Research Articles (Central Analytical Facility)

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    Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture
    (Nature, 2021) Ellero, Andrea Antonio; Van den Bout, Iman; Vlok, Mare; Cromarty, Allan Duncan; Hurrell, Tracey
    Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.
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    Transcriptomic changes associated with husk scald incidence on pomegranate fruit peel during cold storage
    (Elsevier, 2020-05-05) Belay, Zinash A.; Caleb, Oluwafemi J.; Vorster, Alvera; Van Heerden, Carel; Opara, Umezuruike Linus
    Pomegranate fruit is valued for its social, economic, aesthetic and health benefits. The fruit rapidly loses quality after harvest due to continued metabolic responses and physiological disorders under sub-optimal conditions. The incidence of physiological disorder such as husk scald manifests during storage and commercial shipping, which affects the appearance and limits marketability. Despite the importance of pomegranate husk scald, little information is available about the origin and molecular mechanisms. Therefore, the aim of this study was to investigate the scald incidence of pomegranate fruit at molecular level using RNA-Seq (Ion Proton™ Next Generation Sequencing) by analyzing peel transcriptomic changes. The RNA-seq analysis generated 98,441,278 raw reads. 652 Differentially Expressed Genes (DEGs) with a fold change of > |2|, a p value ≤ 0.05 and a false discovery rate (FDR) of <0.05 were identified between healthy and scald fruit peels. An analysis of the gene ontologies of these DEGs revealed the 432 genes were assigned with molecular functions, 272 as cellular components and 205 as part of biological processes. In this analysis, genes (Pgr023188 and Pgr025081) that encode uncharacterized protein and gene (Pgr007593) that encodes glycosyltransferase showed significantly highest fold changes. Genes (Pgr003448, Pgr006024 and Pgr023696) involved in various iron binding and oxidoreductase activities were significantly suppressed. This is the first transcriptome analysis of pomegranate fruit peel related to husk scald development. Results obtained from this study will add valuable information on husk scald related changes on pomegranate fruit at genomic level and provide insight on other related physiological disorders.
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    Serum amyloid A binds to fibrin(ogen), promoting fibrin amyloid formation
    (Nature Research (part of Springer Nature), 2019-02-28) Page, Martin J.; Thomson, Greig J. A.; Nunes, J. Massimo; Engelbrecht, Anna-Mart; Nell, Theo A.; De Villiers, Willem J. S.; De Beer, Maria C.; Engelbrecht, Lize; Kell, Douglas B.; Pretorius, Etheresia
    Complex associations exist between inflammation and thrombosis, with the inflammatory state tending to promote coagulation. Fibrinogen, an acute phase protein, has been shown to interact with the amyloidogenic ß-amyloid protein of Alzheimer’s disease. However, little is known about the association between fibrinogen and serum amyloid A (SAA), a highly fibrillogenic protein that is one of the most dramatically changing acute phase reactants in the circulation. To study the role of SAA in coagulation and thrombosis, in vitro experiments were performed where purified human SAA, in concentrations resembling a modest acute phase response, was added to platelet-poor plasma (PPP) and whole blood (WB), as well as purified and fluorescently labelled fibrinogen. Results from thromboelastography (TEG) suggest that SAA causes atypical coagulation with a fibrin(ogen)-mediated increase in coagulation, but a decreased platelet/fibrin(ogen) interaction. In WB scanning electron microscopy analysis, SAA mediated red blood cell (RBC) agglutination, platelet activation and clumping, but not platelet spreading. Following clot formation in PPP, the presence of SAA increased amyloid formation of fibrin(ogen) as determined both with auto-fluorescence and with fluorogenic amyloid markers, under confocal microcopy. SAA also binds to fibrinogen, as determined with a fluorescent-labelled SAA antibody and correlative light electron microscopy (CLEM). The data presented here indicate that SAA can affect coagulation by inducing amyloid formation in fibrin(ogen), as well as by propelling platelets to a more prothrombotic state. The discovery of these multiple and complex effects of SAA on coagulation invite further mechanistic analyses.
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    Physico-elemental analysis of roasted organic coffee beans from Ethiopia, Colombia, Honduras, and Mexico using X-ray micro-computed tomography and external beam particle induced X-ray emission
    (MDPI, 2019) Cloetea, Karen J.; Smit, Ziga; Minnis-Ndimba, Roya; Vavpetic, Primoz; Du Plessis, Anton; Le Roux, Stephan G.; Pelicon, Primoz
    The physico-elemental profiles of commercially attained and roasted organic coffee beans from Ethiopia, Colombia, Honduras, and Mexico were compared using light microscopy, X-ray micro-computed tomography, and external beam particle induced X-ray emission. External beam PIXE analysis detected P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, Br, Rb, and Sr in samples. Linear discriminant analysis showed that there was no strong association between elemental data and production region, whilst a heatmap combined with hierarchical clustering showed that soil-plant physico-chemical properties may influence regional elemental signatures. Physical trait data showed that Mexican coffee beans weighed significantly more than beans from other regions, whilst Honduras beans had the highest width. X-ray micro-computed tomography qualitative data showed heterogeneous microstructural features within and between beans representing different regions. In conclusion, such multi-dimensional analysis may present a promising tool in assessing the nutritional content and qualitative characteristics of food products such as coffee.
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    Investigation on the hereditary basis of colorectal cancers in an African population with frequent early onset cases
    (Public Library of Science, 2019-10-24) Katsidzira, Leolin; Vorster, Anna; Gangaidzo, Innocent T.; Makunike-Mutasa, Rudo; Govender, Dhiren; Rusakaniko, Simbarashe; Thomson, Sandie; Matenga, Jonathan A.; Ramesar, Raj
    Background: Approximately 25% of colorectal cancer patients in sub-Saharan Africa are younger than 40 years, and hereditary factors may contribute. We investigated the frequency and patterns of inherited colorectal cancer among black Zimbabweans. Methods: A population-based cross-sectional study of ninety individuals with a new diagnosis of colorectal cancer was carried out in Harare, Zimbabwe between November 2012 and December 2015. Phenotypic data was obtained using interviewer administered questionnaires, and reviewing clinical and pathology data. Cases were screened for mismatch repair deficiency by immunohistochemistry and/or microsatellite instability testing, and for MLH1, MSH2 and EPCAM deletions using multiplex ligation-dependent probe amplification. Next generation sequencing using a 16-gene panel was performed for cases with phenotypic features consistent with familial colorectal cancer. Variants were assessed for pathogenicity using the mean allele frequency, phenotypic features and searching online databases. Results: Three Lynch syndrome cases were identified: MSH2 c.2634G>A pathogenic mutation, c.(1896+1_1897–1)_(*193_?)del , and one fulfilling the Amsterdam criteria, with MLH1 and PMS2 deficiency, but no identifiable pathogenic mutation. Two other cases had a strong family history of cancers, but the exact syndrome was not identified. The prevalence of Lynch syndrome was 3·3% (95% CI 0·7–9·4), and that of familial colorectal cancer was 5·6% (95% CI, 1·8–12·5). Conclusions: Identifying cases of inherited colorectal cancer in sub-Saharan Africa is feasible, and our findings can inform screening guidelines appropriate to this setting.