Recent Submissions

  • The Linkage of Yeast Metabolites, Produced Under Hyperosmotic Stress, to Cellular Cofactor Systems During Icewine Fermentation.

    Allie, Robert; Centre for Biotechnology
    Icewine is a dessert wine of critical importance to the Canadian wine industry. The Icewine grapes are frozen on the vine, creating ice crystals, and subsequently concentrating the solutes in the juice. Icewine juice places yeast under increased osmotic stress, resulting in altered metabolism. This includes increased glycerol production, an internal osmolyte, and higher acetic acid production as they are linked to the cytosolic NAD+ and NADP+ cofactor systems. The yeast glycerol transporter Stl1p allows for glycerol uptake, lowering the production of glycerol and therefore acetic acid. Here we compare two Saccharomyces cerevisiae wine yeast strains, K1-V1116 wild type and K1-V1116 Δstl1, with Saccharomyces uvarum CN1, and relate the differences in metabolite production to the cofactor systems. To that end, starter cultures of each strain were established Icewine juice with samples collected at fixed intervals and assayed for acetic acid, glycerol, ethanol, acetaldehyde, sugar, and the NAD+/NADH and NADP+/NADPH cofactor systems. K1-V1116 wild-type, K1-V1116 Δstl1 knockout, and CN1 showed different kinetics of glycerol and acetic acid production. Although glycerol production per unit time did not vary among the three yeast strains, per unit sugar consumed, K1V1116 Δstl1 produced the most glycerol followed by CN1 and then K1-V1116. K1-V1116 Δstl1 was found to produce the highest amount of acetic acid as a function of sugar consumed compared to the wildtype. CN1 produced the lowest amount of acetic acid as a function of sugar despite producing higher glycerol than the K1 V1116 wild-type. While there was no statistical difference in the NAD(H) redox system ratios between the three yeast to account for the differences in glycerol and acetic acid production, S. uvarum CN1 showed statistically lower amounts of oxidized NADP+ to total NADP(H) compared to both of the S. cerevisiae K1 strains. These findings provide further insight about yeast metabolism under hyperosmotic stress.
  • Re-evaluation of analytical chemistry techniques in studying DNA structures

    Vanloon, Jesse; Department of Chemistry
    This work describes the use of analytical chemistry techniques to examine the structural changes that DNA adopts when subjected to a number of external/internal factors. A self-complementary sequence, d(CG)9, and a non-self-complementary sequence (mixed sequence) were used to study the conformational effects displayed by each type of oligonucleotide sequence. The structural changes adopted by DNA was examined using a variety of analytical techniques, such as: nuclear magnetic resonance imaging (NMR), differential scanning calorimetry (DSC), ultra violet visible (UV-Vis) spectroscopy, circular dichroism (CD) spectroscopy, and high-performance liquid chromatography (HPLC). 1) d(CG)9 and a mixed sequence in the B- and Z-DNA conformation was examined by CD and UV-Vis at a concentration of 1mM using a home-made cuvette called a Flexicell with a minimum pathlength of 0.129± 0.015 mm. The CD and UV-Vis spectra’s produced were found to be reliable when compared to commercial cuvettes with a pathlength of 1 cm and sample concentration of 10 µM. 2) d(CG)9 was lyophilized and reconstituted using either water or buffer to determine if d(CG)9 adopts a different structure when reconstituted using different conditions. It was determined that lyophilized d(CG)9 adopts a hairpin conformation when reconstituted with water, and a B-DNA duplex when reconstituted with a buffer containing NaCl. 3) d(CG)9 was thermally denatured using DSC to determine if DSC can be a viable method to study oligonucleotides. It was determined that d(CG)9 undergoes a two-state unfolding pathway. 4) Nuclear Overhauser Effect spectroscopy (NOESY) and correlation spectroscopy (COSY) were used to examine the conformational differences of 2’-deoxyadenosine when incubated in water. From the distance and torsion angle constraints obtained from NOESY and COSY respectively, and from existing crystal structures, it was found the structures that were determined by NMR spectroscopy were misleading because of spectral artifacts. 5) A mixed sequence was treated with organic modifying agents to determine the minimal condition required for DNA denaturation when different modifiers were used. It was determined that urea at a concentration of 8 M and at a pH of 12.5 is sufficient to denature the mixed sequence duplex.
  • Analysis of the effect of genetic heterogeneity on de novo genome assembly using Xylocopa virginica as a model.

    Tang, Haimeng; Centre for Biotechnology
    Next generation sequencing (NGS) technology has revolutionized genomic and genetic research, and as a result, de novo genome sequencing and assembly for non-model organisms has now become a common task in genome research. However, the integral properties of a genome such as ploidy, mutations, and repeat content impose issues for current genome assemblers. In this work, we used Xylocopa virginica (Eastern carpenter bees) as a unique model organism for examining on the effect of sequence heterozygosity on quality of de novo genome assembly. Using two de Bruijn graph genome assemblers, we assembled four bee genomes representing different sex and age (unworn male, worn male, unworn female, worn female) using standard Illumina sequencing and one genome using 10X linked-reads library for an unworn female. We discovered that there is a noticeable difference in a variety of genome assembly quality metrics, with the haploid unworn male genome having the highest quality and the worn diploid female genome having the lowest quality. In fact, the N50 value of the unworn male genome was >100 times higher than that of the worn female genome. The genome quality pattern supports the hypothesis that sequence heterozygosity resulting both from ploidy and somatic variants can affect the result of an assembly with former shown to be a much bigger player than the latter. Furthermore, we observed that the density of variants was moderately correlated to the density of breakpoints in the genome assemblies. Overall, our results indicate that increased ploidy and accumulation of somatic variants both negatively affect the quality of the resulting assembly with the former being much more significant than the latter. When considering a de novo assembly project for a non-model organism, whenever possible, haploid samples at the youngest possible age are to be recommended. Furthermore, use of a long-read platform can lead to better genome quality. However, at least for the 10x linked reads, having too much sequencing data does not necessarily lead to a better genome assembly.
  • Hard Pressed to Find A Difference: Evaluating the total tannin content of Cabernet franc L cv. wines, made using pre- and post-fermentation pressing treatments

    de Felice Renton, Leah Isobel; Centre for Biotechnology
    Total tannin concentrations were monitored in Cabernet franc grapes in 2018 and 2019 from two vineyard areas in the Niagara Peninsula, Ontario. Total tannins were measured using the methyl cellulose precipitation (MCP) assay. In 2018, post-fermentation pressing treatments of 100kPA, 150kPA and 200kPA were applied with a control (no press treatment). In 2019, pre-fermentation pressing treatments in combination with juice removal (saignée) were applied with a control (no pre-fermentation treatment). Free sorting and consumer preference testing of the 2018 wines were evaluated using trained panellists and a consumer panel. Pre- and post-fermentation press treatments had little impact on the tannin concentrations in wines post fermentation. Observed trends over time suggest treatments have an influence in the behavior of tannin polymerization and stability, but these results are specific to vineyard site. This suggests that Cabernet franc varietal wines can be produced using low press-treatments without compromising the concentration of total tannins which may contribute to improved wine quality.
  • An in vivo, ex vivo, and in vitro exploration of the use of chronic hypoxia/physioxia and ROS/RNS-mediated alteration of physiological function in mitochondrial disease

    Messner, Holt; Centre for Biotechnology
    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are by-products of cellular O2 metabolism and participate in cell signalling and normal physiological homeostasis. Although ROS/RNS are normal and important molecules in cell physiology, production in excess or in absence of sufficient cellular antioxidant capacity can lead to critical cellular damage which has shown to contribute to a plethora of disease pathologies. Experimental evidence has also supported the use of chronic limitation of ambient O2 gas as a means to reduce the amount of ROS/RNS in both animals and cell culture. The purpose of this thesis was to explore the use of a regulated O2 environment to determine if chronic hypoxia and/or physioxia may influence ROS/RNS production in animal and cell culture models of various known mitochondrial diseases. The absence of superoxide dismutase 2 (SOD2) has been proven to be extremely lethal, so I attempted to breed and house entire mouse dams in hypoxia (11% O2) to investigate if limiting O2 might reduce the amount of ROS/RNS-mediated damage and extend the life span of SOD2 knockout (KO) mouse pups. Many attempts to rescue these KO mice failed due to premature death and/or maternal cannibalism, however, the SOD2 heterozygote (SOD2+/-) – which experience halved SOD2 expression compared to wildtype controls – were viable and body mass data was examined. Although no main effect was found of genotype on body mass over time, male, but not female, mice housed in chronic hypoxia gained significantly less weight than their normoxia (20% O2) counterparts. After examining live animals, I focused on measuring the effects of regulating the O2 environment on ROS production in a variety of cellular models of different mitochondrial disease. Some measure of structural and/or functional integrity is compromised in mitochondrial disease, typically leading to exacerbated proton leak, subsequent superoxide/hydrogen peroxide (H2O2) formation, and, unsurprisingly, a significant potential for cellular damage. These features of mitochondrial disease are further compounded by the fact that a great deal of published cell culture work does not actively regulate O2 levels and thus cells often experience an environment with O2 levels hyperoxic relative to what is typically experienced in vivo. The purpose of this second study was to investigate whether growing and assaying mito-disease cell lines in a regulated, physiologically-relevant O2 environment would reduce H2O2 output to levels similar to wildtype controls. Measuring H2O2 production from various mito-disease cell lines, almost all cell lines produced more H2O2 when grown in normoxia (18% O2) culture conditions compared to physioxia (5% O2), however, only a few of the mitochondrial disease cell lines tested here produced the expected increase in cellular H2O2 efflux than their wildtype counterparts at 18% O2. As expected, almost all mitochondrial disease cell lines produced H2O2 at a similar level to their respective controls when grown in 5% O2. Furthermore, NADPH Oxidases (NOX) were explored as a potentially significant source of elevated ROS production at 18% O2. However, upon NOX inhibition, no significant measurable changes in H2O2 production were reported in any of the cell lines tested here. Finally, the last study in this thesis explored structural and functional consequences which may accompany halved SOD2 expression in adult SOD2+/- female mice over time. Sarco-endoplasmic reticulum ATP-ase (SERCA) is an enzyme responsible for calcium handling in myocytes and its function is critical for proper muscular relaxation and contraction. The soleus and extensor digitorum longus (EDL) were analyzed to determine whether muscle type (ie. slow-oxidative muscle or fast-glycolytic muscle) would influence the effects of heterozygous SOD2 deletion. Interestingly, the soleus muscle showed significant impairments in SERCA function with a reduction in SERCA’s apparent affinity for calcium, whereas there were no differences between genotypes in the EDL muscle. This corresponded well with the fact that SERCA tyrosine nitration was significantly elevated in the soleus, particularly on SERCA2a. Conversely, there were no signs of elevated SERCA tyrosine nitration on SERCA1a, the predominant SERCA isoform in the EDL. In conclusion, the results from these studies provide some insight to the roles that O2 and ROS generation have on physiological function in vivo and in vitro, though it also prompts further investigation due to mixed results in many cases.
  • The Development of a Time-Resolved, Vesicle-Based, Fluorescence Assay for the Activity of the Lipid Kinase Pik1

    Meehan, Kailey; Centre for Biotechnology
    Pik1 is a yeast phosphatidylinositol-4 kinase that is critical for vesicular traffic to the plasma membrane and endosomes from the trans-Golgi (1, 2). Pik1 is a soluble enzyme, and the small myristoylated, Ca2+ binding, EF hand protein, Frequenin (Frq1) facilitates its membrane localization (3). It has been suggested that Pik1 finds its substrate, phosphatidylinositol (PI), within phospholipid bilayers with assistance from the PI/PC transfer protein Sec14 (4). It is proposed that Sec14 stimulates the activity of Pik1 by partially removing PI from the bilayer and presenting it to the kinase as a more suitable substrate (4). In order to test this hypothesis, the Bellbrook Labs Transcreener ADP2 FI Assay kit, a fluorescence-based enzyme assay kit that reports the production of ADP, was adapted to create a time-resolved, vesicle-based assay. The assay was developed and validated with the catalytic subunit of Protein Kinase A (PKA) and the commercial human PI kinase PIK3C3. An expression system for Pik1 was created in yeast. To preserve the stability of the 125 kDa enzyme it was co-expressed with both Frq1 and the heat shock protein, Cdc37, producing the Pik1-Frq1 complex with a removable 10xhistidine tag on Pik1. The growth of the yeast transformants was investigated using different culture conditions, media, and methods of inducing protein expression. The expression of the Pik1-Frq1 complex was confirmed on a Western blot that showed a band corresponding to the molecular weight of the complex. Next steps will involve optimizing the purification of the Pik1-Frq1 complex on a metal affinity resin, with the ultimate goal being to test the Sec14 presentation mechanism by measuring the kinase activity of Pik1 from purified fractions in the presence and absence of Sec14.
  • Vector Competence and Viral Interactions of Zika Virus

    Shivafard, Shayda; Centre for Biotechnology
    Zika virus (ZIKV) (Flaviviridae: Flavivirus) is a mosquito-borne pathogen that has been linked to life-threatening health complications following its emergence in the Americas. As ZIKV continues its northern expansion, it becomes increasingly important to identify the risk for ZIKV transmission in North America through the determination of competent vector species as well as the potential for flavivirus co-infections. The susceptibility to infection and the potential for ZIKV transmission was investigated in mosquito species local to Southern Ontario, Canada. Wild mosquitoes were exposed to a ZIKV-infected blood meal or sugar meal at a final titer of 105 plaque-forming units. Colony Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) mosquitoes were also fed a ZIKV-infected bloodmeal alongside the wild mosquitoes. ZIKV transmission was not detected among the blood-fed wild mosquitoes, however, low infection rates of 9.7% and 33.3% were observed in Aedes vexans (Meigan) and Coquillettidia perturbans (Walker), respectively. In the sugar-fed wild mosquitoes, a low infection rate (6.1%) and transmission efficiency (1.2%) was observed for Culex pipiens Linnaeus only. Among the colony mosquitoes, Ae. albopictus displayed a higher transmission efficiency. The results indicate that these mosquitoes are not likely to be competent ZIKV vectors. The infection dynamics of ZIKV were further analyzed following simultaneous and sequential exposure to West Nile virus (WNV) (Flaviviridae: Flavivirus) in mammalian and insect cell lines. Cells were co-infected or superinfected with the viruses at a final multiplicity of infection of 0.01. Viral RNA was subsequently extracted and amplified from the supernatant samples. Viral interference was observed in the mammalian cell line but not in the insect cell line. Additionally, the infection order of the viruses had a significant impact on the estimated viral titer. These results may be applicable in areas where the viruses co-circulate and risk of co-infection exists.
  • Analysis of the pattern and trend of human genomic variations in the form of single nucleotide polymorphisms (SNPs) and small insertions and deletions (INDELs)

    chundi, vinay kumar; Department of Biological Sciences
    Single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) are the most common genetic variations in the human genome. They have been shown to associate with phenotype variation including genetic disease. Based on data in a recent version of the NCBI dbSNP database (Build 150), there are 305,651,992 SNPs and 19,177,943 INDELs, and together as all small sequence variants, they represent approximately 11% of the human reference genome sequences. In this study, we aimed first to examine the characteristics of SNPs and INDELs based on their location and variation type. We then identified the ancestral alleles for these variants and examined the patterns of variation from the ancestral state. Our results show that the occurrence of small variants averages at 104 SNPs/kb and 6.5 INDELs/kb for a total of ~11% of the genome. Chromosome 16 and 21 represent the least and most conserved autosomes, respectively, while the sex chromosomes are shown to have a much lower density of SNPs and INDELs being more than 30% lower in the X chromosome and more than 85% lower in the Y chromosome. By gene context, SNPs are biased towards genic regions and INDELs are biased towards intergenic regions, and further, INDELs are biased towards protein-coding genes and intron regions within the genic regions and SNPs are biased towards non-coding genes in the genic regions. Within the coding regions, SNPs and INDELs are biased towards missense and frameshift variations, respectively. Some of the biases were due to biased sources of the variation data targeting at genic regions, while the bias towards intron regions is due to selection pressure. Further, genes with the highest level of variation showed enrichment in functions related to environmental sensing and immune responses, while those with least variation associate with critical processes such as mRNA splicing and processing. Through a comparative genomics approach, we determined the ancestral state for most of these variants and our results indicate that ~0.79% of the genome has been subject to SNP and INDEL variation since the last common human ancestor. Our study represents the first comprehensive data analysis of human variation in SNPs and INDELs and the determination of their ancestral state, providing useful resources for human genetics study and new insights into human evolution.
  • Bioinformatic and morphological characterization of Catharanthus roseus mutants

    Jones, Graham; Centre for Biotechnology
    Catharanthus roseus, a member of the Apocynaceae family, has been studied extensively for its valuable chemotherapeutic monoterpenoid indole alkaloids (MIAs). Ethyl methanesuphonate (EMS) mutagenesis is a screening tool that has been used to look for altered MIA profiles in hope of discovering mutations of crucial MIA biosynthetic genes. Without a high-throughput mutation detection screen for C. roseus sequencing data, a range of techniques must be used to discover the EMS-induced changes within the plant. Bioinformatic and morphological analysis revealed the likely alterations leading to unique MIA profiles in two C. roseus EMS mutants: the high-ajmalicine accumulating line M2-0754 and the low-MIA accumulating line M2-1582. Expression of geissoschizine synthase (GS) was downregulated almost seven-fold in the leaves of M2-0754, leading to the accumulation of an alternate pathway MIA from the labile intermediate. The low-MIA profile and increased auxin sensitivity of M2-1582 is likely due to the expression of a dysfunctional auxin influx transport protein homologue.

    Tongkoua Nkamou, Anne Patricia; Centre for Biotechnology
    Drug delivery systems (DDS) have become an important focus research over the past three decades with the emergence of new therapies based on biomolecules such as peptides, DNA, enzymes etc., with the aim of improving or optimising the potency of these biomolecules. Liposomes are colloidal vesicles derived from amphiphilic phospholipid biomolecules and have the capacity to encapsulate a broad spectrum of molecules. The promise of liposomes in regard to drug delivery relates to an increase in the bioavailability of drugs, a decrease in drug toxicity, an increase in the efficacy of the drug, and targeted delivery to areas of pathology. Different liposomes varying in their phospholipid composition present different characteristics in terms of entrapment capacity of drugs, stability of liposomes and drug release profile. Optimizing any of these characteristics will hence improve the efficiency of liposomes as DDS. This study reveals the potential for siloxane-containing phospholipids to display a higher entrapment capacity than conventional liposomes related to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospcocholine (POPC) and controlled released when using the model compound, calcein. With the siloxane-phospholipids being studied, zero-order release was observed and in some instances appeared to be independent of the pH of the release media.
  • Development of a Transcriptome-Based Genome Assembly Tool and Whole Genome Sequencing for Autism Spectrum Disorders

    Baldwin, Robert; Centre for Biotechnology
    This thesis consisted of two independent projects. The first involved developing a software tool that uses transcriptome data to improve genome assemblies. The second involved processing and analyzing whole genome sequencing (WGS) from the ASPIRE autism spectrum disorder (ASD) cohort. The first project produced the bioinformatics software called RDNA. This free tool was written in Perl and should be valuable for users interested in genome assembly. Comparative assessment between RDNA and the leading transcript based scaffolding software showed that RDNA can significantly improve genome assemblies while making relatively few scaffolding connection errors. RDNA also makes possible the assembly of scaffolding connections, including gap filling, using BLAST. The second project was undertaken with collaborators and involved processing and analyzing whole genome sequencing (WGS) data from the ASPIRE ASD cohort. The ASPIRE ASD cohort consisted of several hundred probands from both simplex and multiplex families. Sequencing occurred for 120 of these individuals who were selected based upon membership in two phenotype clusters (C1 and C2). These individuals had a relatively high rate of intellectual disability (ID) compared to heavily studied ASD cohorts such as the Simons Simplex Collection (SSC), indicating a significant involvement of de novo sequence variants. Analysis of rare single nucleotide variants (SNVs) and insertion/deletions (indels) identified large risk factors for severe neurodevelopmental disorders (NDDs), two of which were previously observed de novo among individuals with severe, undiagnosed NDDs. On this basis, ABCA1 was found to be a novel candidate risk gene. Gene Ontology (GO) analysis of rare loss of function and missense SNVs indicted the importance of lipid metabolic processes and synaptic signalling. Overall, the genetic variation examined by this study pertained to a modest number of cases, consistent with previous findings that ASD is a genetically heterogeneous disorder with a complex genetic architecture.
  • A systematic view on mobile elements’ contribution to human transcriptomes

    Joshi, Aditya; Centre for Biotechnology
    Mobile elements (MEs) are a major component in most higher eukaryotic genomes. MEs account over 50% of the human genome and consist of long interspersed elements (LINEs), Short interspersed elements (SINEs), and SINE-VNTR-Alu (SVAs) and long terminal repeats (LTR). MEs are known to play important roles in genome evolution and gene function and their roles include but are not limited to the generation of alternative splicing, insertional mutations, genomic instability and epigenetic regulation. Nevertheless, a systematic analysis of MEs contribution to transcriptomes in humans has not been conducted. In this study, we examined the MEs’ participation in the human reference transcriptome and the transcriptomes of many human tissues. Our results show that MEs contribute to 16% to the human reference transcriptome and on average ~24% to the full transcriptomes of human tissues. MEs’ contribution to human full transcriptomes varies from tissue to tissues and from person to person. MEs contribute to all exon features, but 60% of them are in non-coding regions. Also, they contribute to the transcripts of 4,402 protein coding genes, including regions, which are conserved among primates, mammals, and even vertebrates. We noticed that while Alus are the most prominent contributors to human transcriptomes involving mostly alternative transcripts, SVAs as the youngest ME class showed most active participation in protein coding genes with 78% contributing to the canonical transcripts. Furthermore, MEs also participate in post-transcriptional regulation by contributing to the formation of 16% miRNAs, ~9% of miRNA target sites, and at least 2,921 double stranded RNA sites for RNA-editing. In conclusion, our data demonstrate that MEs are very active participants in human transcriptomes by contributing to both non-coding RNAs and protein coding transcripts and to post-transcriptional regulation via miRNA and RNA-editing.
  • Mutagenesis and recombinant expression of active site variants of the Arabidopsis CC-type glutaredoxin GRX480 reveal an altered protein expression profile in E.coli and changes in structural integrity of the GRX480 protein and its associated complex formation.

    Murphy, Colleen; Centre for Biotechnology
    Stress conditions such as high temperature, drought, high salinity, metal stress, and pathogenic infection significantly increase production of reactive oxygen species (ROS) in the cell. Glutaredoxins (GRXs) are small redox proteins that possess conserved cysteine (C or Cys) residues in their active site and exhibit oxidoreductase activity to protect vital proteins from oxidative damage. CPYC and CGFS type GRXs can be found in humans, yeast, E.coli, and both lower and higher plants. The CC-type class is entirely plant-specific and is thought to have emerged with the evolution of intricate signalling mechanisms involved in plant disease resistance and floral complexity. The Arabidopsis CC-type glutaredoxin, GRX480, possesses a Cys-Cys-Met-Cys (CCMC) active site and is thought to participate in the salicylic acid (SA) mediated pathways involved in plant Systemic Acquired Resistance (SAR) to confer immediate and long term resistance to biotrophic pathogens. The structural and stoichiometric properties of the GRX480 protein remain uncharacterized and the mystery surrounding the role of the conserved cysteine residue (CCMC) in CC-type glutaredoxins make this protein a prime candidate for mutagenesis studies. The AtGRX480 genetic sequence was codon optimized to allow for improved recombinant expression in E.coli cell cultures. Single (SCMC, CSMC, CCMS), double (SSMC, CSMS, SCMS), and triple (SSMS) GRX480 active site variants were created by site directed mutagenesis and a Strep II tag was added to the C-terminal end of the protein for isolation purposes. Recombinant expression of these proteins in E.coli DE3 cells caused a drastic decrease in total protein concentration when compared to untransformed cultures. Wildtype GRX480 exhibited a 6.5 fold decrease while the active site variants exhibited fold reductions within a range of 1.9 to 12.7, the CSMS and CSMC variants being the lowest and highest fold reductions respectively. All recombinant protein expression caused a decrease in protein bands within the 36-32, 20, 16, and 12 kDa range of the native protein expression profile of E.coli cultures. When isolating the GRX480 proteins on the FPLC adapted Strep-column, a single injection of crude protein solution was ineffective in isolating sufficient amounts of protein to be examined by SDS-PAGE and immunoblot analysis. The utilization of a multiple injection method drastically improved GRX480 protein isolate yield from the column, however the majority of the protein remained bound to the column as desthiobiotin was demonstrated to be a poor eluting substrate. Examination of this isolate by gel filtration chromatography, SDS PAGE, and immunoblot revealed that wildtype GRX480 (CCMC) and the SCMC, CSMS, SSMC, and SSMS variants form a tetrameric complex in vitro. The SCMS, CSMS, and CCMS variants exhibited formation of an extremely large complex, a trimeric complex, or were not observable as an intact protein during gel filtration respectively. The SCMC, CCMS, CSMC, and CSMS variants displayed a distinct degradation pattern in the N-terminal region of the protein with the CSMC and CSMS variants possessing an additional distinct degradation band. Submission of the GRX480 amino acid sequence to the I-TASSER (Iterative Threading ASSEmbly Refinement) server revealed alternative GSH binding amino residues other than the N-terminal active site cysteine. High sequence homology and predicted structural similarities of GRX480 to monothiol and dithiol glutaredoxins known to be involved in iron-sulfur [FeS]-cluster biosynthesis or [Fe-S] mediated redox sensing were also identified by I-TASSER. These results together offer novel and previously unreported features of the GRX480 protein in terms of complex formation, the roles of the active site cysteine residues, and the observed changes in native E.coli protein concentrations with recombinant expression of the GRX480 protein.
  • Investigating Flavivirus Infection, Dissemination, and Transmission Dynamics Using Zika virus, West Nile virus, and their Mosquito Vectors

    Agbulos, Darrell; Centre for Biotechnology
    Flaviviruses (family Flaviviridae, genus Flavivirus) are a group viral pathogens responsible for causing disease and death in both humans and animals. Mosquito saliva potentiates Flavivirus infection in both in vitro and in vivo models; however, it remains unknown whether saliva from different species differentially potentiates infection. By inoculating the saliva of different mosquito species plus WNV onto Vero cells, plaque assays were used to study if saliva could differentially potentiate WNV infection. It was found that while there was no significant difference between Ae. aegypti and Ae albopictus saliva (p=0.19), more interestingly was that both saliva treatments had a significant reduction in plaques formed compared to virus alone (p= 0.01 and p=0.00). The presence of mosquito saliva appears to exert a protective effect in vitro when WNV is present. It also remains to be elucidated as to whether Canadian mosquitoes are able to spread Zika virus. By orally infecting wild caught mosquitoes with a ZIKV infected sugar meal and detecting the presence of virus 10 and 14 days post infection (d.p.i.), the vector competence of Canadian mosquitoes was evaluated. It was found that after 10 (n=50) and 14 d.p.i. (n=32), 2% and 0% of a population of Culex pipiens mosquitoes were found to be able to become infected and transmit the virus, respectively. Although Culex pipiens mosquitoes from the Niagara region may not be vectors of ZIKV, that does not negate other Canadian mosquitoes as being potential vectors.
  • The Synthesis and Reactivity of Vitamin E Quinones

    Krueger, Nicholas W.; Centre for Biotechnology
    Vitamin E has been the subject of numerous studies over the last nine decades since its discovery. Still, the biological activity of vitamin E is not completely understood. Various studies suggest that the primary function of vitamin E is a fat-soluble antioxidant preventing lipid peroxidation in cellular membranes. The antioxidant efficiency across the vitamin E isomers have been shown to be similar in vitro in various organic solvents and aqueous lipid suspensions. Despite these results, significantly different biological effects have been observed in biological assays supplemented with the various tocols. Furthermore, the differences are more pronounced when comparing the biological effects of the α-tocols to non α-tocols. We hypothesized the different biological effects observed were correlated to differences in the chemical reactivity of the products of tocol oxidation: tocol quinones. Herein, an investigation of the adduct formation of tocol quinones with N-acetyl cysteine (NAC) is described. The synthesis of the tocopheryl quinones is described via the two-electron oxidation of the parent tocols with ceric ammonium nitrate supported on silica. The synthesis of α-tocopherol quinone and α-tocotrienol quinone produced the target compounds in 80% yield. The oxidations of the other tocol isoforms yielded para-tocol quinones (29-45% yield) and ortho-tocol quinones (20-35% yield). The rate of reaction of the tocopheryl quinones with NAC was monitored by following the production of the tocopherol hydroquinone adduct by the increase in absorption at 308 nm over time in a 67% methanol/ 33% aqueous Tris/HCl buffer. The curves generated were fit to a one-phase exponential. The rate of reaction increased as the pH increased for the γ- and δ-tocopheryl quinone isoforms. There was no reactivity observed between the α-tocopheryl quinone isoforms and NAC. There was no significant difference in the reactivity between the tocopherol quinones and tocotrienol quinones with the same methylation pattern. At the most physiologically relevant pH in our study (pH = 7.5), the δ-tocopheryl quinones (k ≈ 0.63) reacted approximately 8 times faster than the γ-tocopheryl quinones (k ≈ 0.08). The electrochemistry of the parent tocols was studied using cyclic voltammetry (CV). The formal redox potential increased slightly as the methylation on the chromanol ring decreased while no significant differences between the tocopherols and tocotrienols were established. The CV of all tocols showed two quasi-reversible one-electron oxidation processes; the first oxidation produced a radical cation, which quickly deprotonated to form a tocopheryl radical, which then undergoes a second oxidation to the corresponding phenoxonium cation. The redox activity of the tocopheryl quinones was also studied using CV. The quinones underwent a quasi-reversible two one-electron reduction process. The δ-tocopheryl quinones have the lowest formal redox potential which slightly increases as methylation increases, while there were no significant differences in the electrochemical behavior of the tocopheryl quinones and the tocotrienyl quinones with the same methylation. We have presented reaction rate differences in the reactivity of the tocol quinones which correlates to the differences in biological activity observed by others with respect to the methylation pattern on the chromanol ring, while no differences in the chemical reactivity of the tocopherol and tocotrienol quinones was observed. Still, this presents convincing evidence that the activity of the tocol quinones should be considered in biological assays.
  • Understanding the diamondback moth (Plutella xylostella) performance in plant alternative cropping systems

    Sidhu, Anshul; Centre for Biotechnology
    The diamondback moth (DBM) (Plutella xylostella) (L.) (Lepidoptera: Plutellidae) is one of the most widespread and harmful insect pests almost exclusively targeting plants belonging to the Brassicaceae family. Understanding of DBM’s behaviour and ecology is therefore crucial to develop novel, economically and environmentally friendly methods of control. One of the main objectives of this study was to determine whether the amount of time (# of generations) spent on a particular host plant by the DBM 4th instar larvae influences their preference, when exposed to and reared on alternative host plants. Experiments included rearing three lineages of DBM on three different plant species for three generations and exposing 4th instar larvae from each generation to the test plant species in a choice experiment. Results indicated that the amount of time spent on a particular host plant had no effect on food selection. The 4th instar larvae prefer turnip (Brassica rapa subsp. rapa) the most and kale (Brassica oleracea var. acephala) is the least preferred plant. Another major objective of this is to examine the response of DBM to intercropping as an integrated pest management strategy and whether intercropping changes the sinigrin concentration in kale leaves. Two DBM males and females were exposed to either kale or kale grown with onions (Allium cepa) in controlled conditions to assess their performance and kale damage. Results indicated that fewer 4th instars and pupae were present in the intercropping. There was no change in the sinigrin content between the two treatments. Results suggested that the physical presence of onions may be necessary for the decreased larval numbers observed in the intercropping system. It is hypothesized that onions may release repellent volatiles rendering the environment less suitable for DBM. Our study shows the complexity of DBM’s interaction with its host plants and adds to the mounting amount of evidence towards the effectiveness of an intercropping system in controlling pest infestation.
  • The impact of grape clone, yeast strain and protein on sparkling wine quality

    Onguta, Esther; Centre for Biotechnology
    The foaming properties in sparkling wine are an indicator of quality as it is the first quality perception consumers have upon opening a bottle of sparkling wine. Proteins, which are derived from both grapes and yeast during sparkling wine production, are known to impact the foaming properties in finished sparkling wines. The objectives of this project were to (1) understand the role and relationship that proteins have on the overall foaming properties and overall quality in sparkling wine, (2) determine the role different yeast and grape clones from different varietals have on sparkling wine quality and (3) understand how bentonite affects sparkling wine quality. The protein concentration in sparkling wine produced from Mariafeld Pinot noir appeared to impact the foaming properties. The longest elapsed time for foam dissipation was observed in the control treatment where bentonite was not used to strip protein and the shortest time was observed in the treatment where bentonite was used to remove grape and yeast proteins. In Riesling sparkling wines, the largest protein concentrations were observed in non-bentonite treated juices while the lowest were observed in the bentonite treatments. The prevalence of foam observed in both bentonite treatments, where grape proteins were completely removed, indicated that proteins derived over the course of secondary fermentation were foam forming in Riesling sparkling wine. It was also observed that different yeast, varietals, clones and soil compositions may impact the protein concentrations, chemical compositions and overall quality of sparkling wine. The results of this research aim to better understand sparkling wine quality to optimize production in the Niagara Peninsula.
  • Secondary Transmission Dynamics of the West Nile Virus in Mosquitoes

    Causarano, Jason; Centre for Biotechnology
    West Nile Virus (WNV) is a member of the genus Flavivirus and is transmitted by mosquitoes. It first appeared in North America in New York City in 1999 and was first detected in Windsor Ontario in an infected bird collected in early August 2001. Prior to WNV, flaviviruses were not of major concern in Canada as only 1 short outbreak had occurred in the country. The secondary transmission cycles of WNV are typically overlooked in regards to their role in maintenance and survival of the virus in nature. The first component of this thesis was to investigate transovarial or transovum transmission (TOT) of WNV in Ontario during the 2012 and 2013 epidemic seasons. 89 males from 7 different traps were tested from samples collected in 2012. During the 2013 season individuals across 9 different WNV vector species were collected throughout the Niagara region with 1023 tested for the presence of naturally occurring WNV TOT. None of the samples showed signs of TOT. A single gravid female Culex pipiens tested positive for WNV, but no viral RNA was found in any of her 270 offspring, highlighting the many difficulties in detecting this type of transmission in natural populations. Laboratory testing was conducted to further investigate secondary transmission of WNV. A novel method for infecting adult mosquitoes was developed during this process; it was found more individuals would feed on an infectious sugar meal (ISM) as opposed to an infected blood meal in the lab setting. The ISM showed rates of infection, dissemination, and transmission by the WNV vector Aedes albopictus that were similar to those observed in previous vector competence studies. The ease of infecting adults and the similar rates of infection to previous literature suggests that this method could be used to investigate vector competence of other species for WNV. The ISM was also successful in the infection of male Aedes albopictus and was used to investigate WNV venereal transmission (VT) within the species. Evidence showed that 45% of the uninfected virgin females were positive for WNV after mating with infected males, indicating for the first time that male Aedes albopictus could transmit the virus venereally.
  • Biofilm Formation and Quorum Sensing in Pseudomonas fluorescens Pf0-1

    Bordeleau, Emily; Centre for Biotechnology
    A bacterial biofilm is a community of microorganisms adhering to a surface, exhibiting biochemical and phenotypic differences from their planktonic counterparts. The transition from a free-floating to sessile cell type has been shown to be, in part, mediated by high intracellular levels of the nucleotide second messenger c-di-GMP. It is suggested that one of the environmental cues for biofilm formation, recognized by members of the c-di-GMP network, is local cell density. In areas of high cell density, cells can communicate through a system called quorum sensing. In gram negative bacteria, acyl-homoserine lactone (AHL) molecules are excreted into the surrounding medium and recognized by cells in close proximity. It is hypothesized that upon recognizing AHLs through c-di-GMP signaling, gene expression is altered leading to a sessile lifestyle. Thus, the long-term goal of this research is to provide evidence for the link between c-di-GMP and quorum sensing-mediated mechanisms in biofilm formation in Pseudomonas fluorescens Pf0-1. The first objective towards this goal was to identify the AHLs utilized by P. fluorescens Pf0-1 in quorum sensing mechanisms. Through gas-chromatography mass-spectrometry (GCMS), two AHLs were identified in the supernatant of P. fluorescens Pf0-1; N-butyryl-HSL and Ndecanoyl- HSL. Subsequent work will focus on the identification of AHLs with longer acyl-chain and varying levels of acyl chain oxidation. The second objective towards this goal was to utilize the 96-well static microtiter plate biofilm assay as a platform for studying the relationships between c-di-GMP and AHL-mediated mechanisms in biofilm formation. As a protocol for 96-well static biofilm assays that was previously successful was no longer reproducible, different microtiter plate surfaces were surveyed for their ability to support P. fluorescens Pf0-1 biofilm and to investigate potential factors that could interfere with development on the abiotic surface. Throughout the troubleshooting process, biofilm assay experiments carried out in microtiter plates with the same type of surface chemistry, but from different manufacturers and batches, resulted in variable quantities of biofilm. This observation then inspired the production of a surface that would create more favorable interactions with bacterial cells and offer increased points of attachment to further promote biofilm formation. In this new platform, microtiter plates are pre-treated by abrasive forces such as sandblasting and drilling before biofilm assays, which gives robust biofilm formation that will allow for future investigation into connections between c-di-GMP and AHL-controlled mechanisms of biofilm formation in P. fluorescens Pf0-1.
  • A Catharanthus roseus mutant with trace levels of secologanin and monoterpenoid indole alkaloids does not express BIS1/BIS2 transcription factors and fails to activate iridoid biosynthesis.

    Kidd, Trevor; Centre for Biotechnology
    The Madagascar periwinkle (Catharanthus roseus) is the sole source of the monoterpenoid indole alkaloids (MIAs) that result in several essential anti-cancer chemotherapies as well as being an important source for other MIA derived pharmaceutical agents. Most of the alkaloid and pre-alkaloid iridoid pathway has been elucidated, but some critical areas remain uncharacterized. The early iridoid pathway is localized to internal phloem associated parenchyma (IPAP) cells, with the latter part of the pathway localized to the epidermal cells indicating intercellular transport within the leaf tissue does occur. However, possible transport or translocation of MIAs or pre-MIAs between organs within Catharanthus roseus has not been studied. Previously, 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants had been screened with a simple TLC (thin layer chromatography) to identify mutants with altered MIA profiles yielded one plant with trace MIA production. This trace MIA status was confirmed using UPLC-MS (Ultra Performance Liquid Chromatography and Mass Spectrometry) and the plant had a thick, stocky, short root system, small leaves, a rigid stem, premature senescence and was susceptible to infection resulting in its rescue to in-vitro. Quantitative real-time PCR analysis of iridoid gene expression showed significant downregulation of several iridoid pathway genes as well as the downregulation of transcription factors BIS1 and BIS2. Feeding the trace MIA mutant roots the iridoid secologanin resulted in alkaloid production in the leaves. While grafting trace MIA mutant shoots onto MIA producing WT roots also resulted in alkaloid production in the mutant leaves. This study establishes that MIAs or pre-MIA iridoids, such as secologanin, may be translocated between the plant organs and this may be useful for the identification of novel transporters, enzymes, and transcription factors.

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