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Engineering yeast strains for the expression of South African G9P[6] rotavirus VP2 and VP6 structural proteins
No full textEnglish: In this study, sequences encoding VP2 and VP6 rotavirus structural proteins from rotavirus strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were used in construction of wide-range yeast expression vectors containing the ORFs encoding rotavirus structural proteins VP2 and VP6. VP2 and VP6 sequences were codon-optimized for expression in yeast strains K. lactis, A. adeninivorans and Pichia pastoris/Hansenula polymorpha. Wide-range yeast expression vectors containing either VP6 or VP2 yeast optimized ORFs were constructed for expression of single proteins in different yeast strains. Dual vectors containing both VP6 and VP2 yeast optimized ORFs were constructed to allow simultaneous expression of proteins in different yeast strains and to enable the formation of double-layered rotavirus-like particles.
A total of eight yeast strains namely: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris Candida deformans and Arxula adeninivorans were selected for screening. Saccharomyces cerevisiae was included as a positive control as triple-layered rotavirus virus-like particles (tlRLPs) have been successfully produced in this yeast before. All nine yeast strains were successfully transformed with pKM177 vectors containing yeast codon-optimized VP6 ORFs. However, only six yeast strains indicated positive VP6 ORF integration. The other three yeast strains indicated no VP6 integration but hygromycin B integration suggesting that the vectors successfully integrated in these yeast strains but VP6 ORF was lost. Integration of both rotavirus VP2/6 ORFs was evident in all yeast strains transformed with the dual expression vectors.
A control to monitor rotavirus VP6 protein expression in yeast was successfully prepared in bacterial cells. The positive control indicated specific reaction when polyclonal rotavirus antibody raised against Nebraska calf diarrhoea rotavirus strain. Rotavirus VP6 expression in yeast strains shown no reactions for K. lactis and A. adeninivorans codon-optimized VP6 ORF in all six yeast strains that tested positive for intergration of the VP6 ORF. All yeast strains except K. marxianus showed possible reaction for VP6 expression for P. pastoris/H. polymorpha codon-optimized VP6 ORF although the identity of VP6 should be confirmed.Afrikaans: In hierdie studie, basispaaropeenvolgings wat kodeer vir VP2 en VP6 rotavirus strukturele proteïene van rotavirus stam RVA/Human-wt/ZAF/GR10924/1999/G9P [6] is gebruik in die konstruksie van ‘n wye verskeidenheid gis uitdrukkingsvektore met rotavirus strukturele proteïene VP2 en VP6 oopleesrame. VP2 en VP6 basispaaropeenvolgings is kodon geoptimaliseer vir uitdrukking in gis stamme K. lactis, A. adeninivorans en P. pastoris/H. polymorpha. Wye verskeidenheid gis uitdrukkingsvektore wat geoptimaliseerde oopleesrame van VP6 of VP2 bevat, is gebou vir uitdrukking van enkel proteïene in verskillende gisstamme. Vektore wat beide VP6 en VP2 geoptimaliseerde oopleesrame bevat is gebou vir gelyktydige uitdrukking van proteïene in verskillende gisstamme en om die vorming van dubbellaag rotavirus-agtige partikels toe te laat.
'n Totaal van agt gisstamme naamlik: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Candida deformans en Arxula adeninivorans is gekies vir sifting. Saccharomyces cerevisiae is ingesluit as 'n positiewe kontrole aangesien trippellaag rotavirus-agtige partikels (tlRLPs) voorheen suksesvol in hierdie gis geproduseer is. Al nege gisstamme is suksesvol getransformeer met pKM177 vektore wat gis geoptimaliseerde kodon VP6 oopleesrame bevat. Daar was egter slegs ses gisstamme waarin positiewe VP6 ORF integrasie gevind is. Die oorblywende drie gisstamme het higromisien B integrasie getoon, wat daarop dui dat die vektore suksesvol geïntegreer het in hierdie gisstamme, maar dat VP6 oopleesraam verloor is. Integrasie van beide rotavirus VP2/6 oopleesrame was suksesvol vir al die gis stamme wat met die dubbele uitdrukkingsvektore getransformeer is.
Rotavirus VP6 proteïenuitdrukking is suksesvol uitgevoer in bakteriële selle. Die positiewe kontrole het ʼn spesifieke reaksie getoon met poliklonale rotavirus teenliggaampies wat teen Nebraska calf diarrhoea rotavirus opgewek is, maar geen reaksie is waargeneem wanneer groep-spesifieke muis monoklonale teenliggaampies gebruik is nie. Rotavirus VP6 uitdrukking in gisstamme het geen reaksies getoon vir K. lactis en A. adeninivorans geoptimiseerde VP6 oopleesrame nie in al ses gisstamme wat positief getoets het vir integrasie van die VP6 oopleesrame. Alle gis stamme behalwe K. marxianus het moontlike reaksie vir VP6 uitdrukking vir P.pastoris / H. polymorpha geoptimiseerde VP6 oopleesraam, alhoewel die identiteit van VP6 nog bevestig moet word.National Research Foundation (NRF)Poliomyelitis Research Foundation (PRF
Expression of rotavirus capsid protein, VP6, in various yeasts
No full textRotavirus infection is one of the six leading causes of death among children under the age of five years. Globally it causes more than 215 000 deaths annually of which 65% occur in low- and/or middle-income countries. The two licenced live-attenuated vaccines (Rotarix™ and RotaTeq™) tend to have a lower efficacy in low- and/or middle-income countries. The lower efficacy of rotavirus live-attenuated vaccines could be due to maternal antibodies and oral polio vaccines interfering with rotavirus vaccine uptake. Rotavirus live-attenuated vaccines have been associated with reassortment with circulating genotype strains. As alternative, subunit vaccines such as viral proteins can be considered. Rotavirus VP6 protein is considered as a candidate for subunit vaccine development. Rotavirus VP6 antibodies are responsible for long lasting immunity and the antibodies against VP6 block the release of the viral mRNA. Previous studies showed rotavirus VP6 provide heterologous protection by a significant reduction of virus shedding in mice and gnotobiotic pigs.
Various recombinant yeasts were engineered by a previous MSc student, Mr M.S. Makatsa, using a unique yeast expression vector (pKM177). The recombinant yeasts contained an open reading frame (ORF) encoding rotavirus VP6 for the RVA/Human wt/ZAF/GR10924/1999/G9P[6] strain. The ORF was codon optimised to favour expression in Arxula adeninivorans (AO), Kluyveromyces lactis (KO) and Pichia pastoris/Pichia angusta (PO). However, there was no expression of VP6 optimised for expression in A. adeninivorans and K. lactis due to an additional out-of-frame ATG in the promoter region of the expression vector. In this study, the additional ATG was successfully removed by site-directed mutagenesis and the Kozak sequence was optimised to produce modified delATG_pKM177_AOVP6 and delATG_pKM177_KOVP6 constructs.
The modified delATG_pKM177_AOVP6, delATG_pKM177_KOVP6 as well as delATG_pKM177_POVP6, the modified plasmid containing the VP6 ORF codon optimised for expression in P. pastoris/P angusta and obtained from a colleague, were transformed into 14 different yeast strains. Partial PCR amplification of the VP6 ORFs was conducted to screen for integration of the expression cassettes into the yeast genomes. Debaryomyces hansenii UFS0610 and Yarrowia lipolytica UFS2415 resulted in no colony formation. Integration into the P. angusta genome was efficient for all the VP6 optimised ORFs.
Integration of the VP6 ORFs in the Y. lipolytica PO 1F, UFS0097 and UFS2221 strains were relatively poor as only 16% of the clones screened showed integrated into the genome. The delATG_pKM177_AOVP6 and delATG_pKM177_POVP6 had 75-80% integration in the various yeast genomes, while delATG_pKM177_KOVP6 integration was relatively low at 46%. Expression of VP6 from all the ORFs was effective in P. angusta strains followed by A.
adeninivorans strains from the UNESCO-MIRCEN yeast culture collection (A. adeninivorans UFS1219 and A. adeninivorans UFS1220), S. cerevisiae and K. lactis. High expression of VP6 in P. angusta and P. pastoris has been reported in previous studies. In this study, almost all P. angusta colonies screened, expressed VP6, while only 13% of P. pastoris colonies screened expressed VP6. There was relatively low expression of VP6 in Y. lipolytica strains as well as the prototype A. adeninivorans strain, LS3.
Six yeasts were identified that successfully expressed rotavirus VP6. Rotavirus VP6 has a unique feature of assembling into oligomeric structures depending on the pH and ionic strength. Assembly of nanotubes or nanosphere have only been reported for VP6 produced by insect cells and E. coli, but not in yeast cells. A simple method was adapted to purify and allowed VP6 to assemble in oligomeric structures. Only VP6 produced in A. adeninivorans UFS1219 was able to assemble in both nanotubes and nanospheres, while VP6 produced in Y. lipolytica only assembled in nanospheres. The recombinant A. adeninivorans 1219 strain shows great potential as producer of a rotavirus subunit vaccine candidate.National Research Foundation (NRF)PR
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Exploring carbon cycling in selected micro-organisms exposed to terrestrial carbon sequestration
No full textSouth Africa‘s economy is primarily driven by the utilization of coal to provide electricity, which results in more fossil fuels to be burnt that contributes towards global warming. The average daily temperature is estimated to rise between 1.1 to 6.4˚C by 2100. Carbon sequestration is a technology that can limit CO2 emission into the atmosphere by storing the CO2 away in oceans or the terrestrial subsurface. South Africa is focusing on geological storage at depths of 1 000 m. Limited scientific knowledge is available on the direct impact when large amounts of supercritical CO2 is injected into the subsurface. This includes the diversity of the deep subsurface microbial communities as well as their ecosystems and biogeochemical processes. The main aim of this project was to use selected deep subsurface micro-organisms (T. scotoductus, Geobacillus sp. GE-7 and Geobacillus sp. A12) and an organism that was known to grow under pressure (E. limosum) and introduce them to CCS conditions using a high pressure syringe incubator system. The identities of the selected micro-organisms were verified using molecular techniques, the genomes of these micro-organisms were retrieved and information regarding possible CO2 fixation pathways was verified using the Metacyc database collection. The CO2 fixation pathways of interest were the Calvin cycle, the reductive acetyl Co-enzyme A and the reductive citric acid cycles. Surprisingly, T. scotoductus and E. limosum were able to remain viable and metabolically active even at 100 bar and 100% CO2. This has never been previously reported in literature. However Geobacillus sp. GE-7 and Geobacillus sp. A12 could not remain viable when the pressure was increased from 2 bar to 20 bar or higher. The outcomes of this study indicate that the interactions between supercritical CO2 and the subsurface organisms should be considered as biogeochemical cycling. However, these interactions in the subsurface are still relatively unknown and the availability of interactive metabolic pathways indicate that the subsurface communities could survive and interact with this introduced substrate
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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