Matching Items (27)

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Display of Domain III from Dengue 2 Envelope Protein on HBsAg Virus-like Particles Vectored by Measles Virus

Description

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E)

Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.

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Created

Date Created
  • 2014-05

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How the EnvZ/OmpR Two-component Regulatory System Affects fepA Gene Expression in Escherichia coli

Description

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.

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Date Created
  • 2016-05

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Early Assessment of Phage Communities in Amazon Peatland Soils

Description

Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published

Little is known about the diversity and role of bacteriophages in carbon (C) rich ecosystems such as peatlands in tropical and temperate regions. In fact, there is no currently published assessment of phage abundance on diversity in a key tropical ecosystem such as Amazon peatlands. To better understand phage assemblages in terrestrial ecosystems and how bacteriophages influence organic C cycling to final products like CO2 and CH4, phage communities and phage-like particles were recovered, quantified, and viable phage particles were enriched from pore water from contrasting Amazon peatlands. Here we present the first results on assessing Amazon bacteriophages on native heterotrophic bacteria. Several steps to test for methodological suitability were taken. First, the efficiency of iron flocculation method was determined using fluorescent microscopy counts of phage TLS, a TolC-specific and LPS-specific bacteriophage, and Escherichia coli host pre- and post-extraction method. One-hundred percent efficiency and 0.15% infectivity was evidenced. Infectivity effects were determined by calculating plaque forming units pre and post extraction method. After testing these methods, fieldwork in the Amazon peatlands ensued, where phages were enriched from pore water samples. Phages were extracted and concentrated by in tandem filtering rounds to remove organic matter and bacteria, and then iron flocculation to bind the phages and allow for precipitation onto a filter. Phage concentrates were then used for overall counts, with fluorescent microscopy, as well as phage isolation attempts. Phage isolations were performed by first testing for lysis of host cells in liquid media using OD600 absorbance of cultures with and without phage concentrate as well as attempts with the cross-streaking methods. Forty-five heterotrophic bacterial isolates obtained from the same Amazon peatland were challenged with phage concentrates. Once a putative host was found, steps were taken to further propagate and isolate the phage. Several putative phages were enriched from Amazon peatland pore water and require further characterization. TEM imaging was taken of two phages isolated from two plaques. Genomes of selected phages will be sequenced for identification. These results provide the groundwork for further characterizing the role bacteriophage play in C cycling and greenhouse gas production from Amazon peatland soils.

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Date Created
  • 2016-05

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Effects of Environmental Conditions on Pyocyanin Production in Pseudomonas aeruginosa

Description

Pyocyanin is a pigment produced by Pseudomonas aeruginosa that acts as a virulence factor in helping this pathogen to establish chronic infection in the lungs of persons with cystic fibrosis

Pyocyanin is a pigment produced by Pseudomonas aeruginosa that acts as a virulence factor in helping this pathogen to establish chronic infection in the lungs of persons with cystic fibrosis (CF). Then, as lung infections become chronic, P. aeruginosa tends to down-regulate pyocyanin production. The effects of environmental conditions, particularly temperature change, on pyocyanin production in P. aeruginosa has not been widely studied in the past. The goals of this project were twofold: First, we aim to identify how environmental conditions potentially present in the CF lungs affect pyocyanin pigment production in P. aeruginosa. Second, through the examination of effects of environmental changes, we aim to identify methods to modulate phenotypes of P. aeruginosa in order to identify putative biomarkers through metabolic analysis. This paper also identifies a newly derived pyocyanin culturing and extraction procedure that yields increased sensitivity for pyocyanin detection.
Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.

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Created

Date Created
  • 2016-12

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The Development of Small Molecule Inhibitors of the TWEAK-Fn14 Pathway in Glioblastoma Multiforme

Description

Glioblastoma multiforme is the most common and aggressive primary malignant brain tumor in adults, exhibiting a median survival of only 15 months after diagnosis. A significant challenge in treating GBM

Glioblastoma multiforme is the most common and aggressive primary malignant brain tumor in adults, exhibiting a median survival of only 15 months after diagnosis. A significant challenge in treating GBM is the ability of glioma cells to invade normal brain tissue, escape surgical resection, and resist radiotherapy and chemotherapy. We have previously demonstrated that the TWEAK-Fn14 signaling axis plays an important role in glioma cell invasion and discovered a small molecule, L524-0366, that specifically disrupts the TWEAK-Fn14 interaction. However, low affinity limits L524-0366’s clinical feasibility. By utilizing structure-activity relationship analyses of L524-0366, we identified additional small molecules that may inhibit TWEAK-Fn14 signaling. Here, we identify five additional novel Fn14 signaling inhibitors that specifically inhibited TWEAK-Fn14 NF-κB-dependent signaling and suppressed TWEAK-induced glioma cell migration. Furthermore, we demonstrate that two molecules exhibit improved affinity for Fn14, two molecules showed binding to the TWEAK ligand but not Fn14, and one showed no binding to either TWEAK or Fn14. These molecules will be further tested for in vitro and in vivo functionality, and serve as foundations for additional medicinal chemistry for drug modifications.

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Created

Date Created
  • 2016-12

Characterization of the structure and interactions of the AcrAB-TolC multi-drug efflux pump in Escherichia coli

Description

The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions,

The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.

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Created

Date Created
  • 2016-05

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Reversal of the ΔdegP Phenotypes by a Novel rpoE Allele of Escherichia coli

Description

RseA sequesters RpoE (σ[superscript E]) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane

RseA sequesters RpoE (σ[superscript E]) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ[superscript E] to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ[superscript E] regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ[superscript E] levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ[superscript E]-dependent RybB::LacZ construct showed only a weak activation of the σ[superscript E] pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ[superscript E] and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA[subscript L222Q], it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ[superscript E]-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ[superscript E]-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ[superscript E] may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ[superscript E] levels.

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Date Created
  • 2012-03-16

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Unearthing the Antibacterial Mechanism of Medicinal Clay: A Geochemical Approach to Combating Antibiotic Resistance

Description

Natural antibacterial clays, when hydrated and applied topically, kill human pathogens including antibiotic resistant strains proliferating worldwide. Only certain clays are bactericidal; those containing soluble reduced metals and expandable clay

Natural antibacterial clays, when hydrated and applied topically, kill human pathogens including antibiotic resistant strains proliferating worldwide. Only certain clays are bactericidal; those containing soluble reduced metals and expandable clay minerals that absorb cations, providing a capacity for extended metal release and production of toxic hydroxyl radicals. Here we show the critical antibacterial components are soluble Fe[superscript 2+] and Al[superscript 3+] that synergistically attack multiple cellular systems in pathogens normally growth-limited by Fe supply. This geochemical process is more effective than metal solutions alone and provides an alternative antibacterial strategy to traditional antibiotics. Advanced bioimaging methods and genetic show that Al[superscript 3+] misfolds cell membrane proteins, while Fe[superscript 2+] evokes membrane oxidation and enters the cytoplasm inflicting hydroxyl radical attack on intracellular proteins and DNA. The lethal reaction precipitates Fe[superscript 3+]-oxides as biomolecular damage proceeds. Discovery of this bactericidal mechanism demonstrated by natural clays should guide designs of new mineral-based antibacterial agents.

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Date Created
  • 2016-01-08

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Genetic Manipulation of Pseudomonas aeruginosa Clinical Isolates

Description

Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that is the leading cause of chronic infection in the lungs of adults with cystic fibrosis (CF). During chronic lung infections,

Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that is the leading cause of chronic infection in the lungs of adults with cystic fibrosis (CF). During chronic lung infections, P. aeruginosa populations adapt genetically to the CF lung, selecting several important mutations required for long-term persistence. These genetic adaptations lead to phenotypic changes that are associated with the transition from early-stage to late-stage chronic CF infection.
The goal of this project was to develop tools for gene transfer between P. aeruginosa clinical isolates. These tools will allow shuffling of early/late stage of infection genes to restore wild-type phenotypes in late chronic infection isolates and create single-phenotype mutants in the early infection strains. This will allow isolation and investigation of single phenotypes in the clinical isolates to identify metabolic biomarkers specifically for detecting the target phenotypes.

The gene transfer mechanisms of transformation by electroporation, transformation by heat shock, and conjugation were tested using the plasmid pMQ30 with a construct to create an in-frame deletion of the rhlR gene (rhlR) via allelic exchange. The disruption of the P. aeruginosa wild-type rhlR gene leads to rhamnolipids-deficient mutant strains; therefore, rhamnolipids production was assessed to validate successful in-frame deletion of the rhlR gene in the P. aeruginosa clinical isolates and laboratory strains. Based on the efficiencies determined from the gene transfer mechanisms tested, the conjugation mechanism was determined to be the most efficient method for gene transfer in P. aeruginosa laboratory strains, and was used to investigate gene transfer in the P. aeruginosa clinical isolates.

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Date Created
  • 2018-05

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The Effectiveness of Inhibition and Biofilm Disruption on Antibiotic Resistant E. coli

Description

The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic

The purpose of this study was to observe the effectiveness of the phenylalanyl arginine β-naphthylamide dihydrochloride inhibitor and Tween 20 when combined with an antibiotic against Escherichia. coli. As antibiotic resistance becomes more and more prevalent it is necessary to think outside the box and do more than just increase the dosage of currently prescribed antibiotics. This study attempted to combat two forms of antibiotic resistance. The first is the AcrAB efflux pump which is able to pump antibiotics out of the cell. The second is the biofilms that E. coli can form. By using an inhibitor, the pump should be unable to rid itself of an antibiotic. On the other hand, using Tween allows for biofilm formation to either be disrupted or for the biofilm to be dissolved. By combining these two chemicals with an antibiotic that the efflux pump is known to expel, low concentrations of each chemical should result in an equivalent or greater effect on bacteria compared to any one chemical in higher concentrations. To test this hypothesis a 96 well plate BEC screen test was performed. A range of antibiotics were used at various concentrations and with varying concentrations of both Tween and the inhibitor to find a starting point. Following this, Erythromycin and Ciprofloxacin were picked as the best candidates and the optimum range of the antibiotic, Tween, and inhibitor were established. Finally, all three chemicals were combined to observe the effects they had together as opposed to individually or paired together. From the results of this experiment several conclusions were made. First, the inhibitor did in fact increase the effectiveness of the antibiotic as less antibiotic was needed if the inhibitor was present. Second, Tween showed an ability to prevent recovery in the MBEC reading, showing that it has the ability to disrupt or dissolve biofilms. However, Tween also showed a noticeable decrease in effectiveness in the overall treatment. This negative interaction was unable to be compensated for when using the inhibitor and so the hypothesis was proven false as combining the three chemicals led to a less effective treatment method.

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Created

Date Created
  • 2018-05