (PD), butextended use of L-DOPA causes abnormal involuntary movements (AIMs) called L-DOPA induced dyskinesias (LIDs). The present study aims to investigate alterations in HPA axis stimulation, neuroinflammation, DA signalling, and cholinergic signalling using molecular markers in a rat model of LIDs. A unilateral 6-hydroxydopamine (6-OHDA) lesion in the medial forebrain bundle of male Sprague-Dawley rats was used to model Parkinsonism. The PD rat model was treated with L-DOPA to further model LIDs. L-DOPA treated groups included rodents treated for 14 days and rats that developed AIMs during 28 days of treatment. LIDs severity was rated using the AIMs score. Motor skills were assessed using the elevated beam walking test. Cognitive functions were assessed using the Morris water maze test and the novel object recognition test. The concentrations of tumour necrosis factor-alpha (TNF-α), corticosterone, acetylcholinesterase (AChE), and dopamine (DA), and the expressions of D1 receptor (D1R) and D2 receptor (D2R) were quantified. L-DOPA treatment for 14 days improved the 6-OHDA-induced hypokinesia, incoordination, spatial learning, and spatial memory but did not improve recognition memory impairment. Prolonged (28 days) L-DOPA treatment led to AIMs development and failed to improve 6-OHDA-induced spatial memory impairment. L-DOPA treatment significantly increased striatal TNF-α and striatal DA concentration, cerebellar TNF-α and DA concentration, prefrontal cortex (PFC) DA and AChE concentration, but significantly reduced striatal AChE concentration, the concentration of TNF-α and D1R expression in the PFC, plasma corticosterone, and hippocampal AChE concentration. When treatment was prolonged for 28 days, striatal D2R expression significantly increased, while cerebellar TNF-α and DA concentration significantly decreased. Increased striatal D2R signalling increases motor output since the direct basal ganglia (BG) pathway is activated in LIDs. The present study showed significantly increased cerebellar DA concentration in response to BG hypoactivity; however, as striatal D2R increased cerebellar DA decreased. The connectivity between the BG and cerebellum in PD increases off L-DOPA and lowers On L-DOPA. The cognitive decline in the 6-OHDA lesioned rodents and those treated with L-DOPA results from increased AChE concentration. High AChE concentration leads to increased ACh catabolism which impairs cognitive function.
Alzheimer’s disease dementia risk in post-traumatic stress disorder: identification of common underlying mechanisms using rat models.
(2021) Faborode, Oluwaseun Samuel.; Mabandla, Musa Vuyisile.
Post-traumatic stress disorder (PTSD) is a neuropsychiatric disorder that can develop from exposure to a trauma. Studies have shown that people who have PTSD are susceptible to developing dementia, mostly Alzheimer’s disease (AD), suggesting common underlying risk factors in the comorbidity. Although several molecular pathways have been implicated in AD and PTSD, including oxidative stress, cellular apoptosis, synaptic dysfunction and stress dysregulation, the underlying neurobiological mechanisms linking AD and PTSD are less understood. This study, therefore, investigated the effect of trauma-like exposure in an amyloid-beta (Aβ) rat model of AD. Seventy-two adult male Sprague-Dawley rats were used throughout the study. The animals were randomly divided into four groups where they received either footshocks or Aβ(1-42) injection or were exposed to footshocks and Aβ(1-42) injection or remained naive. Following inductions, the animals were tested for cognitive, locomotor and anxiety-like behaviours. Thereafter, brain samples were collected for further neurochemical analyses. Our results show that footshocks increased anxiety-like behaviour and impaired fear memory extinction in Aβ(1-42) lesioned rats. A combination of footshocks and Aβ(1-42) also reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NAD (P) H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and increased the expression of Kelch-like ECH-associated protein 1 (Keap1) in the amygdala and hippocampus. Prior exposure to footshocks before Aβ(1-42) lesion caused a decrease in the number of crossing in the target quadrant of the Morris water maze test and reduced percentage alternation in the Y-maze test, indicating memory deficits. There was an interactive effect of footshocks and Aβ(1-42) lesion on the downregulation of BIN1 and the upregulation of NR2B in the hippocampus. There was also an interactive effect of footshocks and Aβ(1-42) lesion on the upregulation of FKBP5 in the hippocampus, amygdala, and PFC. Our finding suggests that footshock stress can exacerbate AD-like pathology via dysregulated redox balance, BIN1 downregulation, FKBP5 and NR2B upregulation, and increased apoptosis in the brain of Aβ(1-42)-lesioned rats. These molecular changes were associated with increased anxiety, impaired fear extinction and memory deficits. These findings, therefore, suggest common molecular mechanisms in PTSD and AD. Isifo sengcindezi eba semva kwesehlakalo esibi i-post-traumatic stress disorder (i-PTSD) yisifo sokusebenzelana kwezinzwa nensebenzamqondo esidala wukwehlelwa yisehlakalo esibi. Ucwaningo selukhombise ukuthi abantu abane-PTSD basengcupheni yokuba nesifo sokukhohlwa, ikakhulukazi i- Alzheimer’s (i-AD), okuchaza ukuthi izimo eziyingcuphe enkulu ekubeni nezifo eziyizimbelambela. Nakuba izindlelamigudu eziningi zamamolekhyuli zisoleka kwi-AD ne-PTSD, okufaka nengcindezi ye-oxidative, i-cellular apoptosis, i-synaptic dysfunction kanye nokungalawuleki kwengcindezi, izindlelamigudu zempiliswanozinzwa nomzimba ezixhumanisa i-AD ne-PTSD akuqondwa ngokuphelele. Ngakho-ke lolu cwaningo, luphenye umthelela wokubekeka endaweni ecisho ibe yisehlakalo esinzima kwimodeli yamagundane i-amyloid-beta (Aβ) ye-rat model of AD. Amagundane amadala esilisa angamashumi ayisikhombisa nambili asetshenziswa kulolu cwaningo. Izilwane zehlukaniswa ngokungahleliwe zaba amaqembu amane lapho zanikwa khona amafootshocks noma umjovo we-Aβ(1-42) noma ayengathola i-footshocks nomjovo we-Aβ(1-42) noma ahlale engazi. Ukulandela ukwethulwa, izilwane zahlolelwa ukuziphatha kwazo ngokomcabango, ukuhamba nokuba nexhala. Emva kwalokho, amasampula obuchopho aqoqwa ukuze aphinde acutshungulelwe amanyurokhemikhali. Imiphumela yethu iveze ukuthi ama-footshocks enyuse ukuziphatha sakuba nexhala kanye nokuqeda ukukhumbula ukwesaba okugwegwile kuma-Aβ(1-42) onikwe amalesioned rats. Ingxubevange yefootshocks ne-Aβ(1-42) iphinde yehlisa ukuvela kwe-nuclear factor erythroid 2-related factor 2 (Nrf2), i-NAD (P) H: i-quinone oxidoreductase 1 (NQO1), i-heme oxygenase-1 (HO-1), kanye nokuvela okusezingeni eliphezulu kwe-Kelch-like ECH-associated protein 1 (Keap1) kwi-amygdala ne-hippocampus. Ukusondelana nama-footshocks kwangaphambi kwe-Aβ(1-42) kwdala ukwehla esibalweni sokuhlanganayo kwi-target quadrant yokuhlolwa kwe-Morris water maze test nenguqunguquko yephesenti kwi-Y-maze test, okuveza ukushoda kokukhumbula. Kwaba nomthelela onokungenelana wama-footshocks kanye ne-Aβ(1-42) lesion ekulawulenikwehlisa i-BIN1 nasekulawulenikukhuphula i-NR2B kwi-hippocampus. Kwaphinde kwaba nomthelela ongenelanayo we-footshocks ne- Aβ(1-42) lesion ekulawulenikukhuphula i- FKBP5 kwi-hippocampus, i-amygdala, ne- PFC. Esikutholile kuphakamise ukuthi ingcindezi ye-footshock ingabhebhezela isakhiwosifo esifuze i-AD nge-dysregulated redox balance, i-BIN1 downregulation, i-FKBP5 ne-NR2B upreguation, i-apoptosis ephezulu ebuchosheni be-Aβ(1-42)-ekuma-lesioned rats. Lezi zinguquko kumamolekhyuli zazihlotshaniswe nokwenyuka kwexhala, inqedakwesaba egwegwile nokulahlekelwa wukukhumbula. Ngakho-ke lokhu okutholakele, kuphakamisa izindlela ezejwayelekile zomumo wamamolekhyuli kwi-PTSD ne-AD.
An investigation into kojic acid-associated mitochondrial toxicity and inflammation in melanoma cells (SK-MEL-1).
(2023) Suritham, Tamzin Kimera.; Chuturgoon, Anil Amichund.; Ghazi, Terisha.
ojic acid (KA), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, is used in agriculture, food, and cosmetics. KA is known to have antimicrobial, antifungal, antioxidant, and anti-inflammatory properties. The cosmetic industry's increasing interest in KA is due to its ability to inhibit tyrosinase activity resulting in skin lightening. The mitochondria play a key role in maintaining homeostasis and ensuring efficient melanin production. Therefore, mitochondrial dysfunction has severe effects on the skin. This study investigates mitochondrial stress, antioxidant responses, protein kinase signalling and inflammation in human melanoma (SK-MEL-1) cells. The mitochondria are important in processing metabolites and supplying the cell with energy in the form of ATP. KA interacts with key mitochondrial homeostasis proteins. Our results found an increase in macromolecule damage specifically lipid peroxidation and protein oxidation. Due to oxidative conditions, increased Nrf2 expression was observed. LON protease is ATP-dependent and regulated by Sirtuin 3 expression. Mitochondrial function was affected illustrated by decreased ATP production leading to decreased LON protease and Sirtuin 3 protein expression. Following increased oxidative stress, KA suppressed the expression of protein kinases but increased inflammatory mediators. There was decreased expression of phospho-Akt, Akt, phospho-GSK3β, p38 and ERK1/2. The mediation of the NLRP3 inflammasome involves priming and activation. At concentrations with high proliferation, NFκB gene and protein expression was activated. The protein kinase signalling pathways are known as mediators of inflammation; however, protein and gene expression of inflammatory mediators was increased following KA treatment. The inflammasome was subsequently activated as shown by an increase in intracellular caspase 1 levels as well as NLRP3, ILβ and IL-6 expression. KA induced mitochondrial stress and suppressed mitochondrial homeostasis proteins. The increased Nrf2 expression could have further downregulated LON protease expression and increased macromolecule damage. Oxidative conditions could have activated the inflammasome pathway independent of protein kinase signalling. In conclusion, KA displayed mitochondrial toxicity following acute exposure by suppressing mitochondrial homeostasis, protein kinase pathways and initiating inflammation. 1
An investigation on the effects of a rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet-induced prediabetic rats.
(2023) Siboto, Angezwa.; Khathi, Andile.; Ngubane, Phikelelani Siphosethu.; Sibiya, Ntethelelo Hopewell.
Prediabetes is a metabolic disorder that often precedes the onset of type 2 diabetes mellitus. This development of this asymptomatic condition is associated with chronic consumption of high calorie diets and sedentary lifestyles. Prediabetes results in decreased insulin sensitivity in the peripheral tissues resulting in elevated blood glucose levels that are not high enough for a diagnosis of type 2 diabetes. This moderate hyperglycaemia has been shown to lead to trigger complications such as non-alcoholic fatty liver disease, renal dysfunction and cardiovascular disease which are generally only diagnosed during type 2 diabetes. The current management strategy for prediabetes consists of a combination of pharmacological and lifestyle intervention. The pharmacological agents such as metformin while lifestyle intervention involves dietary modification to lower calorie diets. Studies show that prediabetic patients tend to be more dependent pharmacological intervention and struggle with changing diets thus lowering the efficacy of drugs such as metformin. This often leads to the eventual development of prediabetes. Therefore, there is a need for new pharmacological agents that can remain effective in both the presence and absence of dietary intervention. In our laboratory we have synthesised a novel rhenium (v) compound with uracil-derived ligands that has shown promising biological activities that include anti-hyperglycaemic effects in diet-induced prediabetic rats. This compound was shown to improve insulin sensitivity in peripheral tissues in prediabetic rats. To advance from this knowledge, this study sought to investigate the effects of the rhenium (V) compound with uracil-derived ligands on markers associated with hepatic, cardiovascular and renal complications in diet induced prediabetic rats model.
Analysis of a multidrug resistant acinetobacter SPP. outbreak in the intensive care unit of King Edward VIII Hospital.
(2000) Deedat, Fathima.; Sturm, Adriaan Willem.
The study arose out of a need to investigate and control a nosocomial outbreak caused by multidrug resistant Acinetobacter spp in the fifteen-bed intensive care unit of King Edward VIII Hospital. Following the discovery of the index case, four other patients were found to have a similar strain of Acinetobacter spp. All fifteen patients in the ward were subsequently screened for the organism. Forty-seven isolates were obtained from 12 patients. Eight of the patients were infected with the organism and six of these eight patients subsequently died. Swabs from the ward environment were also screened for the organism, which was found in patients' baths, suction water and urine collection jars. The outbreak was aborted by the use of strict infection control techniques. Minimum inhibitory concentrations (MICs) of 20 of the 47 isolates were determined for the following antimicrobials: imipenem, ciprofloxacin, gentamicin, amikacin, netilmycin,cefotaxime, ceftazidime and tetracycline. The same 20 isolates were further typed using ribotyping. Seven different antibiogram patterns were obtained using the MIC data. The majority of isolates (11) fit into a Single type, and showed resistance to all drugs tested, except for susceptibility to tetracycline and netilmycin only. Ribotyping revealed 5 different types. There were 9 isolates of ribotype a, 2 of ribotype b, 3 of ribotype c, 5 of ribotype d and 1 of ribotype e. In conclusion, this study describes a nosocomial outbreak with a multidrug resistant Acinetobacter spp. in an intensive care unit. The results showed that there was no correlation between the two typing methods used, ribotyping was more discriminatory than antibiogram types, with the majority of strains belonging to two different ribotypes.
Anatomic study of the morphologic relationship between the proximal left and right coronary arteries.
(2016) Singh, Sadhna.; Satyapal, Kapil Sewsaran.; Lazarus, Lelika.; Ajayi, Nasirudeen Oladipupo.
Arising from the aorta, the right (RCA) and left (LCA) coronary arteries provide the arterial supply to the heart. An extensive literature review revealed that most studies have either evaluated the morphology of the RCA or the LCA independently. This study aimed to document the relationship between the morphology of the RCA and LCA using coronary angiograms and fetal dissections. In addition, variations such as split or double RCA and absence of the LCA was documented. A review of 500 coronary angiograms and a fetal dissection of 41 heart specimens was conducted. The RCA and LCA were classified according to their branching patterns and arterial dominance. The embryologic relationship between the RCA and the LCA was also documented including their lengths and diameters. The angiographic review showed that the most prevalent branching pattern of the LCA was bifurcation in 65.8%, while trifurcation and quadrifurcation occurred in 20.4% and 1.6%, respectively. The splitting of the RCA and absence of the LCA occurred in 4.2% and 11.8%, respectively. A significant correlation was found between the split RCA and absent LCA showing that the split RCA was more prevalent in the absence of the LCA. The dissection of the fetal heart specimens (age range 13.13 - 26.95 weeks) found that the RCA arose from the right aortic sinus and provided arterial dominance in all the specimens. The LCA was classified into types according to their branching pattern. The bifurcation, trifurcation and quadrifurcation of the LCA occurred in 68.3%, 29.3% and 2.4% of hearts, respectively. The mean lengths of the RCA and LCA were 0.98 ± 0.54mm and 1.83 ± 0.77mm, respectively. The mean diameters of the RCA and LCA were 0.38 ± 0.12mm and 0.49 ± 0.17mm, respectively. A significant correlation was found between the RCA and LCA length and the fetal age indicating changes in the development of the coronary vasculature with fetal development. A knowledge of the distribution of the RCA and LCA assists in providing information on the area of the myocardium supplied. With the advent of coronary angiography, a comprehensive understanding of coronary arterial anatomy and their variations is necessary.
Anatomical classification of Tessier craniofacial clefts number 3 and number 4 in a South African population.
(2019) Omodan, Abiola Olugbenga.; Madaree, Anil.; Lazarus, Lelika.; Pillay, Pamela.; Satyapal, Kapil Sewsaran.
The craniofacial clefts are rare defects of the face with an incidence of 1.43 to 4.85 per 100,000 live births. In 2016, WHO reported a death rate of 303,000 new-borns before 4 weeks of age due to congenital anomalies of which craniofacial clefts are one. Surviving the defect is associated with long term disabilities which impacts the individual, families, the healthcare system and society. How much we know about these clefts is seriously hampered by the rarity and the variations of these defects, so much so, that its treatment and communication amongst researchers is affected. The understanding of the skeletal defects occurring in the clefts has long been postulated as a key to any successive reconstruction of the face. This study aimed to reveal the extent of our understanding of these clefts, document the anatomical basis for the craniofacial cleft number 3 and number 4 and generating a sub-classification based on this and also document the clinical presentation as well as associated clefts of these craniofacial clefts in our select South African population. The methods used to achieve these included conducting a scoping review of the literature on patients with Tessier cleft number 3 and number 4 using relevant identified studies from 1976 sourced from PubMed, Medline, EBSCOhost, Google Scholar and the Cochrane libraries. The result of the study was reported using the Preferred Reporting Items for Systematic and Meta-analyses (PRISMA). Likewise, CT scans of patients who had been treated for Tessier clefts number 3 and 4 at Inkosi Albert Luthuli Central Hospital in Durban South Africa between 2003 and 2017 were analysed. Measurements of the expected defects in each cleft were taken and compared with the unaffected side as reference points. Emerging patterns of their analysis were then used to generate a sub-classification for these clefts. Lastly the records of 8 patients who had been treated for either Tessier cleft number 3 or number 4 were reviewed and compared with 9 studies sourced from the literature. In addition to the defects recorded, associated clefts and other congenital malformations were also documented, and findings were compared. The scoping review had 33 studies that met the inclusion criteria. The majority were conducted in middle income countries (54.5%) while none were recorded in low income countries. Only 12.1% of the included studies reported on anthropometry. In understanding the skeletal defects, the presence of an alveolar cleft, the emerging patterns of comparison of the measurements of the maxilla and the orbits of the cleft side and the non-cleft side as well as absence of the bone were used to arrive at a sub-classification system using (a), (b). (c), (M+ O+), (M- O-), and (0). Clinical presentation of the patients who had been treated as cases of Tessier cleft number 3 and number 4 were compared to the reviewed literature and the different parameters were documented. In addition, associated clefts were also recorded, and this study found that the association pattern noted for Tessier cleft number 4 did not conform to its traditional counterpart. In conclusion, this study found that the knowledge of Tessier clefts number 3 and number 4 exist albeit not fully documented. Also, the study proposed a sub-classification for Tessier clefts number 3 and number 4 that will allow physicians to anticipate the extent and form of skeletal defect present before even seeing the patient. Lastly, it was concluded that however variable these clefts appear; they have a similar presentation worldwide and also that associated clefts do not conform to the original Tessier classification system.
An anatomical exploration of the extracranial (V1-V3) and intracranial (V4) components of the vertebral arteries in a select KwaZulu-Natal population.
(2021) Omotoso, Bukola Rukayat.; Lazarus, Lelika.; Satyapal, Kapil Sewsaran.; Harrichandparsad, Rohen.
The risk of injury to the vertebral artery is a significant complication of surgery. The presence of anatomical variation in the course of the vertebral artery increases the likelihood of injury. Due to inadequate understanding of the presence and location of anatomical variations in the morphology and morphometry, the vertebral artery can be injured during surgical intervention. Apart from the vascular injury that can occur during surgical intervention, anatomical variations have implications for some pathologies in the posterior circulation territory. These include aneurysm formation, cerebrovascular disorders, posterior circulatory stroke, and some neurovascular problems. In this retrospective observational study, we investigated the anatomical features of the extracranial (V1-V3) and intracranial (V4) components of the vertebral arteries in a South African population. The study is an observational, retrospective chart review of 554 consecutive South African patients (Black, Indian, and White) who had undergone computed tomography angiography (CTA) at Lenmed Ethekwini Hospital and Heart Centre, Durban, South Africa, from January 2009 to September 2019. The vertebral artery exhibited various morphological variations in its course. We report the incidence of variant origin of the left vertebral artery (6.9%). The level of entry into the transverse foramen ranged between C7-C3. We report the incidence of vertebral artery tortuosity at V1, V2: 76.6%, and 32.1%, respectively. We observed fenestration at V3 (0.18%) and V4 (0.4%) segments. We registered the incidence of the persistent first intersegmental artery (1.1%), extradural PICA origin (2.8%), atresia (6.7%), and hypoplastic terminal vertebral artery (13.2%). Average length and diameter at each vertebral artery segment were registered; we also report on hypoplasia of the vertebral artery. Anatomical variations of the vertebral artery are common in the South African population studied in the present study. Imaging of the complete segments of the vertebral artery from the origin to the point of convergence to form the basilar artery may be necessary to decide a treatment strategy for interventions in the vicinity of the vertebral artery. Understanding the patterns of anatomical variations of the vertebral arteries will contribute significantly to the diagnosis of various diseases in the posterior circulatory territory. The average diameter was significantly larger on the left in all the racial groups, but there were no significant gender differences. We registered a left dominance pattern in all the segments (V1-V4). Iqoqa Ingozi yokulimala emithanjeni yomgogodla iyinkinga enzima kakhulu yokuhlinzwa. Ukuba khona kokwehlukahlukana kokwakheka komzimba ekuhambeni komthambo womgogodla kwandisa amathuba okulimala. Ngenxa yokuqonda okunganele kokukhona kanye nendawo yokwehlukahlukana kwesakhikwo somzimba ekwakhekeni nokulinganisa umumo, umthambo womgogodla ungalimala ngesikhathi sokuhlinzwa. Ngaphandle kokulimala kwemithambo yegazi okungenzeka ngesikhathi sokuhlinzwa, ukuhlukahlukana kwemithamdo yomgogodla kunomthelela ngezinye izimbangela ngokuthola umsuka wesifo ngokuhamba kwegazi emigudwini. Lokhu kubandakanya ukwakheka kokuvuvukala komthambo, ukuphazamiseka kokuhamba kwegazi engqondweni, ukushaywa yisifo sohlangothi, nezinye izinkinga ngezinzwa nemithambo. Kulolu cwaningo lokubheka ngokuqhathanisa abanesifo nabangenaso, sibheke ukwakheka komzimba kwamathambo ekhanda ngaphandle (V1-V3) kanye nokwakheka kwawo ngaphakathi (V4) nezingxenye zemithambo yomgogodla emphakathini waseNingizimu Afrikha. Ucwaningo lungukuzibonela ngqo, ukuqhathanisa ngokubuyekeza amashadi eziguli zaseNingizimu Afrikha angama-554 ngokulandelana (abaNsundu, amaNdiya, nabaMhlophe) abafakwe emshinini bahlolwa wonke umzimba ngekhompuyutha ukubona okusemithanjeni (isibonathambomzimba) (CTA) esibhedlela i-Lenmed Ethekwini neSikhungo seNhliziyo, eThekwini, eNingizimu Afrikha, kusukela kuMasingana wowezi-2009 kuya kuMandulo wowezi-2019. Umthambo womgogodla ukhombisa ukwehlukahluka kwesakhiwo ekuthubelezeni kwawo. Sibika isehlakalo semvelaphi esehlukile somthambo womgogodla kwesokunxele (6.9%). Izinga lokungena esikhaleni esiphakathi komthambo womgogodla laliphakathi kwe-C7 ne-C3. Sibika isehlakalo esihambisana nokuguga komthambo womgogodla nomfutho wegazi okulinganiselwa phakathi kuka-V1, V2: 76.6% no-32.1%, ngokulandelana. Sibone ukuhlinzwa kwesakhiwo sendlebe ngaphakathi kwezingxenye ezingu-V3 (0.18%) nezingu-V4 (0.4%). Sabhalisa izehlakalo zomthambo wokuqala ngezingxenye ezilokhu zikhona ngo-1.1%, imvelaphi ye-PICA yamathambo ekhanda (2.8%), isicubu esingenayo embotsheni ngokwemvelo (6.7%), nokungakhuli kwesitho ngokuphelele (13.2%). Isilinganiso sobude nobubanzi engxenyeni ngayinye yomthambo womgogodla yabhaliswa; siphinde sibike ngokungasebenzi ngokwejwayelekile komthambo womgogoda. Ukwehlukahlukana kokwakheka komthambo womgogodla kuvamile kubantu baseNingizimu Afrikha ocwaningweni lwamanje. Ukufanekisa kwezingxenye eziphelele zomthambo womgogodla lapho zihlangana khona ukwenza umthambo ophakathi nendawo ekhanda kungadingakala ukunquma ngamasu okwelapha ngokungenelela endaweni eseduze nomthambo womgogodla. Ukuqonda ukuphiceka kwesakhiwo esahlukahlukene semithambo yomgogodla kuzodlala indima ebalulekile ekuhlonzeni izifo ezahlukahlukene ekuhlinzekweni kokuhamba kwegazi. Isilinganiso sobubanzi besisikhulu kakhulu kwesokunxele kuwo wonke amaqembu ezinhlanga, kodwa kwakungekho mehluko obalulekile phakathi kobulili. Sibhalise indlelakwenza ebihamba phambili kuzo zonke izingxenye ebe ngu-V1-V4.
An anatomical investigation of intracranial meningiomas.
(2021) Anirudh, Ezra Earl.; Lazarus, Lelika.; Harrichandparsad, Rohen.
Meningiomas are generally benign, highly vascularised, slow-growing tumours arising from the arachnoid cap cells of the arachnoid villi. The clinical presentation of these tumours is usually location dependant due to the vast expanse covered by the meninges. Resection of a meningioma is generally performed after preoperative embolisation. A feeder vessel is selected and embolised in an attempt to reduce excessive blood loss and postoperative complications. However, embolisation requires a sound knowledge of the vasculature of the meninges since these vessels supply portions of the cranial nerves. Literature consulted have investigated anatomical features of meningioma’s; however, there is a scarcity of studies investigating patients specifically referred for preoperative embolisation. Therefore, this study aimed to investigate the anatomical features, namely the location, histology, volume and vascularity of intracranial meningiomas referred for preoperative embolisation. This entailed using Magnetic resonance imaging (MRI), Digital subtraction angiography (DSA), and the histological reports obtained from the data bank at the central regional hospital in Durban, South Africa. A retrospective chart review yielded 103 patients that met the inclusion criteria, of which 98 patients (subset 1) presented with a single meningioma and 5 patients (subset 2) presented with multiple meningiomas. The average age of patients (at the time of diagnosis) was reported within the 40–49-year group and primarily within the female population (subset 1: 67.3%; subset 2: 80%). The benign grade of meningiomas was reported as the most common (70.4%), of which the meningothelial subtype (48%) was predominant. Meningiomas were mostly observed within the supratentorial region (subset 1: 57.2%; subset 2: 91.7%) with almost equal incidences in subset 1 and a majority on the right side in subset 2. Regarding tumour volume, subset 1 revealed the largest meningiomas within the supratentorial region (90.9 cm3), and subset 2 revealed an average tumour volume of 43.9 cm3. In terms of meningioma vascularity, within the supratentorial region, the external carotid arteries were noted to be a common primary feeder vessel, for the skull base region the primary arterial supply is the internal carotid artery. This study provides insight into the anatomical basis of intracranial meningiomas within a select South African population as it has introduced a novel methodology of meningioma vascularity. This may assist endovascular surgeons in assessing the feeder vessel contributions of meningiomas and understand the prevalence of these anatomical parameters in this population.
An anatomical investigation of the sympathetic and parasympathetic contributions to the cardiac plexus.
(2011) De Gama, Brenda Zola.; Satyapal, Kapil Sewsaran.; Partab, Pravesh.; Lazarus, Lelika.
The cardiac plexus is “formed by mixed autonomic nerves” that are “described in terms of superficial and deep components, with the superficial located below the aortic arch and anterior to the right pulmonary artery, and the deep located anterior to the tracheal bifurcation (above the division of the pulmonary trunk) and posterior to the aortic arch” (Standring et al., 2008). This investigation aims to review and update the medial cardiac contributions of the cervical and thoracic sympathetic chains to the cardiac plexus and also the contributions from the vagus nerve and its counterpart, the recurrent laryngeal nerve. This study involved the macro and micro-dissection of 100 cadaveric sides of adult and fetal material. The number of ganglia in a cervical sympathetic chain varied from 2 to 5 in this study. This study confirms previous reports on the location of the two components of the cardiac plexus. The origin of the sympathetic contributions to the cardiac plexus in this study were either ganglionic, interganglionic or from both the ganglion and interganglionic chain of the respective ganglia. The superior cervical cardiac nerve had an incidence of 92% while the middle cervical cardiac nerve had an incidence of 65% in the specimens studied. This study also records a vertebral cardiac nerve that arose from the vertebral ganglion in 39% of the cases. The inferior cervical and cervicothoracic cardiac nerves had incidences of 21%, respectively. This investigation records the thoracic caudal limit of the sympathetic contributions to the cardiac plexus as the T₅ ganglion. The findings in this study indicate the importance of understanding the medial sympathetic contributions and their variations to the cardiac plexus as this may assist surgeons during minimal surgical procedures, sympathectomies, pericardiectomies and in the management of diseases like Reynaud’s Phenomenon and angina pectoris (Kalsey et al., 2000; Zhang et al., 2009).
The anatomical study of the osteochondral, vascular and muscular relations of the superficial and deep cervical plexuses.
(2010) Pillay, Pathmavathie.; Satyapal, Kapil Sewsaran.; Partab, Pravesh.
In standard anatomical textbooks, the formation of the cervical plexus is well defined; however the accurate differentiation into superficial and deep plexuses, their emerging patterns, and gross anatomical relations are not documented as expansively. In order to obtain detailed anatomical knowledge of the superficial and deep cervical plexuses, the investigation aimed to clarify the anatomy and variations of these plexuses, define possible anatomical landmarks, and record the relationship of the external jugular vein and muscles of the posterior triangle of neck to the branches of the superficial cervical plexus, and the relationship of the common carotid artery, internal jugular vein, sympathetic chain, cervical verterbrae, and vertebral artery to the deep cervical plexus. The studies utilized the gross anatomical dissection, morphological and statistical analyses of forty fetal and fifteen adult cadaveric, formalinized specimens. The branches of the superficial cervical plexus emerged from the posterior border of the sternocleidomastoid muscle at the great auricular point (situated in the middle third of the muscle) and was described as ascending (lesser occipital, great auricular, transverse cervical nerves) and descending (supraclavicular nerves). Further, these branches were recorded according to their branching patterns, relations to the external jugular vein and variations. The branching patterns are described as single, duplicate and triplicate. The external jugular vein was constantly located inferior to the great auricular nerve, superior to the transverse cervical nerve and intertwined with the branches of the supraclavicular nerves. Variations of the branches of the superficial cervical plexus were observed only in fetuses and classified according to their course, branching patterns and communications. The emerging point of the branches of the superficial cervical plexus on the sternocleidomastoid muscle, were determined according to the seven types of “emerging pattern” classification by Kim et al., (2002). In order to record the deep cervical plexus, the sternocleidomastoid muscle was reflected with the following observation: the ventral rami of the second and third cervical nerves emerged between the scalenus anterior and scalenus medius muscles, and the third and fourth cervical nerves was located at the lateral edge of scalenus medius muscle. The deep cervical plexus was described as communicating, muscular, ansa cervicalis, and phrenic nerves. The superior cervical ganglion constantly communicated with the ventral rami of the cervical nerves; and the hypoglossal communicated with the superior root of the ansa cervicalis. The muscular branches were observed to the scalenus anterior and scalenus medius muscles with an anomalous branch to the sternocleidomastoid muscle. The ansa cervicalis demonstrated a degree of variation with regard to its origin, course and formation of the loops. The phrenic nerve arose from the ventral rami of the third, fourth and fifth cervical nerves and descended on the lateral border of the scalenus anterior muscle. The precise understanding of the anatomy of the superficial and deep cervical plexuses together with variations may assist anesthetists and surgeons to accurately identify the vascular, neural and muscular structures and reduce the risks of complications when performing neural blocks in regional anesthesia, facial rejuvenation surgery and parotidectomies.
Anterior synostotic plagiocephaly: a quantitative analysis of craniofacial features using computed tomography.
(2021) Mohan, Nivana.; Lazarus, Lelika.; Madaree, Anil.; Harrichandparsad, Rohen.
Anterior synostotic plagiocephaly (ASP) is caused by the premature fusion of one coronal suture, which results in severe craniofacial asymmetry that can be challenging to correct. The various methods of the surgical procedures, as well as the distinctive facial characteristics of ASP, have been well documented. However, there is a paucity of literature pertaining to the quantitative analysis of the craniofacial features that are affected in ASP. This study used preoperative computed tomography (CT) scans to document and compare the morphometry of the anterior cranial fossa (ACF), orbit, and ear on the ipsilateral (synostotic) and contralateral (non-synostotic) sides in a select South African population of patients diagnosed with ASP. The dimensions of the ACF, orbit and the position of the ear on the ipsilateral and contralateral sides were measured using a set of anatomical landmarks on two-dimensional (2D) CT scans of 18 consecutive patients diagnosed with non-syndromic ASP. The differences between the ipsilateral and contralateral sides were computed and expressed as a percentage of the contralateral side. The findings of this study revealed that there was side-to-side asymmetry in the ACF, orbit, and ear. All ACF parameters decreased significantly (t-test; p<0.001) on the ipsilateral side when compared to the contralateral side, resulting in the volume of the ACF being the most affected (-27.7%). In terms of the orbit, on the ipsilateral side, the length-infraorbital rim (IOR), height, and surface area parameters increased significantly (t-test; p<0.001), with the height being the most affected (24.6%). The remaining orbital parameters (length-supraorbital rim (SOR), breadth and volume) decreased significantly (t-test; p<0.001), with the length-SOR parameter being the most affected (-10.8%). Furthermore, the ipsilateral SOR was noted to be displaced more cranially by an average of 3.89mm from the contralateral SOR. With regards to the position of the ipsilateral ear, it was found to be displaced anteriorly (9.33mm) and caudally (5.87mm) from the contralateral ear. This study augments the existing literature by providing actual values to corroborate the hallmark characteristics of ASP. These measures may help surgeons plan the technique and extent of surgical correction of the affected craniofacial structures during corrective surgery as it will provide them with an indication of the extent of the deformity on the ipsilateral side as compared to the contralateral side. The results of this study have the potential to propose a grading system in ASP patients according to severity of the condition if the sample size is increased.

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