INVESTIGATING THE EFFECTS OF IONIC LIQUIDS ON DNA GQUADRUPLEX AND PROTEIN STRUCTURE USING MOLECULAR DYNAMICS SIMULATIONS

Nucleic acids and proteins have huge implications in biomedicine and bioengineering, however their storage instability limits their applicability and current storage protocols are expensive and globally-inaccessible. Finding an alternative biocompatible media to store nucleic acids and proteins would reduce costs and increase their applicability. Ionic liquids (ILs) are molten salt compounds that have been shown to modulate the stability and activity of nucleic acids and proteins. In this thesis, molecular modeling studies of DNA/RNA and protein structure in ILs will be discussed (Chapter 1) and this method will be used to study the IL effects on the structure on the Pu22 c-MYC DNA G-quadruplex (Chapter 2) and the azurin protein (Chapter 3). ILs have been observed to stabilize/destabilize DNA G-quadruplexes linked to cancer oncogene expression, however the structural effects of imidazolium-based ILs on G-quadruplexes remain unknown. Bioengineering of azurin is attractive for soil bioremediation, thus understanding the structural changes induced by TMG amino acid-based ILs will mediate future IL design for enhancing azurin\u27s activity. In Chapter 2, molecular dynamics (MD) simulations will elucidate the stabilizing mechanism of four imidazolium-based ILs of increasing hydrophobicity to Pu22, using the G-quadruplex stabilizer TMPyP4 as a molecular probe. In Chapter 3, conventional and replica-exchange MD simulations will provide insight into the enthalpic and entropic change induced by two TMG-AA based ILs on the folded and unfolded azurin conformations

Near-Neutral Balanced Selection Theory (NNBST): Implication on the molecular evolution of SARS-CoV-2 and Zika virus

Virus pandemics have significantly impacted human welfare and development. Understanding the true molecular evolution of viruses is critical for preventing future pandemics and developing the next-generation drugs and vaccines. Unfortunately, viruses exhibiting constant genomic substitution rates (GSRs) despite increasing vaccinations and human infections (SARS-CoV-2) and viruses under seemingly extreme conservation (ZIKV) paints a confusing picture of viral evolution, and the main evolutionary theories (Selectionist Theory, Kimura’s Neutral Theory) cannot explain their molecular evolution. Here, we developed a first-principle model, c/u, to characterize reproductive fitness changes in viruses based on variations in the nucleotide/codon mutation rate (c) with respect to the global genomic mutation rate (u). c/u is nucleotide-centric, since phenotypic changes in proteins are reflected by genotypic changes in genes. c/u can assign selection types to coding regions (virus function) and non-coding untranslated regions (UTRs, virus gene regulation). Additionally, c/u can handle conserved sites (c/u=0) and sites with few mutations under false positive selection (c/u\u3c\u3c\u3e\u3e1). The constant GSRs of SARS-CoV-2 and ZIKV are seemingly under effective neutral selection (c/u=1), indicating no advantageous/disadvantageous fitness change. Yet, the L-shaped probability distribution of c/u for a NT site indicates high sequence conservation, with higher c/u values being increasingly uncommon. Varying nucleotide and segment substitution rates were observed in both viruses, indicating a mixture of advantageous selection (c/u\u3e1) and purifying selection (c/

Probing the Activation Mechanisms of Agonist DPI-287 to Delta-Opioid Receptor and Novel Agonists Using Ensemble-Based Virtual Screening with Molecular Dynamics Simulations.

Pain drugs targeting mu-opioid receptors face major addiction problems that have caused an epidemic. The delta-opioid receptor (DOR) has shown to not cause addictive effects when bound to an agonist. While the active conformation of the DOR in complex with agonist DPI-287 has been recently solved, there are still no FDA-approved agonists targeting it, providing the opportunity for structure-based virtual screening. In this study, the conformational plasticity of the DOR was probed using molecular dynamics (MD) simulations, identifying two representative conformations from clustering analysis. The two MD conformations as well as the crystal conformation of DOR were used to screen novel compounds from the ZINC database (17 million compounds), in which 69 drugs were picked as potential compounds based on their docking scores. Notably, 37 out of the 69 compounds were obtained from the simulated conformations. The binding stability of the 69 compounds was further investigated using MD simulations. Based on the MM-GBSA binding energy and the predicted drug properties, eight compounds were chosen as the most favorable, six of which were from the simulated conformations. Using a dynamic network model, the communication between the crystal agonist and the top eight molecules with the receptor was analyzed to confirm if these novel compounds share a similar activation mechanism to the crystal ligand. Encouragingly, docking of these eight compounds to the other two opioid receptors (kappa and mu) suggests their good selectivity toward DOR

Novel Inhibitors to MmpL3 Transporter of Mycobacterium tuberculosis by Structure-Based High-Throughput Virtual Screening and Molecular Dynamics Simulations

Tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis (Mtb) utilizes mycolic acids for building the mycobacterial cell wall, which is critical in providing defense against external factors and resisting antibiotic action. MmpL3 is a secondary resistance nodulation division transporter that facilitates the coupled transport of mycolic acid precursor into the periplasm using the proton motive force, thus making it an attractive drug target for TB infection. In 2019, X-ray crystal structures of MmpL3 from M. smegmatis were solved with a promising inhibitor SQ109, which showed promise against drug-resistant TB in Phase II clinical trials. Still, there is a pressing need to discover more effective MmpL3 inhibitors to counteract rising antibiotic resistance. In this study, structure-based high-throughput virtual screening combined with molecular dynamics (MD) simulations identified potential novel MmpL3 inhibitors. Approximately 17 million compounds from the ZINC15 database were screened against the SQ109 binding site on the MmpL3 protein using drug property filters and glide XP docking scores. From this, the top nine compounds and the MmpL3-SQ109 crystal complex structure each underwent 2 × 200 ns MD simulations to probe the inhibitor binding energetics to MmpL3. Four of the nine compounds exhibited stable binding properties and favorable drug properties, suggesting these four compounds could be potential novel inhibitors of MmpL3 for M. tuberculosis

Effects of Ionic Liquids on Metalloproteins.

In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins

Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery1,2, spreading efficiently via low-dose fecal-oral transmission3,4. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries, but is now emerging as a problem in the developing world, apparently replacing the more diverse S. flexneri in areas undergoing economic development and improvements in water quality4-6. Classical approaches have shown S. sonnei is genetically conserved and clonal7. We report here whole-genome sequencing of 132 globally-distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and has diversified into several distinct lineages with unique characteristics. Our analysis suggests the majority of this diversification occurred in Europe, followed by more recent establishment of local pathogen populations in other continents predominantly due to the pandemic spread of a single, rapidly-evolving, multidrug resistant lineage

A randomized cross over trial of tolerability and compliance of a micronutrient supplement with low iron separated from calcium vs high iron combined with calcium in pregnant women [ISRCTN56071145]

BACKGROUND: Prenatal micronutrient combinations with high iron content are associated with high rates of gastrointestinal symptoms. This coupled with nausea and vomiting of pregnancy results in women often discontinuing their multivitamins. A new prescription supplement (PregVit(®)) that separates iron from calcium in two tablets – morning and evening, has lower elemental iron content (35 mg), but results in similar extent of iron absorption when compared to another supplement containing (60 mg) of elemental iron (Materna(®)). The objectives of this study were to compare tolerability and compliance with PregVit(® )vs. a supplement with high iron content (Materna(®)), in pregnant women. METHODS: Randomized, crossover open labeled study in 135 pregnant women attending outpatient clinics in Ontario and Quebec. RESULTS: Use of PregVit(® )was associated with a 30% reduction in constipation rate as compared to Materna(®). Both products demonstrated similar compliance rates. Compliance of Materna(® )was negatively associated with the severity of nausea and vomiting of pregnancy. No such correlation was found for PregVvit(®). CONCLUSION: PregVit(®), a supplement with lower iron content (35 mg), has significantly decreased constipation rates as compared to 60 mg iron- Materna and has similar compliance rates. High iron content in multivitamin supplements is associated with adverse effects in pregnancy

Evolution of the insecticide target Rdl in African Anopheles is driven by interspecific and interkaryotypic introgression.

The evolution of insecticide resistance mechanisms in natural populations of Anopheles malaria vectors is a major public health concern across Africa. Using genome sequence data, we study the evolution of resistance mutations in the resistance to dieldrin locus (Rdl), a GABA receptor targeted by several insecticides, but most notably by the long-discontinued cyclodiene, dieldrin. The two Rdl resistance mutations (296G and 296S) spread across West and Central African Anopheles via two independent hard selective sweeps that included likely compensatory nearby mutations, and were followed by a rare combination of introgression across species (from A. gambiae and A. arabiensis to A. coluzzii) and across non-concordant karyotypes of the 2La chromosomal inversion. Rdl resistance evolved in the 1950s as the first known adaptation to a large-scale insecticide-based intervention, but the evolutionary lessons from this system highlight contemporary and future dangers for management strategies designed to combat development of resistance in malaria vectors

Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species

Background Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group. Results Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes, genes present in not all, but more than one strain, was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant 13 evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence. Conclusion Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan genome guarantees the species a quick and economical response to diverse environments

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens--Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44) and the complete gene of pvcsp (n = 47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines