Browsing by Subject "biology"

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    Open Access
    Electrospun Tissue Engineered Vascular Grafts
    (2019) Hülk, Vincent Martin; Bezuidenhout, Deon
    ePTFE and Dacron vascular grafts are successful in large diameter applications but provide poor outcomes in small diameter (<6 mm) applications. Occlusion, poor cell ingrowth and a mismatch of compliance compared to the native vessel, cause failure to the current synthetic grafts. Spontaneous transmural endothelialisation can be enhanced in non-degradable scaffolds with heparin-mediated growth factor delivery via heparin surface modification, as well as induce an anti-thrombotic lumen. Electrospinning of vascular grafts offer a means to mimic the natural extracellular matrix (ECM) with improved porosity and pore for increased cell ingrowth, incorporation of drugs for sustained release, and tailoring mechanical properties match that of the native vessel. This study aims to produce a small diameter biodegradable vascular graft with sufficient porosity/pore size and incorporated heparin as an angiogenic/anti-thrombotic agent. DegraPol® (DP30), a degradable polyurethane was dissolved in different ratios of chloroform/HFIP (24%w/w) and electrospun at 30%, 40%, and 50% rH to obtain a small diameter vascular graft with improved porosity/pore size and mechanical properties. The sodium salt of heparin (HepNa+ ) was modified to heparin tributylamine (HepTBA) to ensure the solubility in organic solvents and incorporated into the electrospinning solution at 3% and 5% wt/wt (w HepTBA/w polymer). The grafts were analysed for morphological (fibre diameter, porosity/pore size, fibre alignment) and mechanical (hoop stress, strain, burst pressure, compliance) properties. After in vitro elution and degradation studies, grafts (DP30 and DP30+3%HepTBA) were evaluated in an in vivo pilot study using a rat infrarenal aortic interposition model (28 days). Relative humidity did not significantly influence the scaffold morphology or the mechanical properties, for solvent systems used. However, the addition of HFIP to the solution had on average a 2.9-fold decrease in the circumferential UTS and strain from 0.63±0.16 MPa and 90±16%, respectively. The grafts showed theoretical compliance in the physiological range of 6-8%/100mmHg and showed a significant amount of drug release in the first 5 days and a cumulative release of 62% and 36% (respectively) by day 28. The DP30 and DP30+3%HepTBA grafts lost 46% (P> 0.5) and 50% (P< 0.01) of its circumferential UTS respectively, whereas the loss in maximum strain for the same groups was 66% (P< 0.0001) and 76% (P< 0.01). A porosity of 64.5±2.7% and 54.9±2.2% (P< 0.01) was achieved for the DP30 and DP30+3%HepTBA with 52% and 29% of the pore sizes larger than 10 µm. The in vivo pilot study showed patent grafts with tissue ingrowth and endothelium on the lumen for DP30 as well as the DP30+3%HepTBA group. The DP30 grafts show promise for the replacement of small diameter vessels. Heparin-eluting grafts will be further evaluated in long-term isolated loop models to determine their capacity for spontaneous transmural endothelialisation.
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    Open Access
    Electrospun Tissue Engineered Vascular Grafts
    (2019) Hülk, Vincent Martin; Bezuidenhout, Deon
    ePTFE and Dacron vascular grafts are successful in large diameter applications but provide poor outcomes in small diameter (<6 mm) applications. Occlusion, poor cell ingrowth and a mismatch of compliance compared to the native vessel, cause failure to the current synthetic grafts. Spontaneous transmural endothelialisation can be enhanced in non-degradable scaffolds with heparin-mediated growth factor delivery via heparin surface modification, as well as induce an anti-thrombotic lumen. Electrospinning of vascular grafts offer a means to mimic the natural extracellular matrix (ECM) with improved porosity and pore for increased cell ingrowth, incorporation of drugs for sustained release, and tailoring mechanical properties match that of the native vessel. This study aims to produce a small diameter biodegradable vascular graft with sufficient porosity/pore size and incorporated heparin as an angiogenic/anti-thrombotic agent. DegraPol® (DP30), a degradable polyurethane was dissolved in different ratios of chloroform/HFIP (24%w/w) and electrospun at 30%, 40%, and 50% rH to obtain a small diameter vascular graft with improved porosity/pore size and mechanical properties. The sodium salt of heparin (HepNa+ ) was modified to heparin tributylamine (HepTBA) to ensure the solubility in organic solvents and incorporated into the electrospinning solution at 3% and 5% wt/wt (w HepTBA/w polymer). The grafts were analysed for morphological (fibre diameter, porosity/pore size, fibre alignment) and mechanical (hoop stress, strain, burst pressure, compliance) properties. After in vitro elution and degradation studies, grafts (DP30 and DP30+3%HepTBA) were evaluated in an in vivo pilot study using a rat infrarenal aortic interposition model (28 days). Relative humidity did not significantly influence the scaffold morphology or the mechanical properties, for solvent systems used. However, the addition of HFIP to the solution had on average a 2.9-fold decrease in the circumferential UTS and strain from 0.63±0.16 MPa and 90±16%, respectively. The grafts showed theoretical compliance in the physiological range of 6-8%/100mmHg and showed a significant amount of drug release in the first 5 days and a cumulative release of 62% and 36% (respectively) by day 28. The DP30 and DP30+3%HepTBA grafts lost 46% (P> 0.5) and 50% (P< 0.01) of its circumferential UTS respectively, whereas the loss in maximum strain for the same groups was 66% (P< 0.0001) and 76% (P< 0.01). A porosity of 64.5±2.7% and 54.9±2.2% (P< 0.01) was achieved for the DP30 and DP30+3%HepTBA with 52% and 29% of the pore sizes larger than 10 µm. The in vivo pilot study showed patent grafts with tissue ingrowth and endothelium on the lumen for DP30 as well as the DP30+3%HepTBA group. The DP30 grafts show promise for the replacement of small diameter vessels. Heparin-eluting grafts will be further evaluated in long-term isolated loop models to determine their capacity for spontaneous transmural endothelialisation.
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    Open Access
    Evaluation of a nutrition and health education program on components of metabolic health on women from under-resourced South African communities
    (2022) Guillén, Pujol-Busquets Georgina; Lambert, Estelle V; Larmuth, Kate; Smith, James; Faig, Anna Bach
    Background: Diet-related non-communicable diseases (NCDs) pose a substantial burden in terms of financial cost, morbidity, and mortality. In South Africa there is currently a double burden of infectious diseases and NCDs. Overconsumption of sugar, refined carbohydrates, and poor-quality fats, increases the risk for developing chronic diseases. Families from poor communities are often forced to eat these harmful foods due to a lack of nutrition education, or because they cannot afford or don't know how to access healthy foods. While poverty is an important barrier to health and education for both men and women, it tends to yield a higher burden in women. There is evidence that low-carbohydrate high-fat (LCHF) diets can improve metabolic health in well-controlled clinical trials where quality food is either provided or where participants have the financial and logistical means to access the foods promoted by this diet. However, one cannot assume that the same nutritional advice will translate to residents of underserved communities, who may not understand the advice nor be able to afford or access the foods promoted by this diet. Women from these communities are often the gatekeepers to healthy food choices for their families, but they are at a particular disadvantage. Eat Better South Africa (EBSA) runs nutrition education programs to teach – predominately women – how to choose affordable healthier foods that are lower in refined carbohydrates and higher in healthy fats to prevent or manage metabolic conditions. Aims: This research aims to optimise the EBSA program for women from underresourced communities and to evaluate its effectiveness for changing dietary behaviour and improving metabolic health. The objectives were: 1) to explore women's perceptions of the EBSA program and the barriers and facilitators that they faced to change their dietary habits and adhere to the EBSA recommended diet; 2) to conduct community assessments in the under-resourced communities that EBSA planned to run programs, better address women's needs and explore their willingness to participate in a nutrition and health education program; 3) to assess the effects of the EBSA program on women's metabolic health and wellbeing through mixed-methods and to explore EBSA's team perceptions of the program, and 4) to explore health practitioners' perceptions of the health and nutritional advice recommended by EBSA. Methods: The first part of this project consisted of a qualitative study through focus group discussions (FGDs) with women from previous EBSA programs (n=18) and naïve EBSA participants (n=60). The second part of the project consisted of a mixed-method evaluation (n=32) of a pilot study on an EBSA intervention to assess health status changes. These methods included qualitative methods (in-depth individual (IDIs) interviews with both EBSA participants and EBSA team members and FGDs with the EBSA participants), and quantitative methods (diet assessment, metabolic health markers and physical activity behaviour) to assess changes before and after the pilot intervention program. The last part of this project consisted of a qualitative IDI study on health practitioners' perceptions and understanding of a LCHF diet (n=16). Thematic analysis of the qualitative data was conducted using NVivo 12 software. Descriptive and statistical analysis of the quantitative data was done using Stata 16 and Jamovi. Results: The first formative study indicated that the EBSA participants' greatest facilitators and barriers revolved around understanding the educational content and on how to implement the dietary advice. The mixed methods results of the second study, the pilot, indicated that, overall, the women experienced improvements in dietary behaviours and biomarkers related to inflammation, lipids, and glycaemic profiles. Furthermore, the participants waist circumference, weight, blood pressure, triglycerides and HbA1c were significantly reduced, and those changes were sustained six months after the EBSA intervention. Women's carbohydrate intake was significantly reduced, and their health markers improved despite a slight increase in sedentary behaviour. The qualitative results found that the major reason women enrolled in the program was because they suffered from NCDs. Most women found that the EBSA diet made them feel less hungry, more energetic and they felt that their health had improved. Most women spoke of socioeconomic challenges which made it difficult for them to follow EBSA's recommendations, such as employment status, safety issues in the community, and lack of support from relatives. Women felt that some health practitioners were not supportive of the diet, but that they became more positive after observing the related health outcomes. Quantitative results converged with qualitative results except for physical activity behaviour. EBSA's team perceptions of the program and participants' challenges matched participants' experiences. The final qualitative study indicated that health practitioners' approaches to LCHF diets seemed to be supportive but not advocating. Conclusion: Although, currently, some international food guidelines endorse LCHF diets, there is still a considerable amount of confusion and lack of knowledge regarding this diet. This study provided data on the dietary intake and health risk status of women from under-resourced South African communities, and the facilitators and challenges of a LCHF education program to change their dietary behaviour. Results suggested that most participants followed EBSA's dietary recommendations and experienced health improvements as a result. Follow up data at six months suggested that those changes could be sustainable. For people with diet-related chronic diseases, LCHF diets should be supported by experienced health care professionals who can facilitate optimal nutritional intake. This is the same for any other diet; the evidence for long-term compliance and the sustainability of carbohydrate restriction is currently not yet established. In the absence of this evidence, existing data suggest that it is a legitimate and potentially effective treatment to adopt a LCHF diet as an option for patients to manage and prevent NCDs in under-resourced communities.
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    Open Access
    Geographic variation in the phenotype of an African horseshoe bat species, Rhinolophus damarensis, (Chiroptera: Rhinolophidae)
    (2018) Maluleke, Tinyiko; Jacobs, David; Winker, Henning
    Studies involving geographic variation in the phenotypes of bats help scientists to explain why these mammals are the most species rich mammalian order second only to rodents, with well more than 1 300 species occurring worldwide. Such species richness or high diversity is the manifestation of the generation of biodiversity through the splitting of lineages within bat species. A lineage of bat species can diversify into several lineages which then differentiate from each other in allopatry. Thus, the spatial separation of a lineage into several lineages could be attributed to geographical, ecological and environmental factors across the distributional range of the species. Similarly, vicariant events may also play a role in separating lineages within species. The Damara horseshoe bat species, Rhinolophus damarensis, is widely distributed but restricted to the western half of southern Africa, where it occurs across several major biomes. Formerly regarded as the subspecies, R. darlingi damarensis, it was elevated to full species status on the basis of genetic and phenotypic differences between it and R. darlingi darlingi. Rhinolophus damarensis is itself made up of two ecologically separated genetic lineages. A total of 106 individuals of R. damarensis were sampled from seven localities across its distributional range, with a view to determining and documenting the extent of geographic variation in body size, echolocation parameters, wing parameters, cranial shape and postcranial morphology of individuals from populations of R. damarensis across the distributional range of the species. Firstly, an investigation into geographic variation in resting echolocation frequency (RF) of the horseshoe bat species, R. damarensis was carried out in the western half of southern Africa (Chapter 2). Three hypotheses were tested. The first one, James’Rule (JR), states that individuals occurring in hot humid environments generally have smaller body sizes than conspecifics that occur in cooler, dryer environments, and the largest are expected to occur in cool, dry areas. On this basis and because of the known relationship between body size and RF, it was predicted that there should be a correlation between body size and climatic factors and between body size and RF. The second hypothesis was Isolation by Environment (IbE) mediated through sensory drive, which proposes that diversification of lineage may be driven by environmentally-mediated differences in sensory systems. Under this hypothesis, it was predicted that call frequency variation should be correlated with climatic variables. The third hypothesis was that Isolation by Distance (IbD) can influence phenotypic trait variation by restricting gene flow between populations. Under the Isolation by Distance (IbD) Hypothesis, it was predicted that call frequency variation should be partitioned in accordance with geographic distance between populations. To investigate the probability of the JR, IbE and IbD, the Akaike’s information criterion AICc candidate models were evaluated with different combinations of environmental (annual mean temperature and relative humidity), spatial (latitude and region) and biological (forearm as a proxy for body size) predictor variables to determine their influence on resting frequency (RF) across the distributional range of R. damarensis. Linear mixed effects models (LMEs) were employed to analyse the relationship between the response variable (RF) and the environmental, spatial and biological predictor variables. The influence of prey detection range and atmospheric attenuation was also investigated. The results showed no evidence for JR or for random genetic drift. Body size was neither correlated with RF nor environmental variables, suggesting that variation in RF was not the result of concomitant variation in body size as proposed by JR. Similarly, the Mantel test showed no IbD effect and there was therefore no evidence that genetic drift was responsible for the variation in RFs. In contrast, the LMEs showed that there was support for IbE in the form of an association between RF and region (in the form of the variable “Reg”) which was based on the two geographically separated genetic lineages. Furthermore, RF variation was also associated with the climatic variable AMT. The taxonomic status of R. damarensis was investigated using ecological traits and phenotypic characters including skulls, wings and echolocation (Chapter 3) and three dimensional (3D) scanned skulls and mandibles (Chapter 4). The main objective (Chapter 3 and Chapter 4) was to test whether previously reported genetic divergence between the two R. damarensis lineages was associated with phenotypic divergence. Morphometric and echolocation measurements were taken from hand held individual bats in the field, and skull measurements were taken from field collected voucher specimens as well as museum specimens. Discriminant Function Analyses (DFA) revealed that there was geographic variation among populations and lineages of R. damarensis. Multivariate Linear Regressions (MLV) and Linear models (LM) on the basal parts of bacula revealed significant differences between the southern and northern lineages of R. damarenis. The bacula of the two lineages of R. damarensis appear to have different shapes. Diversification through shape analyses (Chapter 4) was investigated using three dimensional (3D) geometric morphometric analyses based on X-ray microcomputed tomography (µCT) scanning of dried skulls and mandibles of R. damarensis. Procrustes Anova results of both mandibles and skulls indicated that there were no significant differences between sexes but that the shape of skulls and mandibles varied across different localities (Chapter 4). Canonical Variate Analysis (CVA) suggested that geographic variation in R. damarensis mandibles was based on the shape and thickness of the alveolar bone. Geographic variation in the skulls was based on changes in the rostrum, anterior medial swelling and brain case. Some populations had slightly deeper rostra, slightly larger anterior medial swellings and smaller braincases, whilst others had slightly shallower rostra, slightly smaller anterior medial swellings and larger braincases. The northern lineage was found to be separated from the southern lineage based on the changes in skull and mandible shape. Therefore, separation of lineages within R. damarensis (Chapter 4) could be associated with the foraging and feeding behaviour of the species under different ecological conditions due to ecological opportunity. Overall, differences in the RF were found to be associated with Isolation by Environment mediated through sensory drive and this has led to the formation of two regional (northern and southern) groupings in RF (Chapter 2). The two lineages were supported by both the phenotypic divergence (Chapter 3) and shape variation within R. damarensis skulls and mandibles (Chapter 4). Thus, phenotypic differences corresponded to genetic differences between the two lineages and provide support for IbE.
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    Leveraging big data resources and data integration in biology: applying computational systems analyses and machine learning to gain insights into the biology of cancers
    (2020) Sinkala, Musalula; Martin, Darren; Mulder, Nicola; Barth, Stefan
    Recently, many "molecular profiling" projects have yielded vast amounts of genetic, epigenetic, transcription, protein expression, metabolic and drug response data for cancerous tumours, healthy tissues, and cell lines. We aim to facilitate a multi-scale understanding of these high-dimensional biological data and the complexity of the relationships between the different data types taken from human tumours. Further, we intend to identify molecular disease subtypes of various cancers, uncover the subtype-specific drug targets and identify sets of therapeutic molecules that could potentially be used to inhibit these targets. We collected data from over 20 publicly available resources. We then leverage integrative computational systems analyses, network analyses and machine learning, to gain insights into the pathophysiology of pancreatic cancer and 32 other human cancer types. Here, we uncover aberrations in multiple cell signalling and metabolic pathways that implicate regulatory kinases and the Warburg effect as the likely drivers of the distinct molecular signatures of three established pancreatic cancer subtypes. Then, we apply an integrative clustering method to four different types of molecular data to reveal that pancreatic tumours can be segregated into two distinct subtypes. We define sets of proteins, mRNAs, miRNAs and DNA methylation patterns that could serve as biomarkers to accurately differentiate between the two pancreatic cancer subtypes. Then we confirm the biological relevance of the identified biomarkers by showing that these can be used together with pattern-recognition algorithms to infer the drug sensitivity of pancreatic cancer cell lines accurately. Further, we evaluate the alterations of metabolic pathway genes across 32 human cancers. We find that while alterations of metabolic genes are pervasive across all human cancers, the extent of these gene alterations varies between them. Based on these gene alterations, we define two distinct cancer supertypes that tend to be associated with different clinical outcomes and show that these supertypes are likely to respond differently to anticancer drugs. Overall, we show that the time has already arrived where we can leverage available data resources to potentially elicit more precise and personalised cancer therapies that would yield better clinical outcomes at a much lower cost than is currently being achieved.
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    Mechanisms determining the coexistence of open- and closed-canopy biomes
    (2018) Power, Simon C; Cramer, Michael; Bond, William; Verboom, Tony
    Open- (e.g. grassland, savanna, shrubland) and closed-canopy (e.g. forest) biomes frequently coexist in the same landscape, where open environments tend to be fire-prone with higher light, but lower nutrient and water availability than closed environments. Environmental heterogeneity could select for divergent floristic assemblages and adaptive traits, from which emergent differences in resource availability and fire incidence contribute to excluding species from the alternate habitat. In this thesis, I investigated whether the coexistence of open–closed canopy biomes, such as forest and fynbos in the Cape Floristic Region, is contingent on environmental heterogeneity coupled with contrasting species traits. Given the heterogeneity in multiple environmental properties between open- and closed-canopy biomes, I hypothesized that boundaries between open- and closed-canopy biomes will display greater floristic turnover compared to boundaries between structurally similar biomes (e.g. open- and opencanopy biomes). To explore this, genus- and family-level turnover were correlated with climate, fire, leaf area index (LAI: proxy for understorey light) and soil properties across biome boundaries in South Africa. Both genus- and family-level turnovers were highest across open–closed boundaries and most strongly predicted by increased differences in LAI, suggesting that contrasting light regimes provide significant adaptive challenges for plants. The potential effect of contrasting light regimes is highlighted by the absence of open-canopy species from forest understoreys, where low, dynamic light could limit the ability of plants to acquire sufficient carbon. This apparent shade intolerance led to the hypothesis that open-canopy species lack the traits to maintain a positive carbon balance under low and dynamic light. To test this, leaf traits and photosynthetic response to continuous or dynamic light were compared between forest and fynbos species grown under three light treatments. Fynbos species experienced high mortality under shade treatments, produced leaves that were thicker, up to 1000 times smaller, had lower photosynthetic rates (0.8 versus 3.4mol m-2 s -1 ) under continuous low light (400 mol m-2 s -1 ) and lower light-use efficiency during dynamic light sequences than forest species. These differences imply that shade intolerance in fynbos species is associated with traits that are inefficient at harvesting light and require relatively continuous high intensity light for carbon assimilation. Moreover, these inefficiencies would make it difficult to support the carbon intensive traits (e.g. cluster roots, lignotubers, sclerophyllous leaves) that facilitate fire survival and nutrient acquisition/conservation in open habitats. In contrast, forest species are able to colonize open habitats during the long-term absence of fire, implying that they are able to tolerate high light and low nutrient conditions. Given that plants frequently cope with contrasting conditions through the expression of phenotypic plasticity, it was hypothesized that closed-canopy species possess greater plasticity than open-canopy species. To assess this, the response of leaf traits and foliar nutrition to changes in LAI and soil nutrition were compared between forest and fynbos species in the field. Leaf size and specific leaf area in forest species correlated positively with LAI and soil nutrition, whereas fynbos species response was weak, suggesting that forest species are more plastic. This plasticity may be realised by the variable light conditions forest species experience through their canopy and the occupation of higher nutrient soils, which alleviate belowground constraints. By comparison, the occupation of low nutrient soils by fynbos may inhibit plasticity given the selection of inflexible, conservative leaves. Consequently, I propose that the coexistence of open- and closed-canopy biomes arises from the steep turnover in selective regimes, which together with the contrasting adaptive traits and degrees of phenotypic plasticity they require, act together to competitively exclude species from the alternate habitat.
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    Noise reduction during diffusion tensor imaging of infants
    (2019) Jordaan, Johannes Petrus; Meintjies, Ernesta; van der Kouwe, Andr´e
    Acoustic noise produced during echo planar imaging (EPI) has been known to reach excessive levels. In addition to causing general patient discomfort and anxiety, this level of noise makes the scanning of young children and infants particularly difficult. Infants are typically scanned while sleeping to minimise motion as they cannot ethically be sedated for research purposes. The extreme noise during MRI acquisitions often cause them to wake before the end of the scanning session. This problem is exacerbated by particularly noisy acquisitions, such as the single shot echo planar diffusion tensor imaging sequence. The main aim of this project was to reduce the noise of this particular acquisition specifically for the scanning of newborn infants. Acoustic noise during MRI acquisitions mainly originates from mechanical vibrations in the gradient coil assemblies due to interactions between the rapidly changing currents applied to the coils and the main static field. A transfer function relating the output acoustic noise spectrum to the gradient excitation input spectrum was developed and used to identify resonant peaks which would amplify coinciding gradient waveform harmonics. In addition to resonant peaks, the transfer function showed significant amplification of frequencies above 1 kHz. In this work, noise reduction was achieved by implementing digital low-pass filters to reduce high-frequency harmonics of the standard trapezoidal gradient waveforms, focusing on the EPI readout portion of a diffusion tensor imaging (DTI) sequence. For comparison purposes, an EPI readout using sinusoidal frequency encoding waveforms and a constant phase encoding blip was also implemented. In addition to reducing produced noise, a passive noise reduction enclosure was built from open cell polyurethane foam mounted in a PVC frame to surround the sleeping infant and act as an acoustic insulation box. Lastly, the effectiveness of introducing pink noise from an external source to mask the abrupt changes in scanner noise, was also investigated. The altered k-space trajectories due to the modifications made to the EPI readout gradient waveforms were corrected through a custom one-dimensional regridding procedure applied along the frequency encoding axis in k-space. Noise reduction was measured with an Optimic 1155 optical microphone from Optoacoustics, attached on top of a cylindrical water phantom inside a 16 channel infant head coil in the isocenter along the Z-direction, facing the bore in the rightleft direction (similar to the orientation that the ears of a sleeping infant would be). Signal-to-noise ratio (SNR) and fractional anisotropy (FA) within the corpus callosum (CC) were compared for images acquired using the standard and modified (filtered and sinusoidal readouts) DTI sequences, the latter each for regridding kernel window sizes of 2 and 4, respectively. The acoustic noise spectra of the filtered and sinusoidal EPI sequences demonstrated a significant reduction in EPI harmonics compared to the standard sequence, but very little difference between each other. Without the foam enclosure, the filtered acquisition with filtered crushers reduced peak sound pressure levels (SPL) by 3.4 and 4 dBA for strong and no fat suppression, respectively, and A-weighted equivalent continuous sound levels (LA,eq) by 2.5 and 2.8 dBA, respectively. Adding the foam enclosure increased peak SPL reduction to 4.8 dBA with fat suppression and 7 dBA without. The sinusoidal sequence performing similarly or marginally (no more than 0.5%) worse than the filtered on all outcomes. SNR measurements in the CC were higher for all volumes of the filtered acquisition compared to the standard, while those of the sinusoidal were similar or slightly lower compared to the standard acquisition. FA values in the CC of the sinusoidal and filtered acquisitions did not differ from those of the standard acquisition (pairwise student’s t-test, all p’s >0.2). For the 16 channel head coil, image reconstruction time increased by only 45 seconds for a regridding kernel width W = 2. Filtering gradient waveforms is an effective technique to reduce acoustic noise during DTI without increasing acquisition time, reducing image quality, or altering FA measures. The proposed method has the potential to be generalized to most gradient waveforms across a variety of sequences. With the addition of the passive noise reduction enclosure, the combined noise reduction could greatly reduce infant anxiety and startling, leading to an increase in the number of infants in whom the acquisition protocol is completed. Acoustic noise produced during echo planar imaging (EPI) has been known to reach excessive levels. In addition to causing general patient discomfort and anxiety, this level of noise makes the scanning of young children and infants particularly difficult. Infants are typically scanned while sleeping to minimise motion as they cannot ethically be sedated for research purposes. The extreme noise during MRI acquisitions often cause them to wake before the end of the scanning session. This problem is exacerbated by particularly noisy acquisitions, such as the single shot echo planar diffusion tensor imaging sequence. The main aim of this project was to reduce the noise of this particular acquisition specifically for the scanning of newborn infants. Acoustic noise during MRI acquisitions mainly originates from mechanical vibrations in the gradient coil assemblies due to interactions between the rapidly changing currents applied to the coils and the main static field. A transfer function relating the output acoustic noise spectrum to the gradient excitation input spectrum was developed and used to identify resonant peaks which would amplify coinciding gradient waveform harmonics. In addition to resonant peaks, the transfer function showed significant amplification of frequencies above 1 kHz. In this work, noise reduction was achieved by implementing digital low-pass filters to reduce high-frequency harmonics of the standard trapezoidal gradient waveforms, focusing on the EPI readout portion of a diffusion tensor imaging (DTI) sequence. For comparison purposes, an EPI readout using sinusoidal frequency encoding waveforms and a constant phase encoding blip was also implemented. In addition to reducing produced noise, a passive noise reduction enclosure was built from open cell polyurethane foam mounted in a PVC frame to surround the sleeping infant and act as an acoustic insulation box. Lastly, the effectiveness of introducing pink noise from an external source to mask the abrupt changes in scanner noise, was also investigated. The altered k-space trajectories due to the modifications made to the EPI readout gradient waveforms were corrected through a custom one-dimensional regridding procedure applied along the frequency encoding axis in k-space. Noise reduction was measured with an Optimic 1155 optical microphone from Optoacoustics, attached on top of a cylindrical water phantom inside a 16 channel iv infant head coil in the isocenter along the Z-direction, facing the bore in the rightleft direction (similar to the orientation that the ears of a sleeping infant would be). Signal-to-noise ratio (SNR) and fractional anisotropy (FA) within the corpus callosum (CC) were compared for images acquired using the standard and modified (filtered and sinusoidal readouts) DTI sequences, the latter each for regridding kernel window sizes of 2 and 4, respectively. The acoustic noise spectra of the filtered and sinusoidal EPI sequences demonstrated a significant reduction in EPI harmonics compared to the standard sequence, but very little difference between each other. Without the foam enclosure, the filtered acquisition with filtered crushers reduced peak sound pressure levels (SPL) by 3.4 and 4 dBA for strong and no fat suppression, respectively, and A-weighted equivalent continuous sound levels (LA,eq) by 2.5 and 2.8 dBA, respectively. Adding the foam enclosure increased peak SPL reduction to 4.8 dBA with fat suppression and 7 dBA without. The sinusoidal sequence performing similarly or marginally (no more than 0.5%) worse than the filtered on all outcomes. SNR measurements in the CC were higher for all volumes of the filtered acquisition compared to the standard, while those of the sinusoidal were similar or slightly lower compared to the standard acquisition. FA values in the CC of the sinusoidal and filtered acquisitions did not differ from those of the standard acquisition (pairwise student’s t-test, all p’s >0.2). For the 16 channel head coil, image reconstruction time increased by only 45 seconds for a regridding kernel width W = 2. Filtering gradient waveforms is an effective technique to reduce acoustic noise during DTI without increasing acquisition time, reducing image quality, or altering FA measures. The proposed method has the potential to be generalized to most gradient waveforms across a variety of sequences. With the addition of the passive noise reduction enclosure, the combined noise reduction could greatly reduce infant anxiety and startling, leading to an increase in the number of infants in whom the acquisition protocol is completed.
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    Open Access
    The Immunogenicity of Plant-produced Human Papillomavirus (HPV) Virus-like particles (VLPs) in Mice
    (2019) Naupu, Paulina Ndinelago; Hitzeroth, Inga; Van Zyl Alta
    Cervical cancer is caused by infection with high-risk Human papillomaviruses (HPVs). It is ranked fourth among the top cancers in women worldwide, with ~87% of the global cervical cancer cases reported in developing countries. The HPV L1 capsid protein can self-assemble into virus-like particles (VLPs) that are structurally like native virions, which is the foundation on which commercially available vaccines have been developed. There are 3 commercially available HPV vaccines that are effective at preventing HPV infections, but are expensive, therefore limiting their use in the poorer developing countries where they are most needed. Thus, there is a need for more cost-effective HPV vaccines for use in these countries. Over the years, the use of plants to produce vaccines has begun to be more favourably looked upon as a costeffective alternative to conventionally used expression systems. The aim of this study was to evaluate the plant-based transient expression system as a tool to produce potentially cost-effective HPV L1 VLP-based vaccines, particularly for developing countries. Firstly, the L1 proteins of the 8 most common high-risk Human papillomavirus types in Africa (HPV 16, 18, 31, 33, 35, 45, 52, and 58) and 2 low risk types (HPV 6 and 34) were transiently expressed in Nicotiana benthamiana. The proteins were purified via isopycnic ultracentrifugation using sucrose and Optiprep™ density gradients, and the assembly of VLPs assessed by transmission electron microscopy (TEM). To further assess whether the VLPs are immunogenic, HPV 35, 52 and 58 were selected for mice studies. These were selected in particular, as HPV 35 is the fifth most prevalent type in Africa, and HPV 52 and 58 are among the most frequently reported high-risk types in Sub-Saharan Africa. VLPs representing the 3 HPV types were quantified and prepared for immunization in mice. The commercially available Gardasil® HPV VLP vaccine was used as a positive control. The immunogenicity of the vaccines was evaluated by testing for the presence of anti-L1 antibodies in sera from immunized mice using enzyme-linked immunosorbent assays (ELISAs) and western blots. Sera from immunized mice were also tested for the presence of neutralizing antibodies using pseudovirion based neutralization assays (PBNAs). L1 proteins of all 10 HPV types tested were successfully expressed in N. bethamiana, and TEM analysis showed that expression resulted in the successful formation of fully assembled VLPs (40-60nm) as well as small VLPs and/or capsomeres (25-39nm). The analysis of the immune response showed that type-specific L1-specific antibodies were produced which were able to successfully neutralize homologous pseudovirions (PsVs) in PBNAs. Sera from mice immunized with plant-produced VLPs were further tested against heterologous HPV 6, 16, 18, 31, and 45 PsVs. However, none of the tested heterologous HPVs were neutralized, suggesting that plant-made VLPs induced type-specific neutralizing antibodies only. In conclusion, this study successfully demonstrated the potential for using plant-based transient expression systems to produce affordable and immunogenic HPV vaccines, particularly for developing countries. This is the first study describing the expression of 10 HPV L1 proteins in plants, marking a step towards the development of cheaper HPV vaccines which could be combined to generate an effective multivalent vaccine against HPVs.
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    Third-line antiretroviral therapy in Africa: effectiveness in a Southern African retrospective cohort study
    (2015) Meintjes, Graeme; Dunn, Liezl; Coetsee, Marla; Hislop, Michael; Leisegang, Rory; Regensberg, Leon; Maartens, Gary
    Background An increasing number of patients in Africa are experiencing virologic failure on second-line antiretroviral therapy (ART) and those who develop resistance to protease inhibitors (PI) will require third-line ART, but no data on the outcomes of third-line are available from the region. We assessed the virologic outcomes and survival of patients started on salvage ART in a Southern African private sector disease management programme.Methods Retrospective observational cohort study with linkage to the national death register. Adults (≥18years) who started salvage ART between July 2007 and December 2011 were included. Salvage ART was defined by inclusion of darunavir or tipranavir in an ART regimen after having failed another PI. For Kaplan–Meier (KM) analysis, patients were followed up until event, or censored at death (only for virologic outcomes), leaving the programme, or April 2014.Results152 patients were included. Subtype was known for 113 patients: 111 (98%) were infected with subtype C. All 152 had a genotype resistance test demonstrating major PI resistance mutations. Salvage drugs included were: darunavir/ritonavir (n=149), tipranavir/ritonavir (n=3), raltegravir (n=58), and etravirine (n=8). Median follow-up was 2.5years (IQR=1.5–3.3). 82.9% achieved a viral load ≤400 copies/ml and 71.1% ≤50 copies/ml. By the end of the study 17 (11.2%) of the patients had died. The KM estimate of cumulative survival was 87.2% at 2000days.ConclusionsVirologic suppression was comparable to that demonstrated in clinical trials and observational studies of salvage ART drugs conducted in other regions. Few deaths occurred during short term follow-up. Third-line regimens for patients with multidrug resistant subtype C HIV in Africa are virologically and clinically effective.
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    Unravelling the biology of the Southern African Sauropodomorph dinosaurs, Plateosauravus and the 'Maphutseng dinosaur'
    (2019) Krupandan, Emil Darius; Chinsamy-Turan, Anusuya; Pol, D
    The nomen dubium “Euskelosaurus ” has functioned as a waste - basket taxon for decades, with large bodied sauropodomorph material from South Africa as signed to it without proper analysis.