The Effects of Various Combinations of Different Classes of Anticancer Drugs and Tyrosine Kinase Inhibitors on the Human MCF-7 Breast carcinoma Cell Line
Beynon Abrahams1
Prof Donavon Hiss2, Dr Sahar Abdul-Rasool2
University of the Free State1, University of the Western Cape2
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Introduction and aim: In breast cancer, the altered expression of epidermal growth factor receptors (EGFR and HER-2) parallels an aggressive clinical course and the development of resistance to anticancer therapies. EGFR inhibitors that specifically target the intracellular and extracellular domains of EGFR include monoclonal antibodies (e.g., trastuzamab) and tyrosine kinase inhibitors (TKIs): (e.g., gefitinib) are amongst the most effective agents that are currently used in clinical practice.
Objective: In this study we examined the effects of doxorubicin (DOX), cisplatin (CPL) and three investigational TKIs: EGFR inhibitor I, EGFR Inhibitor II/BIBX1382 and EGFR/ErbB-2/ErbB-4 Inhibitor, individually and in combination, on human MCF-7 breast carcinoma cells.
Methodology: Analyses of MCF-7 cells exposed to DOX, CPL and TKIs, alone and in combination, included dose-response curves (cytotoxicity assays), drug synergy analysis, morphological staining of apoptotic cells with haematoxylin and eosin and Annexin V-FITC. Combination drug effect analysis was used to study the efficacy of EGFR inhibitor combinations on MCF-7 cells.
Results: MCF-7 cells were exposed to different concentrations of TKIs alone and in combination with each other. The combination of the TKIs demonstrated a significant reduction in cell growth, compared to the inhibitors used individually. Synergistic as well as antagonistic effects were observed in combinations with DOX, CPL and the TKIs with resultant decreases in dose reduction indices (DRIs) implying greater efficacies with the respective combinations.
Conclusion: Our findings coincide with previous assertions that concurrent blocking of the ErbB family of RTKs and other drug targets in cancer cells may be of particular benefit to breast cancer patients. Elucidation of the mechanisms involved in the combined cytotoxicity of DOX, CPL and TKIs and optimizing their synergistic concentrations to enhance efficacy could add significant value to cancer drug discovery and development.

Aqueous extracts of orange fleshed sweet potato ameliorate oxidative stress in insulin resistant C2C12 myotubes
Taiwo Betty Ayeleso1
Khosi Ramachela1, Emmanuel Mukwevho1
Northwest University Mafikeng Campus, Mmabatho Northwest1
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Oxidative stress has been implicated in the development of insulin resistance and its progression into type 2 diabetes and its complications. Natural antioxidants from edible plants are source of dietary components that can help to manage insulin resistance. Sweet potato, which is very rich in antioxidants, is a global food crop that has been used as a folk remedy for diabetes mellitus. This study investigated the effect of aqueous extracts of orange sweet potato (leaves and tubers) on the indices of oxidative stress in insulin resistant skeletal muscle cells. Insulin resistance was induced in C2C12 myotubes with sodium palmitate. Oxidative stress biomarkers (total glutathione, GSH and lipid peroxidation, LPO) and activities of antioxidant enzymes (catalase, CAT and glutathione peroxidase, GPx) were measured using established techniques. Antioxidant capacity using trolox equivalence absorbance capacity (TEAC) and ferric reducing antioxidant power (FRAP) assays were also carried out. An antidiabetic drug-metformin was used as the standard control.
The results showed that there was a significant increase in GPx activity with a corresponding decrease in total glutathione in palmitate-treated group. However, treatment with the extracts and metformin improved glutathione status in all the groups. No significant difference was shown in CAT activity between the control and the palmitate-treated groups while there was upregulation of CAT activity by both the extracts and metformin. There was a significant reduction of lipid peroxidation in all the groups treated with the extracts and metformin. Furthermore, FRAP and TEAC were improved following treatment with the extracts and metformin. These findings showed that the extract of orange fleshed sweet potato has the potential to manage insulin resistance through its antioxidant capacity.

Schools as Sites for Social Change: Applying the TEARS Principle
Peet du Toit1
Ronél Ferreira2, William Fraser3, Gerda Gericke4, Karien Botha5, Evangeline Nortje6, Michael Kleynhans6, Morne Ferreira6, Garises M6, van der Merwe M6
Department of Physiology, Associate of the Institute for Food, Nutrition and Well-being, Associate of the Institute for Cellular and Molecular Medicine, Associate of Sport, Exercise Medicine and Lifestyle Institute, University of Pretoria1
Department of Educational Psychology,University of Pretoria2
Department of Science, Mathematics and Technology Education,University of Pretoria3
Department of Human Nutrition,University of Pretoria4
Department of Educational Psychology,University of Pretoria5
Department of Physiology, University of Pretoria6
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Abstract: In order to truly be innovative in education, it is sometimes necessary to break away from conventional practices while, at the same time, keeping the students’ best interests in mind. We support the notion that innovation in teaching and learning plays a vital role in the success of an institution and as such have launched innovative initiatives amongst various institutions. These initiatives present various benefits, such as empowering children and supporting well-being through innovative educational wellness indicators and health promoting intervention. Based on the results obtained in this study, and the needs and knowledge based on the teachers and learners, an intervention plan was developed to support positive change within the schools and the community.
This intervention plan includes the development of learning content that can be included in the curriculum without adding any additional burden to the teachers as well as the training of teachers to better implement this curriculum. The intervention further aims to assist with social change by guiding the schools in improving their learner feeding scheme and physical activity programme. The TEARS principle is an innovative 5-step enterprise that has been developed and implemented in order to pioneer the necessary changes in education.
Keywords: TEARS, Wellness, Intervention, Social Change

Case study: investigation into the impact of the limitless you peak performance program on overall wellbeing
Du Toit, PJ1
Kleynhans, MJ1; Kalmeier, G1; Nortje, E1; Balt, K1,2; Bester, J1; Grobbelaar, C1; Stander, A1; Vermeulen, A3;  Vermeulen, T; Ferreira, M1; Garises, M1; van der Merwe, M1; Bjorkman, D4
Department of Physiology, Faculty of Health Sciences, University of Pretoria, Associate of the Institute for Food, Nutrition and Well-being, Associate of the Institute for Cellular and Molecular Medicine, Associate of Sport, Exercise Medicine and Lifestyle Institute1
Neurofeedback2, Neurolink3, Neuro Business Institute4
Department of Anatomy, Faculty of Health Sciences, University of Pretoria, Associate of the Institute for Food, Nutrition and Well-being, Associate of the Institute for Cellular and Molecular Medicine, Associate of Sport, Exercise Medicine and Lifestyle Institute5
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Introduction: The Limitless You Peak Performance Program (LYPPP) has been meticulously compiled to serve as a multifaceted intervention program. The three core objectives of this holistic program are to improve cognitive ability; reduce stress beliefs; and enhance performance. This tripod of outcomes supports augmented brain-body balance and ultimately pledges improved overall wellbeing.
Case History: The case study involved a male participant, 27 years of age. At the time of the study the participant was unemployed and his highest level of education was a National Senior Certificate (matric). The participant was brought to the Limitless You Academy by his father who expressed concerns about his son’s general state of wellbeing. The father saw the need for an intervention in his son’s life due to various issues, namely: lack of self-esteem and self-worth; bad habits; inability to cope with current life situation; lack of motivation and drive; difficulty sleeping; anger and aggression problems; anxiety attacks; depression; withdrawal from previously loved hobbies/interests; reduced physical activity/ sedentary lifestyle; and poor general lifestyle choices.
Aim: The purpose of the case study was to investigate the impact of the LYPPP on overall wellbeing of the participant.
Methods: The case study was based on a pretest - posttest design, where a wide range of measurements were taken before and after the intervention program. The measurements were divided into three sub-categories, each comprising a battery of tests to assess the following: health-related fitness index; skill-related fitness index; and brain fitness index.
Based on the pretest results an individualized intervention program was developed. The intervention program was followed for a total of 4-weeks. The program was designed to include intervention exercises concerning all three categories of assessment. Throughout the 4-week intervention phase health-related fitness interventions were completed for 20 – 90 min per day and skill-related fitness interventions were completed for 18 – 30 min per day. The intervention schedule also included prescribed brain training exercises which were performed for 18 – 30 min per day. Intervention sessions were scheduled in advance and conducted on-site under supervision of a Limitless You consultant. After successful adherence to the 4-week intervention program, a posttest battery of assessments was completed to compare results.
Discussion & conclusion: The intervention program endorsed several essential improvements in various components related to all three assessment categories. Health-related fitness components with notable improvements include reduced CSI%, which relates to reduced cardiovascular risk as well as stabilized and improved blood coagulability. Of the skill-related fitness components, major post-intervention improvements were seen for all of the visual skills assessments as well as the coordination tests. Both major components of the brain-fitness index revealed excellent improvements in overall psychological performance and functionality. Below is a description of important psychological and related problems reported before and after the neurofeedback training.
Before training the client reported sleep problems. He especially had difficulty falling asleep, waking up, nightmares and Bruxism. He complained of constant fatigue, ringing in the ears and stomach pain. He also reported having a big problem with concentration and distractibility and was taking Conserta for this. There was also complaints of anger and aggression problems, risk taking behaviors and binge drinking over weekends. Mood swings, anxiety and depression was also reported.
After the fourth neurofeedback session he reported that he was sleeping much better and did not feel as tired. The parents also reported that his mood had improved a lot and that they were able to have better conversations with him again. After session 6 he reported that he is now able to go to bed at 10 or 11 at night instead of 1:00 or 2:00 in the morning. He was also now able to sleep for 7 or 8 hours and get up refreshed and ready for the day. He was feeling more efficient and doing things around the house instead of sleeping until 10:00 or 11:00 am. By session 8 he reported that he was not using his Conserta anymore but was sleeping very well and feeling motivated and more focused. He started exploring the idea of continuing his studies again. By the end of the 15 sessions all sleep issues initially reported had resolved. He was feeling much more relaxed and focused. He wasn’t experiencing the depressive and anxious symptoms anymore. His concentration and focus was much better even without the medication. His parents also reported that they were getting along much better. They also experienced a large improvement in his mood and with aggressive behaviour.
It can be concluded that the holistic approach of the LYPPP leads to augmented brain-body balance and an enhanced state of overall wellbeing.

In vitro Chemo-preventative activity of Strelitzia Nicolai Aril extract containing Bilirubin
Veneesha Thaver1, Mickey Naidu2, Neil A. Koorbanally3
School of Laboratory Medicine and Medical sciences, Discipline of Physiology, University of KwaZulu-Natal1
School of Health Sciences, Discipline of Pharmacology, University of KwaZulu Natal2
School of Chemistry and Physics, University of KwaZulu-Natal3
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Introduction: The discovery of the only animal pigment, bilirubin, in the plant Strelitzianicolai (Regel and Körn.) has triggered a vast number of questions regarding bilirubin’s formation and its role in the human body. Recent studies have confirmed that bilirubin at certain levels have many medical benefits. Various case studies have revealed that bilirubin is a potent antioxidant. Cervical cancer is one of South Africa’s largest womens’ health crises. It is estimated that it affects one out of 41 South African women and kills approximately 8 women in the country every day. Thus, the aim of this study was to investigate if the aril extract of S. nicolai containing bilirubin possesses anti-cancer activity and to determine its effect on the induction of apoptosis.
Materials and methods: The DPPH activity was firstly used to determine the antioxidant effect of the extract. Thereafter, the cytotoxic effect was tested using the XTT assay. Apoptosis was confirmed and quantified using the Annexin V-PE kit and the morphology was studied using acridine orange and ethidium bromide. Results: The aril extract decreased cell viability by 52% and induced apoptosis in HeLa cells; as shown by the Annexin V-PE Apoptosis detection kit and morphological studies with acridine orange/ethidium bromide staining. Conclusion: The activity of the extract as a potent antioxidant was immensely enhanced as compared to the bilirubin standard. These results suggest that S. nicolai aril extract containing bilirubin works synergistically as opposed to bilirubin on its own. Furthermore, this extract might be a good candidate for the therapeutic intervention of cervical cancer.
Keywords: Bilirubin, Strelitzianicolai, apoptosis, aril extract, antioxidant

Investigation into the impact of the limitless you peak performance program on performance of athletes
Morne Ferreira
Garises, M1; van der Merwe, ML1; Kleynhans, MJ1; Kalmeier, G1; Nortje, E1; Balt, K1,2; Bester, J1; Grobbelaar, C1; Stander, A1; Cornelius, D3; Du Toit, PJ1.
Department of Physiology, Faculty of Health Sciences, University of Pretoria, Associate of the Institute for Food, Nutrition and Well-being, Associate of the Institute for Cellular and Molecular Medicine, Associate of Sport, Exercise Medicine and Lifestyle Institute1; Neurofeedback2; Tuks Athletics3
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Introduction: The Limitless You Peak Performance Program (LYPPP) has been meticulously compiled to serve as a multifaceted assessment program. The three core objectives of this holistic program are to improve cognitive ability; reduce stress beliefs; and enhance performance. This tripod of outcomes supports augmented brain-body balance and ultimately pledges improved overall performance.
Aim: The purpose was to determine the performance levels of professional athletes by using the LYPPP.
Methods: The research undertook an observational, cross-sectional design. The method including performing test on 40 professional athletes. The measurements were divided into three sub-categories, each comprising a battery of tests to assess the following: health-related fitness index; skill-related fitness index; and brain fitness index. Data and statistical analysis were done by means of descriptive statistics, whereby performance means and standard deviations were determined.
Discussion and conclusion: The main contributing factors to high level performance were divided into health related fitness, skill related fitness and brain fitness and were deemed to encompass anthropometry, sports vision techniques, sports specific testing, six brain performance drivers, physical and motor skills, respectively. It can be concluded that the holistic approach of the LYPPP leads to augmented brain-body balance assessment.

Novel method to decrease core temperature in hyperthermic athletes
CC. Grant1
Jansen Van Rensburg1, A. Janse Van Rensburg1 P, Cele-Zondi, Z. Mahomed1, M. Bester1, E. Kramer1, N. Claassen1
University of Pretoria1
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Background: Athletes from several sporting disciplines train and compete in hot environments that do not only impair performance but also pose a potential risk to athlete health. In endurance sports, for every serious cardiac adverse event, there may be 10 serious events related to heat stroke.
Purpose: This study investigated a carbon based compound/cooling system which was designed for therapeutic hypothermia or temperature reduction after acute ischemic events. The aim of this study was to investigate the effect of using this method, directly after athletes exercised for a maximum period of 2 hours in a hot/humid environment, or until their core temperature reached 39°C. It was hypothesized that this method can significantly decrease the time to return to the normal baseline core temperatures.
Methods: Fifteen un-acclimatized healthy male athletes (20.3y) were asked to perform an exercise protocol in a climatic chamber (dry-bulb temperature= 39.0°C and a natural ventilated wet bulb temperature of 28.0°C). A workload of 80 Watts was achieved by means of a block stepping exercise on and off a 30.5cm stepping block (stepping rate=24 steps.min-1). During the recovery period, athletes were placed in a supine position and cooling pads were fitted on chest, back, neck and thigh area until core temperature returned to baseline. A telemetric system was used for continuous core temperature monitoring. The system included a pre-calibrated temperature sensor (radio pill) and data recorder which was inserted rectally, similar to the application of a rectal suppository. The protocol was repeated twice; once with the aid of the cooling pads and once without.
Results: The non-parametric Wilcoxon’s signed rank test indicated a significant decrease (p=0.0023) in cooling time, indicating that the cooling aid accelerated reestablishment of the athlete’s normal core temperature with 13 minutes.
Conclusion: This technology may provide a new alternative to accelerate the rapid cooling of athletes that experience hyperthermia, thereby avoiding catastrophic injury and adverse cardiovascular outcomes in the collapsed endurance athlete. New sport applications to be investigated include pre-/post-exercise cooling as well as during the half time break in team sports to manage the athlete’s core temperature.

Arterial Wave Reflection and Subclinical Atherosclerosis in Rheumatoid Arthritis
Chanel Robinson1,Aletta Millen1, Patrick Dessein1
University of Witwatersrand1
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Background/purpose: Rheumatoid arthritis (RA) increases cardiovascular disease risk. Wave reflection occurs at arterial branching points, which are particularly prone to atherosclerosis. We hypothesised that wave reflection as assessed in separation analysis, represents atherosclerosis in RA.
Methods: Confounder adjusted associations of SphygmoCor determined arterial stiffness (pulse wave velocity), wave reflection (augmentation index, reflected wave pressure and reflection magnitude) and pressure pulsatility (central systolic blood pressure, central pulse pressure, peripheral pulse pressure , pulse pressure amplification and forward wave pressure) with carotid plaque and intima-media thickness (c-IMT) on ultrasound were assessed in 163 RA patients (110 white, 31 Asian, 17 black and 5 of mixed ancestry). As age, sex, race, heart rate, body height, body weight and brachial mean blood pressure are established potential confounders in the present context, these characteristics were entered into each of the initial multivariable models. Subsequently, other baseline characteristics that had bivariate associations with arterial function variables were additionally entered as potential confounders in fully adjusted models on atherosclerosis.
Results: One SD increase in reflected wave pressure (OR (95% CI) = 2.54 (1.41-4.44), p = 0.001), reflection magnitude (OR (95% CI) = 1.84 (1.17-2.89), p = 0.008), central pulse pressure (OR (95% CI) = 1.89 (1.12-3.22), p = 0.02) and peripheral pulse pressure (OR (95% CI) = 2.09 (1.23-3.57), p = 0.007) were independently associated with plaque. The association of wave reflection with plaque was further independent of arterial stiffness and pressure pulsatility, and was present in both hypertensive and normotensive RA patients. In receiver operator characteristic curve analysis, the optimal cutoff value for reflected wave pressure in predicting plaque presence was 25 mm Hg with a sensitivity, specificity, positive predictive value and negative predictive value of 45.2%, 89.3%, 78.6% and 66.2%, respectively; a high reflected wave pressure increased the odds ratio for plaque 6.3 and 13.7 fold in univariate and adjusted analysis, respectively. Central systolic blood pressure (partial r = 0.161, p = 0.05) was independently related to c-IMT.
Conclusion: Increased wave reflection is associated with high risk atherosclerosis in RA. Consideration and therapeutic targeting of wave reflection may improve cardiovascular disease prevention in RA.

Effects of CXC chemokine receptor-4 (CXCR-4) blockade on melanoma and endothelioma cell growth and on the expression of TGF-β, EGFR and PDGFR in vitro
Yvette Hlophe
Annie Joubert, Peaceful Mabeta
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Tumour cells overexpress lymphatic growth factors, vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor-D (VEGF-D). Overexpression of VEGF-C and VEGF-D upregulates the expression of a chemokine receptor CXCR-4 on the tumour cell. The ligand CXCL12 to CXCR-4 is expressed by lymphatic endothelial cells (LECs) and by stromal cells in the tumour micro-environment. The gradient that is established between CXCR-4 and CXCL12 enhances tumour cell metastasis. Therefore the aim of the study was investigate the effects of blocking CXCR-4 on tumour cell survival and to determine changes in the expression of the biomarkers, transforming growth factor-beta (TGF-β), platelet deriving growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR) in the B-16 melanoma and End-2 endothelioma cells.
CTCE-9908, a chemokine inhibitor specific to the CXCR-4 receptor was used to inhibit CXCR-4 in the B-16 and the End-2 cell lines. Control cells were treated with H2O and Sunitinib, a tyrosine kinase inhibitor with antiangiogenic and antitumour properties was used as a positive control at 2.6µg/ml. Crystal violet (CV) assay was used to determine cell cytotoxicity. Cell morphological changes were studied using transmission electron microscopy (TEM). Confocal analysis was used to detect the expression of TGF-β, VEGF-C, EGFR and PDGFR. Flow cytometry was used for cell cycle analysis and the expression of PDGFR-β, TGF-β, and VEGF-C was further assessed using western blotting.
The results from CV studies showed that the IC50 after 24 hours of treatment was 10µg/ml for the End-2 cells and 100µg/ml for the B-16 cells. Cell cycle indicated that End-2 (>60%) and the B-16 (>70%) cells exposed to the chemokine inhibitor CTCE-9908 had their highest cell concentration in the S phase. TEM analysis indicated apoptotic features. Confocal analysis of CTCE-9908 at 10µg/ml for the End-2 cells and 100µg/ml for the B-16 cells had no effect on the metastatic biomarkers. Western Blot analysis revealed that CTCE-9908 had no effect on End-2(10µg/ml) cells but inhibited expression of PDGFR and TGF-β in the B-16(100µg/ml) cells.
CTCE-9908, inhibited PDGFR and TGF-β expression in melanoma B-16 cells. Therefore CTCE-9908 may be useful in the therapeutic management of melanomas expressing PDGFR and TGF-β.

Contribution of Backward and Forward Wave Pressures to Age-Related Increases in Aortic Pressure in a Community Sample Not Receiving Antihypertensive Therapy
Bryan Hodson1
Gavin R Norton1, Imraan Ballim1, Pinhas Sareli1
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology,  Faculty of Health Sciences,  University of the Witwatersrand1
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Background. Reports on the contribution of aortic forward (Pf) and backward (Pb) wave pressures to age-related increases in aortic pulse pressure (PPc) have been confounded by the inclusion of participants receiving antihypertensive therapy. We therefore aimed to assess the relative contribution of Pf and Pb to age-related increases in PPc in community participants not receiving antihypertensive therapy.
Methods. Aortic pressures were determined using radial applanation tonometry and SphygmoCor software using an assumed triangular wave for wave separation analysis) in 892 participants not receiving antihypertensive therapy. We validated our results using aortic flow waves (echocardiography) for wave separation analysis in 254 of these participants.
Results. In multivariate regression models in those s in PPc, but Pb effects were markedly diminished by adjustments for Pf (0.709±0.002 vs 0.257±0.002 mm Hg per year, pntribute to age-related increases in aortic PPc across the adult lifespan, but at an older age, this effect may be attributed to a large extent to the impact of forward on backward wave pressures (Newton’s 3rd Law of Motion).

In vitro effects of the GPR120 agonists, TUG-891 and docosahexaenoic acid on RANKL-mediated migration of DU145 prostate cancer cells
Kayla Howard1
Bernadette van Heerden1, Abe Kasonga1, Magdalena Coetzee1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
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Bone remodelling is a process whereby osteoclasts resorb bone and osteoblasts form new bone. Osteoclast precursors express the receptor activator of NF-кB (RANK) receptor. Osteoblasts express RANK ligand (RANKL) which binds to RANK to promote osteoclast formation and activity. Several other cell types such as prostate, liver, colon and immune cells are known to express RANKL. Metastatic prostate cancer cells express high levels of parathyroid hormone-related protein (PTHrP) which may activate pre-osteoblasts and osteoclasts causing an increase in RANKL expression and osteoclast formation leading to increased bone destruction. Several types of prostate cancer cells express RANK and activation by RANKL may promote metastasis to bone. Docosahexaenoic acid (DHA), a GPR120 agonist, has shown beneficial effects in patients diagnosed with prostate cancer. GPR120 receptor is present in many different cells types and has been shown to affect downstream NF-кB pathways.
The study investigated the in vitro effects of the GPR120 agonists, TUG-891 and DHA on RANKL-mediated migration of DU145 prostate cancer cells.
DU145 prostate cancer cells were cultured at a density of 15 000 cells/cm² in the presence of RANKL (30 ng/mL), in combination with TUG-891 or DHA (1-100 uM) for 24 hours. Three separate experiments were done in 5-fold. Alamar blue staining was performed to determine the effects of the agonists on cell viability in these cells. For scratch assays, cells were seeded at 155 000 cells/cm² and the rate of migration of the cancer cells was measured at 0, 6, 24 and 30 hours. PCR was performed to determine gene expression of β-catenin and PTHrP.
DU145 prostate cancer cells were shown to express RANK and GPR120. TUG-891 and DHA did not affect cell viability of the cancer cells at the tested concentrations. Reduced migration of DU145 cells was observed when exposed to TUG-891 and DHA. Both agonists were shown to down regulate genes involved in the migration of prostate cancer and cancer-induced bone loss.
The study shows that GPR120 agonists may reduce metastasis of prostate cancer by affecting RANKL-mediated migration pathways.

Moringaoleifera protects growing male Sprague Dawley rats from high fructose diet-induced hepatic lipid accumulation but may predispose them to visceral obesity
Kasimu Ghandi Ibrahim1,2
N.Muhammad1, 3, A.R. Ndhlala4 and K.H. Erlwanger1
School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa 1
Department of Physiology, College of Health Sciences, UsmanuDanfodiyo University, P.M.B. 2254, Sokoto, Nigeria2
Department of Physiology, College of Health Sciences, Federal University BirninKebbi, P.M.B. 1157, BirninKebbi, Nigeria3
Vegetable and Ornamental Plants, Agricultural Research Council – Roodeplaat, Pretoria, South Africa4
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Introduction: Consumption of fructose-rich diets has been implicated in the rising incidence of metabolic disorders. There is a worldwide increase in the use of natural products in the treatment of diseases due to their perceived safety and affordability. Moringaoleifera extracts have beneficial effects in ameliorating dyslipidaemia, obesity and diabetes mellitus. The benefits of early prophylactic interventions in promoting good health later in life are well documented.
Aim: To investigate the effects of a methanolic extract of Moringaoleifera leaves (MEMO) on high fructose-induced metabolic dysfunction in growing male Sprague Dawley rats.
Methods: A total of fifty one 21 day old weaned male Sprague Dawley rats were used. The rats were randomly allocated to six treatment groups. A negative control group (n= 9) that received plain tap water, and others that received either fructose (20%, w/v) alone (n=9) with plain gelatine cubes, of MEMO alone (n=8), of MEMO with fructose (20%, w/v) (n=9), of fenofibrate alone (n=8), or of fenofibrate with fructose (20%, w/v) (n=8). While all the rats had ad libitum access to commercial chow, the MEMO and fenofibrate were suspended in gelatine cubes. The interventions lasted for 10 weeks after which the rats were terminated and tissues collected for biochemical analysis.
Results: MEMO (p<0.0001, ANOVA) and fenofibrate (p<0.05, ANOVA) significantly prevented the increased hepatic lipid deposition caused by the administration of fructose. The fasting blood glucose concentration (mmol.L-1) of rats that had fructose alone (4.00±0.42) or with MEMO (4.10±0.34), was significantly lower (p<0.0001, ANOVA) than those that had fenofibrate alone (4.80±0.23) or with fructose (5.00±0.26). Rats that had MEMO alone and fructose alone had significantly higher (p< 0.05, ANOVA) absolute and relative to tibial length visceral fat pad mass compared to the other treatment groups.
Conclusion: MEMO protected the rats against abnormal hepatic lipid deposition induced by fructose but may have predisposed them to the development of visceral obesity.

Investigating the effect of HIV exposure on immunological and neurological development in exposed but uninfected infants through head circumference and interferon-beta measurements
Jacobus Johannes Le Roux1
Theresa Rossouw1, Peet Du Toit1
Department of Physiology, University of Pretoria1
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Objective: Conduct a pilot study to assess the association among HIV exposure, head circumference (HC) and Interferon-Beta (IFN-β). HC has been shown to correlate with brain size and cognitive function in later life. IFN-β is known to play a significant role in a host’s acute anti-viral response. Elevated levels of IFN-β have been found in certain neurological abnormalities, suggesting that sustained elevation of IFN-β levels might lead to neurodevelopmental delay and a decrease in HC.
Method: Fifty-five women and their infants were recruited at Kalafong hospital and paired blood samples taken at birth and 10 weeks post-partum. Twenty mother-infant pairs with adequate samples were included in a sub-analysis exploring IFN-β levels in infants, tested by means of manual ELISA. HC was categorised according to percentiles and z-scores. Analysis was performed in STATA 14 and significance set at 5%.
Results: Fifteen mothers were HIV-infected and 5 uninfected. Of the infected mothers, all were on antiretroviral therapy and 60% had an undetectable HIV viral load. Exploratory statistics showed no difference in terms of maternal age, parity, body-mass index, or gestational duration between the groups. HIV-exposed infants were similar in weight and length, but had significantly smaller HC than their unexposed counterparts at birth (p=0.026) but no longer at 10 weeks (p=0.26). HC was associated with infant weight at birth (p=0.02) but not with APGAR scores at 1 and 5 minutes. No associations were found between HIV-exposure status and IFN-β or between HC and IFN-β at either time point.
Conclusion: HIV-exposed infants had a significantly smaller HC at birth than unexposed infants. No associations were found between IFN-β levels in infants and exposure status or HC.
Explanation: These results confirm previous studies that have shown lower HC in HIV-exposed infants. This could, however, not be explained by the hypothesis that increased levels of IFN-β are responsible for this observation. A low HIV viral load and hence limited viral antigen exposure of the infant, the influence of other concomitant viral infections that were not tested for e.g. cytomegalovirus, or the lack of test sensitivity could be some factors that affected the results.

Functional Characterisation of Gonadotropin-Releasing Hormone-Estrogen Conjugates as Potential Therapeutics for Prostate Cancer
Stacey Leijenaar1
Dr Claire Newton1, Dr Ross Anderson, Prof. Robert Millar1
Centre for Neuroendocrinology, University of Pretoria1
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Background:  Prostate cancer is estimated to be increasing at a rate of 2-3%. It is a common cause of cancer related deaths, with a higher prevalence in developed countries as well as in Southern Africa, making it highly relevant in the South African context.
Prostate cancer is an androgen-dependent disease and androgen deprivation therapy (ADT) is an effective treatment for prostate cancer allowing avoidance of drastic measures including orchiectomy. The hypothalamic–pituitary–gonadal axis is responsible for the production and regulation of androgens from the testes. Gonadotropin releasing hormone (GnRH) is the main hormone controlling this axis. GnRH agonists (which downregulate this axis and reduce the level of androgens produced) are the foremost therapeutic option for ADT. ADT is associated with negative side effects, such as loss of trabecular bone mass, hot flushes and loss of libido, due to a concomitant decrease in estrogens as well as androgens. It is important to find novel treatments that remain effacious against the disease but ameliorate the associated negative side effects and provide patients with an improved quality of life.
Methods:  Estradiol and the phytoestrogen genistein were conjugated to a GnRH agonist. Conjugates have been tested to determine whether the GnRH and the estrogens moieties retain their respective activities. The ability of the conjugate to stimulate production of inositol phosphate in cells stably expressing GnRH receptor (GnRHR) was used as an indication to measure agonist activity at the GnRHR. Cells expressing estrogen receptor and stably expressing an estrogen response element (ERE) driving the expression of a luciferase reporter gene were stimulated by the conjugates. Response was monitored by a luminometer to assess estrogen receptor activity.
Results:  The Genistein-GnRH analogue and the 17β-estradiol-GnRH analogues were able to achieve comparable levels of stimulation to the GnRH analogue alone in cells expressing the GnRH receptor. Similarly, it was found that estrogen receptor activity is stimulated by both conjugates to the same degree as unconjugated estrogens.
Conclusion:  Results demonstrate that the conjugates retain GnRH and estrogen activity. We now intend to examine their potential to alleviate ADT side effects associated with the treatment of PC.

In vitro effects of cowpea (Vignaunguiculata) and soy bean (Glycine max) digests on osteoclastogenesis in RAW264.7 murine macrophages
Sumari Marais1
Abe Kasonga1, Shaakirah Moosa1, Prof Marlena Kruger2
Department of Physiology, University of Pretoria1; Institute of Food, Nutrition & Human Health, Massey University, Palmerston North, New Zealand, Extraordinary Professor Department of Human Nutrition and Associate of the Institute for Food, Nutrition and Well-being, University of Pretoria2
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Bone is a dynamic organ undergoing constant turnover in response to mechanical strain and damage. Bone cells, namely osteoclasts, are responsible for bone resorption while osteoblasts deposit new bone. Dysregulation of bone remodelling could lead to various bone diseases. Dietary factors could influence bone turnover, therefore dietary modification can be used to slow down bone loss. It has been shown that phytoestrogens in soy beans might have a bone protective effect in vitro. Cowpea is a bioactive rich legume consumed as a staple in many parts of the world including Africa. The aim of this study was to determine the effects of both cowpea and soy digests on osteoclastogenesis in receptor activator of nuclear factor kappa-B ligand (RANKL)-stimulated RAW264.7 murine macrophages in vitro.
Cowpea and soy digests were tested in vitro using undifferentiated RAW264.7 murine macrophages and cell viability was assessed by Alamar Blue assay. Murine macrophages were seeded at a density of 10 000 cells/cm2 and treated with cowpea or soy digest and differentiated with RANKL for five days. The effects of both digests were subsequently tested on a variety of factors that are characteristic of osteoclasts such as cell morphology, osteoclast formation, tartrate-resistant acid phosphatase activity, actin ring formation, bone resorption and nuclear factor kappa B (NF-κB) expression. Three independent experiments were conducted in triplicate for each test.
Cowpea digest increased cell viability but soy digest had no effect on cell viability in undifferentiated cells. Both digests inhibited osteoclastogenesis with soy showing a more pronounced effect. Tartrate resistant acid phosphatase (TRAP) positive osteoclasts were decreased significantly in both digest treated cells. Cowpea and soy digest treated cells had compromised actin rings and bone resorption decreased in soy but not in cowpea-treated osteoclasts. An LDH assay did not show any increase in necrosis in the digest treated cells. NF-κB expression was not altered with cowpea or soy digest treated cells. These finding illustrate the inhibitory effect of cowpea and soy digests on RANKL-induced osteoclastogenesis in vitro. Changes in gene and protein expression levels of key signalling molecules involved in osteoclastogenesis will be investigated to elucidate the pathways affected.

Adiponectin regulation of AMPK on oleanolic acid treated Sprague Dawley rats
Mashudu Given Matumba1
Trevour Nyakudya2, Emmanuel Mukwevho1
North-West University1;     University of Johannesburg2
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AMPK is known to control both glucose and lipid metabolism, two main candidates critical in the development of type-2 diabetes (T2D). Studies have shown that AMPK can be activated by adiponectin. Patients suffering from T2D are known to have low adiponectin concentration in their blood plasma. In this study we have assessed one of the anti-diabetic compound Oleanolic acid (OA) if it could produce desirable effect in upregulating adiponectin concentration and the subsequent regulation of AMPK. Sprague Dawley rats were fed with high fructose diet (HFD) to induce T2D, and the rats that developed insulin resistance were considered as diseased, they were then treated with OA. Analysis of adiponectin concentration in blood plasma was done, AMPK gene expression and subsequent genes that play vital role in glucose and lipid metabolism (GLUT-4 and CPT-1) in skeletal muscle tissue was also performed. The results showed 1 fold increase in blood plasma adiponectin concentration after OA administration.
Furthermore AMPK gene expression showed 4 fold increase and GLUT-4 gene expression was increased with 1.5 fold whereas CTP-1 gene expression was increased with 1.5 fold. These results clearly indicate that OA produced good effects in ameliorating insulin resistance since it was able to upregulate all the genes and adiponectin concentration which are well known to be abnormally suppressed in a situation of T2D. In conclusion these study further confirms that OA can be used as an effective therapeutic agent to ameliorate T2D and these study also suggest that OA’s mechanism of action it could be through AMPK pathway.Key words: Type-2 diabetes, Insulin resistance, AMPK, Adiponectin, Oleanolic acid

Ex vivo effects of novel anti-cancer agents on the human haematopoietic system
Anne Mercier1
Micahel Pepper2, Chrisna Durandt2, Carlo Jaclson2
Department of Physiology, University of Pretoria1; Department of Immunology, Institute for Cellular and Molecular Medicine, and SAMRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria2
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Aim:Among the most serious unwanted effects of chemotherapy are myelosuppression and blood dyscrasias. Novel sulphamoylated oestrone analogues have been shown to induce programmed cell death in various cancer cell lines. The aim of these ex vivo experiments was to assess cytotoxicity of 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10) 15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) on haematopoietic multipotent CD34+ progenitor cells and the effects of these compounds on their differentiation. Additionally, cytotoxicity on circulating leukocytes and erythrocytes was determined.
Methods:  Haematopoietic stem- and progenitor cells (HSPCs) were isolated from umbilical cord blood units using a CD34+ magnetic bead isolation kit. Peripheral blood was drawn from fasting female adults. Isolated CD34+ cells and circulating leukocytes were exposed to ESE-15-one and ESE-16 at 2 concentrations for 24 hours; namely the concentration which decreases proliferation of HeLa cancer cells by 50% (IG50), as well as half of thereof. Annexin V and the viability dyes, propidium iodide and 7-amino-actinomycin D (7-AAD), were used to quantify cell proliferation and apoptosis by flow cytometry. Absolute cell numbers were simultaneously obtained using Flow CountTM counting beads. Exposed HSPCs were seeded onto growth factor-supplemented medium to assess colony forming unit (CFU) ability. Scanning electron microscopy was done on erythrocytes to monitor the drug-effect on cell morphology.
Results: Both drugs were mildly toxic to CD34+ HSPCs at the determined IG50 concentration. No cytotoxic effects were observed at the lower concentrations. The compounds had no effect on CFU number or type, although the absolute number of cells was decreased compared to the controls. The drugs had no effect on the morphology of circulating erythrocytes or platelets and demonstrated only a mild toxicity in compound-exposed circulating leukocytes.
Conclusion:  Multipotent CD34+ HSPCs displayed relative resistance to the novel anti-cancer compounds, exposure to which did not affect their differentiation. Comparative sparing of circulating leukocytes, erythrocytes and platelets from the cytotoxic effects of ESE-15-one and ESE-16 was observed. Future in vivo studies will aim to assess whether these novel microtubule disrupting agents cause myelosuppression.

Effects of a newly synthesized bromodomain 4 inhibitor on a metastatic (MDA-MB-231) breast cell line
Thandi VuyelwaMqoco
Department of Physiology, University of Pretoria
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Background and aim: Cancer is one of the leading causes of death worldwide. Recent studies have shown that breast cancer is associated with genetic instability and abnormal epigenetic changes. Since epigenetic changes can be reversible, compounds that can repair the epigenetic changes represent exciting potential therapeutic targets for cancer. The aim of this study was to evaluate the in vitro effects of a bromodomain 4 inhibitor (ITH-47) in a metastatic MDA-MB-231 breast cancer cell line.
Methods: The cytotoxic effects of a bromodomain 4 inhibitor (ITH-47) was evaluated using a crystal violet staining assay. Morphological changes in MDA-MB-231 cell line was investigated using polarization-optical transmitted light differential interference contrast, light microscopy and transmission electron microscopy. In addition, the effects of cytotoxic concentrations of ITH-47 on cell cycle progression and apoptosis markers (annexin V-FITC, mitochondrial membrane potential (MMP)) were studied.
Results: Crystal violet staining results demonstrated the GI50 (48 h) for ITH-47 was 15μM on MDA-MB-231 cells. Data from microscopy techniques showed compromised cell density and characteristics of apoptosis in the morphology of ITH-47-treated cells. Cell cycle progression analysis revealed a statistically significant increase in the number of compound treated cells that were in the G1 phase. Results from the annexin V-FITC assay and MMP further confirmed the induction of apoptosis by this compound.
Conclusion: Data from this study revealed that exposure to ITH-47 affected the proliferation, morphology and induced apoptosis in metastatic (MDA-MB-231) breast cell line. Future studies will be conducted to further elucidate the action mechanism/s of ITH-47 within this cell line.

Methanolic leaf extracts of Moringa oleifera prevent high fructose diet-induced dyslipidaemia and hepatic lipid accumulation in Growing Female Rats
N. Muhammad1, 3
Kasimu Ghandi Ibrahim1,2 A.R. Ndhlala4 and K.H. Erlwanger1
School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, Johannesburg, South Africa 1
Department of Physiology, College of Health Sciences, Usmanu Danfodiyo University, P.M.B. 2254, Sokoto, Nigeria2
Department of Physiology, College of Health Sciences, Federal University Birnin Kebbi, P.M.B. 1157, Birnin Kebbi, Nigeria3
Vegetable and Ornamental Plants, Agricultural Research Council - Roodeplaat, Pretoria, South Africa4
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Aim: We investigated the protective effects of a methanolic leaf extract of Moringaoleifera on fructose-induced metabolic dysfunction in growing female rats.
Methods: 21-day old weaned female Sprague Dawley rats (N=51) were randomly divided into six treatment groups. In addition to commercial rat feed ad libitum, the growing pups received either plain drinking water (group I, negative control), or 20% fructose solution (group II), or Moringaoleiferamethanolic leaf extract (400 mg/kg) with or without fructose solution (groups III and V), or fenofibrate (100 mg/kg) with or without fructose solution (groups IV and VI). After ten weeks of intervention, body mass gain, circulating triglycerides, cholesterol concentrations and hepatic lipid storage were assessed.
Results: Consumption of the fructose solution significantly increased circulating (mmol/L) triglycerides [2.30±0.43 (Group II) vs 1.70±0.30 (Group I); P<0.01, ANOVA] and hepatic lipid stores [11.00±0.21% (Group II) vs 9.60±0.21% (Group I); P<0.05, ANOVA]. The methanolic leaf extracts of Moringaoleifera prevented the fructose-induced elevation in triglycerides [2.20±0.38 (Group III) vs 2.30±0.43 mmol/L (Group II); P<0.05, ANOVA] and hepatic lipid stores [9.80±0.77% (Group III) vs 11.00±0.21% (Group II); P<0.05, ANOVA]. No significant differences were observed in the body mass gain (P>0.05, ANOVA) and plasma cholesterol concentrations (P>0.05, ANOVA) between the different treatment groups.
Conclusion: Moringaoleiferamethanolic leaf extract is beneficial in preventing the hypertriglyceridaemia and abnormal hepatic lipid deposition secondary to high fructose intake in female rats.

The use of a novel, cost effective method of measuring lower limb weight bearing in patients following a stroke-a case report
Pooveshni Naidoo1
Dr Karien Mostert1, Prof Joyce Mothabeng1
University of Pretoria1
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Introduction: A major impairment faced following a stroke is the ability to bear weight or transfer weight equally through the paretic lower limb. It has been widely noted in literature that individuals with stroke bear only 25% to 43% body weight on the paretic lower limb (Brière, Nadeau, Lauzière& Gravel, 2012; Brière, Lauzière, Gravel & Nadeau, 2010; Eng& Chu, 2002; Mercer, Freburger, Chang & Purser, 2009; Pèrennou 2005). This poses a major hurdle for stroke sufferers, since the ability to contain a body mass over the base of support under different tasks and environmental conditions is one of the requirements of daily life (Ikai, Kamikubo, Takehara, Nishi &Miyano, 2003). With advancements in technology, the use of force plates have been widely used as outcome measures (Brière et al., 2012; Eng et al., 2002; Chern, Lo, Chen, Yang & Tang, 2010; Stoller, Rosemeyer, Baur, Hunt, Radlinger& Schuster-Amft, 2015) as they boast high levels of validity and reliability (Mercer et al., 2009; Nichols, 1997).
However in community settings and government hospitals, this equipment is rarely available, due to high costs, inability to train staff (Paliwal, 2013) or difficulty to update necessary software. Therefore, the researcher has undertaken developing a novel, cost effective method to measure lower limb weight bearing in patients with stroke. Method: A case study was done on a 45 year old female, who suffered her first stroke. Mrs M showed poor postural alignment, assymetrical weight bearing and lacked right knee control on evaluation. A substance made from water, flour, salt and boric acid (commonly known as Playdough) was placed with a two centimetre height on a non-slip surface. Mrs M was asked to place both feet on the substance and manoever into standing from sitting three times. Thereafter measurements were taken by a ruler on the three weight bearing areas of the foot, namely the calcaneus, meta-tarsal head of big toe and meta-tarsal head of last digit (Kaplan, 2015).
Data Analysis: Observations have shown changes in the appearance of foot imprints of the right (paretic) to the left (healthy) lower limbs. Furthermore, measurements of the specific weight bearing points indicate increased depth in the left foot imprint when compared to the right. Conclusion: This case report indicated that weight bearing of the lower limbs can be measured using a novel, cost effective method. Recommendation: It is proposed that validity and reliability of weight bearing using a novel, cost effective method be compared to the force plate, which is being conducted by the researcher currently.

In the Presence of Modest Independent Effects on Blood Pressure, Obesity Markedly Modifies the Impact of Age on Ambulatory Pulse Pressure in a Community Sample
Glenda Norman1
Gavin R Norton1, Monica Gomez1, Olebogeng HI Majane1, Pinhas Sareli1, Angela J Woodiwiss1
Cardiovascular Pathophysiology and Genomics Research Unit, School of Physiology Faculty of Health Sciences, University of the Witwatersrand, Johannesburg1
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Background: The major factor responsible for age-related increases in blood pressure, is an increases in pulse pressure (PP). The extent to which obesity, which is part of the aging process, contributes to age-related increases in pulse pressure (PP) is uncertain.
Methods: In 850 adult participants of an African community (age>16 years, 39.9% obese, 43.8% abdominal obesity), we determined the extent to which adiposity indices modify the impact of age on 24-hour, day and night ambulatory pulse pressure (PP).
Results: Independent of confounders, a 1 SD increase in waist circumference (WC)(17.2 cm) translated into only a 1.03 to 2.60 mm Hg increase in 24-hour, day or night diastolic (DBP) and systolic BP (SBP) respectively and only a 1.11 to 1.44 mm Hg increase in 24-hour, day or night pulse pressure (PP).  Moreover, whilst a 1 SD increase in age (18.2 years) showed a strong independent relationship with hypertension (OR=3.69 CI=2.99 to 4.60, p=0.001), a I SD increase in WC was only modestly associated with hypertension (OR=1.38 CI=1.14 to 1.68, p<0.0001). Although no interaction between adiposity indices and age were  independently associated with either ambulatory SBP or DBP, marked interactions between WC, BMI or the homeostasis model of insulin resistance and age were independently associated with 24 hour, day and night PP (p<0.0005). This translated into a greater independent relationship between age and 24-hour (β-coefficient=0.22±0.03, p<0.0001), day and night PP in those with an increased as compared to those with a normal (β-coefficient=0.05±0.02, p<0.01 for comparison of β-coefficient) WC. Similar findings were noted with BMI as opposed to WC.
Conclusions: A major impact of obesity on blood pressure may not be through independent effects on steady-state or pulsatile pressures, but rather through a marked ability to modify the effect of age on PP.

Impact of Dietary Salt Intake on Arterial Stiffness in Overweight or Obese Women of African Ancestry
Mercy Mashao, Muzi Joseph Maseko, Edgar Phukubje
SefakoMakgatho Health Sciences University, Department of Pre-Clinical Sciences
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Background: An increased dietary salt intake can result in increased arterial stiffness independent of blood pressure or the renin-angiotensin-aldosterone system. However studies conducted in our community have failed to show a direct relationship between salt and arterial stiffness. Our study population has a high percentage of women who are overweight or obese and given the independent association between arterial stiffness and the indices of obesity, it is possible that these indices mask the relationship between arterial stiffness and dietary salt intake.
Methods: We recruited 346 women of African ancestry from the south western township of Johannesburg South Africa. We measured conventional blood pressure, 24-h ambulatory blood pressure and collected 24-hour urine samples on all the participants. Anthropometric measurements were taken and a standard questionnaire was issued to determine lifestyle habits and history of medication. To assess arterial stiffness we measured pulse wave velocity using applanation tonometry.
Results: The mean age of participants was 43.9±18.4 with 64% of participants being women. The mean BMI of the group was 29.2±7.9, and 67% of participants were either overweight (24%) or obese (43%). Thirty-seven percent (37.1%) of the sample population was hypertensive, 23.0% consumed alcohol regularly, 16.9% smoked, and 12.2% had diabetes mellitus or an HbA1C >6.1%. People with increased adiposity, whether classified by body mass index or waist circumference had an increased pulse wave velocity compared to those with normal adiposity. After correcting for covariates, there was a significant relationship between pulse wave velocity and 24-hour urinary sodium excretion in the total sample of women and those with a normal BMI. However no relationship was found between salt and pulse wave velocity in the overweight and obese group. A similar trend was observed when WC was used as an index of adiposity. A significant relationship was only found in women with normal waist circumference.
Conclusion: In a population sample of women of African ancestry, overweight/obese individuals have increased arterial stiffness compared to normal weight participants and dietary salt intake is associated with arterial stiffness however this relationship is masked by indices of obesity.

Naringin protects against HIV-1 protease inhibitors-induced glucose intolerance and impaired insulin signalling in vivo
Sanelisiwe Nzuza1
Dr Peter Owira1

Molecular and Clinical Pharmacology Research Laboratory, Department of Pharmacology, Discipline of Pharmaceutical Science, School of Health Sciences, University of KwaZulu –Natal, Durban1
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Insulin resistance, glucose intolerance and overt diabetes are known metabolic complications associated with chronic use of HIV-Protease Inhibitors. Naringin is a grapefruit-derived flavonoid with anti-diabetic, anti-dyslipidemia, anti-inflammatory and anti-oxidant activities. The study investigated the protective effects of naringin on glucose intolerance and impaired insulin secretion and signaling. Male Wistar rats were divided into six groups (n = 6) and were daily orally treated with distilled water {3.0 ml/kg body weight (BW)}, atazanavir (133 mg/kg BW), saquinavir (333 mg/kg BW) with or without naringin (50 mg/kg BW), respectively for 56 days. Body weights and water consumption were recorded daily. Glucose tolerance tests were carried out on day 55 of the treatment and thereafter, the rats were sacrificed by halothane overdose.
Atazanavir- or saquinavir-treated rats exhibited significant weight loss, polydipsia, elevated Fasting blood glucose (FBG), reduced Fasting Plasma Insulin (FPI) and expression of phosphorylated, Insulin Receptor Substrate-1 (IRS-1) and Akt proteins compared to controls, hepatic and pancreatic glucokinase levels and also increasing pancreatic caspase-3 and -9 as well as UCP2 protein expression compared to controls, respectively. These effects were completely reversed by naringin treatment. Naringin prevents PI-induced glucose intolerance and impairment of insulin signalling and as nutritional supplement it could, therefore, alleviate metabolic complications associated with antiretroviral therapy

Vitamin D3 pro-hormone exerts growth-modifying effects on an in vitro metastatic epidermoid cervical carcinoma cell line
Rivak Punchoo1
Sumari Marais1, Anton Stoltz1, Annie Joubert1
University of Pretoria1
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Introduction:  An increasing body of research supports the hypothesis that the active form of vitamin D (VD) exerts protective anti-cancer properties. Cervical carcinomas demonstrate a locally active VD metabolism however the action of VD in this cancer is poorly investigated.
Aim:  To investigate the effects of VD on growth in an epidermoid metastatic in vitro cancer line (CaSki).
Objectives:  1. Assess VD’s action on cell viability and cytotoxicity in CaSki cultures.
Assess VD’s action on the gross morphology and ultrastructure in CaSki cultures.
Methods: CaSki cells were cultured and treated with clinically relevant 25-hydroxycholecalciferol D3 pro-hormone (10-1000 ng/ml) and incubated for 96 hours. The cultures were assessed at 24 hour intervals and the percentage viability and cytotoxicity measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays respectively. Microscopy was performed on cultures using haemotoxylin and eosin bright field microscopy, polarization-optical transmitted light differential interference contrast microscopy (PlasDIC) and scanning and transmission electron microscopies. Data analyses of three independent experiments performed in triplicate for each incubation were expressed as a mean ± standard deviation for percentage viability and cytotoxicity. Statistical analysis was performed by one-way analysis of variance followed by Bonferroni post-hoc testing (P Results: A significant increase in percentage cell viability was detected at the clinically deficient VD treatment concentration (10ng/ml) in comparison to VD sufficiency treatment (40 ng/ml) at 48 hour and 72 hour incubations. Supra-therapeutic treatments of VD paradoxically caused a significant increase in cell viability at 1000 ng/ml in comparison to 250 ng/ml at 48 hour, 72 hour and 96 hour incubations. Cell proliferation analysis identified similar trends in growth although these were not statistically significant.
The percentage cytotoxicity was significantly decreased at clinically sufficient VD treatment (40 ng/ml) in comparison to VD deficient treatment (10ng/ml) at incubations 72 hours and 96 hours. A significant decrease in cytotoxicity at 1000 ng/ml in comparison to other supra-therapeutic treatments (100 ng/ml, 250 ng/ml and 500 ng/ml) was observed at 72 hours and 96 hours.
Gross and ultrastructural morphology of CaSki cultures incubated at 72 hours (10 ng/ml) showed cells with extensive cytoplasmic vacuolation, abnormal nuclear:cytoplasmic ratio and abnormal intra-cellular cytoplasmic distribution. Cultures incubated at 72 hours (at 40 ng/ml) showed polyhedral cell clusters with uni-nucleated and multi-nucleated cells containing single or multiple nucleoli with intense eosinophilic cytoplasmic staining. An increased cell density at 10ng/ml VD treatments in comparison to 40 ng/ml VD treatments at 48 hours and 72 hours was also identified. Microscopy did not identify classical features of apoptosis or necrosis.

Prevalance of Musculoskeletal Disorders among employees in a welding company within mangaung metropolitan municipality
France Raphela
Central University of Technology, Free State
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Introduction:  Work-related musculoskeletal disorders pose a major challenge to the public health and socioeconomic development. The purpose of this study was to investigate and report the prevalence of musculoskeletal disorders (MSDs) among employees in a welding company and make recommendations to assist in successful management.
Methods:  A cross-sectional survey was conducted in a welding company in the Mangaung Metropolitan Municipality in the Free State Province, South Africa. The study population consisted of full-time welders and fitters (exposed group) and office workers (unexposed group). The exposed group comprised of 37 welders and 21 fitters while the unexposed group comprised of 30 office workers. The participants completed self-administered questionnaires consisting of open and close-ended questions.
Results and discussions:  The median ages for the exposed and unexposed groups were 33 and 37 years respectively. Back pain was the most common musculoskeletal disorder reported by the participants. Forty percent and 17% of the exposed and unexposed groups, respectively, suffered from pain on the hands. The prevalence of pain on the neck was similar in both group groups accounting for 45% in the exposed group and 43% for the unexposed group. The prevalence of pain on the hands among the exposed group (40%) was significantly higher than the unexposed group (17%). The exposed group (29%) and unexposed group (13%) experienced muscle weakness.

Physiological stress and burnout in crossfit participantsJenna Robinson1
Prof Peet du Toit1, Dr Priyesh Bipath1
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria1
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Introduction: Burnout is primarily classified as a syndrome of emotional exhaustion and physical fatigue. Longstanding burnout is said to carry an increased risk for negative health consequences such as cardiovascular, metabolic and inflammatory diseases. In addition to the effects on physical health, burnout may negatively impact on productivity, quality of life and psychological well-being in general.
Crossfit training involves the high intensity workouts of a combination of elements from several sports such as gymnastics, powerlifting, rowing and running. Mental strength and resistance to fatigue is essential, especially during competitions. Physical fatigue and emotional exhaustion due to overtraining may lead to exercise-related burnout. Apart from proper training regimens, research necessitates investigating the physiological effects of burnout to help improve quality of health.
Aim: The aim of this study was to investigate physiological stress and burnout in crossfit participants undergoing a workout programme.
Methods: Male participants (n=15; >18 years) with a mean BMI of 26.61±1.69 kg/m2 were recruited from Crossfit Brooklyn in Pretoria. The participants were required to perform a pre-determined crossfit workout in order to test the sympathetic response. Measurements included skin conductance (electrodermal response), electromyography, blood volume pulse, skin temperature, blood pressure and anthropometric indices. Physiological measurements were taken at both baseline and post workout. The Shirom-Melamed Burnout Scale and Short-form 36 (SF36) questionnaires were used to assess burnout and physical well-being respectively.
Results: When comparing the sympathetic overload for the post workout versus baseline, there was a significant increase in blood volume pulse (126.5±1.2 vs. 73.5±9.4 beats/min; p<0.001) and electrodermal response (9.2±4.7 vs. 4.9±1.9 µSiemans; p=0.006) but not in electromyography activity (4.8±5.2 vs. 2.9±2.8 µV; p=0.241). For the physical facet 50% of participants reported feeling physically exhausted while only 21% presented with burnout on the frequency scale. The emotional exhaustion and cognitive weariness facets were distributed unevenly across the questionnaire scales. The results warrant future investigations.
Conclusion: The results present a new dynamic for understanding physiological stress and burnout in crossfit. The research is aligned to help improve the future well-being of crossfit participants in order to avoid burnout.

Angry platelets and tissue factor:  unsuspected mediators in the pathogenesis of cardiovascular disorders in type II diabetics
Prashilla Soma1
Thandi Mqoco1, Albe Swanepoel1, Janette Bester1
University of Pretoria1
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Introduction:Type II diabetes mellitus is a pandemic associated with disturbance in haemostasis that could contribute to the development of diabetic vascular disease and accelerated atherosclerosis. The aims are therefore to describe and compare platelet function and to measure tissue factor levels in type II diabetes (with and without cardiovascular disease) and healthy individuals using scanning electron microscopy, flow cytometry and ELISA respectively.
Methods:Diabetic subjects were recruited from the diabetic clinic at Steve Biko Academic
Hospital, Pretoria. 30 diabetics with cardiovascular disease and 30 without were enrolled to participate and informed consent was obtained from all participants. Blood
samples were drawn from all participants in citrated whole blood so that platelet specific antigens, tissue factor levels and platelet rich plasma could be analysed. The platelet parameters were identifiers (CD41 and CD42) and markers of activation (CD62 and CD63).
Results:Microscopy analysis of the platelets confirmed features of pseudopodia with spreading and extensive clumping. This hyper-activation was seen in platelets from individuals with diabetes with and without cardiovascular disease. However, diabetic patients with cardiovascular disease are characterised by an increased presence of hyper-activation and microparticle formation. We found that the ultrastructural results fully supported the flow cytometry results. The results of the flow cytometry showed that, compared to healthy individuals, both diabetic groups showed a significant difference in both platelet identifiers (CD41-PE, CD42b-PE) as well as markers indicating platelet activation (CD62P-PE and CD63-PE). TF levels were significantly elevated in both diabetic groups when compared to the controls.
Conclusion:Analysis of ultrastructural findings in diabetic platelets reveal remarkable findings which may add to a better understanding of the pathogenesis of atherosclerosis and thrombosis. The flow cytometric data shows that the platelet surface receptors and platelet activation are statistically elevated. This is suggestive of enhanced platelet activation and it appears as if platelets are displaying ‘angry’ behaviour. The finding in our study of elevated TF levels in both diabetic groups compares favourably with other studies where high TF levels are found in type 2 diabetic subjects. However, we show that cardiovascular complications in diabetes further increases TF levels.

Effects of an in silico-designed Estradiol analogue on induction of autophagy in a breast cancer cell line
Danielle Sandra Tatchum1 
Iman Van den Bout1, Annie Joubert1
University of Pretoria1
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Background: 2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17-β-estradiol and potential anti-tumour- and anti-proliferative agent that induces apoptosis in vitro and in vivo. Owing to 2ME2’s rapid metabolism and low oral bioavailability, a 2ME2 analog, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) was in silico-designed in our laboratory. This study aimed at investigating whether a 24 h exposure to 0.2 μM ESE-16 could induce autophagy in breast adenocarcinoma cells. The presence of acidic vacu9oles was determined via monodansylcadaverine (MDC) staining and quantification of acidic vacuoles were assessed by spectrophotometry.
Materials and Methods: Possible induction of autophagy was assessed by flow cytometric analysis of cyto ID staining of autophagosomes. Microtubule-associated protein light chain (LC3) II levels were determined by western blotting. A possible disruption in lysosomal activity was also assessed via confocal microscopy.
Results: An increase in acidic vacuoles was observed by MDC staining. A statistically significant 1.7-fold increase in autophagosome accumulation was also noted by flow cytometric analysis of cyto ID. Western blot analysis revealed that LC3-II protein levels were not statistically significantly increased. Lysotracker staining revealed a disruption in lysosomal activity following enlarged lysotracker-positive vacuoles spread throughout the cell without any concentration close to the nucleus in MCF-7 cells after exposure to ESE-16 for 24 h.
Conclusions: This study revealed that 24 h of exposure to 0.2 μM ESE-16 resulted in an increase in MDC staining of acidic vacuoles, as well as Cyto ID staining of autophagosomes indicating induction of autophagy. The non-significant increase in LC3-II protein levels suggested that there was no fusion between autophagosome and lysosome; thus the increase in autophagosome accumulation observed by Cyto ID staining.

The Effect of Lithium on Excitotoxicity and Cellular Proliferation following N-Methyl D-Aspartic acid-Induced Seizure Activity in Rat Organotypic Hippocampal Slice Cultures: A Model of West Syndrome
Sylvia van Belle1
Vinogran Naidoo1, Clare Phillips1
University of Cape Town1
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West Syndrome (WS) is a devastating form of epilepsy that begins in infancy and results in lifelong mental impairment if not death. In South Africa the lack of an effective treatment for WS is compounded by structural and social barriers to health care. A drug which preserves cognitive function and decreases seizure severity would greatly impact the lives of WS sufferers. The hippocampus is particularly susceptible to seizure damage which decreases neurogenic ability leading to cognitive deficits. Lithium (Li) has been found to ameliorate neuronal injury in numerous brain injury models but has never been investigated pre- and post-seizure. We first tested the effect of Li on cytotoxicity and cell proliferation post N-Methyl D-Aspartic acid (NMDA)-induced seizures in organotypic hippocampal slice cultures obtained from postnatal 6-7 Wistar rat pups. Li (0.8 mM) decreased NMDA (100 µM)-induced cytotoxicity three days post-injury (DPI) and less so at DPI-7, but decreased excitotoxic injury below control levels at both time points. Li, administered post-NMDA-treatment, increased cell proliferation in the dentate gyrus (DG) in NMDA-exposed slices at DPI-3. When slices were treated chronically with Li for 21 days and then exposed to NMDA on culture day 22, a significant reduction in cytotoxicity was also observed. Here, slices analyzed at DPI-10 revealed that Li increased the numbers of immature doublecortin- and Prox-1-positive neurons in the DG.
Interestingly, at DPI-10, a greater number of NeuN-positive cells in the DG within the NMDA+Li group was also found, compared to NMDA-only treated tissues. Moreover, chronic Li-pretreated slices showed a greater number of resting, Iba-1-positive, microglia than activated microglia, compared to NMDA-only exposed slices which had a greater number of morphologically amoeboid-like Iba-1-positive microglia. Future studies are needed to determine whether the hippocampal stem-cell (nestin-positive) population is protected by Li. Equally important will be to investigate whether microglia-activation via the alternative M2-pathway may contribute to tissue repair and control of inflammation in excitotoxic-hippocampal slices exposed to Li. Our study demonstrates the potential of Li as a therapeutic agent against NMDA-induced seizures ex vivo, suggesting further investigation in vivo allowing for cell lineage tracing, electrophysiological assessments and determination of cognitive outcomes.

A bis-sulphamoylatedestradiol derivative induces ROS-dependent cell cycle abnormalities and subsequent apoptosis
Michelle Helen Visagie1
Iman van den Bout1, Anna Margaretha Joubert1
Department of Physiology, Faculty of Health Sciences, University of Pretoria1
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Introduction:While 2-methoxyestradiol (2ME2) shows potential as an anticancer agent, clinical trials revealed limitations due to low bioavailability. Subsequently, derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) were synthesised. EMBS and other derivatives possess improved antiproliferative-, antimitotic- and pro-apoptotic activities in several tumourigenic cell lines. However, the mode of action exerted by EMBS in tumorigenic cells remains elusive. In this study the mode of action of EMBS as a representative of the sulphamoylated 2ME2 derivatives was investigated.
Methods:Hydrogen peroxide production was quantified using dichlorofluorescein diacetate. Cell proliferation was investigated using crystal violet staining. Cell cycle progression was assessed using propidium iodide staining. Effects on H2A phosphorylation was visualised using confocal microscopy and a phospho H2A antibody.
Results:EMBS exposure led to increased reactive oxygen species (ROS) production which was correlated to the loss of cell proliferation apoptosis induction. Hydrogen peroxide levels increased to 1.7 fold after 4h followed by a decrease to 0.5 fold at 10h. Hydrogen peroxide levels subsequently increased to nearly 3-fold after 24h exposure to EMBS. EMBS exposure resulted in an initial arrest at the G2/M border while endoreduplication (DNA content >4N) appeared and cell numbers decreased to 50% after 24h. Confocal microscopy revealed that EMBS induced DNA double-stranded breaks which is indicative of endoreduplication and oxidative-induced DNA damage. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated antiproliferative activity, cell cycle arrest and cell death suggesting that ROS production is essential for the ability of EMBS to induce cell death. Inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS induced apoptosis via the JNK pathway.
Conclusion:Our data suggests that EMBS targets a pathway resulting in increased ROS production as an early event culminating in cell cycle arrest and apoptosis induction in tumourigenic cells. This study establishes a novel ROS/JNK-dependent mode of action for sulphamoylated derivatives. Unravelling of ROS-dependent mechanisms of action for cell death induction contributes to development of improved biological targets and more efficient chemotherapeutic compounds, as well as an understanding of the role of ROS production in cell cycle arrest and apoptosis.