Matching Items (33)

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Evolution-informed modeling improves outcome prediction for cancers

Description

Despite wide applications of high-throughput biotechnologies in cancer research, many biomarkers discovered by exploring large-scale omics data do not provide satisfactory performance when used to predict cancer treatment outcomes. This

Despite wide applications of high-throughput biotechnologies in cancer research, many biomarkers discovered by exploring large-scale omics data do not provide satisfactory performance when used to predict cancer treatment outcomes. This problem is partly due to the overlooking of functional implications of molecular markers. Here, we present a novel computational method that uses evolutionary conservation as prior knowledge to discover bona fide biomarkers. Evolutionary selection at the molecular level is nature's test on functional consequences of genetic elements. By prioritizing genes that show significant statistical association and high functional impact, our new method reduces the chances of including spurious markers in the predictive model. When applied to predicting therapeutic responses for patients with acute myeloid leukemia and to predicting metastasis for patients with prostate cancers, the new method gave rise to evolution-informed models that enjoyed low complexity and high accuracy. The identified genetic markers also have significant implications in tumor progression and embrace potential drug targets. Because evolutionary conservation can be estimated as a gene-specific, position-specific, or allele-specific parameter on the nucleotide level and on the protein level, this new method can be extended to apply to miscellaneous “omics” data to accelerate biomarker discoveries.

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Agent

Created

Date Created
  • 2016-10-21

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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

Created

Date Created
  • 2016-12

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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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Agent

Created

Date Created
  • 2013-05

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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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Agent

Created

Date Created
  • 2016-12

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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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Agent

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

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Strategies to enhance RNA-origami-based immunotherapeutics for an induction of long-term tumor-regression

Description

Recently, we have demonstrated that a novel RNA origami (RNA-OG) nanostructure functions as a TLR3 agonist both in vitro and in vivo. This RNA nanostructure could induce effective antitumor immunity

Recently, we have demonstrated that a novel RNA origami (RNA-OG) nanostructure functions as a TLR3 agonist both in vitro and in vivo. This RNA nanostructure could induce effective antitumor immunity in a CT26-OVA-iRFP tumor model that expresses both ovalbumin (OVA) and near infrared protein (iRFP), rendering a significant delay in tumor growth or complete tumor-regression. However, in a similar tumor line that expresses iRFP but not OVA, i.e. a CT26-Neo-iRFP model, RNA-OG induced responses that were consistently inferior to those observed in CT26-OVA-iRFP. Interestingly, the antitumor immunity initially generated against CT26-OVA-iRFP was found to render the mice immune to a challenge with the more malignant CT26-Neo-iRFP line. In addition to OVA expression, the two cell lines also showed different levels of MHC-I. Ongoing research has been focused on deciphering the molecular nature of the different responses. Then, we can search for strategies that increase the tumor immunogenicity, and therefore improve the therapeutic efficacy of RNA-OG for inducing long-term tumor-regression.

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Agent

Created

Date Created
  • 2019-05

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Improving expression vectors for recombinant protein production in plants

Description

Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems

Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.

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Agent

Created

Date Created
  • 2012

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Developmental plasticity: the influence of neonatal diet and immune challenges on carotenoid-based ornamental coloration and adult immune function in mallard ducks

Description

Conditions during development can shape the expression of traits at adulthood, a phenomenon called developmental plasticity. In this context, factors such as nutrition or health state during development can affect

Conditions during development can shape the expression of traits at adulthood, a phenomenon called developmental plasticity. In this context, factors such as nutrition or health state during development can affect current and subsequent physiology, body size, brain structure, ornamentation, and behavior. However, many of the links between developmental and adult phenotype are poorly understood. I performed a series of experiments using a common molecular currency - carotenoid pigments - to track somatic and reproductive investments through development and into adulthood. Carotenoids are red, orange, or yellow pigments that: (a) animals must acquire from their diets, (b) can be physiologically beneficial, acting as antioxidants or immunostimulants, and (c) color the sexually attractive features (e.g., feathers, scales) of many animals. I studied how carotenoid nutrition and immune challenges during ontogeny impacted ornamental coloration and immune function of adult male mallard ducks (Anas platyrhynchos). Male mallards use carotenoids to pigment their yellow beak, and males with more beaks that are more yellow are preferred as mates, have increased immune function, and have higher quality sperm. In my dissertation work, I established a natural context for the role that carotenoids and body condition play in the formation of the adult phenotype and examined how early-life experiences, including immune challenges and dietary access to carotenoids, affect adult immune function and ornamental coloration. Evidence from mallard ducklings in the field showed that variation in circulating carotenoid levels at hatch are likely driven by maternal allocation of carotenoids, but that carotenoid physiology shifts during the subsequent few weeks to reflect individual foraging habits. In the lab, adult beak color expression and immune function were more tightly correlated with body condition during growth than body condition during subsequent stages of development or adulthood. Immune challenges during development affected adult immune function and interacted with carotenoid physiology during adulthood, but did not affect adult beak coloration. Dietary access to carotenoids during development, but not adulthood, also affected adult immune function. Taken together, these results highlight the importance of the developmental stage in shaping certain survival-related traits (i.e., immune function), and lead to further questions regarding the development of ornamental traits.

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Created

Date Created
  • 2012

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Breaking the senescence: inhibition of ATM allows S9 cells to re-enter cell cycle

Description

The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid

The Philadelphia chromosome in humans, is on oncogenic translocation between chromosomes 9 and 22 that gives rise to the fusion protein BCR-Abl. This protein is constitutively active resulting in rapid and uncontrolled cell growth in affected cells. The BCR-Abl protein is the hallmark feature of chronic myeloid leukemia (CML) and is seen in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) cases. Currently, the first line of treatment is the Abl specific inhibitor Imatinib. Some patients will, however, develop resistance to Imatinib. Research has shown how transformation of progenitor B cells with v-Abl, an oncogene expressed by the Abelson murine leukemia virus, causes rapid proliferation, prevents further differentiation and produces a potentially malignant transformation. We have used progenitor B cells transformed with a temperature-sensitive form of the v-Abl protein that allows us to inactivate or re-activate v-Abl by shifting the incubation temperature. We are trying to use this line as a model to study both the progression from pre-malignancy to malignancy in CML and Imatinib resistance in Ph+ ALL and CML. These progenitor B cells, once v-Abl is reactivated, in most cases, will not return to their natural cell cycle. In this they resemble Ph+ ALL and CML under Imatinib treatment. With some manipulation these cells can break this prolonged G1 arrested phenotype and become a malignant cell line and resistant to Imatinib treatment. Cellular senescence can be a complicated process requiring inter-play between a variety of players. It serves as an alternate option to apoptosis, in that the cell loses proliferative potential, but does not die. Treatment with some cancer therapeutics will induce senescence in some cancers. Such is the case with Imatinib treatment of CML and Ph+ ALL. By using the S9 cell line we have been able to explore the possible routes for breaking of prolonged G1 arrest in these Ph+ leukemias. We inhibited the DNA damage sensor protein ataxia telangiectasia mutated (ATM) and found that prolonged G1 arrest in our S9 cells was broken. While previous research has suggested that the DNA damage sensor protein ataxia-telangiectasia mutated (ATM) has little impact in CML, our research indicates that ATM may play a role in either senescence induction or release.

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Agent

Created

Date Created
  • 2011