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Item Open AccessPrediction of the femoral length from markers on its proximal and distal ends(University of the Free State, 2003-11) Tegegn, Walelign Nega; Gous, A. E. F.An estimation of stature from the whole length of limb bones is well documented. However, skeletal remains available for forensic work are often fragmentary. This study presents a prediction of the femoral length using markers on its proximal and distal ends. A total of 400 South African White and Black adult dried femora, devoid of gross pathology, and grouped by sex were obtained from the Raymond Dart Collection of Human Skeletons in the Department of Anatomical Sciences at the University of Witwatersrand, Johannesburg. The Maximum Femoral Length (FL), Neck-Shaft Angle (NSA), Neck Length (NL), Maximum Vertical Diameter of the Head of Femur (VDH), Intertrochanteric Apical Axis Length (ITAAL), Upper Breadth of the Femur (VHA), and Lateral Condyle Height (LCH) were measured. The data were statistically analysed using the various components of a PC version of SAS soft ware program. The student's t-test was used to calculate the significant differences of means between the sexes and races within the study sample as well as with other studies. The critical value for statistical significance was placed at the 0.05 level. Correlation coefficients between femoral length and the other variables were calculated. The length of femur significantly and positively correlated with all segment measurements in both races and sexes. Femoral length was regressed on segment measurements individually and in combination and simple as well as multiple linear regression equations were developed for White and Black South Africans. Stepwise selection procedure was employed to formulate the multilinear regression equations. Most of the models developed in the present study are significant at p< 0.0001, r² values are high, and standard errors of the estimates (S. E.E.) are very low. Therefore, the equations developed in this study present a reasonable degree of accuracy for the estimation of femoral length from its proximal and distal segments in South African Whites and Blacks. Once the length of femur is established, it is possible to calculate living stature of the individual with a reasonable degree of precision. The necessity of population and sex specific regression models is addressed. Item Open AccessCharacterisation of ß-lactamases implicated in resistance to ß-lactem antibiotics in urinary tract infections(University of the Free State, 2005-11-29) Ramainoane, 'Matabane; Theron, M. M.South Africa is not excluded from the problems encountered world-wide in the treatment of nosocomial urinary tract infections, commonly caused by enzyme-producing Enterobacteriaceae. These enzymes include the ß-lactamases and extended-spectrum ß-lactamases (ESBLs) capable of hydrolysing the ß-lactam agents and in particular the expanded-spectrum cephalosporins frequently used. The study was designed to determine the role of ß-lactamases in resistance development in commonly encountered pathogens implicated in urinary tract infections and to characterise the enzymes involved. Resistance to the ß-lactam agents amoxicillin, ceftriaxone, ceftriaxone, piperacillin and cefoxitin was suspected to involve the presence of one or more β-lactamases in the isolates from Bloemfontein hospitals. Diverse and complex β-lactamases were identified and ESBLs were detected in 80% of the isolates. These β-lactamases were characterised by isoelectric focusing (IEF) and genetic analysis (DNA amplification by PCR) to investigate the presence of possible genes responsible for resistance development. The production of blaTEM and blaSHV type genes was demonstrated. Isolates harbouring these genes were highly resistant to amoxicillin and piperacillin, with MIC90s of >128μg/ml. Resistance to these antibiotics was shown to be readily transferred between strains and there was an indication that the resistance genes are carried on plasmids and was transferred by conjugation. A plasmid of 9-10 kb was detected in 83% of the isolates and could be one of the mechanisms implicated in the transfer of ESBLs in uropathogenic bacteria. ß-Lactam resistance could be attributed to the presence and action of ß-lactamases such as the TEM and SHV type enzymes and this resistance can be transmitted between bacteria, causing problems specifically in the hospital environment. Further and continuous investigations are required to find a solution for this ever increasing problem. Item Open AccessClinically relevant ex vivo fatty acid profiles from a lipid model for colorectal adenocarcinoma(University of the Free State, 2007-11) Nel, Amanda; Louw, L.; Badenhorst, P. N.The challenge we face today is identification of the mechanisms through which dietary factors perturb fundamental fatty acid (FA) pathways in cancer cells. In principle, insight into a lipid model for the cancer entity under assessment is required. This prompted and investigation into FA role-players that drive cellular over-production and apoptotic pathways under circumstances where environmental factors impede essential fatty acid metabolism (EFAM) that contributes to colorectal cancer (CRC). Ultimately, better understanding of FA metabolic pathways followed under colorectal pathological conditions can contribute to improved therapies by which growth and recurrence of this disease may be obviated. PURPOSE OF STUDY: The primary goal of this study was to construct clinically relevant ex vivo FA profiles from a lipid model for colorectal adenocarcinoma, not previously reported in the literature. Aims were to identify prominent FA role-players and to debate their involvement in different signaling pathways. The final purpose of this study was to present a rationale for adjuvant FA therapies to improve the management of CRC. MATERIAL AND METHODS: The study group consisted of CRC biopsies (TNM staging, mostly T2/T3) and normal colorectal mucosal biopsies, (n8 of each group). Biopsy selection was hampered by difficult circumstances, since most patients received treatment prior to surgery. All the lipid analyses were personally conducted at the Biolipid Division of the Department Biochemistry at the University of the Free State (UFS) as follows: lipids were extracted; total lipids (TLs) were fractionated into neutral lipids (NLs) and phospholipids (PLs); phosholipid subclasses, i.e. phosphatidylcholine, (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), were separated; and fatty acid methyl esters of all the classes and subclasses were analyzed. Lipid analyses were done according to well established and internationally accepted gaschromatography (GLC) and thin layer chromatography (TLC) techniques. Statistical analyses were performed by a biostatistician attached to the UFS, and p-values and 95% confident intervals (95% CI) for median of differences, as well as specific ratios were calculated. MAIN RESULTS AND DISCUSSION: The ex vivo phospholipid FA profile for CRC cells revealed linoleic acid (LA), arachidonic acid (AA), palmitic acid (PA) and oleic acid (OA) as main role-players involved in CRC. An extensive literature study revealed that: prolonged inflammatory responses and oxidative events, marked by the up-regulation of cyclooxygenases (COXs) and inducible nitric oxide synthase (iNOS), contribute to colorectal carcinogenesis; and prolonged cytokine mediated responses, orchestrated by nuclear factor-kappa Beta (NF-κβ) with consequent immunodeficiency, are prevalent in CRC patients. Past research revealed without doubt that high dietary AA intake (meat) and COX-2 metabolism play a pivotal role during colorectal carcinogenesis. CRC is a stress-related cancer and it is plausible that high iron intake and lipids contribute to chronic inflammation and radical-mediated cell damage in this age-related disease. Current research directions in CRC concern LOX metabolism and peroxisome proliferator activator receptors (PPARs) are receiving much attention, especially PPARδ/β that was an enigma and elicited controversial debate in adenomas (polyps) and adenocarcinoma. Taken together at this point in time, the impact of environmental factors on essential fatty acid metabolism (EFAM) that impede LA conversion to AA via delta-6 and -5 desaturase (Δ6d and Δ5d) pathways with up-regulation of the fatty acid synthase (FAS) and Δ9d pathways, and the down-regulation of LA oxidative metabolism via the 15 LOX-1 pathway with up-regulation of the COX-2 and 15- LOX-2 pathways of AA, is characteristic of colorectal carcinogenesis. Seen in context, the lipid findings of this study integrated with evidence-based information in the literature encompasses the following: excessive dietary linoleic acid (LA) intake that can contribute to cell proliferation, radical-mediated cell damage, and the production of conjugated linoleic acid (CLA) in the gut; high dietary AA intake and COX-2 over-expression that play a pivotal role during carcinogenesis and immune responses with consequent exhaustion of the AA source; enhanced FAS and Δ9d activities that contribute to, respectively, enhanced palmitic acid (PA) and oleic acid (OA), is prevalent in CRC cells. The high PA content in CRC cells apparently activates PPARδ/β and thereby suppresses apoptosis, a crucial factor in the pathobiology of CRC. The link between OA and CRC still needs proper clarification. It is conceivable that the high saturated fatty acid (SFA) content observed in CRC cells, exogenous intake with red meat and endogenous production by enhanced FAS activity, contribute to colorectal carcinogenesis and immunodeficiency in CRC patients, since it can stimulate COX-2 expression (and PGE2 activity) and downregulate the Th1 immune pathway that make CRC a Th2 dominant disease. Taken in consideration that lipid rafts rich in SFAs down-regulate the Th1 cytokine subset, particularly interleukin-2 (IL-2) that stimulates lymphocyte production, it is plausible that management of the CRC patient is hampered by immunodeficiency. All the altered cell signaling pathways in CRC debated in this study served as sound foundation for clinical intervention with adjuvant FA therapeutic strategies to improve CRC management. CONCLUSION: The identification of prominent FA role-players in CRC cells that serve as ligands for specific PPAR family members contributed to the assessment of FA driven proliferation and apoptotic pathways characteristic of CRC. It is conceivable that enhanced PA (and LA) contributes to PPARδ/β over-expression that can suppress PPARγ (and PPARα) activity and then tilts the scale in favor of apoptotic resistance and survival of CRC cells. From an epidemiological viewpoint, excessive iron and SFAs seem to be important co-factors in the multifactorial etiology of CRC. The biohydroxygenation of LA to different CLA products in the gut and to what extent harmful CLA products may pose a danger need to be fully explored, especially since the uptake of beneficial products is apparently limited. Among current therapeutic options in the field of lipids, concurrent therapy with a COX-2 inhibitor (Celecoxib) and docosahexaenoic acid (DHA) to improve membrane fluidity and the impact of drug therapy entered clinical trials and although the outcome is still awaited, the impact of DHA on lymphocyte production may be a concern. Based on a mountain of evidence presented in this study, it is suffice to say that there is a rationale for additional adjuvant FA therapies that can be included in the existing therapeutic regime for CRC management. Firstly, CLA and EPA therapy for prevention of polyp recurrence and improvement of immunocompetence in CRC patients and secondly, GLA and EPA therapy in the management of patients with a history of polyp growths is proposed. Item Open AccessEvaluation of a commercial radiation oncology treatment planning system against Monte Carlo simulated dose distributions(University of the Free State, 2007-11) Shaw, William; Du Plessis, F. C. P.English: A method is described in this study whereby dose distributions calculated by a treatment planning system (TPS) were evaluated by using dose distributions calculated with Monte Carlo (MC) simulations. The MC calculated dose data were used as a benchmark. A generic Siemens MD 2 linear accelerator was simulated with the BEAMnrc MC code to obtain beam specific dynamic variables in a phase space file (PSF) related to particle fluence in a plane at a known distance from a water phantom. Dose distributions from various field sizes were produced by simulations with the DOSXYZnrc MC code. Two datasets were produced consisting of percentage depth dose (PDD), profiles and diagonal profile data for 6 and 15MV x-ray beams. The CadPlan TPS was commissioned with these datasets for both energies. Analyses of TPS calculated dose distributions were done in a water phantom and dose distributions for various clinical cases on patient CT data. Patient CT datasets were transformed into patient CT models that were suitable for dose calculations with DOSXYZnrc. These models consisted of various media with various densities for which interaction cross section data is available. Dose distributions for a number of clinical treatment plans could be devised on both the TPS and DOSXYZnrc. These included head and neck, breast, lung, prostate, oesophagus and brain plans. Calculations on the TPS were done for the Single Pencil Beam (SPB) and in some cases the Double Pencil Beam (DPB) convolution algorithms in combination with the Batho and ETAR (Equivalent Tissue-air ratio) inhomogeneity correction algorithms. Dose distributions were normalized to the depth of maximum dose (dmax) for single fields and to the ICRU reference point in full treatment plans. The location of these points was the same for the TPS and DOSXYZnrc distributions. PDD curves, beam profiles, dose-volume histograms (DVHs) and equivalent uniform doses (EUDs) were produced to aid in the evaluation of the TPS dose calculation accuracy. Results demonstrated that the assumptions in the convolution models used to produce beam penumbra regions, especially in blocked field cases, fail to account for scattered dose contributions outside the treatment field and overestimated the dose underneath small or thin shielding blocks. The PB algorithms in combination with the inhomogeneity corrections show total disregard for lateral and longitudinal electron transport through heterogeneous media. This effect is pronounced in regions where electronic equilibrium is not found, like low density lung. This region, in combination with high density bone nearby, proved even larger discrepancies as dose absorption decreases in low density media and increases in high density media. A small 15 MV field passing through lung tissue exhibited large dose calculation errors by the PB algorithms. The dataset produced here is flexible enough to be used as a benchmark for any TPS utilizing commissioning measurements in water. This method can address commissioning results as well as any clinical situation requiring dose calculation verification. Item Open AccessThe effect of the embalming fluid, used by the Department of Basic Medical Sciences (UFS), on the viability of Mycobacterium TB in human cadaver lung tissue(University of the Free State, 2012-06) Correia, Janine Carla; Steyl, J. L.; De Villiers, H. C.Embalming fluid contains substances such as formalin, ethanol, phenol, and other solvents to prevent decomposition temporarily. These agents disinfect, preserve, and/or sanitize. The risk of contracting a disease such as tuberculosis (TB) among persons, who are in close contact with recently deceased people, is high and the risk varies according to occupation. Workers at Anatomy Departments and embalmers are some of those people who are at a greater risk of contracting tuberculosis carried by cadavers. The question thus arises whether the penetration of formalin and other embalming agents into the tissue infected with Mycobacterium tuberculosis (MTB) is sufficient to render the bacilli non-infectious. The aim is to test the efficacy of the embalming fluid used at the department of Basic Medical Sciences (UFS) on eliminating Mycobacterium tuberculosis in human cadaver lung tissue. The cadavers were accompanied by their death certificates indicating the cause of death. Only cadavers whose death certificate indicated that the cause of death was TB, was selected to be included in the study. Closed needle biopsies were performed on 20 cadavers to obtain lung tissue from the apical and hilar areas. With the use of a pro-cut biopsy needle, a sample of lung tissue was obtained by inserting the needle through the 3rd intercostal (hilar sample) and the supraclavicular space (apical sample). The first sample was taken before embalming. The second sample 3 weeks after embalming. Tissue was then retrieved and deposited into a sterile specimen container, with saline as transport medium, and transported to Pathcare Laboratory (Drs Dietrich, Voigt, Mia, and partners) in Bloemfontein. The following diagnostic tools were used by Pathcare: direct microscopy from aspirates (lungs in the case of Pulmonary TB or from granulomatous lesions), MGIT culture, identification using PCR techniques, if positive. Before embalming 50% of the apical samples tested positive for MTB and 3 weeks after embalming none tested positive for MTB. Before embalming 40% of the samples taken from the area close to the hilus (perihilar), tested positive for MTB, 3 weeks after embalming none tested positive. The results show that 3 weeks after embalming none of the tested lung samples contained viable MTB. Thirteen of the 20 cadavers tested did have a viable strain of MTB before embalming occurred. It is of special interest to mention that one cadaver still had viable MTB 36 days after death. According to previous studies, after death, MTB can remain infectious for about 8 days in unembalmed lung tissue and up to 14 days if stored between 2 - 4oC. From this result, it is clear that MTB can survive in dead bodies with significant post-mortem intervals. It is evident from the results that the embalming fluid used at the department of Basic Medical Sciences (UFS) renders the bacilli non-infectious, because no growth was indicated 3 weeks after embalming. Item Open AccessThe prevalence of anatomical variations in the intraorbital part of the ophthalmic artery and its branches in cadavers(University of the Free State, 2017) Mpolokeng, Kentse Sana; Potgieter, J. H.; Labuschagne, M. J.; Louw, G. J.English: The human orbit contains various important structures that may show variations relating to their anatomy. This study focused on the intraorbital part of the ophthalmic artery in a South African cadaver sample in the Free State (UFS) and Western Cape provinces (UCT). Meyer pioneered the study of the ophthalmic artery as far back as 1887, with a main focus on its branches and their variations. Very limited investigation has been carried out in this field and available literature has little information on this. Currently no published data exists on the South African population with regard to intraorbital variations within the ophthalmic artery and its branches. Original research was conducted to address the problem of the lack of data. Dissections of the eyes were done to investigate and document the possible variations of the intraorbital part of the ophthalmic artery and its branches. The aim of the study was to determine the prevalence of anatomical variations in the intraorbital part of the ophthalmic artery and its branches in a cadaver sample. The results of the study will be of value to surgical interventionists treating patients with vascular diseases within the orbital region and also to the ophthalmology students studying the orbital vascular anatomy. A total number of 59 cadavers were utilised, and 118 eyes were dissected under the lighted magnifier and observed for variations. The sample consisted of 23 cadavers (46 eyes) from the Department of Basic Medical Sciences of the University of the Free State, and 36 cadavers (72 eyes) from the Department of Human Biology of the University of Cape Town. Sixteen types of variations were observed and documented. The ophthalmic artery crossed below the optic nerve in the left eye in 7.63% of cadavers at both institutions. No ophthalmic artery crossed below the optic nerve in the right eye in the UCT group, whereas 17.39% in the UFS group crossed below the optic nerve. Statistical analyses determined the frequencies of the variations. In certain individuals there were more than one type of variation which is in agreement with published literature. The majority of variations in branching patterns occurred bilaterally and in most cases, the variation in the left eye differed from the variation in the right eye. Males showed a higher frequency of variations. These findings may well contribute to clinical application in ophthalmology and radiology while it will also inform anatomy students studying the blood supply to the eye and surrounding structures. Item Open AccessAssessing the sexual dimorphism of the upper limb in a Free State skeletal population using univariate measurements(University of the Free State, 2020-11) Smal, Sunette Amanda; Maass, P.; Potgieter, H.Increasing numbers of incomplete unidentified skeletal remains requiring anthropological analysis have created the need for studies of bones other than the pelvis and cranium for sex estimation purposes. This study evaluated the use of 20 measurements of the radius, ulna, humerus, scapula and clavicle of 30 males and 30 females from a Free State skeletal collection. Measurements were assessed for side differences and sexual dimorphism. Sectioning points were calculated for each measurement and used to classify the sex of each individual with the accuracies of classifications also being calculated and compared to those obtained using existing classification standards. All measurements showed a significant positive correlation to each other, ranging from moderate (R=0.6) to very strong (R=0.99), reflecting the proportional biological relationship between body dimensions. Males were larger than females for all measurements (p<0.001), despite large overlaps in the ranges, reflecting the genetic and adaptive differences between the sexes. The largest proportional difference between the means of the males and females was found in the sagittal diameter at the midshaft and the acromial epiphyseal width of the clavicle, as well as in the anterior-posterior diameter at the midshaft of the radius, while the smallest was found in the maximum lengths of the radius, ulna, humerus and clavicle. Classification of sex based on the Free State derived sectioning points of each measurement was most accurate for measurements taken at joints or muscle attachment sites, such as the vertical head diameter of the humerus and the sagittal diameter at the midshaft of the clavicle with accuracies of more than 90%. The least accurate measurement for classification of sex was the acromial epiphyseal width (71.67%), which may have been as a result of the different type and rates of ossification of the clavicle affecting the variability of its morphology beyond sexual differences. For most measurements, the Free State-derived sectioning points performed better than those of the existing sectioning points and the existing classification standards, suggesting that regionally specific standards for classification may be useful. These results indicate that there is sufficient dimorphism in the upper limb bones to allow for accurate classification of sex in this Free State skeletal sample, but that differences in expression of dimorphism may exist between regional South African populations and may need to be considered when deciding which measurements to use for sex estimation. Further studies on the need for such regionally specific standards would be useful in the South African context. Item Open AccessMutational analysis of a South African haemophilia B population(University of the Free State, 2023) Bester, Chené; Janse van Rensburg, W. J.; Kloppers, J. F.Introduction and Aim: Haemophilia B is an X-linked recessive bleeding disorder characterised by a deficiency of coagulation factor IX (FIX), due to a wide spectrum of causative mutations in the FIX encoding gene (F9). Based on the plasma concentration of normal FIX coagulant activity (FIX:C), haemophilia B can be classified as mild (>5 – <40 international units per decilitre (IU/dL)), moderate (1 – 5 IU/dL) or severe (<1 IU/dL). Genetic testing is an important aspect in the haemophilia B diagnostic approach and appropriate patient management. Given the low prevalence, haemophilia A and B are considered orphan diseases; however, because haemophilia B is much rarer than haemophilia A, it is a disorder that is often neglected in terms of basic research, resulting in suboptimal disorder management. The aim of this study was to screen haemophilia B patients in our region for known and novel F9 causative gene variants, as well as determine the genotype/phenotype relationship for each study participant. Methods: A total of 21 participants were enrolled in this study. All the participants were screened using conventional PCR assays to amplify F9 exon 1 – 8, followed by direct Sanger sequencing to analyse and confirm causative F9 gene variations. The F9 variants identified were compared to various F9 gene variants databases to confirm known and novel variants. Furthermore, for the functional analysis a FIX one-stage and an enzyme-linked immunosorbent assay (ELISA) assay were done to measure the FIX:C. Discrepancies between the ELISA and one-stage assay were determined by calculating the p-value. The genotype/phenotype relationship was determined for the study participants and subsequently compared to previously published data. Results and Discussion: A total of eight pathogenic F9 variations were identified throughout the FIX protein domains: p.Ser⁴º⁶Leu, p.Glu²⁴¹*, p.Asn³⁹³del, p.Asn¹º⁴Metfs*31, p.Phe¹²¹Leufs*3, p.Lys⁴⁵⁹Serfs*24, p.Val²⁴³Phefs*2, and Thr⁸⁴Glufs*20. Approximately 63% of the variants detected were novel. The previously published variants identified in our study did correlate with previously published data. Conclusion: The discovery of novel F9 pathogenic variations in our South African population is an exciting finding, proving that genetic analysis of people with Haemophilia B is an important undertaking to better understand the pathogenesis of Haemophilia B in our population, and the wider sub-Saharan African population. Item Open AccessMoringa oleifera and autophagy: evidence from in vitro studies on chaperone-mediated autophagy in HepG₂ Cancer cells(Taylor and Francis Group, 2023) Bopape, Matlola; Tiloke, Charlette; Ntsapi, ClaudiaHepatocellular carcinoma (HCC) is the most prevalent primary liver cancer in Sub-Saharan African countries, including South Africa (SA). Given the limitations in current HCC therapeutics, there is an increasing need for alternative adjuvant therapeutic options. As such, several cell survival mechanisms, such as autophagy, have been identified as potential adjuvant therapeutic targets in HCC treatment. Of the three most established autophagic pathways, the upregulation of chaperone-mediated autophagy (CMA) has been extensively described in various cancer cells, including HCC cells. CMA promotes tumor growth and chemotherapeutic drug resistance, thus contributing to HCC tumorigenesis. Therefore, the modulation of CMA serves as a promising adjuvant target for current HCC therapeutic strategies. Phytochemical extracts found in the medicinal plant, Moringa oleifera (MO), have been shown to induce apoptosis in numerous cancer cells, including HCC. MO leaves have the greatest abundance of phytochemicals displaying anticancer potential. However, the potential interaction between the pro-apoptotic effects of MO aqueous leaf extract and the survival-promoting role of CMA in an in vitro model of HCC remains unclear. This review aims to summarize the latest findings on the role of CMA, and MO in the progression of HCC.