Matching Items (43)

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High-throughput identification of miRNA targets in the 3`UTRs of the soil nematode C. elegans

Description

miRNAs are short non-coding regulatory RNAs that have an important roles in a wide range of biological processes. Dysfunction of miRNA regulation has also been shown to occur in diseases

miRNAs are short non-coding regulatory RNAs that have an important roles in a wide range of biological processes. Dysfunction of miRNA regulation has also been shown to occur in diseases such as cancer. Despite the widespread influence of miRNAs in these contexts, the vast majority of miRNA targets are poorly characterized. The aim of this research project was to gain a better understating of miRNA targeting by using the model organism C. elegans. In order to do this I adapted a novel high-throughput assay to detect miRNA targets for use with the C. elegans 3`UTRome. As a proof of principle I performed this assay on 96 C. elegans 3`UTRs using high-throughput techniques. The results revealed miRNA interactions with two predicted 3`UTR targets for the miRNA lin-4 and ten unpredicted targets. The results also corroborated previous findings that certain worm miRNAs require special modifications to be expressed in human cells.

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Created

Date Created
  • 2013-12

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Alternative Treatments for Endocrine Resistant Breast Cancer

Description

The focus of this project was to look at alternative treatments for endocrine resistant breast cancer (ERBC), which are breast cancers that have become resistant to hormone therapies such as

The focus of this project was to look at alternative treatments for endocrine resistant breast cancer (ERBC), which are breast cancers that have become resistant to hormone therapies such as Tamoxifen or aromatase inhibitors. The first part of this project involves investigating the relationship between histone de-acetylase inhibitor Vorinostat and Tamoxifen in MCF7 G11 cells, Tamoxifen resistant sub-clones, according to the PSOC Time grant. The second part involves targeting the androgen receptor (AR) in MCF7 sub-clones with AR antagonists, Bicalutamide and MDV3100, and investigating the possible usage of AR as a biomarker, due to over-expression of AR in ERBC, in accordance with the Mayo ASU Seed Grant.
The synergistic effects between Vorinostat and Tamoxifen observed through a phase II study on breast cancer patients resistant to hormone therapy may involve more than the modulation of ER-alpha to reverse Tamoxifen resistance in ERBC cells. RT-qPCR of genes expressed in Tamoxifen resistant cells, trefoil factor 1(TFF1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC), were evaluated along with ESR1 and Diablo as a control. MYC was observed to have increased expression in the treated cells, whereas the other genes had a decrease in their expression levels after the cells were treated for 3 days with Vorinostat IC30 of 1 µM. As for targeting the AR, MCF7 Tamoxifen sensitive and resistant cells were not affected by the AR antagonists to determine an IC50. The cell viability for all MCF7 sub-clones only decreased for high concentrations of 5.56 µM - 50 µM in Bicalutamide and 16.67 µM – 50 µM of MDV1300. Furthermore, hormone depletion of MCF7 G11 Tamoxifen resistant sub-clones did not show a great response to DHT stimulation or the AR antagonists. In the RT-qPCR, the MCF7 G11 cells showed an increase in mRNA expression for ER, AR, and PR after 4 hours of treatment with estradiol. As for the DHT treatment, ER, AR, PR, and PSA had a minimal increase in the fold change, but the fold change in AR was less than in the estradiol treatment. The Mayo Clinic will investigate the possible usage of AR as a biomarker through immunohistochemistry.

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Created

Date Created
  • 2014-05

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HPV Antibodies as Novel Biomarkers for the Detection of Cervical Neoplasia

Description

Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV

Background: The human papillomavirus (HPV) is the cause of virtually all cervical cancer, with over 520,000 new cases and 275,000 deaths annually. Although there are at least 200 unique HPV strains, only “high-risk” types, may progress to cancer. Serum antibodies to HPV oncoproteins are stable and specific markers that may be able to detect high-grade cervical intraepithelial neoplasia (CIN3). Biomarkers have potential as a rapid, point-of-care HPV screening tool for low resource areas in the way that traditional cytology cannot, and HPV DNA testing is not yet able to.
Methods: We have designed a multiplexed magnetics programmable bead ELISA (MagProBE) to profile the immune responses of the proteins from 11 high-risk HPV types and 2 low-risk types—106 genes in total. HPV genes were optimized for human expression and either built with PCR or commercially purchased, and cloned into the Gateway-compatible pANT7_cGST vector for in vitro transcription/translation (IVTT) in a MagProBE array. Anti-GST antibody (Ab) labeling was then used to measure gene expression.
Results: 53/106 (50%) HPV genes have been cloned and tested for expression of protein. 91% of HPV proteins expressed at levels above the background control (MFI = 2288), and the mean expression was MFI = 4318. Codon-optimized genes have also shown a 20% higher expression over non-codon optimized genes.
Conclusion: Although this research is ongoing, it suggests that gene optimization may improve IVTT expression of HPV proteins in human HeLa lysate. Once the remaining HPV proteins have been expression confirmed, the cDNA for each gene will be printed onto slides and tested in serologic assays to identify potential Ab biomarkers to CIN3.

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Agent

Created

Date Created
  • 2013-05

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TWEAK functions as chemotactic factor for glioma cells via Lyn activation

Description

The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance

The long-term survival of patients with glioblastoma multiforme is compromised by the tumor's proclivity for local invasion into the surrounding normal brain. These invasive cells escape surgery and display resistance to chemotherapeutic- and radiation-induced apoptosis. We have previously shown that tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion and survival via binding to the fibroblast growth factor-inducible 14 (Fn14) receptor and subsequent activation of the Rac1/NF-kappaB pathway. In addition, we have reported previously that Fn14 is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Here we demonstrate that TWEAK can act as a chemotactic factor for glioma cells, a potential process to drive cell invasion into the surrounding brain tissue. Specifically, we detected a chemotactic migration of glioma cells to the concentration gradient of TWEAK. Since Src family kinases (SFK) have been implicated in chemotaxis, we next determined whether TWEAK:Fn14 engagement activated these cytoplasmic tyrosine kinases. Our data shows that TWEAK stimulation of glioma cells results in a rapid phosphorylation of the SFK member Lyn as determined by multiplex Luminex assay and verified by immunoprecipitation. Immunodepletion of Lyn by siRNA oligonucleotides suppressed the chemoattractive effect of TWEAK on glioma cells. We hypothesize that TWEAK secretion by cells present in the glioma microenvironment induce invasion of glioma cells into the brain parenchyma. Understanding the function and signaling of the TWEAK-Fn14 ligand-receptor system may lead to development of novel therapies to therapeutically target invasive glioma cells.

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Agent

Created

Date Created
  • 2013-05

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Detection of antibodies to HPV16-associated oropharyngeal cancer using custom bead arrays

Description

Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens

Oropharyngeal cancer (OPC) is the world's sixth most common cancer and in many cases is associated with infection with human papillomavirus (HPV) type 16. Antibodies (Abs) to HPV16 viral antigens are potential diagnostic biomarkers of HPV-associated OPC (HPV OPC). A custom multiplexed bead array assay was used to detect Abs to HPV16 antigens E1, CE2, NE2, E4, E5, E6, E7, L1, and L2. Following extensive optimization of the assay, these genes were expressed as GST-fusion proteins and captured onto anti-GST magnetic beads. Serum was obtained from 256 OPC patients at the time of diagnosis and from 78 healthy controls. The median fluorescent intensity (MFI) was determined for each antigen and ratios of MFI to control GST-fusion protein were determined for each serum sample. Cutoff values were set as the mean + 3 SD of the MFIs of healthy controls and p-values were calculated using Wilcoxon unpaired and Fisher's exact test. Results of this experiment showed that HPV16 E1, CE2, NE2, E4, E6, and E7 Ab levels were elevated in OPC patients compared to controls (p<0.001), as were Ab levels to L1 (p = 0.013) and L2 (p = 0.023), per Fischer's exact test. Abs to CE2, NE2, E6, and E7 were identified as a potential biomarker panel for early detection of HPV OPC. For the 111 patients with known HPV+ tumors as measured by tumor PCR of E6 and/or E7, this assay had a sensitivity of 90% and specificity of 87% (AUC = 0.96). From these results, we conclude that custom bead array assays can be used to detect HPV16 Abs in patient sera, and we have identified a 4-Ab biomarker panel for the early detection of HPV OPC.

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Agent

Created

Date Created
  • 2013-05

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Transcriptome gene expression analysis of breast cancer using RNA-Seq

Description

Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of

Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths in females worldwide, accounting for 23% of all new cancer cases and 14% of all total cancer deaths in 2008. Five tumor-normal pairs of primary breast epithelial cells were treated for infinite proliferation by using a ROCK inhibitor and mouse feeder cells. Methods: Raw paired-end, 100x coverage RNA-Seq data was aligned to the Human Reference Genome Version 19 using BWA and Tophat. Gene differential expression analysis was completed using Cufflinks and Cuffdiff. Interactive Genome Viewer was used for data visualization. Results: 15 genes were found to be down-regulated by at least one log-fold change in 4/5 of tumor samples. 75 genes were found to be down-regulated in 3/5 of our tumor samples by at least one log-fold change. 11 genes were found to be up-regulated in 4/5 of our tumor samples, and 68 genes were identified to be up-regulated in 3/5 of the tumor samples by at least one-fold change. Conclusion: Expression changes in genes such as AZGP1, AGER, ALG11, and S1007 suggest a disruption in the glycosylation pathway. No correlation was found between Cufflink's Her2 gene-expression and DAKO score classification.

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Agent

Created

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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Created

Date Created
  • 2013-05

Point-of-Care Testing for Detection of Measles and Mumps Immunity

Description

Measles and mumps are highly contagious, vaccine-preventable diseases with cases continuing to persist in high two-dose vaccinated populations. Recent outbreaks on university and college campuses across the United States prompt

Measles and mumps are highly contagious, vaccine-preventable diseases with cases continuing to persist in high two-dose vaccinated populations. Recent outbreaks on university and college campuses across the United States prompt a need for further understanding of the immunity levels afforded by the MMR vaccine which has significantly decreased incidence rates of measles and mumps since it was introduced.
Current methods for IgG antibody detection include enzyme immunoassays (EIA) such as the commercially available Diamedix Immunosimplicity® Measles IgG test kit and the Diamedix Immunosimplicity® Mumps IgG test kit. EIAs generally provide high sensitivity and strong specificity, however, there is a need for rapid screening of measles and mumps specific immunity in outbreak and resource-limited areas which could be solved by use a point-of-care (POC) platform.
This study aims to optimize a point-of-care device for the multiplexed detection of MeV, MuV, and RuV IgG antibodies in sera and to compare the sensitivity to commercial enzyme immunoassays. The IgG antibody levels to MeV and MuV were measured using EIA test kits for a total of 44 healthy serum samples. Of the samples, 6% were seronegative for MeV-specific IgG antibodies and 75% were seronegative for MuV-specific antibodies, showing low correlation of IgG antibody levels between both viruses.
To improve the sensitivity of the POC device, multiple conjugated fluorescent secondary antibodies were tested with different surface chemistries. Signal detection was measured using the pre-developed four-site slide reader. Preliminary data show that Nile Red microspheres provide robust signal detection and should be the secondary antibody of choice when sera are tested for IgG antibodies using the POC platform in future work.

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Agent

Created

Date Created
  • 2017-05

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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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Agent

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Date Created
  • 2016-12

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Understanding the Biochemistry of Different P53 Mutants Having Different Sensitivities to Simvastatin

Description

The p53 gene functions as a tumor suppressor that inhibits proliferation, regulates apoptosis, DNA repair, and normal cell cycle arrest. Mutation of the p53 gene is linked to be prevalent

The p53 gene functions as a tumor suppressor that inhibits proliferation, regulates apoptosis, DNA repair, and normal cell cycle arrest. Mutation of the p53 gene is linked to be prevalent in 50% of all human cancers. In this paper, we are exploring triple negative breast cancer and the effects of simvastatin on tumor growth and survival. Simvastatin is a drug that is primarily used to treat high cholesterol and heart disease. Simvastatin is unique because it is able to inhibit protein prenylation through regulation of the mevalonate pathway. This makes it a potential targeted drug for therapy against p53 mutant cancer. The mechanism behind this is hypothesized to be correlated to aberrant activation of the Ras pathway. The Ras subfamily functions to transcriptionally regulate cell growth and survival, and will therefore allow for a tumor to thrive if the pathway is continually and abnormally activated. The Ras protein has to be prenylated in order for activation of this pathway to occur, making statin drug treatment a viable option as a cancer treatment. This is because it acts as a regulator of the mevalonate pathway which is upstream of protein prenylation. It is thus vital to understand these pathways at both the gene and protein level in different p53 mutants to further understand if simvastatin is indeed a drug with anti-cancer properties and can be used to target cancers with p53 mutation. The goal of this project is to study the biochemistry behind the mutation of p53's sensitivity to statin. With this information we can create a possible signature for those who could benefit from Simvastatin drug treatment as a possible targeted treatment for p53 mutant cancers.

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Agent

Created

Date Created
  • 2016-12