Aptamers against viral proteins as a tool to answer biological questions

2019 ◽  
Author(s):  
◽  
Carolina Camargo
Keyword(s):  
Lentiviral Vector ◽  
Rna Aptamers ◽  
Target Cells ◽  
University Of Missouri ◽  
Practical Applications ◽  
Intracellular Expression ◽  
The University ◽  
Further Development ◽  
Viral Surface

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] There are two experimental chapters in this dissertation in which the fundamental questions center around aptamers and viruses and how these two concepts interlace. The first experimental chapter (chapter two) seeks to utilize previously characterized aptamers against HIV-1 Reverse Transcriptase (RT) that will be delivered by a lentiviral vector and which intracellular expression from different human promoters was evaluated. The goal of this study was to identify important elements of vector design that will impact transgene expression in target cells. This study was based on the hypothesis that intracellular expression of RNA aptamers delivered by a lentiviral vector could offer a platform to enable adequate aptamer expression that would translate into viral suppression. And the second experimental chapter (chapter four) describes an in vitro 2'FY-RNA selection against Filoviral glycoproteins and outlines three different strategies that were followed to achieve selection of specific aptamers. Aptamers described in this chapter were able to recognize Ebolavirus glycoprotein ectodomain as well as in its native conformation displayed on the viral surface. Taking the observations obtained in this dissertation, aptamer technology could be expanded into further development for practical applications.

Advances in aptamer evolution and engineering

2016 ◽  
Author(s):  
◽  
Khalid Kamal Alam
Keyword(s):  
High Throughput Sequencing ◽  
Target Selection ◽  
Three Dimensional ◽  
Rna Aptamers ◽  
Hiv Reverse Transcriptase ◽  
University Of Missouri ◽  
Selection Approach ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.

Sequence and secondary structural analysis of a novel class of RNA aptamers that inhibit reverse transcriptase

2012 ◽  
Author(s):  
◽  
Angela Whatley
Keyword(s):  
Reverse Transcriptase ◽  
Rna Aptamers ◽  
University Of Missouri ◽  
Hiv Rna ◽  
Cellular Assays ◽  
Subtype B ◽  
Single Target ◽  
Ic50 Values ◽  
The University ◽  
Major Target

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] RNA aptamers are synthetic RNAs selected for their ability to bind a molecular target. Reverse Transcriptase (RT) is the major target of antiretroviral therapy used in the treatment of HIV. RNA aptamers were selected to bind to RT of HIV-1 subtype B. Previous work showed aptamers from this selection inhibited various RT enzymatic activities, and have also been shown to inhibit viral replication in cellular assays. Most of the aptamers from this selection have predicted pseudoknot structures. The aptamers were classified into Pseudoknot families. Family 1 Pseudoknots (F1Pk) have a conserved UCCG sequence in stem one. Family 2 Pseudoknots (F2Pk) aptamers have CYGG (Y is pyrimidine either C or U). Aptamer secondary structural families are correlated with inhibitory behavior. F2Pk aptamers inhibit a broader range of RT subtypes compared to F1Pk. In the present work we present results of a screen into the inhibitory behavior of a novel family of anti RT aptamer from a previously well- studied pool. We found a group of aptamers that inhibit RT from subtype B very well and have novel secondary structure. These aptamers fold into stem loops with a highly conserved UCAA bulge. Aptamers in the UCAA family inhibit RT very well in primer extension assays and have IC50 values as low as 1.6nM. This work highlights the diversity of RNA secondary structures that are able to bind to a single target.

Ribozymes and aptamers in the RNA world, and in synthetic biology

2016 ◽  
Author(s):  
◽  
Raghav Raj Poudyal
Keyword(s):  
Synthetic Biology ◽  
Genetic Information ◽  
Rna World ◽  
Information Storage ◽  
Rna Aptamers ◽  
Darwinian Evolution ◽  
University Of Missouri ◽  
Biologically Relevant ◽  
Functional Rnas

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The RNA world hypothesis postulates that Ribonucleic Acids (RNA) may have provided functions of catalysis and genetic information storage during the origin of life on earth. An RNA based life is hypothesized to have undergone Darwinian evolution to ultimately lead into extant biology, where DNA is used as the repository for genetic information and proteins are used as biological catalysts. The discovery of functional RNAs such as catalytic RNAs, regulatory RNAs, and ligand-binding RNA aptamers further strengthen this hypothesis. These functional RNAs are also used as tools for synthetic biology and therapeutics. This work highlights strategies used by RNA enzymes (Ribozymes) for catalysis of chemical reactions, and explores new chemistries catalyzed by ribozymes. We also engineered an in vitro evolved ribozyme to control activities of other functional RNA molecules. Finally, this work explores innovative approaches to discover new RNA enzymes that catalyze biologically relevant reactions. Findings from these studies have revealed potential roles of RNA enzymes during the primordial earth, and also opened doors to build RNA-based tools that regulate biological processes.

Investigating the role of arginine 21 in the structure and function of human [alpha]a-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh Shripad Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N- terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [subscript]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

254 DIFFERENTIAL TRANSCRIPTION AND CYSTOPLASMIC POLYADENYLATION ELEMENTS IN PORCINE GERMINAL VESICLE AND METAPHASE II OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 234
Author(s):  
S. Korte ◽  
G. Springer ◽  
W. Spollen ◽  
R. Patel ◽  
K. Whitworth ◽  
...  
Keyword(s):  
Expressed Sequence Tag ◽  
Germinal Vesicle ◽  
Differentially Expressed ◽  
Cdna Libraries ◽  
University Of Iowa ◽  
Cytoplasmic Polyadenylation ◽  
University Of Missouri ◽  
Exact Test ◽  
The University

Identification of transcripts produced during porcine oocyte maturation is one of the first steps in defining pathways important to development. Practically, this information will aid in the refinement of in vitro culture processes, allowing for more efficient in vitro embryo production. To this end, mRNA was isolated from 50 in vitro-matured sow metaphase II stage oocytes (Bomed, Inc., Madison, WI, USA) (MIIs), 50 in vitro-matured gilt metaphase II-stage oocytes (MIIg), and 50 gilt germinal vesicle stage oocytes (GVO) by using the Dynabead® system (Dynal, Inc., Lake Success, NY, USA) and amplified by using the SMART system (Clontech, Palo Alto, CA, USA). The PCR products were ligated into a pSport vector and transformed into electro-competent E. coli. Colonies were randomly picked and sequenced at the University of Missouri DNA Core. Sequences were clustered with similar sequences derived from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Swine/) by using the tlcluster program developed at the University of Iowa. Following clustering, individual clusters in the cDNA libraries were compared by using Fisher's exact test (P < 0.01) to determine if they were differentially represented. Two sets of comparisons were performed, one between the MIIs and MIIg libraries, and another between the GVO library and the combination of both metaphase II libraries (MII). The number of clusters per number of clones in the library was 966/1668 (GVO), 458/820 (MIIg), and 158/819 (MIIs). There were 15/419 clusters that were different between the MIIs and MIIg libraries, and 26/1269 that were different between the MII and GVO libraries. Potential cytoplasmic polyadenylation elements (CPEs) identified from the literature were found in the GVO and MII libraries by using a custom pattern-matching program. Of the clusters with differential expression, 4/15 (MIIs vs. MIIg) and 7/26 (MII vs. GVO) contained CPEs. Table 1 contains a partial list of differentially expressed genes and the sequence of their cytoplasmic polyadenylation elements. Many genes were found to be differentially expressed in both (MII vs. GVO and MIIs vs. MIIg) comparisons. Collectively, these findings will facilitate the elucidation of important developmental pathways in swine and other animals. Table 1. Comparison of mRNA expression and cytoplasmic polyadenylation elements This work was partially funded by a University of Missouri Life Sciences Mission Enhancement grant and Food for the 21st Century.

379 Initial safety, efficacy, and product attributes from the SURPASS trial with ADP-A2M4CD8, a SPEAR T-cell therapy incorporating an affinity optimized TCR targeting MAGE-A4 and a CD8α co-receptor

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A404-A404
Author(s):  
David Hong ◽  
Jeffrey Clarke ◽  
Tanner Johanns ◽  
Partow Kebriaei ◽  
John Heymach ◽  
...  
Keyword(s):  
T Cells ◽  
Head And Neck Cancer ◽  
T Cell ◽  
Head And Neck ◽  
Neck Cancer ◽  
Lentiviral Vector ◽  
Cell Subset ◽  
Target Cells ◽  
T Cell Therapy

BackgroundThe ongoing SURPASS trial (NCT04044859) evaluates safety and efficacy of next-generation ADP-A2M4CD8 SPEAR T-cells co-expressing the CD8α co-receptor with the engineered MAGE-A4c1032T cell receptor (TCR).MethodsFirst-in-human trial in HLA-A*02 positive patients (pts) with advanced cancers expressing MAGE-A4 antigen by immunohistochemistry. Eligible pts undergo apheresis, T-cells are isolated, transduced with a Lentiviral vector containing the MAGE-A4c1032 TCR and CD8α co receptor, and expanded. Expansion, transduction level, cellular composition and function of the manufactured product (MP) are assessed in vitro. Prior to infusion, pts receive lymphodepletion with fludarabine 30 mg/m2/day for 4 days and cyclophosphamide 600 mg/m2/day for 3 days.ResultsAs of 16 July 2020, 5 pts (1 with MRCLS, 2 with esophagogastric junction [EGJ] cancers, 1 with ovarian cancer, and 1 with head and neck cancer) were treated with ADP-A2M4 CD8 (range ~1 to 5.7 billion transduced cells). No DLTs or SAEs have been reported. To date, 1 pt with EGJ cancer had a partial response (PR per RECIST) and has had progression-free survival >6 months. One pt with head and neck cancer also had a PR. All other pts have had best overall response of stable disease.MP expanded by an average of 15.3 fold during manufacturing (range 5.9 to 25.6-fold). On average, 43% of T-cells in the MP expressed the TCR (range 23 to 63%). The fraction of CD4+ cells in the final MP varied (range 45 to 84%). Co-expression of the MAGE-A4 TCR and CD8α in CD4+ T-cells in the patient MP enabled CD4+ T-cells to kill tumor target cells directly in vitro. MAGE-A4 expression in tumor biopsies varied (H-score range 55 to 300). Transduced T-cells were detected in peripheral blood of all pts. IFN-gamma increased transiently in the serum of 1 pt who responded.ConclusionsADP-A2M4CD8 SPEAR T-cells have shown an acceptable safety profile and pts with EGJ cancer and head and neck cancer have demonstrated evidence of antitumor activity. Translational data and early clinical results indicate that co-expression of the CD8α co-receptor on CD4+ SPEAR T-cells may increase the potency of the product by conferring additional killing activity to the helper T-cell subset. This dose escalation trial is ongoing and updated clinical and translational data will be presented.Trial RegistrationNCT04044859Ethics ApprovalThe trial was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and was approved by local authorities. An independent ethics committee or institutional review board approved the clinical protocol at each participating center. All the patients provided written informed consent before study entry.

An exploration of mechanisms for Arabidopsis TCP transcription factor activity at an interface of development and immunity

2018 ◽  
Author(s):  
◽  
Benjamin J. Spears
Keyword(s):  
Receptor Gene ◽  
Direct Interaction ◽  
Sequence Motifs ◽  
Crop Species ◽  
University Of Missouri ◽  
Agriculture And Food ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] "The work presented in this dissertation aimed to characterize new functions and regulatory targets for TCP8, potentially in both defense and development signaling networks. Using in vitro and in vivo promoter interaction screens in yeast and Arabidopsis, respectively, the PTI-related immune receptor gene EFR as well as a set of growth-related BR signaling genes were identified as regulatory targets of TCP8; these findings were verified through direct interaction assays characterization of tcp mutants for associated phenotypes. Additionally, SRFR1-interacting TCPs were shown to be post-translationally modified by SUMO proteins, alongside data suggesting that SRFR1 sequence motifs that facilitate interactions with SUMO are critical to its function. Together, these data describe novel roles for TCP8 and other class I TCPs, as well as novel regulatory mechanisms for their activities in the context of their interactions with SRFR1 and other TPR proteins. As a highly-conserved TF family among plants, including economically relevant crop species, advancing our understanding of TCP regulatory activities could eventually yield translational benefits to agriculture and food production worldwide."--Page 36-37.

Investigating the role of arginine 21 in the structure and function of human [alpha]A-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh S. Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N-terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [Alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [Alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [gamma]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

Ultrastructural Changes in Tumor Cells During Human Peripheral Blood Monocyte-Mediated Cytotoxicity

1981 ◽  
Vol 39 ◽  
pp. 452-453
Author(s):  
K. E. Muse ◽  
D. G. Fischer ◽  
H. S. Koren
Keyword(s):  
Target Cell ◽  
Human Peripheral Blood ◽  
Mononuclear Phagocytes ◽  
Ultrastructural Changes ◽  
Target Cells ◽  
Limited Information ◽  
Blood Monocytes ◽  
Tumoricidal Activity ◽  
Activated State

Mononuclear phagocytes, a pluripotential cell line, manifest an array of basic extracellular functions. Among these physiological regulatory functions is the expression of spontaneous cytolytic potential against tumor cell targets.The limited observations on human cells, almost exclusively blood monocytes, initially reported limited or a lack of tumoricidal activity in the absence of antibody. More recently, freshly obtained monocytes have been reported to spontaneously impair the biability of tumor target cells in vitro (Harowitz et al., 1979; Montavani et al., 1979; Hammerstrom, 1979). Although the mechanism by which effector cells express cytotoxicity is poorly understood, discrete steps can be distinguished in the process of cell mediated cytotoxicity: recognition and binding of effector to target cells,a lethal-hit stage, and subsequent lysis of the target cell. Other important parameters in monocyte-mediated cytotoxicity include, activated state of the monocyte, effector cell concentrations, and target cell suseptibility. However, limited information is available with regard to the ultrastructural changes accompanying monocyte-mediated cytotoxicity.

TEM study of boron additions to cobalt aluminide

1988 ◽  
Vol 46 ◽  
pp. 546-547
Author(s):  
Gerald B. Feldewerth
Keyword(s):  
High Temperature ◽  
Turbine Engines ◽  
Major Drawback ◽  
University Of Missouri ◽  
Specific Strength ◽  
Aerospace Applications ◽  
Wide Range ◽  
Ordered Alloys ◽  
The University ◽  
Very High

In recent years an increasing emphasis has been placed on the study of high temperature intermetallic compounds for possible aerospace applications. One group of interest is the B2 aiuminides. This group of intermetaliics has a very high melting temperature, good high temperature, and excellent specific strength. These qualities make it a candidate for applications such as turbine engines. The B2 aiuminides exist over a wide range of compositions and also have a large solubility for third element substitutional additions, which may allow alloying additions to overcome their major drawback, their brittle nature.One B2 aluminide currently being studied is cobalt aluminide. Optical microscopy of CoAl alloys produced at the University of Missouri-Rolla showed a dramatic decrease in the grain size which affects the yield strength and flow stress of long range ordered alloys, and a change in the grain shape with the addition of 0.5 % boron.

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