Matching Items (44)

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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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Role of RAG2 C-terminal region in enforcing appropriate recombination cleavage directed at legitimate DNA targets

Description

V(D)J Recombination is the mechanism responsible for generating diversity in the repertoire of antigen receptors of T and B cells. This recombination process proceeds in two steps: site-specific cleavage mediated

V(D)J Recombination is the mechanism responsible for generating diversity in the repertoire of antigen receptors of T and B cells. This recombination process proceeds in two steps: site-specific cleavage mediated lymphocyte-specific recombinase known as Recombination Activating Genes 1 and 2 complex (RAG) at the junction of coding gene segments and their flanking recombination signal sequence (RSS) and then followed by rejoining of the double strand broken DNA by the non-homologous end joining (NHEJ) complex. Mutations and truncations of the RAG-recombinase have been found associated with genomic instability and chromosomal translocation. It has been hypothesized that these RAG mutants may have abnormality in their interactions with recombination intermediates, ultimately causing premature release of the ends for aberrant joining. Additionally, these mutations have an increase in targeting non-B type DNA instead of legitimate recombination substrates that contain RSSs. To directly test these hypotheses, we have developed a fluorescence-based detection system to monitor in real time the recombination cleavage reaction from the pre-cleavage to the post-cleavage stages and to compare RAG-DNA interactions between wild type and mutant RAG1/2 during this process. Our study provides important insight into the ability of the C-terminus of RAG to regulate RAG recombinase activity.

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Created

Date Created
  • 2014-12

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Importance of cholesterol-rich membrane microdomains in measles virus

Description

Lipid microdomains play a vital role in a number of biological processes. They are often a target of diseases and viruses. Viruses in particular utilize lipid microdomains to gain entry

Lipid microdomains play a vital role in a number of biological processes. They are often a target of diseases and viruses. Viruses in particular utilize lipid microdomains to gain entry and fuse with the host-cell membrane. Measles virus (MV) a human pathogen, spread from cell to cell by inducing fusion of cellular membranes. This causes the formation of large multinucleated cells, syncytia. It has been previously reported that lipid microdomains are essential for measles virus infection/replication. In this study we used methyl beta cyclodextrin (MBCD), a cholesterol-sequestering agent to disrupt lipid microdomains. Through transfection of Vero h/SLAM cells, we found that Measles virus fusion was dependent on lipid microdomains integrity. Indeed, a dose dependent fusion inhibition was documented with increasing concentrations of MBCD resulting in reduced formation of syncytia.

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Agent

Created

Date Created
  • 2013-05

Entrepreneurship Initiative at Westward Ho (DIY Bench)

Description

The goal is to develop a long term collaborative partnership that benefits the four main stakeholders: Arizona State University, The City of Phoenix, Westward Ho residents, and Westward Ho ownership.

The goal is to develop a long term collaborative partnership that benefits the four main stakeholders: Arizona State University, The City of Phoenix, Westward Ho residents, and Westward Ho ownership. Arizona State University gains unique access to a research and learning environment for faculty and students of a variety of health disciplines. The City of Phoenix receives stability and safety to the neighborhood and protects the city's investment in the Westward Ho. The residents gain needed services through participation in ASU programs and initiatives. They acquire new life skills that contribute to their independence, thereby reducing the demand for costly emergency services and adding to their quality of life. The owners gain a more stable resident population and ASU's investment allows them to continue to upgrade the property, benefitting the city, the residents, and ASU.

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Created

Date Created
  • 2013-05

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CLONING AND EXPRESSION OF FLAVIVIRUS (YELLOW FEVER VIRUS AND DENGUE VIRUS) RECOMBINANT ENVELOPE PROTEINS IN E. COLI

Description

As research progresses in the field of vaccinology, momentum has been gained to develop an efficacious and efficient dengue virus (DV) vaccine for all four serotypes. Dengue viral outbreaks across

As research progresses in the field of vaccinology, momentum has been gained to develop an efficacious and efficient dengue virus (DV) vaccine for all four serotypes. Dengue viral outbreaks across the world have called for a vaccine campaign. However, due to anti--"body dependent enhancement of infection, dengue virus has provided Researchers with challenges in developing a safe vaccine. Currently, there are a handful of vaccine candidates in clinical trial, but live chimeric attenuated vaccines dominate them. There are associated risks with using a live chimeric attenuated vaccine, but they are less expensive to generate and seem to provide a high immune response. Subunit vaccines are safer to use and can provide full protection for several years with then use of adjuvants and a booster shot. As a result, our lab is interested in pursuing this route to produce an effective dengue vaccine. The main target for a dengue subunit vaccine is the envelope protein, which is known to be an important recognition site by neutralizing antibodies. Therefore, expression of a recombinant envelope protein in a prokaryotic expression system is useful to study the immune response in vivo. This could be taken a step further and recombinant dengue envelope proteins can be expressed by a eukaryote to help generate hypotheses and insight to create a successful dengue virusn subunit vaccine.

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Date Created
  • 2013-05

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In Vitro Display of Major Histocompatibility Complex (MHC)-Complexes on Luminex Platform Beads

Description

Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein

Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein Expression by Thermo Scientific in the presence of β2 microglobulin (β2m) and antigenic peptide.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.

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Date Created
  • 2013-05

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Targeted Delivery DNA-Tetrahedron Assembled Therapeutics

Description

As advanced as current cancer therapeutics are, there are still challenges that need to be addressed. One of them is the non-specific killing of normal cells in addition to cancerous

As advanced as current cancer therapeutics are, there are still challenges that need to be addressed. One of them is the non-specific killing of normal cells in addition to cancerous cells. Ideal cancer therapeutics should be targeted specifically toward tumor cells. Due to the robust self-assembly and versatile addressability of DNA-nanostructures, a DNA tetrahedron nanostructure was explored as a drug carrier. The nanostructure can be decorated with various molecules to either increase immunogenicity, toxicity, or affinity to a specific cell type. The efficiency of the specific binding and internalization of the chosen molecules was measured via flow cytometry. Using a murine B cell lymphoma as the model system, several targeting molecules have been evaluated for their specific binding and induced internalization of DNA nanostructures, including an anti-Igκ antibody, an idiotype-binding peptide, and a g-quadruplex nucleolin specific aptamer. It was found that adding the anti-Igκ antibody appeared to provide increased binding and facilitated cellular internalization. Also, it was found that the presence of CpG appeared to aid in the binding of nanostructures decorated with other molecules, as compared to nanostructures without CpG. The g-quadruplex aptamer thought to specifically bind cancer cells that overexpress nucleolin was tested and found to have better binding to cells when linked to the nanostructure than when alone. The drug doxorubicin was used to load the DNA-nanostructure and attempt to inhibit cancer cell growth. The DNA-nanostructure has the benefit of being self-assembled and customizable, and it has been shown to bind to and internalize into a cancer cell line. The next steps are to test the toxicity of the nanostructure as well as its specificity for cancerous cells compared to noncancerous cells. Furthermore, once those tests are completed the structure’s drug delivery capacity will be tested in tumor bearing mice. The DNA-nanostructure exhibits potential as a cancer specific therapeutic.

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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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Date Created
  • 2016-12

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Measles Virus Vectoring Hepatitis C Non-structural Protein 3: Towards a Hepatitis C Vaccine

Description

Hepatitis C virus (HCV) is a globally prevalent infection which is a main contributor to the global burden of liver disease. Due to its ability to establish a chronic infection,

Hepatitis C virus (HCV) is a globally prevalent infection which is a main contributor to the global burden of liver disease. Due to its ability to establish a chronic infection, and the lack of usefulness of traditional neutralizing antibody vaccine design in producing a protective immune response, a preventative vaccine has been notoriously difficult to produce. To overcome this, a vaccine using non-structural protein 3 (NS3) as a target to elicit a T cell specific immune response is thought to be a possible strategy for eliciting a protective immune response against hepatitis C infection. In this paper, a recombinant strain of measles virus (MV) that expresses HCV NS3 protein was analyzed. The replication fitness of this recombinant virus also indicates that this construct replicates at a higher rate than parental measles strain. It is also demonstrated through western blot analysis of protein expression and immunofluorescence that this recombinant virus expresses both the inserted HCV NS3 protein, as well as native measles proteins.

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Date Created
  • 2015-05

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Expression of the measles virus proteome by RAPID ELISA for serological assays

Description

Background: Measles virus (MV) infections are the main cause of vaccine-preventable death in children younger than 5 years. The World Health Organization (WHO) has estimated there are over 20 million

Background: Measles virus (MV) infections are the main cause of vaccine-preventable death in children younger than 5 years. The World Health Organization (WHO) has estimated there are over 20 million cases of measles every year. Currently, diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. These commercial assays measure reactivity against the immunodominant N antigen and can have a false negative rates of 20-30%. Centralized testing by clinical labs can delay rapid screening in an outbreak setting. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to five individual MV proteins representing 85% of the measles proteome. Methods: MV genes were subcloned into pANT_cGST vector to generate C-terminal GST fusion proteins. Single MV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured using a sandwich ELISA for GST expression using relative light units (RLUs) as readouts. Single MV antigens were used as bait to determine the IgG-dependent reactivity in 12 serum samples obtained from immunized animals with previously determined neutralization titer (NT) and the correlation between NT and ELISA reactivity was determined. Results: Protein expression of five measles genes of interest, M, N, F, H, and L, was measured. L exhibited the strongest protein expression with an average RLU value of 4.34 x 10^9. All proteins were expressed at least 50% greater than control (2.33 x 10^7 RLU). As expected, reactivity against the N was the highest, followed by reactivity against M, F, H and L. The best correlation with NT titer was reactivity against F (R^2 = 0.62). Conclusion: These data indicate that the expression of single MV genes M, N, F, H, and L are suitable antigens for serologic capture analysis of measles immunity.

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Created

Date Created
  • 2015-05