Expression, solubilisation, purification and characterisation of recombinant bluetongue virus viral protein 7

Bluetongue virus belongs to the Orbivirus genus from the Reoviridae family. It infects predominantly domestic and wild ruminants and is economically significant worldwide. Bluetongue virus VP7 forms the intercepting layer between the outer capsid (VP2 and VP5) and VP3 which surrounds the genomic material. BL21(DE3), NiCo21(DE3), C43(DE3) pLysS and KRX Escherichia coli cells were transformed with a pET28a plasmid with the cDNA sequence encoding Bluetongue virus VP7. Expression of Bluetongue virus VP7 was tested at post induction temperatures between 16˚C and 37 ˚C, at inducer concentrations between 0.1 mM and 1.0 mM isopropyl-β-D-thiogalactopyranoside in BL21(DE3), NiCo21(DE3) and C43(DE3) pLysS cells and 0.05 % and 0.15 % rhamnose for KRX cells, in two types of growth media (LB and 2xYT) and post-induction growth times between two and 16 hours. Under all conditions tested; Bluetongue virus VP7 expression was found to be predominantly in the insoluble fraction (pellet). BL21(DE3) and NiCo21(DE3) cells were chosen and grown for five hours post induction, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown at a post-induction temperature of 37 ˚C. Bluetongue virus VP7 in bacterial cell inclusion bodies was solubilised using urea and a freeze-thaw step. Solubilisation was tested with urea concentrations between 2 M and 8 M, with solubilisation efficiency not increasing past 5 M urea. Solubilized Bluetongue virus VP7 was purified using nickel-affinity chromatography. Purified Bluetongue virus VP7 was then probed with far-UV circular dichroism and intrinsic fluorescence in several buffer conditions including different urea and guanidinium chloride concentrations as well as in the presence of glycerol and sodium chloride. Guanidinium chloride was able to cause Bluetongue virus VP7 unfolding, and the unfolding transition had 94 % and 89 % reversibility at 218 nm and 222 nm respectively. Bluetongue virus VP7 was shown to contain a native-like structure in 20 % glycerol and in up to 8 M urea and was found to be stable till at least 55 ˚C, even in the presence of 5 M urea. Glycerol and sodium chloride influenced the conformation of the protein resulting in different unfolding transitions. Thermal unfolding of Bluetongue virus VP7 was found to be irreversible.Life and Consumer SciencesM. Sc. (Life Sciences

Information management and the need for cataloging in fanfiction communities

Fanfiction communities are not formal libraries nor are they traditional archives. They are staffed almost entirely by volunteers who are anonymous writers and readers of the content. These communities are a fascinating look at the organic ways information management and homegrown folksonomies can come about. With fanfiction growing in popularity, one can expect to see growth coming in the needs of information management within the movement

What's up with all the AI hype?

The current rise of large language models (LLM) like ChatGPT and Bard and image-generating apps like Stable Diffusion, DALL-E 2, and Midjourney have led to a surge in hype and media coverage. This presentation was an attempt to cut through the hype and clarify the ways in which our unit might make use of the new generative AI tools available while highlighting the pitfalls and risks associated with their use

Structural characterisation of the p53 binding domain of RBBP6 and its mechanism of interaction with p53

Abstract in English, Afrikaans, ZuluWorldwide, cancer is a major health issue, causing millions of deaths every year. Retinoblastoma binding protein 6 (RBBP6) is thought to facilitate the interaction between p53 and its prototypical negative regulator, MDM2. RBBP6 has been found to be overexpressed in several cancers and its ability to interact with p53 and cause its degradation, makes it a prospective biomarker and drug target in cancer therapy. In order to target this interaction, a better understating of the p53 binding domain of RBBP6 is needed. In this thesis, expression trials were undertaken for the recombinant p53 binding domain of RBBP6 (namely RBBP6 p53BD) under several conditions. These included different bacterial cell lines, inducer concentrations and post-induction growth time, as well as the domain with and without a polyhistidine tag. The best expression conditions found were NiCo21 (DE3) cells, 37 °C, 0.1 M IPTG and 16 hours post-induction growth. A three-step purification protocol is presented for the polyhistidine-tagged RBBP6 p53BD, utilising hydrophobic interaction and nickel IMAC chromatography. RBBP6 p53BD was purified to approximately 95 % homogeneity. The purified recombinant domain was shown to have structure and be functional as it could bind endogenous p53. The domain was characterised using clear native PAGE and far-UV CD and was found to exist in a single monomer form. Its secondary structure was predicted to be largely intrinsically disordered with 59 % random coil, 19 % alpha-helices, 9 % beta-strands, and 13 % turns. The stability of the domain was also investigated using far-UV CD when the protein was exposed to increasing temperature or known denaturants. The spectrum produced by RBBP6 p53BD with increasing temperature showed a gain in secondary structure and high percentage recovery of its native state after returning to starting temperature. Greater structural changes were seen in the presence of denaturants, with the greatest structural change and lowest percentage recovery seen in the presence of guanidinium chloride. High purity and stability are important for future investigations into the structure of RBBP6 p53BD and drug interaction studies. Localisation images produced from immunocytochemistry experiments are presented here that show different subcellular localisation of RBBP6 and p53 in the cancer cell lines A549, MCF7 and MDA-MB-231 and compared to the normal cell line, HEK293 T. With normal cells having the highest localisation of RBBP6 in the nucleus and negligible localisation in the cytoplasm. Cancer cells still showed the high localisation in the nucleus but higher localisation in the cytoplasm than normal cells. Colocalisation images of p53 and RBBP6 in cancer cell lines are also presented, indicating that p53 and RBBP6 localise in similar cell regions. Using the plugin JACoP from ImageJ, the Manders coefficient of p53 to RBBP6 was predicted to be 0.761 in MCF7 cells and 0.784 in A549 cells. The Manders coefficient of RBBP6 to p53 was found to be 0.904 for MCF7 and 0.834 for A549 cells. The Pearson’s coefficient was found to be 0.759 for MCF7 and 0.630 for A549 cells. This Indicates positive colocalisation of p53 and RBBP6 in both MCF7 and A549 cells, suggesting that these proteins could interact in vivo. Co-immunoprecipitation assays were performed and showed the ability of endogenous p53, RBBP6 and MDM2 to interact and form a complex in vitro in cancer and normal cell lines. Thus supporting RBBP6 as a facilitator to p53 and MDM2’s interaction in human cells as well as supporting RBBP6 as a potential cancer therapy drug target.Kanker is wêreldwyd 'n groot gesondheidskwessie, wat jaarliks miljoene sterftes veroorsaak. Daar is gevind dat retinoblastoombindende proteïen 6 (RBBP6) ooruitgedruk word in verskeie kankers. Daar word vermoed dat RBBP6 die interaksie tussen p53 en sy belangrikste negatiewe reguleerder, MDM2, fasiliteer. RBBP6 se vermoë om met p53 te reageer en die agteruitgang daarvan te veroorsaak, maak dit 'n voornemende biomerker en geneesmiddelteiken in kankerterapie. Eerstens, in hierdie tesis, is uitdrukkingsproewe onderneem vir die rekombinante p53-bindingsdomein van RBBP6 (naamlik RBBP6 p53BD) onder verskeie toestande. Bevindinge toon dat die beste uitdrukkingstoestande was die domein met 'n polihistidienmerker in NiCo21 (DE3) selle, 37 °C, 0.1 M IPTG en 16 uur na-induksie groei. 'n Drie-stap suiweringsprotokol word aangebied vir die polihistidien-gemerkte RBBP6 p53BD, deur gebruik te maak van hidrofobiese interaksie en nikkel IMAC chromatografie. Daar is getoon dat die gesuiwerde rekombinante domein struktuur het en funksioneel is aangesien dit endogene p53 kon bind. Die domein is gekarakteriseer deur gebruik te maak van clear native-PAGE en ver-UV circular dichroism daar is gevind dat dit in 'n enkele monomeervorm bestaan en 'n groot hoeveelheid intrinsieke versteuring bevat. Die stabiliteit van die domein is ook ondersoek deur gebruik te maak van ver-UV CD wanneer die proteïen aan toenemende temperatuur of bekende denaturante blootgestel is, en daar is gevind dat dit relatief min verandering in struktuur en 'n goeie hoeveelheid herstel onder alle toestande het. Hoë suiwerheid en stabiliteit is belangrik vir toekomstige ondersoeke rondom die struktuur van RBBP6 p53BD en geneesmiddelinteraksiestudies. Tweedens word lokaliseringsbeelde wat uit immunositochemie-eksperimente geproduseer word hier aangebied wat verskillende uitdrukkingsvlakke van RBBP6 en p53 in die kanker- en normale sellyne toon. Wanneer die kolokaliseringsbeelde bestudeer word, het die gegenereerde Manders en Pearson se koëffisiënte gedui op positiewe kolokalisering tussen p53 en RBBP6 in beide MCF7 en A549 selle, wat daarop dui dat hierdie proteïene in vivo interaksie kan hê. Ko-immunopresipitasietoetse is uitgevoer en dit het die vermoë van interaksie tussen endogene p53, RBBP6 en MDM2 getoon en om 'n kompleks in vitro te vorm in kanker en normale sellyne. Dit ondersteun dus RBBP6 as 'n fasiliteerder in p53 en MDM2 se interaksie in menslike selle, asook RBBP6 as 'n potensiële kankerterapie-teiken.Emhlabeni wonke, umdlavuza uyinkinga enkulu yezempilo, ebulala izigidi zabantu minyaka yonke. I-Retinoblastoma ebopha amaprotheni 6 (RBBP6) kutholakale ukuthi iningi kakhulu kumangqamuzana omdlavuza. I-RBBP6 kucatshangwa ukuthi yenza kube lula ukusebenzisana phakathi kwe-p53 nesilawuli sayo esikhulu i-MDM2. Ikhono le-RBBP6 lokusebenzelana ne-p53 futhi libangele ukuwohloka kwayo liyenza iRBBP6 ibe i-biomarker kanye nokusetshenziswa yemithi ekwelapheni umdlavuza eqondiswe kuyo. Okokuqala, kule thesis, ngiqale ngezivivinyo zokukhiqiza esibophezelayo se-p53 se-RBBP6 (okungukuthi i-RBBP6 p53BD) ngaphansi kwezimo ezimbalwa. Izimo ezinhle kakhulu zokukhiqhiza iRBBP6 p53BD ezitholiwe kwakuyisizinda esinomaka we-polyhistidine kumangqamuzana e-NiCo21 (DE3), ezingeni lokushisa i-37 °C, kanye ne 0.1 M IPTG kumahora angu-16 okukhula kumangqamuzana. Iphrothokholi yokukhishwa kwe-RBBP6 p53BD okulandela ukukhiqhizwa kwayo enezinyathelo ezintathu, kusetshenziswa ukusebenzisana kwe-hydrophobic kanye ne-nickel IMAC chromatography. I-RBBP6 p53BD ekhiqhiziwe ibonakale inesakhiwo futhi siyasebenza njengoba singahlanganisa ne-p53 etholakala kumangqamuzana. Ukuzinza kwe-RBBP6 p53BD ekhiqhiziwe kuphenyisiswe nge-PAGE kanye ne-CD ye-UV ekude futhi kwatholakala ukuthi ikhona kufomu elilodwa le-monomer futhi iqukethe inani elikhulu lokuphazamiseka kwangaphakathi. Ukuzinza kwesizinda (i-RBBP6 p53 BD) kuphinde kwaphenywa kusetshenziswa i-CD ye-UV ekude kwatholakala ukuthi kunoshintsho oluncane kwisakhiwo se-RBBP6 p53BD ezingeni lokushisa elikhulayo noma ama-denaturants aziwayo ahloliwe. Ukuhlanzeka okuphezulu nokuzinza kubalulekile ophenyweni oluzayo mayelana nesakhiwo se-RBBP6 p53BD kanye nezifundo zokusebenzisana nemithi. Okwesibili, izithombe ezibonisa amazinga ahlukene we-RBBP6 kanye ne-p53 kumdlavuza kanye kumangqamuzana avamile zikhiqizwe ekuhlolweni kwe-immunocytochemistry. Lapho utadisha izithombe ze-colocalisation ama-coefficients ka-Manders kanye ne-Pearson akhiqizwa abonisa ukuhlangana okuhle phakathi kwe-p53 ne-RBBP6 kuwo womabili amangqamuzana omdlavuza e-MCF7 kanye ne-A549, okuphakamisa ukuthi lawa maprotheni angasebenzisana emzimbeni. Ukuhlolwa kwe-Co-immunoprecipitation kwenziwa futhi babonisa ikhono le-endogenous p53, i-RBBP6 ne-MDM2 ukusebenzisana nokwenza i-complex kumangqamuzana omdlavuza kanye navamile. Ngakho-ke imiphumela isekela i-RBBP6 njengomgqugquzeli ekusebenzisaneni kwe-p53 ne-MDM2 kumangqamuzana omuntu kanye nokusekela i-RBBP6 njengethagethi yemithi esingaba khona yokwelapha umdlavuza.National Research FoundationLife and Consumer SciencesPhD. (Life Science

Advance Placement and The Achievement Gap in the 21st Century: A Multiple Linear Regression of Marginalized Populations in AP Enrollment

The analysis of the College Board's Advanced Placement (AP) enrollment focused on marginalized populations' (i.e., African American, Hispanic, and poor students) limited access and the schools' efforts to bridge the gaps. Little research has been done on marginalized populations' AP passage rates. The researchers of this correlational study investigated AP enrollment and passage rates in public and charter high schools in Florida (n = 355) by comparing the enrollment and passage proportions amongst Caucasian, African American, and Hispanic students as well as the proportions of students who received free or reduced lunch. The results showed a weak, positive relationship between proportions of Hispanic, African American, and Caucasian students passing AP exams and the proportion of AP students enrolled. There was a strong, positive relationship amongst the proportions of African-American, Hispanic, and Caucasian students who passed the exam. However, a weak, negative relationship was found between the proportions of students who were enrolled in AP classes and those who received free or reduced lunch. There was also a negative relationship between Hispanic, African American, and Caucasian students passing the AP exam and the percentage of the school's population enrolled in free or reduced lunch. The findings suggest that schools with high poverty rates have a low enrollment rate of students in AP courses. The focus on AP enrollment rates for minority students has led to increased rates of minorities successfully completing advanced coursework, but there is still a need for similar focus on high poverty schools

An update on non-invasive urine diagnostics for human-infecting parasitic helminths: what more could be done and how?

Reliable diagnosis of human helminth infection(s) is essential for ongoing disease surveillance and disease elimination. Current WHO-recommended diagnostic assays are unreliable in low-endemic near-elimination settings and typically involve the invasive, onerous and potentially hazardous sampling of bodily fluids such as stool and blood, as well as tissue via biopsy. In contrast, diagnosis by use of non-invasive urine sampling is generally painless, more convenient and low risk. It negates the need for specialist staff, can usually be obtained immediately upon request and is better accepted by patients. In some instances, urine-based diagnostic assays have also been shown to provide a more reliable diagnosis of infection when compared to traditional methods that require alternative and more invasive bodily samples, particularly in low-endemicity settings. Given these relative benefits, we identify and review current research literature to evaluate whether non-invasive urine sampling is currently exploited to its full potential in the development of diagnostic tools for human helminthiases. Though further development, assessment and validation is needed before their routine use in control programmes, low-cost, rapid and reliable assays capable of detecting transrenal helminth-derived antigens and cell-free DNA show excellent promise for future use at the point-of-care in high-, medium- and even low-endemicity elimination settings

Future schistosome hybridizations: Will all Schistosoma haematobium hybrids please stand-up!

nterrogating the genetic make-up of schistosome larvae (i.e. eggs, miracidia and cercariae) originating from definitive or intermediate snail hosts with molecular DNA methods has, by noting unexpected inter-species hybrids, started a revolution in our appraisal of African schistosomiasis [1-4]. Here, two dominant species of human schistosome exist, Schistosoma haematobium and S. mansoni, which are transmitted by specific intermediate freshwater snails, Bulinus spp. for the former and Biomphalaria spp. for the latter. The two schistosomes cause either urogenital or intestinal schistosomiasis, respectively [5] and depending on local snail distributions, schistosome transmission zones in the aquatic habitat may or may not overlap [6]. Within the S. haematobium group, a further 8 sister species are described with S. intercalatum and S. guineensis of medical importance, causing intestinal schistosomiasis while others, such as S. bovis, S. curassoni and S. mattheei occur in livestock, with the remaining species infecting wildlife. Schistosoma mattheei is also of medical interest for occasional infection and associated disease [7]. In contrast, S. mansoni has a single sister species, S. rodhaini, typically found in small rodents which can hybridise with S. mansoni, if given sufficient opportunity [2]

Publishing Values-based Scholarly Communication

Publishing Values-based Scholarly Communication is a collaboration between members of the HuMetricsHSS (Humane Metrics in Humanities and Social Science) initiative and the Publishing and the Publicly Engaged Humanities working group. In this contribution to the Scholarly Communication Notebook (SCN), we have created a resource that will increase understanding of how a values-based approach to scholarly communication can address the challenges of publishing publicly engaged scholarship, with particular emphasis on humanities and social sciences. The OER can be found online at https://publiclyengagedpublishing.org