Matching Items (44)

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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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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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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

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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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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

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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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Date Created
  • 2016-10-21

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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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Created

Date Created
  • 2019-05

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Addicted to autophagy: Ph+ B-ALL may acquire iImatinib-resistance and enhanced malignancy through a highly-active autophagy pathway

Description

The majority of chronic myeloid leukemia (CML) and some of acute lymphocytic leukemia (ALL) cases are associated with possessing the BCR-Abl fusion protein from an oncogenic translocation, resulting in a

The majority of chronic myeloid leukemia (CML) and some of acute lymphocytic leukemia (ALL) cases are associated with possessing the BCR-Abl fusion protein from an oncogenic translocation, resulting in a constantly active form of Abl and rapid proliferation. CML and ALL cells that possess the BCR-Abl fusion protein are known as Philadelphia chromosome positive (Ph+). Currently, Imatinib (selective Abl inhibitor) is used as therapy against CML and ALL. However, some patients may have malignancies which show resistance to Imatinib. Previous work displays that the transformation of progenitor B cells with the v-Abl oncogene of Abelson murine leukemia virus results in cell cycle progression, rapid proliferation, and potentially malignant transformation while preventing any further differentiation. Progenitor B cells transformed with the temperature-sensitive form of the v-Abl oncogene have served as a model to study cellular response to Imatinib treatment. After some manipulation, very few cells were forced to progress to malignancy, forming tumor in vivo. These cells were no long sensitive to v-Abl inactivation, resembling the Imatinib resistant ALL. Autophagy is the process by which proteins and organelles are broken-down and recycled within the eukaryotic cell and has been hypothesized to play a part in cancer cell survival and drug-resistance. LC3 processing is a widely accepted marker of autophagy induction and progression. It has also been shown that Imatinib treatment of Ph+ leukemia can induce autophagy. In this study, we examined the autophagy induction in response to v-Abl inactivation in a Ph+-B-ALL cell model that shows resistance to Imatinib. In particular, we wonder whether the tumor cell line resistant to v-Abl inactivation may acquire a high level of autophagy to become resistant to apoptosis induced by v-Abl inactivation, and thus become addicted to autophagy. Indeed, this tumor cell line displays a high basal levels of LC3 I and II expression, regardless of v-Abl activity. We further demonstrated that inhibition of the autophagy pathway enhances the tumor line's sensitivity to Imatinib, resulting in cell cycle arrest and massive apoptosis. The combination of autophagy and Abl inhibitions may serve as an effective therapy for BCR-Abl positive CML.

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Date Created
  • 2011

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Molecular chaperones of the endoplasmic reticulum promote hepatitis C virus E2 protein production in plants

Description

Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver

Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation.

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Agent

Created

Date Created
  • 2011

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Avian retinal carotenoid accumulation: ecophysiological constraints and behavioral consequences

Description

The elaborate signals of animals are often costly to produce and maintain, thus communicating reliable information about the quality of an individual to potential mates or competitors. The properties of

The elaborate signals of animals are often costly to produce and maintain, thus communicating reliable information about the quality of an individual to potential mates or competitors. The properties of the sensory systems that receive signals can drive the evolution of these signals and shape their form and function. However, relatively little is known about the ecological and physiological constraints that may influence the development and maintenance of sensory systems. In the house finch (Carpodacus mexicanus) and many other bird species, carotenoid pigments are used to create colorful sexually selected displays, and their expression is limited by health and dietary access to carotenoids. Carotenoids also accumulate in the avian retina, protecting it from photodamage and tuning color vision. Analogous to plumage carotenoid accumulation, I hypothesized that avian vision is subject to environmental and physiological constraints imposed by the acquisition and allocation of carotenoids. To test this hypothesis, I carried out a series of field and captive studies of the house finch to assess natural variation in and correlates of retinal carotenoid accumulation and to experimentally investigate the effects of dietary carotenoid availability, immune activation, and light exposure on retinal carotenoid accumulation. Moreover, through dietary manipulations of retinal carotenoid accumulation, I tested the impacts of carotenoid accumulation on visually mediated foraging and mate choice behaviors. My results indicate that avian retinal carotenoid accumulation is variable and significantly influenced by dietary carotenoid availability and immune system activity. Behavioral studies suggest that retinal carotenoid accumulation influences visual foraging performance and mediates a trade-off between color discrimination and photoreceptor sensitivity under dim-light conditions. Retinal accumulation did not influence female choice for male carotenoid-based coloration, indicating that a direct link between retinal accumulation and sexual selection for coloration is unlikely. However, retinal carotenoid accumulation in males was positively correlated with their plumage coloration. Thus, carotenoid-mediated visual health and performance or may be part of the information encoded in sexually selected coloration.

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Created

Date Created
  • 2011