Relativistic astrophysical models of perfect and radiating fluids.

Doctor of Philosophy in Applied Mathematics. University of KwaZulu-Natal, Durban, 2019.Abstract available in PDF file.The author acknowledged Professor K.S. Govinder for his contribution on conformed mappings and Lie symmetries

New technologies accelerate the exploration of non-coding RNAs in horticultural plants.

Non-coding RNAs (ncRNAs), that is, RNAs not translated into proteins, are crucial regulators of a variety of biological processes in plants. While protein-encoding genes have been relatively well-annotated in sequenced genomes, accounting for a small portion of the genome space in plants, the universe of plant ncRNAs is rapidly expanding. Recent advances in experimental and computational technologies have generated a great momentum for discovery and functional characterization of ncRNAs. Here we summarize the classification and known biological functions of plant ncRNAs, review the application of next-generation sequencing (NGS) technology and ribosome profiling technology to ncRNA discovery in horticultural plants and discuss the application of new technologies, especially the new genome-editing tool clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, to functional characterization of plant ncRNAs

Expression and characterization of full length and truncated versions of major outercapsid protein VP2 of bluetongue virus in bacterial and insect cells

The spread of bluetongue virus (BTV) to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines (Ferrari, De Liberato et al. 2005; Veronesi, Hamblin et al. 2005). Subunit vaccines are one of the attractive alternative strategies. Subunit vaccines against BTV would target the outercapsid protein VP2, the main neutralization-specific antigen (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). A subunit vaccine based on the use of BTV-VP2 may be achieved by either using VP2 by itself or by means of virus-like particles (VLPs) on which VP2 proteins are exposed. In VLPs, the VP2 is co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV. The BTV-VLP vaccine strategy is advantageous since it presents the neutralizing epitopes of more than one viral protein in a more authentic manner as found on the virus itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However there are difficulties associated with large scale production and a decrease in the stability of the particles over time (Berg, Difatta et al. 2005; Wang, Zhao et al. 2006). Studies have already demonstrated the vaccine potential of BTVVP2 by itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However if BTV-VP2 is to be used by itself as a single subunit vaccine, it is important that the protein is expressed under conditions where it is correctly folded and soluble. Solubility refers to the capacity of the expressed antigen to fold into an ordered tertiary structure that authentically exposes the neutralizing epitopes to the immune system (Dinner, Sali et al. 2000; Dobson 2003). However non-native interactions within and between in vitro synthesized viral proteins such as BTV-VP2 often leads to protein aggregation or insolubility. The immune response against aggregated or insoluble proteins is generally very poor. This problem of aggregation and insolubility may be alleviated to an extent by generating truncated versions of the protein from which hydrophobic regions that promote aggregation have been deleted leaving only the major neutralizing epitopes of the antigen (Fukumoto, Xuan et al. 2003; Bonafe, Rininger et al. 2009; Liu, Zeng et al. 2009; Seo, Pyo et al. 2009). The focus of the research presented in this dissertation was to evaluate the solubility of full-length BTV(10)-VP2 and truncated versions thereof after expression in a prokaryotic and baculovirus-Sf9 expression system. The full-length BTV(10)-VP2 (956 amino acids) gene and genes encoding truncated versions of BTV(10)-VP2 i.e. BTV(10)-VP2(aa450) (amino acid 1 to 450) and BTV(10)- VP2(aa650) (amino acid 1 to 650) were cloned into the bacterial expression vector pET160-DEST and the baculovirus expression vector pDEST™8. The C-terminal hydrophobic regions which might contribute to aggregation or insolubility of the protein when expressed in vitro were deleted from these truncated BTV(10)-VP2 proteins. The truncated proteins however still contained BTV neutralizing epitopes that were predicted from literature. The prokaryotic expression of the full-length BTV(10)-VP2 and the other truncated recombinant BTV(10)-VP2 proteins was carried out in E. coli BL21 Star DE3 expression strain. The initial pilot expression study confirmed high level expression of the recombinant proteins. The study also revealed that these proteins were insoluble. The optimization of the prokaryotic expression in order to increase the yield of soluble proteins by means of differential inducer concentrations, fermentation temperature and harvesting times did not produce soluble BTV(10)-VP2 and truncated BTV(10)-VP2 proteins. Previous studies have demonstrated the role of L-arginine in the recovery of soluble proteins from aggregation by reversing aggregation (Tsumoto, Umetsu et al. 2003). However in the current study, arginine treatment of the inclusion body and bacterial lysate containing the BTV(10)- VP2 and truncated recombinant proteins did not release soluble proteins. No soluble recombinant BTV(10)-VP2 proteins were detected when the recombinant proteins were expressed in BL21 host cells over-expressing heat-shock proteins (hsps) and chemical chaperones. However when the different recombinant proteins were co-expressed with the molecular chaperones dnaK-dnaJ-GrpE, it resulted in a fraction of soluble recombinant BTV(10)-VP2 proteins. In particular, approximately 50% of the total expressed BTV(10)-VP2(aa450) protein was soluble while approximately 20% of the total expressed BTV(10)-VP2(aa650) and full-length BTV(10)-VP2 were found soluble when coexpressed with dnaK-dnaJ-GrpE chaperones. These recombinant proteins could be eluted from a nickel affinity column further confirming that these proteins are in fact soluble. Interestingly the coexpression of the BTV(10)-VP2(aa450) protein with the above chaperones in combination with chaperones groEL-groES or only groEL-groES did not produce any soluble proteins. Baculovirus-insect expression of the aforementioned BTV(10)-VP2 recombinant proteins was carried out in Spodoptera frugiperda 9 (Sf9) cells. High level expression of the recombinant proteins was confirmed by an initial pilot expression study conducted at 42 hours post infection (p.i.). The pilot study also revealed that the recombinant proteins were insoluble. Arginine treatment of the lysate released a small fraction of soluble BTV(10)-VP2(aa450) and BTV(10)-VP2(ORF) proteins only detectable with immunoblot analysis using the anti-BTV(10) IgY antibodies. The amount of solubilized proteins was however too small to justify the cost associated with this expression system.Dissertation (MSc)--University of Pretoria, 2010.Geneticsunrestricte

eCALIBRATOR : a comparative tool to identify key genes and pathways for eucalyptus defense against biotic stressors

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under long-term nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection

Immune control of viral infections is heavily dependent on helper CD4(+) T cell function. However, the understanding of the contribution of HIV-specific CD4(+) T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4(+) T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4(+) T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4(+) T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4(+) T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4(+) T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = −0.5, P = 0.02). These data identify an association between HIV-specific CD4(+) T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4(+) T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4(+) T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4(+) T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4(+) T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4(+) T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4(+) T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections

eCALIBRATOR : a comparative tool to identify key genes and pathways for Eucalyptus defense against biotic stressors

Many pests and pathogens threaten Eucalyptus plantations. The study of defense responses in this economically important wood and fiber crop enables the discovery of novel pathways and genes, which may be adopted to improve resistance. Various functional genomics experiments have been conducted in Eucalyptus-biotic stress interactions following the availability of the Eucalyptus grandis genome, however, comparisons between these studies were limited largely due to a lack of comparative tools. To this end, we developed eCALIBRATOR http://ecalibrator.bi.up.ac.za, a tool for the comparison of Eucalyptus biotic stress interaction. The tool, which is not limited to Eucalyptus, allows the comparison of various datasets, provides a visual output in the form of Venn diagrams and clustering and extraction of lists for gene ontology enrichment analyses. We also demonstrate the usefulness of the tool in revealing pathways and key gene targets to further functionally characterize. We identified 708 differentially expressed E. grandis genes in common among responses to the insect pest Leptocybe invasa, oomycete pathogen Phytophthora cinnamomi and fungus Chrysoporthe austroafricana. Within this set of genes, one of the Gene Ontology terms enriched was “response to organonitrogen compound,” with NITRATE TRANSPORTER 2.5 (NRT2.5) being a key gene, up-regulated under susceptible interactions and downregulated under resistant interactions. Although previous functional genetics studies in Arabidopsis thaliana support a role in nitrate acquisition and remobilization under longterm nitrate starvation, the importance of NRT2.5 in plant defense is unclear. The T-DNA mutants of AtNRT2.5 were more resistant to Pseudomonas syringae pv. tomato pv tomato DC3000 inoculation than the wild-type counterpart, supporting a direct role for NRT2.5 in plant defense. Future studies will focus on characterizing the Eucalyptus ortholog of NRT2.5.Supplementary Material: Figure S1 : Selection of atnrt2.5 T-DNA mutants. (A) Diagram of the AtNRT2.5 gene consisting of three exons and two introns with the positions of the T-DNA insertions in GK213H10 (AtNRT2.5-A) and GK046H04 (AtNRT2.5-B) in the second and first exons, respectively. (B,C) PCR detection of T-DNA in AtNRT2.5-A (B) and AtNRT2.5-B (C), respectively, using a combination of the T-DNA left border oligonucleotide and gene-specific oligonucleotides. No amplification was observed in AtNRT2.5-A and AtNRT2.5-B using gene-specific oligonucleotides spanning the T-DNA insertion sites.Table S1 : Summary of the mapping of RNA-seq libraries per Eucalyptus–pathogen interaction sample.Table S2 : Functionally enriched terms of the gene ontology biological processes category in common between the resistant and susceptible defense responses.Table S3 : Functionally enriched terms of the gene ontology biological processes category in the unique set of differentially expressed genes between the resistant and susceptible interactions.The Department of Science and Technology grant for Forest Genomics and Biotechnology, the South African National Research Foundation Grant for Y-rated researchers (UID105767) Incentive funding for rated researchers (UID95807), Technology and Human Resources for Industry Program (THRIP, Grant ID 96413) and the Technology Innovation Agency (TIA) Forest Molecular Genetics Cluster Program.http://www.frontiersin.org/Microbiologyam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 (MWL-2), the most closely related gene to MWL-1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.S1 Fig. Phylogenetic and bioinformatics analysis of all members of the Arabidopsis domain of unknown function 1218 (DUF1218) family, and expression profiling of the candidate members, MODIFYING WALL LIGNIN-1 (MWL-1, At1g31720) and MWL-2 (At4g19370). (A) Neighbor-joining phylogenetic tree of Arabdiopsis DUF1218-containing proteins. ClustalW was used to align protein sequences from TAIR and the alignment thereafter used to construct the tree using p-distance and pairwise deletion with 1000 bootstrap replicates in MEGA5 [16]. Prediction of subcellular localization, signal peptide and number of transmembrane domains was done using SUBA3 [31], Signal-3L [18] and TMHMM[19] respectively, with default settings. Highlighted in pink are the related MWL-1 and 2 sequences. (B) Arabidopsis expression profiles for MWL-1 and MWL-2 across different tissues during development, exctracted from The Bio-Analytic Resource for Plant Biology (http://bar. utoronto.ca/welcome.htm) [20]. Preferential expression is seen at distinct developmental stages, however, there is overlap in the secondary cell wall depositing, 2nd internode region. (DOCX)S2 Fig. Gene ontology enrichment of MWL-1 top 300 co-expressed genes in Arabidopsis. Co-expressed genes were extracted from ATTED-II [10]. GO-full was conducted in Cytoscape 2.8.2 [22] using BiNGO 2.44 [21], while overrepresentation summary enrichment was performed with the REVIGO server [23]. (DOCX)S3 Fig. Phenotypic analysis of At1g31720 (MWL-1) single T-DNA knockout line mutants and MWL-1 overexpression lines. (A) RT-PCR detection of endogenous MWL-1 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-1 overexpression lines 1 to 3 showing detection of MWL-1 transgene in the transgenic lines. Actin2 was used as a control gene and RT-PCR was performed on cDNA from stem tissue. Actin2 and MWL-1 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-1 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-1 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error. Scale bar, 3 cm. Based on a two-tailed Student’s t-test (P-value 0.05) no significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls.S4 Fig. Phenotypic analysis of At4g19370 (MWL-2) single T-DNA knockout line mutants and MWL-2 overexpression lines. (A) RT-PCR detection of endogenous MWL-2 transcript in the wildtype (WT) plants and absence in the single knockout mutant. (B) Semi-quantitative RT-PCR analysis of MWL-2 overexpression lines 1 to 3 showing detection of MWL-2 transgene in the transgenic lines except for OE1 which could be indicative of positional effect (position in the genome), or co-suppression dominant repression. Actin2 was used as a control gene and RT-PCR was performed on stem tissue. Actin2 and MWL-2 gene-specific oligonucleotide sequences can be found in S1 Table. Rosette size (C) and mass (D) of MWL-2 single T-DNA knockout line and overexpression lines 1–3 relative to (WT) control line at four weeks. Qualitative (E) and quantitative (F) stem length of MWL-2 single T-DNA knockout line and overexpression lines relative to WT control at six weeks. For rosette mass n = 3 and for quantitative stem length n = 66. Error bars indicate the standard error while significant difference from the WT based on a two-tailed Student’s t-test (P-value 0.05) is indicated by . Scale bar, 3 cm. No significant differences were seen in the growth and development of the single mutant and transgenic OE lines in comparison to the WT controls except for OE-Line 2. (DOCX)S5 Fig. Transverse sections of six-week-old stem tissue stained with phloroglucinol from At1g31720 (MWL-1) and At4g19370 (MWL-2) T-DNA knockout mutant and overexpression (OE) lines. Transverse sections from wildtype (WT) (A), At1g31720 mutant (B), At4g19370 mutant (C), At1g31720 x At4g19370 double knockdown mutant (D), OEAt1g31720 line 1 (E), line 2 (F), line 3 (G), OEAt4g19370 line 1(H), line 2 (I), line 3 (J). Scale bar, 100μm (indicated in red). No discernible differences were seen in the transverse sections of the single and double mutant as well as the transgenic OE lines in comparison to the WT controls. (DOCX)S1 Table. List of oligonucleotides used in the study. (DOCX)S2 Table. Top 300 Arabidopsis co-expressed genes for MWL-1 (At1g31720) from ATTED-II represented as MR value. (DOCX)S3 Table. Top 300 Arabidopsis co-expressed genes for MWL-2 (At4g19370) from ATTED-II represented as MR value. (DOCX)S4 Table. Structural cell wall carbohydrates and lignin content from MWL-1 and MWL-2 overexpression, single and double knockout lines compared to its respective wildtype (WT) control. (DOCX)Sappi through the Forest Molecular Genetics (FMG) Programme, the Technology and Human Resources for Industry Programme (THRIP, UID 80118), and the National Research Foundation (NRF, UID 71255 and 86936) of South Africa. RM acknowledges an NRF Ph.D. Prestige and Equity Scholarship.http://www.plosone.orgam2016Chemical EngineeringGenetic

Conservation and Diversification of Circadian Rhythmicity Between a Model Crassulacean Acid Metabolism Plant Kalanchoë fedtschenkoi and a Model C3 Photosynthesis Plant Arabidopsis thaliana

Crassulacean acid metabolism (CAM) improves photosynthetic efficiency under limited water availability relative to C3 photosynthesis. It is widely accepted that CAM plants have evolved from C3 plants and it is hypothesized that CAM is under the control of the internal circadian clock. However, the role that the circadian clock plays in the evolution of CAM is not well understood. To identify the molecular basis of circadian control over CAM evolution, rhythmic gene sets were identified in a CAM model plant species (Kalanchoë fedtschenkoi) and a C3 model plant species (Arabidopsis thaliana) through analysis of diel time-course gene expression data using multiple periodicity detection algorithms. Based on protein sequences, ortholog groups were constructed containing genes from each of these two species. The ortholog groups were categorized into five gene sets based on conservation and diversification of rhythmic gene expression. Interestingly, minimal functional overlap was observed when comparing the rhythmic gene sets of each species. Specifcally, metabolic processes were enriched in the gene set under circadian control in K. fedtschenkoi and numerous genes were found to have retained or gained rhythmic expression in K. fedtsechenkoi. Additonally, several rhythmic orthologs, including CAM-related orthologs, displayed phase shifts between species. Results of this analysis point to several mechanisms by which the circadian clock plays a role in the evolution of CAM. These genes provide a set of testable hypotheses for future experiments