MacEwan Open Journals (MacEwan University)
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Nucleotide Specificity Analysis in Universally Conserved NTPases
No full textP-loop GTPases and related ATPases play important roles in many cellular processes. These processes include translation, motility and ribosome biogenesis. P-loop NTPases often serve to regulate these processes by acting as “molecular switches” that change between active NTP-bound states and inactive NDP-bound states (1,2). The YchF subfamily of the Obg family of P-loop GTPases is characterized based on its homology to bacterial YchF (1). This subfamily is notable because it differentially binds and hydrolyzes ATP and/or GTP based on small changes to the G4 motif, or “nucleotide differentiation motif”. This motif varies between protiens and it has been shown that changes to its sequence affect the nucleotide specifity of the protein(1). Most protiens of the YchF family have not been characterized as ATPases or GTPases. To this end we intend to characterize the nucleotide binding and hydrolysis activity the G4 motif of multiple YchF protiens. Characterizing the G4 motifs will help to predict the nucleotide specificity of the protiens as a whole. An understanding of how changes to the G4 motif affect nucleotide binding is important for building understanding of ATPase/GTPase activity. This will be useful in the development of synthetic purine binding proteins and the examination of other similar proteins. Examination of the changes to the G4 motif in YchF homologues in multiple organisms will also help to develop an understanding of what evolutionary pressures cause the unusual variation in substrate for these proteins. Previously created Escherichia coli (E. coli) BL21-DE3 cells containing pET28a plasmids coding for YchF G4 variants were used to analyze the purine binding and hydrolysis properties. These G4 varriants consist of E. coli YchF with a G4 motif altered to match a different YchF homologue. Protien variants were analyzed using fluorescence titration and multiple turnover NTPase assays. Data were analyzed and used to determine differences in the binding and hydrolysis of purine nucleotide phosphates for each G4 motif variant. Analysis showed that each variant displayed differential binding and hydrolysis of purine phosphates. Furthermore the variant based on a previously characterized YchF homologue (OsYchF) showed dual ATPase and GTPase activity matching the original protein (3).
References:
Bourne, H. R. (1995)
Scott, J., et al. (2000)
Cheung, M., et al. (2016)
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Identifying Biomarkers for Assessing an Athlete’s Readiness to Return to Play Following a Sport-Related Concussion Using 1H NMR Spectroscopy
No full textBackground: Sport-related concussion (SRC) is a very common form of mild traumatic brain injury (mTBI). Presently, there is no official assessment used to objectively diagnose SRC and to prognosticate recovery of patients. Several tools have been developed to aid in these aspects, however, due to issues such as restricted specificity, cost and validity, the use of these assessment tools is limited and remains highly subjective. Metabolomics provides a quantitative measurement of the metabolites within a living organism and can be used to measure the metabolic response of an organism to different stimuli. Thus, urinary metabolites that have been altered as a result of an SRC provide a potential avenue for biomarker discovery as it relates to diagnosis and recovery.
Hypothesis: The 1H NMR spectroscopy detected urinary metabolome of athletes will reflect changes in the brain caused by both SRC and the recovery process.
Methods: Samples were obtained from athletes at the beginning of the season, within 72 hours after injury and following medical clearance to return to play. Urine samples were prepared with NMR buffer, centrifuged, the supernatant was pipetted into 5mm NMR tubes. These samples were then analyzed using a 700 MHz Bruker Avance III HD NMR spectrometer. Multivariate statistical analyses were applied to spectral data obtained to determine if there was a significant distinction between baseline, post-SRC and asymptomatic samples. Permutation and cross-validation testing were applied to all Partial Least Squares Discriminant Analysis (PLS-DA) tests for each comparison to ensure the validity of the results. Metabolites that contributed to the observed differences between the metabolomic profiles were identified using Chemometric software.
Results: Statistical data analyses revealed several metabolites that were significantly altered between groups. The identified metabolites were then correlated to biochemical pathways using pathway topology analysis.
Conclusion: This work shows that 1H-NMR based metabolomics is a powerful platform for conducting sport-related concussion analysis. Understanding the lasting metabolic changes that persist even after an athlete has returned to play is key for monitoring individuals following SRC and ultimately preventing cumulative and permanent brain damage.
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Examining Correlates and Consequences of Implicit Theories of Social Anxiety
No full textA small but growing literature concerns the application of implicit theories (i.e., incremental and entity mindsets regarding the stability or malleability of attributes, respectively) to disorders and their symptoms. Implicit theories of social anxiety were explored through a cross-sectional study (N =274 undergraduate participants) examined correlations among implicit theories of social anxiety and social anxiety symptoms, emotional regulation, help-seeking, and behavioural engagement. As predicted, incremental mindsets were correlated with lower social anxiety, greater use of emotional reappraisal, more help-seeking behaviour, greater behavioural activation and less behavioural inhibition. In a regression analysis, incremental mindsets predicted social anxiety symptoms above and beyond the remaining variables. Overall, the current findings warrant additional research of implicit theories of social anxiety, in clinical populations with social anxiety disorder to determine if the found correlates will be replicated and exacerbated within a clinical population.
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Using cortical thickness and elevated plus maze as a means to assess brain and behaviour in adolescent rodents: Paternal preconception nicotine exposure and enriched housing
No full textThe effect of maternal experience on the prenatal environment is well researched, however, comprehensive examination of father experience is lacking. This project aims to gain insight into paternal nicotine exposure and complex housing on offspring brain and behaviour. In this study, four experimental groups were used; nicotine enriched, nicotine pair housed, control enriched and control pair housed. Male Long Evans rats were either kept in pair houses or enriched in complex housing at P45 for 96 days and administered the appropriate solution (nicotine/control) during the last 48 days. Rats were then immediately mated with female Long Evan rats following cessation of enrichment and treatment administration. Offspring were then born to the mothers, never coming into contact with the fathers. A subset of the offspring were perfused on P21 for anatomical analysis while the remaining underwent behavioral testing on P36 and were subsequently perfused. Cortical thickness measurements were completed on the animals perfused at P21 and elevated plus maze (EPM) behavioral task - a measure of anxiety - was conducted on the remaining animals at P36. An increased amount of time spent in the closed arms of the EPM is used as a measure of anxiety in these rodents. Nicotine pair housed males as well as control enriched males were found to have significantly thicker cortexes. However, when these treatments were combined in the nicotine enrichment males, this observed thickness in the cortex seemed to be nullified as the cortex thickness was now comparable to the control pair housed males. Male and female offspring of enriched fathers and male offspring of nicotine exposed fathers spent significantly more time in the closed arms of the EPM, thus being considered more anxious than the controls.
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Modification of the Stripe Rust Resistance Gene Yr10 in Triticum aestivum
No full textStripe rust is a disease in Triticum aestivum (bread wheat) that is caused by the fungal pathogen Puccinia striiformis. The pathogen has evolved to defeat an R gene in T. aestivum known as the Yr10 gene. The Yr10 gene was found to encode an evolutionary-conserved sequence known as the CC-NBS-LRR. This conserved sequence was found to be involved in producing resistance to various pathogens. Within this sequence the coiled coil (CC) and leucine rich repeat (LRR) domains are thought to be important to the protein’s function. This project made use of PCR overlap-extension mutagenesis to mutagenize the CC and LRR domains in an attempt to create modified constructs of the Yr10 gene. PCR reactions gave fragments of the expected sizes which were then assembled into the pANIC6D vector to be used in transformation. Transformation of the vector with inserted sequences into Escherichia coli will be done to confirm the successful insertion of the fragments. There is hope that in the future the modified constructs could be transformed into wheat.
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An aerial inventory of rock glaciers in the Southern Alberta Rocky Mountains
No full textRock glaciers are large lobes of rocky debris commonly found within alpine permafrost in the Alberta Rocky Mountains. Alpine permafrost is of significant scientific interest as it encompasses a large area and exhibits unique responses to climate change. Rock glacier falls from these increasingly unstable mountain slopes can represent significant geohazards, which makes mapping the spatial distribution of these features and classifying their morphology important. They can also be used to identify and understand past climatic change in these alpine regions, with relict (fossilized) rock glaciers indicating a past climate formerly able to create and support these features. Although a common feature within the Alberta Rocky Mountains, information on rock glacier characteristics and distribution within this region is rather limited. The inventory is based on the aerial classification of rock glaciers using high- resolution satellite imagery available through Google Earth and in ArcMap, a geospatial processing program. Geospatial mapping and remote sensing techniques were used for the quantitative and spatial analysis of these features to create an inventory that classifies, quantifies, and characterizes the rock glaciers within this region. Classification of talus-derived and glacier-derived rock glaciers was done using morphological characteristics as the elemental method for identification. Digital Elevation Models (DEM) and optical satellite imagery were used to quantify various attributes such as shape, state, slope, aspect, and elevation. A digital database with the findings from this research will be made publically available for future research through the Alberta Geological Survey.
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Mathematical Modelling of a Measles Outbreak in Pre-vaccine England and Wales
No full textWe present a spatial variant of the time series susceptible-infectious-recovered (TSIR) stochastic population-based model to capture the spatial transmission dynamics of a measles outbreak across the landscape of England and Wales during the pre-vaccine era. Specifically, we explore how the basic dynamical features of a measles outbreak with a seasonal forcing of transmission acts as a major driver of a long-term epidemic behavior. We use a 20-year pre-vaccination era biweekly time series data (births by year and incidence of measles for the years 1944-1966) from 60 towns and cities in England and Wales to capture the spatial spread of measles.
In England and Wales prior to vaccination, measles was endemic in large cities, but in smaller cities disease fadeout occurred. Reappearance of the disease would then occur only after a case was imported from a surrounding city where measles was endemic. To capture spatio-temporal dynamics, multi-city models must be developed, but these models can become very large requiring more memory and processing power than a single computer can deliver.
Rather than represent the population as a linked set of cities, we represent the population as a gridded map. Each grid cell can transmit infectious disease to its neighbors, with probabilities that decline exponentially with distance. We present a stochastic spatial model with six compartments. We call this the kids-susceptible- infectious-recovered-adults-dead (KSIRAD) model.
From the simulation, we recover spatiotemporal maps of the incidence of the infection. We compare simulated time-series graphs with real data compiled by Grenfell and others. Our future work includes testing of our spatial model for measles outbreaks reported in the modern era, for example, in conflict affected areas of the Republic of the Niger in Western Africa in 2016. Socioeconomic disparities in a country like Niger presents significant challenges to reporting and real-time tracking of human infectious diseases.
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Investigation of myo-inositol Phosphates on Human Cells by Phenotypic Cell-Based Assays
No full textMyo-inositol phosphates (IPs) are a large family of compounds that are ubiquitous in eukaryotes. They are involved in many critical cellular functions, from signal transduction to DNA repair in eukaryotes. Myo-inositol hexakisphosphate (InsP6), commonly known as phytic acid, is the most abundant cellular IP and previous studies have suggested that exogenous InsP6 has anti-proliferative effects on multiple cancer cell lines. By contrast, derivatives of InsP6 with lower phosphate number, such as myo-inositol (1,2,4,5,6)-pentakisphosphate (InsP5), have not been studied due to their costly preparation. Here we investigated the effects of InsP6 and InsP5 on the human bone osteosarcoma cell line, U2OS, by phenotypic assays. After 48 h after treatment with either InsP6 and InsP5 at 15 or 50 µM, cells display a striking vacuole formation, as observed by light microscopy. By staining with acridine orange and observing by fluorescence microscopy, the vacuolated cells showed acidic vesicle accumulation, indicative of increased autophagy. By staining with the non-permeant fluorescent dye Lucifer yellow and fluorescence microscopy we observed that vacuolated cells showed higher levels of the fluorescent dye, indicative of endocytosis, than non-treated cells. Vacuoles observed by phase contrast microscopy are frequently stained with Lucifer yellow and acridine orange. These findings suggest that InsP5 and InsP6 affect endocytosis, which subsequently leads to increased lysosomal degradation the engulfed material. Understanding the effects of IPs on human cells is important in understanding inositol signalling pathways and may lead to the discovery of novel anticancer compounds.
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Investigating the Interaction Between DDX17 and The Rift Valley Fever Virus 3’ Terminal Region Using a Nano-scale Structural Approach
No full textRift Valley Fever (RVF) is a currently an untreatable disease that affects both humans and livestock in sub-Saharan Africa. This disease is viral in origin and the Rift Valley Fever virus (RVFV) is transmitted primarily through mosquito vectors Aedes and Culex. RVFV is a part of the Bunyaviridae family, and the genus Phlebovirus. Recent studies have indicated that RVFV interacts with the human DEAD-box RNA helicase protein DDX17. There is minimal research on the specific interactions between this host protein and the viral RNA, although DDX17 has been shown to inhibit RVFV replication. The DDX17 helicase performs a critical role in viral replication, and we are trying to dissect the specific role by which it inhibits RVFV replication. In addition, we are also studying two regions of the RVFV which forms a stem-loop structure that is proposed to interact with DDX17. Using selective 2’-hydroxy acylation analyzed by primer extension (SHAPE) and small-angle x-ray scattering (SAXS) techniques we aim to gain both secondary and 3-dimensional structural information. To identify specific domains of DDX17 that interact with both viral constructs, we require a highly pure preparation of full-length DDX17 and its truncated domains. Thus, the primary objective of my project is to establish protocols for large scale preparation of full-length and individual domains of DDX17 to identify a specific domain of DDX17 that interacts with the RVFV RNA using Electrophoretic mobility shift assays (EMSA) and study interactions using microscale thermophoresis (MST).
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Consequences of 1,3-butadiene DNA adducts – R,R-N6,N6-DHB-dA and R,S-1,N6-g-HMHP-dA – on DNA base pairing
No full textDNA is the hereditary material in humans and almost all other organisms. Unfortunately, upon exposure to carcinogenic species and other compounds, our DNA can be damaged in a variety of ways. An example of DNA damage includes the formation of bulky nucleobase addition products (adducts), which may give rise to cancer. This poster focuses on adducts formed upon exposure to 1,3-butadiene – a human carcinogen that has been associated with an increased incidence of leukemia (cancer of the blood cells). Sources of 1,3-butadiene exposure include automobile exhaust and tobacco smoke, as well as polluted water and air at or near chemical, plastic or rubber facilities. In order to understand the consequences of DNA exposure to 1,2,3,4-diepoxybutane – a metabolite of 1,3-butadiene – this study examines the hydrogen-bonding properties of two adenine (dA) adducts formed upon addition of a bulky moiety to the N6 position, namely the R,R-N6,N6-DHB-dA and R,S-1,N6- g-HMHP-dA adducts. Previous studies have shown that human translesion DNA polymerases hand k preferentially incorporated A, C, G or T opposite R,R-N6,N6-DHB-dA, and A, G or T opposite R,S-1,N6-g-HMHP-dA. This study seeks to explain the observed mutagenicity of these adducts and is the first step in determining the effect of these adducts on the DNA duplex.
Figure 1. 1,3-butadiene adducts R,R-N6,N6-DHB-dA (left) and R,S-1,N6-g-HMHP-dA (right