Browsing by Author "Olson, John S"

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    Amniotic Fluid-Derived Stem Cells as a Source of In Situ Vascularization within Fibrin/Poly(Ethylene Glycol) Hydrogels
    (2014-12-04) Benavides, Omar M; Jacot, Jeffrey G; Fraser, Charles D; Moake, Joel L; Olson, John S
    One of the greatest challenges in regenerative medicine is providing a significant source of vascularization within engineered tissues. Successful vascularization requires both a scaffold that supports vessel formation and a reliable source of vascular cell types. Broad potential for differentiation, high proliferation rates, and autologous availability for neonatal applications make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. We utilized chemical-mediated differentiation of AFSC into endothelial-like cells (AFSC-EC), which expressed key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability while retaining key benefits of fibrin-based scaffolds – quick polymerization, high biocompatibility, and vasculogenic stimulation. AFSC-EC as a vascular cell source and AFSC as a perivascular cell source were compared to established sources of these cell types – human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. In vitro, cell-seeded hydrogels were assessed based on network formation, including parameters such as vessel thickness, length, and area. The development of robust vessels required the presence of both an endothelial and a perivascular cell source and was seen in AFSC co-cultures. Additionally, the co-culture of AFSC with AFSC-EC resulted in a synergistic effect on network parameters similar to MSC. Based on this data, we hypothesized that subcutaneously injecting similar hydrogels in immunodeficient mice would both induce a fibrin-driven angiogenic host response and promote in situ AFSC-derived neovascularization. Two weeks post-injection, AFSC-seeded hydrogels demonstrated significantly higher vascular lumen formation versus those without cells or those seeded with endothelial cells alone; a subset of these lumen were characterized by the presence of red blood cells, suggesting anastamosis with host vasculature. In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydroge In support of the Pediatric Cardiovascular Bioengineering Lab’s global vision, this research demonstrates that AFSC-seeded fibrin/PEG hydrogels have the potential to serve as a vascularized platform for the development of an engineered cardiac patch to be used in autologous repair of congenital heart defects.
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    Cellular fitness as a proxy to determine the physicochemical parameters of an antibiotic efflux pump
    (2016-12-02) Perez, Anisha M; Shamoo, Yousif; Olson, John S
    Determining the quantitative link between protein function and cellular fitness can be challenging as even the modestly sized genome of Escherichia coli is comprised of thousands of genes. Using an appropriate survey of fitness across a range of selective conditions, we can reduce the complexity of this system by tightly linking cellular fitness to the function of one protein essential for growth within that selective environment. Several model proteins have been studied in this fashion whereby in vitro protein parameters are used to predict cellular fitness as a function of selection strength. Underlying this approach, however, is the idea that the reverse relationship is also true: analysis of cellular fitness can be used to predict protein physicochemical properties. In this study I present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the tetracycline efflux pump, TetB, and a family of strategically chosen single amino acid variants. TetB is a member of the Major Facilitator Superfamily (MFS) of transporters which have a conserved protein fold and for which we have a general understanding of how protein structure relates to function. We first performed growth rate analysis on our host strain without tet(B) at a wide range of drug concentrations to obtain global parameters that describe the baseline response of our cellular system. Growth analysis was also performed on strains expressing a chromosomal copy of tet(B) or variant allowing for a quantitative measurement of the fitness effects produced by TetB. Using both sets of fitness data and in vivo protein concentration, our model was able to predict physicochemical pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants which match the current knowledge of how MFS transporter structure influences function. Taken together, this study shows that cellular fitness in strong selective conditions can be used to characterize efflux pumps, a class of proteins which are classically challenging to characterize using classical in vitro biochemistry techniques. Additionally, this analysis opens up the possibility of characterizing protein libraries from high-throughput growth rate assays.
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    Initiation of RNA polymerization and polymerase encapsidation by a Picobirnavirus
    (2015-01-15) Collier, Aaron Michael; Tao, Yizhi Jane; Stewart, Charles R; Olson, John S; Prasad, B.V. V
    During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How these RdRPs molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions that have highly variable answers depending on the virus family being examined. In this study, we have determined a 2.4 Å resolution structure of an RdRP from a human infecting strain of picobirnavirus (PBV). In addition to a conserved polymerase fold, the PBV RdRP possesses a unique, highly flexible 24-aa loop structure (aa495-518) located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays have shown that the wild-type RdRP is capable of initiating RNA synthesis using a de novo mechanism, while a mutant RdRP lacking the loop structure could only synthesize RNA through back-priming, suggesting that the loop likely functions as a platform for the priming nucleotide to bind. Unexpectedly, co-expression of the PBV RdRP with its respective capsid protein (CP) indicated that the PBV RdRP could not be incorporated into recombinant capsids in the absence of the viral genome. Additionally, the PBV RdRP exhibited a high affinity towards the conserved 5’-terminal sequence of the viral genome, suggesting that PBV RdRP molecules are likely packaged through their specific binding to viral RNA during assembly.
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    Understanding dynamic activities of the yeast galactose utilization network under environmental changes
    (2016-01-27) Nguyen, Truong Huu; Bennett, Matthew R; Beckingham, Kathleen M; Olson, John S; Segatori, Laura; Tao, Yizhi Jane
    Cellular adaptability to environmental changes depends on the collective actions of genes, mRNA, proteins and ligands, all of which are components of a ''genetic network''. To understand the dynamics of a gene network in response to temporally and spatially environmental changes, we focus on the galactose utilization network in the yeast Saccharomyces cerevisiae. This network allows yeast cells to metabolize galactose in the absence of glucose and is tightly repressed when glucose is available in the environment. The main question is how the Gal network is activated when glucose is depleted since both sugars cannot be metabolized simultaneously. Using a microfluidic device, we supplied yeast cells with both glucose and galactose before linearly depleting glucose at different rates. We tracked the onset and accumulation of a yellow fluorescent reporter-tagged Gal1p, the first enzyme of the Gal network. Our data shows that the glucose-depletion rate plays an important role in the activation of the Gal network. The onset of the network's activation depends on the time it takes to pass a specific threshold of the glucose concentration. On the other hand, the full induction of the Gal network, represented by the Gal1-accumulation time, is strongly influenced by the depletion rates. In particular, the mean of the Gal1-accumulation time increases significantly when glucose is depleted instantaneously. Furthermore, the variability of the Gal1-accumulation time also increases in short depletion rates and achieves a minimum at intermediate depletion rates. Using a mathematical simulation, we demonstrate that the increase in the accumulation time is due to the loss of energy when glucose is instantaneously depleted. This loss of energy also correlates with the length of diauxie, a period of catabolic transition from glucose to a secondary carbon source. Thus, changes in the glucose-depletion rate not only affect the dynamics of the Gal network's activation, but can also affect the phenotypic outcomes of the single cells within the population. Our results contribute to growing sets of evidence that a gene network can exhibit complex, dynamic behaviors under environmental changes to shape the fitness and survival of individual and collective members of a microbial population.