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  • Undergraduate Poster Abstracts
  • Bioengineering/Biomedical Engineering

    THU-716 CARDIAC PROPAGATION ON THE HEART-ON-A-CHIP

    • Adrian Ledesma-Mendoza ;
    • Francesco Pasqualini ;
    • Kevin Parker ;

    THU-716

    CARDIAC PROPAGATION ON THE HEART-ON-A-CHIP

    Adrian Ledesma-Mendoza1, Francesco Pasqualini2, Kevin Parker2.

    1The University of New Mexico, Albuquerque, NM, 2School of Engineering and Applied Sciences, Harvard University, Boston, MA.

    Arrhythmia, or irregular heartbeat, is caused by an alteration in the electrical conduction system in the heart. Healthy cardiac tissue is composed of aligned cardiac muscle cells, or cardiomyocytes, that are activated sequentially. In diseased tissue, cardiomyocytes change their structure and organization into a misaligned pattern that leads to conduction disturbances. The effects of cell alignment on cardiac impulse propagation are difficult to investigate in traditional in vitro systems where cardiomyocytes are cultured in random orientations. We previously utilized micro-contact printing techniques to control the structural organization of cardiomyocytes in the heart-on-a-chip assay. Here, we coupled this engineered in vitro platform with a computational model of cardiac propagation to investigate how the distribution of gap junctions, protein complexes involved in electrical coupling, change with tissue alignment, and modulate electrical propagation. Taken together, our results indicate that the heart-on-a-chip is an agile platform to investigate the functional implications of changes occurring in cardiac tissue structure.

    FRI-712 ISOLATION OF SALT-TOLERANT CELLULASE-PRODUCING HALOPHILES FOR BIOBUTANOL PRODUCTION IN HALOFERAX VOLCANII

    • Jocelyn Simlick ;
    • David Bernick ;

    FRI-712

    ISOLATION OF SALT-TOLERANT CELLULASE-PRODUCING HALOPHILES FOR BIOBUTANOL PRODUCTION IN HALOFERAX VOLCANII

    Jocelyn Simlick1, David Bernick2.

    1Hartnell College, Salinas, CA, 2University of California, Santa Cruz, Santa Cruz, CA.

    Production of butanol via microbiological metabolic processes is being investigated as an alternative for fossil fuels. Haloferax volcanii is an ideal microbe that can be utilized in breakdown and fermentation of saline-treated plant matter (cellulose rich biomass) for butanol biofuel production. Our study will focus on finding halophiles with cellulases that can fold and work in high saline environments. The International Genetically Engineered Machine (iGEM) field team has collected samples from the salt ponds near Fremont, California, and performed single species isolations from colonies that have shown robust growth. The inoculation and enrichment process was performed to select for cells that will produce a cellulase using microcrystalline cellulose as the sole carbon source in their growth media. Next, we will test for glucose abundance in order to select for high activity cellulases. From there, we will identify the species by using its small subunit rRNA and perform polymerase chain reaction (PCR) to amplify regions that are specific for archaea. The amplified DNA will be sequenced to identify species. If species have previously been identified, we will use BLAST (a database that compares and correlates similar sequences) to identify target cellulase enzymes and attempt to transform Haloferax volcanii. If we find a novel organism, we can then use whole-genome sequencing to produce a draft sequence of its genome and learn about its cellulose and carbohydrate metabolizing capabilities.

    FRI-728 ACOUSTO-MAGNETOPHORETIC ENRICHMENT FOR MULTIPLEX BIOSEPARATIONS

    • Robert Gutierrez ;
    • Korine Ohiri ;

    FRI-728

    ACOUSTO-MAGNETOPHORETIC ENRICHMENT FOR MULTIPLEX BIOSEPARATIONS

    Robert Gutierrez1, Korine Ohiri2.

    1University of California, Irvine, Irvine, CA, 2Duke University, Durham, NC.

    Magnetic bio-sensing beads have been used in lab-on- chip technologies for decades, leading to breakthroughs in areas such as personalized medicine, cell biology, and biotechnology. Many existing magnetophoretic devices, however, perform bioseparations using a single mechanism (i.e., a non-uniform magnetic field), thus causing the simultaneous sorting of multiple targets at high levels of purity, recovery, and throughput to remain a challenge. To address this problem, we have designed a magneto-elastomeric bio-sensing particle (mNACP) with tunable mechanical and magnetic properties. These particles exhibit a negative acoustic contrast factor (i.e., agglomerate to the antinodes of a pressure standing wave) and are thus capable of being differentially sorted using magnetic trapping and an acoustic standing wave from non-labeled cells and commercial magnetic labeling beads (mPACP) that exhibit positive acoustic contrast factors. To demonstrate this, we separated and enriched a complex mixture of unlabeled U-937 white blood cells, 10 wt. % magnetite mNACP, and mPACP by serially sorting with Magnetic- Activated Cell Sorting (MACS®) technology and an acoustofluidic chip driven at 2.43 MHz. Sorting purities were determined using fluorescence imaging.

    THU-714 PATTERNING VCAM-1 ACROSS GLASS SUBSTRATES TO STUDY MONOCYTE CAPTURE

    • Shane Hoang ;
    • Levi Gheber ;
    • Scott Simon ;

    THU-714

    PATTERNING VCAM-1 ACROSS GLASS SUBSTRATES TO STUDY MONOCYTE CAPTURE

    Shane Hoang1, Levi Gheber2, Scott Simon1.

    1University of California, Davis, Davis, CA, 2Ben-Gurion University of the Negev, Beersheba, IL.

    Atherosclerosis, a condition of heart disease where arteries become clogged by plaques of fatty material, is one of the leading causes of death worldwide. One of the precursors to this condition is believed to be the recruitment of monocytes, a subset of white blood cells that are crucial to the innate immune system. During atherosclerotic events, monocytes are quickly recruited to the blood vessel by binding to VCAM-1, a cell adhesion molecule, leading to their activation. Once activated, the monocytes differentiate into macrophages and elicit an immune response to the arterial plaque. However, monocytes have been shown to bind to the arterial lining much longer than expected. Studying the details of monocyte binding to VCAM-I may shed light on the mechanisms involved in this abnormal recruitment. We are approaching this study by patterning spots of VCAM-1 with various distances and concentrations on a glass cover slip, shearing whole blood across the surface, and analyzing the number of monocytes captured on the VCAM-1 pattern. The purpose of our study is to isolate specific parameters that govern monocyte adherence to the substrate. Preliminary results have shown that we have been able to deposit VCAM-1 onto a cover slip in a desired pattern that will be suitable for shearing blood across. Future studies may provide insight on the correlation between monocyte recruitment and the stages of heart disease.

    THU-706 CREATING UNIFORM CELL-EMBEDDED MATRIX SPHERES FOR A NEW TUMOR MODEL

    • Jason Velasquez ;
    • James Freyer ;

    THU-706

    CREATING UNIFORM CELL-EMBEDDED MATRIX SPHERES FOR A NEW TUMOR MODEL

    Jason Velasquez, James Freyer.

    Center for Biomedical Engineering, The University of New Mexico, Albuquerque, NM.

    The in vitro cell culture models used to study cancer do not monitor 3D chemical gradients in real time. Obtaining chemical microenvironment information from the in vitro tumor spheroid model requires destructive techniques, and the spatial resolution of these measurements is poor. Therefore, we have designed a new tumor model that will overcome these limitations. Our model uses a perfusion chamber and cell-embedded matrix spheres with integrated chemical nano-sensors (CEMS) to generate chemical gradients across larger distances. We use an aqueous 2-phase separation system consisting of alginate, polyethylene glycol, dextran, Pluronic F-127, and calcium chloride to generate CEMS. Fluid containing cells flows through a nozzle at the end of a capillary, and acoustic modulation generates approximately 20,000 droplets per second. The alginate droplets are then solidified by crosslinking with calcium, producing CEMS. The sphericity of these droplets is crucial for representing the system with a mathematical model and for maintaining uniform packing density. Improvement in the sphericity of the droplets has been observed by adjusting concentrations of the chemicals used to stabilize the interface between the chemical phases. In order to quantify the uniformity of ALG droplets, samples were imaged and analyzed using ImageJ to estimate the mean and standard deviation of the CEMS volume. We are functionalizing the ALG with fluoresceinamine using carbodiimide chemistry, yielding CEMS whose fluorescence intensity is proportional to pH. This allows us to measure pH gradients across the perfusion device based on changes in fluorescence intensity as a function of distance from the nutrient supply.

    FRI-709 MEASURING THE TEMPERATURE FIELD OF AN OPTICAL THERMOCAVITATION BUBBLE USING PLANAR LASER-INDUCED FLUORESCENCE

    • Vicente Robles Jr. ;
    • Darren Banks ;

    FRI-709

    MEASURING THE TEMPERATURE FIELD OF AN OPTICAL THERMOCAVITATION BUBBLE USING PLANAR LASER-INDUCED FLUORESCENCE

    Vicente Robles Jr.1, Darren Banks2.

    1Pomona College, Santa Ana, CA, 2University of California, Riverside, Riverside, CA.

    Cavitation has developed from an undesirable, damaging phenomenon toward one with applications in the biomedical and fluid-control fields. Optical thermocavitation is a process in which a laser is focused into an absorptive liquid, causing it to superheat. This leads to the creation, growth, and collapse of a vapor bubble. Applications such as laser-assisted surface cooling enhancement and skin-poration by optical cavitation are being developed in the lab of Dr. Aguilar (University of California, Riverside). The former seeks to achieve cooling through induced mixing while the latter is a drug delivery method that punctures the outer layer of skin. To explore the dynamics of bubble formation and effects on surroundings, a technique is being developed to measure the temperature field around a cavitation bubble. The non-intrusive technique is called planar laser-induced fluorescence. It uses rhodamine B in an aqueous copper nitrate (CuNO4, 24% by mass in water) solution. Rhodamine B fluoresces when exposed to a 440 nm wavelength laser. We developed a calibration curve relating the measured intensity with temperature. The fluorescence intensity was measured against temperature in 10 ℃increments from 25 ℃ to 75 ℃, and a linear relationship was found. Fluorescent intensity decreases by approximately 10% for each interval. Then, the optimal concentration of rhodamine B in the solution was determined. At room temperature, we found that, as the concentration went from 1 droplet to 5 droplets per 10 mL of aqueous CuNO4, the average fluorescence increased by about 50 (RGB-pixel average). The next step is implementation of a carbon-nanotube solution as the absorption medium and perfecting the image analysis algorithm.

    THU-720 CHARACTERIZATION OF A PROTEIN BLOCK COPOLYMER FOR DRUG DELIVERY APPLICATIONS

    • Kyle Okino ;
    • Jing Chen ;
    • Joseph Frezzo ;
    • Cynthia Xu ;
    • Jin Kim Montclare ;

    THU-720

    CHARACTERIZATION OF A PROTEIN BLOCK COPOLYMER FOR DRUG DELIVERY APPLICATIONS

    Kyle Okino1, Jing Chen2, Joseph Frezzo2, Cynthia Xu2, Jin Kim Montclare2.

    n/a

    The protein block copolymer CE2-RGD was developed in order to create a novel vehicle for drug delivery applications. This protein block copolymer is comprised of 2 functional domains: 1) a coiled-coil domain (C) derived from the cartilage oligomeric matrix protein (COMP) capable of encapsulating small hydrophobic molecules; and 2) sequential elastin-like peptide domains (E) that impart concentration-dependent thermoresponsiveness. The protein block copolymer has also been modified by site-directed mutagenesis to display the integrin-binding tripeptide sequence (Arg-Gly-Asp) for tumor cell targeting. The protein was expressed recombinantly by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and purified by immobilized metal ion affinity column chromatography. The thermoresponsive behavior of the protein was measured by UV-Vis spectroscopy yielding a transition temperature of 60.65 oC. The secondary structure was confirmed by circular dichroism (CD) spectroscopy. Finally, G25 sephadex size exclusion chromatography results confirmed the consistent encapsulation of the chemotherapeutic doxorubicin at 20.56 +/-0.54 nanograms drug per total milligram of protein block copolymer.

    FRI-718 MULTIPLEXED MEMBRANE BILAYERS ON CARBOXYLATED POLYSTYRENE MICROSPHERES FOR FLOW CYTOMETRY

    • Mirella Galvan-De La Cruz ;
    • Nadiezda Fernandez Oropeza ;
    • Nesia Zurek ;
    • Steven Graves ;
    • Andrew Shreve ;

    FRI-718

    MULTIPLEXED MEMBRANE BILAYERS ON CARBOXYLATED POLYSTYRENE MICROSPHERES FOR FLOW CYTOMETRY

    Mirella Galvan-De La Cruz, Nadiezda Fernandez Oropeza, Nesia Zurek, Steven Graves, Andrew Shreve.

    The University of New Mexico, Albuquerque, NM.

    Lipid membranes hold the contents of cells, help maintain a homeostatic environment within the cell, play an important role in cell signaling, and contain membrane-bound proteins that are the targets of most drugs. The main components of lipid membranes are lipids, sterols, and proteins, all of which work together to provide biological functions. Thus, in the context of developing assay and drug-screening technologies, it is important to study all the components of membranes. The systems currently available to study membranes and membrane proteins in these types of applications have limitations: they are not multiplexable or high throughput and they are not fully biomimetic since they often present portions of membrane proteins removed from their natural membrane environment. These limitations can lead to false positives and negatives during the drug-screening process. For all these reasons, we are developing a high-throughput, multiplexable, biomimetic, membrane-protein assay for flow cytometry. We will present initial work toward these goals showing development and characterization of a lipid bilayer on top of neutrally buoyant, multiplexed, carboxylated polystyrene microspheres. In ongoing work, we are continuing to develop more complex lipid bilayer assemblies for use in multiplexed, peripheral membrane-protein binding assays. This work will serve as a building block to develop multiplexed biomimetic membrane-protein assays for flow cytometry that can be used for drug discovery.

    THU-709 GETTING PAST THE GUARDS: A NOVEL LOOK INTO THE ENDOCYTIC MECHANISMS OF PARTICLES ACROSS THE BLOOD BRAIN BARRIER

    • Gauree Chendke ;
    • Tyler Brown ;
    • Samir Mitragotri ;

    THU-709

    GETTING PAST THE GUARDS: A NOVEL LOOK INTO THE ENDOCYTIC MECHANISMS OF PARTICLES ACROSS THE BLOOD BRAIN BARRIER

    Gauree Chendke, Tyler Brown, Samir Mitragotri.

    University of California, Santa Barbara, Santa Barbara, CA.

    Current treatments for neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, still have not reached their full potential. With most of these treatments, the drug cannot reach the brain in high enough concentrations to provide a significant therapeutic effect. One major obstacle for treating diseases of the brain is the presence of the blood brain barrier (BBB), composed of a tightly connected network of endothelial cells. To overcome this barrier, nanoparticles can be used. Nanoparticles are ideal drug delivery agents since it is relatively easy to manipulate their size, flexibility, and surface chemistry. However, the ideal nanoparticle geometry for penetrating each biological barrier may vary. Therefore, we wish to investigate how the shape and size of nanoparticles affects the mechanism by which the nanoparticles cross the BBB. Using mouse brain endothelial cells (bEnd.3), we blocked different pathways of internalization to understand how a variety of fluorescently labeled nanoparticles of different shape and size are initially endocytosed. Sucrose, nystatin, and amiloride hydrochloride were each used to induce a different type of endocytosis, including clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Particle internalization was assessed quantitatively with fluorescence spectroscopy and confirmed visually with confocal microscopy. Herein, we have outlined a platform of particle geometries for optimum internalization and hope to understand the effects of attaching targeting ligands, such as transferrin receptor antibodies, to the surface of the nanoparticles in the future.

    THU-719 INVERSE FINITE-ELEMENT ANALYSIS OF COMPRESSED MURINE LENSES FOR MECHANICAL INSIGHTS INTO PRESBYOPIA

    • Andre Cleaver ;
    • Luis Rodriguez ;
    • Matthew Reilly ;

    THU-719

    INVERSE FINITE-ELEMENT ANALYSIS OF COMPRESSED MURINE LENSES FOR MECHANICAL INSIGHTS INTO PRESBYOPIA

    Andre Cleaver, Luis Rodriguez, Matthew Reilly.

    The University of Texas at San Antonio, San Antonio, TX.

    Presbyopia is the gradual loss of near vision with age. The ability of the ocular lens to accommodate for near vision declines by midlife. This decline is caused by lens stiffening. Lens stiffness can be directly measured using compression testing. However, stiffness is an extrinsic metric: it depends on lens size and shape and cannot be used to compare lenses from animals of different species, ages, or genotypes. To overcome this difficulty, we propose a mechanical model of lens compression to determine the elastic modulus of the lens, which is an intrinsic measurement of stiffness. To test this proposal, a mechanical model was developed to determine the elastic moduli of the lens’ nucleus and cortex by analyzing the force-displacement data from compression testing of 17-week-old mouse lenses. Both experimental data and mechanical model predictions showed similar trends. Improved agreement between experimental data and modeling predictions was achieved by solving the model using the inverse finite-element method in which regression analysis was used to optimize the elastic moduli of the lens’ nucleus and cortex to match the force required to achieve 10% axial compression. This model will be used to provide better insight on how specific proteins contribute to developing presbyopia. (UTSA supports this study, partially funded by GM060655.)

    THU-729 IDENTIFICATION OF CELLULOSE DEGRADING HALOPHILES FOR BUTANOL PRODUCTION VIA HALOFERAX VOLCANII

    • Adriana Landeros ;
    • David Bernick ;

    THU-729

    IDENTIFICATION OF CELLULOSE DEGRADING HALOPHILES FOR BUTANOL PRODUCTION VIA HALOFERAX VOLCANII

    Adriana Landeros, David Bernick.

    University of California, Santa Cruz, Santa Cruz, CA.

    Today our energy needs are met by burning ancient deposits found deep within the earth: fossil fuels. Haloferax volcanii is a potential host microorganism that can metabolize simple sugars found in plant, paper, and food waste that may be usable for butanol production. However, the enzymes that can break down cellulose, a complex sugar, have not been found in this organism. The focus of this study is to find novel isolates and develop enriching processes for those that can make use of cellulose as an energy source. In our project, 10 samples were collected from hypersaline ponds at a site at the southern end of San Francisco Bay, a part of the Don Edwards National Wildlife refuge. Once collected, samples were grown in different types of media to test for their robustness and ability to use soluble and insoluble microcrystalline cellulose as their primary carbon source. Next, samples were analyzed using microscopy to confirm cell growth after approximately 14 days. We will then identify the species of our isolates and follow that with whole genome sequencing as a means to find the sequences of their salt-tolerant cellulases. These sequences can then be used to transform H. volcanii to enhance its capabilities in butanol production.

    THU-717 NEW DESIGN OF A PNEUMATICALLY POWERED MRI-COMPATIBLE DEVICE FOR STROKE REHABILITATION

    • Jesus Partida ;
    • Melih Turkseven ;
    • Jun Ueda ;

    THU-717

    NEW DESIGN OF A PNEUMATICALLY POWERED MRI-COMPATIBLE DEVICE FOR STROKE REHABILITATION

    Jesus Partida, Melih Turkseven, Jun Ueda.

    George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA.

    Hemiparesis is muscular weakness on one side of the body and is often experienced by stroke survivors when brain cells are deprived of oxygen. This condition impacts their quality of life, and rehabilitation must be sought to improve the symptoms. The previous project achieved a pneumatically powered magnetic resonance imaging (MRI) compatible robot that practiced a specific rehabilitation procedure. This procedure is meant to promote the connection between the individual’s motor cortex and damaged upper limb. This is done by delivering mechanical stimuli to the individual’s upper limb. However, the previous prototype was not space-efficient. This summer a new device was prototyped to address this critical issue. The new device is composed of 2 MRI-compatible pneumatic cylinders. In addition, the device is MRI compatible due to the use of incorporating exclusively nonmagnetic material into the design. These parts include 3D-printed parts made of acrylonitrile butadiene styrene (ABS), bearings made of plastic and glass, a hammer tip composed of synthetic rubber, a plastic shaft, and nylon gears and screws. Space efficiency is achieved through a new mechanical transmission where 2 cylinders are positioned horizontally and used to rotate a bar on a shaft to deliver a blow to the individual’s upper limb. The effectiveness of the design will be experimentally confirmed in the future.

    FRI-720 CELL SIFTER: TOWARD HIGH-THROUGHPUT, SEMIAUTOMATED NANOBIOPSY AND NANOMANIPULATION OF SINGLE T CELLS

    • Rolando Cruz Perez ;
    • Nader Pourmand ;

    FRI-720

    CELL SIFTER: TOWARD HIGH-THROUGHPUT, SEMIAUTOMATED NANOBIOPSY AND NANOMANIPULATION OF SINGLE T CELLS

    Rolando Cruz Perez, Nader Pourmand.

    University of California, Santa Cruz, Santa Cruz, CA.

    The nanopipette, a quartz capillary tube with a nanometer-sized pore, has shown great promise for single-cell analysis with sub-cellular spatiotemporal resolution, but has only been used with adherent cells. A method of capture and immobilization is needed to interrogate nonadherent cells with the nanopipette. The focus of this research is a microfluidic device called the cell sifter. The device enables the capture and release of nonadherent single cells floating in cell media while maintaining viability. The device was fabricated using semiconductor processing techniques and consists of a membrane perforated by a 6 x 6 array of 2-micrometer diameter through-holes. Applying a negative pressure to the device captures and immobilizes single cells at the through-holes. Trypsinized HeLa cells and T cells were stained to confirm post-capture viability via fluorescence microscopy while supplying negative pressures ranging from 0.5 - 2 kilopascals (kPa) for a period of 20 minutes. Subpopulations of mitochondria were nanobiopsied from captured HeLa cells, mitochondrial DNA was amplified via polymerase chain reaction (PCR), and gel-electrophoresis confirmed the presence of the desired products. Nanobiopsies were taken from captured T cells, followed by complementary DNA synthesis. PCR was performed with primers for CD4, CD8, and beta-actin genes. Gel-electrophoresis confirmed the presence of the desired products. To our knowledge, single-cell nanomanipulation and nanobiopsy of non-adherent cells has not been reported. This system will help further elucidate single-cell biology and has many attractive applications such as cellular engineering for cell therapies.

    THU-713 REAL-TIME, LENSFREE, DIGITAL HOLOGRAPHIC MICROSCOPY

    • Kyrollos Yanny ;
    • Patrick Wolf ;
    • Hardik Patel ;
    • Zoltan Gorocs ;
    • Aydogan Ozcan ;

    THU-713

    REAL-TIME, LENSFREE, DIGITAL HOLOGRAPHIC MICROSCOPY

    Kyrollos Yanny, Patrick Wolf, Hardik Patel, Zoltan Gorocs, Aydogan Ozcan.

    University of California, Los Angeles, Los Angeles, CA.

    We demonstrate a novel instrument for real-time digital holographic microscopy on a chip. Our lensfree color holographic video-microscope is capable of performing volumetric imaging of fluidic samples that are pumped through a microfluidic chamber. The chamber is positioned directly on the top of a color CMOS-image sensor chip and is illuminated by a multimode fiber optic cable that is coupled to a high-power RGB LED, creating a uniform illumination that is spatially and temporally partially coherent. Lensfree holograms are captured as a function of time, imaging approximately 24 microliters of liquid per frame, and are then reconstructed to obtain visual information from the flowing objects located at different distances from the image sensor chip. By implementing our reconstruction algorithm in CUDA C, we have achieved a 55-fold speed increase compared to a CPU, yielding color reconstruction of the whole field of view (approximately 30 mm2) at a desired height in less than 30 ms, thereby allowing real time screening of our sample volume at a rate of 5 frames per second. We also designed an illumination circuit that emits a flash pulse to control the high power RGB LED, which allows us to image constantly moving objects in liquid samples without motion blur artifacts, thus allowing us to examine approximately100 mL of liquid sample per hour.

    THU-715 APPLICATION OF NANOPARTICLE CATALYSTS IN BIOMEDICINE: HETEROGENEOUS PHASE PARA-HYDROGEN INDUCED POLARIZATION IN WATER

    • Roberto Naranjo Jr. ;
    • Louis Bouchard ;

    THU-715

    APPLICATION OF NANOPARTICLE CATALYSTS IN BIOMEDICINE: HETEROGENEOUS PHASE PARA-HYDROGEN INDUCED POLARIZATION IN WATER

    Roberto Naranjo Jr., Louis Bouchard.

    University of California, Los Angeles, Los Angeles, CA.

    Magnetic resonance imaging (MRI) is a medical imaging technique featuring excellent soft-tissue contrast but suffers from low sensitivity, preventing the detection of metabolites in low concentrations. Para-hydrogen induced polarization (PHIP) is a signal enhancement technique that creates nonequilibrium nuclear spin polarizations from para-hydrogen. It can be used to develop contrast agents for in vivo molecular imaging. An area of interest is the study of cell metabolism. In order to use the PHIP technique as efficiently as possible, a nontoxic heterogeneous catalyst is needed that can catalyze reactions in a biocompatible solvent and be separated from the polarized product. Without a biocompatible environment, animal or human studies are not possible. Our lab has synthesized both platinum (Pt) and palladium (Pd) nanoparticles (NPs) capped with glutathione ligands that are both water soluble and in a heterogeneous phase. We have achieved proton (1H) polarization enhancement of P = 0.25% with the hydrogenation of 2-hydroxyethyl acrylate to hydroxyethyl propionate using the Pt NPs. We are currently exploring the possible difference in reaction rate between Pt and Pd to achieve higher 1H polarization levels. This could lead to an effective method of monitoring metabolism in cells and to new capabilities for diagnostic imaging.

    THU-712 PREVENTING GENETIC SUSCEPTIBILITY TO CEREBRAL MALARIA: NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE GENE RENDERS MICE RESISTANT TO CEREBRAL MALARIA

    • Nasim Eshragh Nia ;
    • Pedro Cabrales ;

    THU-712

    PREVENTING GENETIC SUSCEPTIBILITY TO CEREBRAL MALARIA: NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE GENE RENDERS MICE RESISTANT TO CEREBRAL MALARIA

    Nasim Eshragh Nia, Pedro Cabrales.

    University of California, San Diego, La Jolla, CA.

    Cerebral malaria (CM), the most severe and potentially deadly neurological complication of infection by the malaria parasite Plasmodium falciparum, is characterized by hypoxia, increased inflammatory responses, and leukocyte adhesion. Although the exact pathogenesis of CM is still unknown, previous research on mice has shown that resistance to CM is particularly affected by gene products that pertain to immune responses. One such gene is Nicotinamide nucleotide transhydrogenase (NNT) which has been shown to have modulatory effects on immune and inflammatory responses. C57BL/6J mice have a gene deletion at the NNT locus, while the C57BL/6NJ strain does not have this gene deletion and, thus, we hypothesize it is resistant to CM infection. Using a closed cranial window model, intravital microscopy techniques were used to quantify hemodynamic changes, oxygen tension (pO2), and blood pH in vivo in infected and uninfected models. Preliminary analysis showed marked decrease in blood flow, vascular pO2, and hemoglobin levels in both models, with a lower percent decrease in all the aforementioned parameters for C57BL/6NJ. C57BL/6J mice showed greater extent of leukocyte adherence in postcapillary venules than did C57BL/6NJ. Neurological scores and survival rates were also higher for C57BL/6NJ, which can be attributed to better oxygen delivery and preservation of brain tissue perfusion in this strain. In conclusion, quantitative evaluation of pial hemodynamics in vivo and resistance of C57BL/6NJ to CM can be a strong indication of the role of the NNT gene and its protein products in regulation of inflammatory responses, complementing our understanding of cerebral malaria pathogenesis.

    THU-718 COMPARATIVE GENOMIC ANALYSIS OF HUMAN NAIVE AND MEMORY B CELL IMMUNOGLOBULIN SWITCH REGIONS

    • Theron Palmer Jr ;
    • Christopher Vollmers ;

    THU-718

    COMPARATIVE GENOMIC ANALYSIS OF HUMAN NAIVE AND MEMORY B CELL IMMUNOGLOBULIN SWITCH REGIONS

    Theron Palmer Jr, Christopher Vollmers.

    University of California, Santa Cruz, Santa Cruz, CA.

    Isotype class switching must occur in order to adapt to the various biological elements of pathogens. The constant regions of B cells’ immunoglobulin heavy chain loci contain several repetitive elements, called switch regions, which are located upstream from the isotype exons. These switch regions are used as recombination sites that save isotype genetic information from being lost due to the DNA cleavage induced by the RAG protein complex during isotype-class switching. Improper recombination of B-cell switch regions could result in the loss of isotype information which will prevent further isotype class switching necessary for the body to adapt to the biological components of pathogens. The recombination of these repeating switch regions that occurs during isotype class switching has not yet been analyzed genetically. In order to better understand the nature of isotype class switching in relation to switch regions, we created a novel assay that amplified the naïve B-cell constant region and the alpha, gamma, and epsilon memory B-cell isotype-switched constant regions. Next, we will sequence the constant region amplicons using the Oxford nanopore MinIon sequencer in collaboration with the lab of Dr. Akeson. By computational analysis, we can then infer the switch regions of both naïve and memory (before and after isotype class switching) B cells looking for switch region recombination patterns during isotype class switching.

    FRI-707 DNA MOLECULAR LOGIC CIRCUITS FOR PATHOGEN DETECTION IN SERUM SAMPLES

    • Dominic Medina ;
    • Adan Myers y Gutierrez ;
    • Matthew Lakin ;
    • Darko Stefanovic ;
    • Steven Graves ;

    FRI-707

    DNA MOLECULAR LOGIC CIRCUITS FOR PATHOGEN DETECTION IN SERUM SAMPLES

    Dominic Medina1, Adan Myers y Gutierrez2, Matthew Lakin3, Darko Stefanovic2, Steven Graves1.

    1Center for Biomedical Engineering, The University of New Mexico, Albuquerque, NM, 2The University of New Mexico, Albuquerque, NM.

    RNA viruses are one of the largest classes of pathogenic organisms in humans. Current viral detection largely uses variants of PCR as the primary mode of detection, making it a relatively expensive process and difficult to perform. To overcome these limitations, we are adapting modular DNA-based molecular logic circuits for RNA virus detection, and in particular, dengue virus. These circuits are protein free, isothermal, and only require synthetic nucleic acids to function making them inexpensive and convenient for use as pathogen detection assays. The circuits work by toehold mediated strand displacement in which a catalytic DNA is released from inhibition and cleaves a FRET reporter molecule. The resulting fluorescence is then easily, quickly, and inexpensively detected by portable machines. Many technical hurdles need to be overcome before these circuits can be used in real-world assays. Methods must be developed to allow the circuits to run in samples containing increasing amounts of serum. In order to mimic infection, we are adding target to bovine serum that will be treated through the same processes that human samples are treated. The assay will not run unless zinc has been added to the system. To ensure specificity, we are conducting a cross-talk test in which the specific target is removed from the system and similar targets are added. Thus far, our system has demonstrated high specificity. We will then make biochemical changes to our system in order to make the biosensor’s reactions more favorable in this real-world simulation.

    FRI-713 NOVEL OSTEOGENIC SCAFFOLDS WITH BIOMIMETIC MINERALIZATION FOR BONE REGENERATION

    • Jasmine King ;
    • Sergio Montelongo ;

    FRI-713

    NOVEL OSTEOGENIC SCAFFOLDS WITH BIOMIMETIC MINERALIZATION FOR BONE REGENERATION

    Jasmine King, Sergio Montelongo.

    The University of Texas at San Antonio, San Antonio, TX.

    The theory of bone regeneration after a fracture is consequential because it can alleviate the problems of bone replacements and can potentially lead to the regeneration of other tissues. Bone regeneration is a natural, physiological process that is observed in the healing of mundane fractures. This project is investigating ways of accelerating this process by scaffolding. For efficient bone regeneration, a sufficient amount of stem cells need to be recruited to fracture sites. Osteogenic stem-cell activity will be quantified in the periosteum versus endosteum of the cortical bone in an established rat model. Periosteal cells derive primarily from mesenchymal stem cells (MSCs), while endosteal cells come from adjacent bone marrow cells (BMCs). In this study, we used a mineralized versus nonmineralized approach for the 2 different types of scaffolds: collagen + hydroxyapatite scaffold (CHS) and collagen scaffold (CS). A scaffold was placed in the femur and MSC and BMC recruitment will be quantified 4 and 8 weeks post implantation. The density of stem cells recruited for periosteum versus endosteum will be calculated and compared for 4-week CSs, 4-week CHSs, 8-week CSs, and 8-week CHSs. The expected outcome of this experiment is that the periosteum at 8-weeks of CHS groups will have the greatest density of stem cells. This finding could be monumental in the world of orthopedics and other applications where fractures are recurrent. (Partially funded by GM007717.)

    FRI-714 SELF-AMPLIFYING ANTIBODY-DRUG CONJUGATES

    • Chantel Charlebois ;
    • Jessica McCombs ;
    • Shawn Owen ;

    FRI-714

    SELF-AMPLIFYING ANTIBODY-DRUG CONJUGATES

    Chantel Charlebois1, Jessica McCombs2, Shawn Owen2.

    1Kate Gleason College of Engineering, Rochester Institute of Technology, Rochester, NY, 2The University of Utah, Salt Lake City, UT.

    Antibody-drug conjugates (ADCs) are a novel form of biotherapeutics, desirable for their ability to couple the targetability of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. Limitations of current ADCs prevent therapeutic levels of drug reaching the cytosol and cause a loss of drug potency due to conjugation. To overcome this challenge, we have engineered an ADC that will induce a self-amplifying feedback loop, therefore allowing for greater drug accumulation in the lysosome. Our ADC uses the mAb trastuzumab (Herceptin®) that binds to HER2, an abundant surface antigen present on target cancer cells. We chose the drug geldanamycin (GA), a potent Hsp90 inhibitor. Hsp90 is a molecular chaperone that is crucial to the endosomal recycling pathway that returns internalized HER2 to the cell surface. By interrupting endosomal recycling of HER2 with GA, we postulate that trastuzumab-GA (Tras-GA) conjugates will induce a self-perpetuating feedback loop allowing for greater drug accumulation in the lysosome. A noncleavable Tras-GA was prepared by conjugating the linker SMCC to native lysines on trastuzumab followed by reacting tras-SMCC with modified, thiol-icontaining GA. Preliminary cell trafficking studies suggest that after a 24 hour treatment, our noncleavable Tras-GA is internalized more than trastuzumab alone. We are building additional ADCs with cleavable linkers to further augment GA efficacy and incorporating second drugs to induce pathways of synthetic lethality.

    FRI-716 THE DESIGN AND OVEREXPRESSION OF ALDEHYDE-ALCOHOL FUSION GENES IN HALOFERAX VOLCANII FOR THE CONVERSION OF BUTYRYL-COA TO BUTANOL

    • Dominic Schenone ;
    • David Bernick ;

    FRI-716

    THE DESIGN AND OVEREXPRESSION OF ALDEHYDE-ALCOHOL FUSION GENES IN HALOFERAX VOLCANII FOR THE CONVERSION OF BUTYRYL-COA TO BUTANOL

    Dominic Schenone, David Bernick.

    University of California, Santa Cruz, Santa Cruz, CA.

    Using fossil fuels as the world's dominant source of energy is no longer sustainable and has led to lasting negative effects on the environment and irreversible changes to our climate. As a result, the development of energy-dense carbon neutral biofuels has gained increased research interest. Butanol is one possible solution. Butanol is a 4-carbon alcohol that can be metabolized from glucose and from cellulose, a glucose polymer. In this study, we continue work done previously to engineer a glucose to butyryl-CoA pathway in the halophilic archaeon Haloferax volcanii. Research is now being done to quantify the presence of butyryl-CoA and butanol in the engineered mutant, and to design aldehyde-alcohol fusion genes to convert butyryl-CoA to butanol. We are developing several approaches for the design and quantification of the fusion genes. These include the identification, optimization, and overexpression of fusion genes found in other halophilic organisms and the creation of fusion genes using native aldehyde and alcohol genes (aldy3, aldy5, and adh2) from H. volcanii. There is evidence that H. volcanii under acidogenic conditions of pH = 6.5 activates these same native genes and we are working to detect the existence of butanol under those conditions. We are developing plasmid constructs using an overexpression plasmid via Gibson assembly which will be transformed into H. volcanii. Once successful transformants are found, an in vitro protein assay will be performed on the expressed proteins to quantify their activity with butyryl-CoA as the substrate.

    THU-727 UNDERSTANDING THE PARACRINE ROLE OF ADIPOSE STEM CELLS ON MACROPHAGE PHENOTYPE

    • Alejandra Preciado ;
    • Kabir S Dhada ;
    • Laura J Suggs ;

    THU-727

    UNDERSTANDING THE PARACRINE ROLE OF ADIPOSE STEM CELLS ON MACROPHAGE PHENOTYPE

    Alejandra Preciado1, Kabir S Dhada2, Laura J Suggs2.

    1University of California Merced, Merced, CA, 2The University of Texas at Austin, Austin, TX.

    Peripheral artery disease is a type of cardiovascular disease caused by the narrowing of arterial veins, which prevents blood flow and oxygen from reaching certain extremities of the body. Progression of the disease leads to a high risk of critical limb ischemia, which can ultimately result in the permanent loss of a limb. Although revascularization procedures promote the restoration of blood flow, it invokes additional oxidative stress and acute vascular damage to neighboring muscle. This calls for an effective treatment for acute vascular damage caused by revascularization procedures for peripheral artery disease. A promising treatment is implantation of stem cells. Mesenchymal stem cells (MSCs), which are multipotent stem cells derived from bone marrow (BM-MSCs) and adipose tissue (A-MSCs), are known to promote the secretion of angiogenic growth factors. Macrophages are divided into 2 essential phenotypes that describe the immune system regulators: M1 phenotype promotes inflammation and M2 phenotype promotes anti-inflammation and modulates the healing process. The main objective of the present study is to understand the cross talk and mechanistic role that MSCs play in the infiltration of the macrophage phenotype. We hypothesize that stem cells can control the phenotypic switch towards M2 macrophages by delivering stem cells at the right conditions. The present study demonstrates how an in vitro 3D hypoxic environment influences the secretion of factors that promote macrophage chemotaxis when compared to previous in vitro 2D studies. PCR extraction will be conducted in order to test for specific M1 (IL-1β, TNF-alpha, i-Nos, IL-12) and M2 (IL-10, Arg-1, PPAR, IGF-1) cytokines.

    FRI-719 SKELETAL BONE LOSS IN RESPONSE TO REMOTE BONE DEFECT HEALING

    • Alejandro Morales Betancourt ;
    • Mark Appleford ;

    FRI-719

    SKELETAL BONE LOSS IN RESPONSE TO REMOTE BONE DEFECT HEALING

    Alejandro Morales Betancourt, Mark Appleford.

    The University of Texas at San Antonio, San Antonio, TX.

    In the U. S., 2.4% of the population suffers bone fractures annually. During the fracture healing process, the lacunar-canalicular network (LCN) triggers basic multicellular units (BMUs) that are composed of osteoclasts and osteoblasts. BMUs are responsible for the activation-resorption-formation sequence, also called remodeling. Initial fractures could lead to secondary fractures due to loss of bone density, but the underlying mechanism relating them to the healing process is unclear. This study strives to find a correlation between bone density reduction and LCN activity to maintain skeletal homeostasis. A critical-sized (6 mm) defect was created in the right femur of 42 rats. The animals were treated by 1 of 3 implants with varying mineral concentration and evaluated after 2 and 4 weeks. Eight bones were harvested from each rat at different skeletal sites. An early assessment of the 42 samples using microCT revealed that bone loss occurred primarily in long bones. Samples were treated with histological techniques. Cellular mechanisms will be evaluated by quantifying bone remodeling parameters using BioQuant software. The expected results are that remodeling activity will be greater in those areas that presented a reduction in bone density. This will support the hypothesis of a correlation between local bone defect healing and loss of bone density due to comprehensive skeletal remodeling activity performed through the LCN. This investigation will provide the possibility of anticipating mineral deficit at certain skeletal sites as a result of fracture healing and the potential ability to alleviate remote secondary fracture risks through techniques such as calcium supplementation.

    THU-728 GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) PROMOTION OF SPERMATOGENIC SURVIVAL OR REGENERATION AFTER HIGH-DOSE ALKYLATING CHEMOTHERAPY

    • Travis Kotzur ;
    • Jennifer Mecklenburg ;
    • Brian Hermann ;

    THU-728

    GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) PROMOTION OF SPERMATOGENIC SURVIVAL OR REGENERATION AFTER HIGH-DOSE ALKYLATING CHEMOTHERAPY

    Travis Kotzur, Jennifer Mecklenburg, Brian Hermann.

    The University of Texas at San Antonio, San Antonio, TX.

    Though chemotherapy and radiation treatments for cancer are lifesaving, they introduce a lifetime of side-effects for survivors, including male infertility. Male infertility occurs after cancer because the therapeutics for cancer also kill the spermatogonial stem cells (SSCs) responsible for ongoing sperm production in adults. Fertility in adults can be preserved by cryopreserving sperm before sterilizing therapies, but there are no approaches for preserving fertility in pre-pubertal male cancer patients who are not yet making sperm. We recently reported a novel approach that preserves spermatogenesis after alkylating chemotherapy (busulfan) using injections of granulocyte colony-stimulating factor (G-CSF). The purpose of the current study is to investigate mechanisms by which G-CSF promotes spermatogenic recovery after busulfan treatment, either by promoting survival of SSCs or stimulating regeneration of spermatogenesis from surviving SSCs and to determine the dosage which promotes maximal spermatogenic recovery. Five-week old male C57BL/6 mice were injected with G-CSF at varying dosages before and/or after busulfan (or vehicle control) treatment and the testes were evaluated 10 weeks later for the degree of spermatogenesis. Histological analysis of spermatogenesis in these testes is ongoing. We expect the results of this study will help clarify how G-CSF protects SSCs from damage by alkylating chemotherapy, which is critical to developing its use as a clinical adjuvant treatment for infertility after cancer in boys. [Supported by NIH grants HD062687 and HD078916 to BPH, the Minnie and Max Tomerlin Voelcker Fund, the Helen Freeborn Kerr Charitable Foundation, UTSA, and UTSA MARC U*STAR research training program (NIH GM007717).]

    FRI-706 STRUCTURAL CHARACTERIZATION OF THE HUMAN METAPNEUMOVIRUS FUSION PROTEIN

    • Jocelyn Campos ;
    • Rebecca DuBois ;

    FRI-706

    STRUCTURAL CHARACTERIZATION OF THE HUMAN METAPNEUMOVIRUS FUSION PROTEIN

    Jocelyn Campos, Rebecca DuBois.

    University of California, Santa Cruz, Santa Cruz, CA.

    Human metapneumovirus (HMPV) is a human pathogen and is one of the most prevalent causes of acute respiratory tract infection worldwide. HMPV is a single-stranded negative sense RNA virus that contains a lipid envelope studded with three surface glycoproteins (F, SH, G). The F protein exists in 2 conformations classified as the pre-fusion and post-fusion forms. Upon triggering cell attachment, the F protein irreversibly shifts to its post-fusion form and performs the membrane fusion event between virus and host cell membranes, resulting in the delivery of the viral genome inside the host cell. Our hypothesis is that engineering a stabilized pre-fusion F protein will allow us to analyze its crystal structure and can be used as an effective vaccine against HMPV. We have produced genetic constructs of the HMPV F gene, and cloned them into expression plasmids. The plasmids will be transfected into human embryonic kidney (HEK) 293 cells as a eukaryotic cell system is preferred in folding the HMPV F protein, which forms trimer containing disulfide bonds and glycosylation modifications. Expression will be confirmed by western blot and the protein will be purified using affinity chromatography on strep-tactin columns. We expect to successfully express and purify the F protein stabilized in its pre-fusion conformation. Validating the presence of pre-fusion F protein will be done through several biochemical analysis tools, including electron microscopy (EM), small angle X-ray scattering (SAXS), and X-ray crystallography. HMPV has a major impact on global health; therefore, it is a good target for preventive and therapeutic measures.

    FRI-729 CAMOUFLAGED LUMBRICUS TERRESTRIS EARTHWORM HEMOGLOBIN: A POTENT HUMAN RED BLOOD CELL SUBSTITUTE WITH UNIQUE BIOPHYSICAL PROPERTIES

    • Alborz Jelvani ;
    • Pedro Cabrales ;

    FRI-729

    CAMOUFLAGED LUMBRICUS TERRESTRIS EARTHWORM HEMOGLOBIN: A POTENT HUMAN RED BLOOD CELL SUBSTITUTE WITH UNIQUE BIOPHYSICAL PROPERTIES

    Alborz Jelvani, Pedro Cabrales.

    University of California San Diego, La Jolla, CA.

    In the U. S., the need for human red blood cells (RBCs) transfusion has steadily increased over the years. Statistically in the U. S., about 12 million RBC transfusions are performed each year; however, transfusion of RBCs is not yet the best method for replacing lost blood components since it may increase the risk of many disease transmissions. Lumbricus terrestris is a species of earthworm that has a type of acellular hemoglobin (Hb) known as erythrocruorin (LtEc) naturally assembled with a high molecular weight of 3.6 MDa, which is very stable, resistant to autoxidation, has lower nitric oxide (NO) dioxygenation activity, and efficiently transports oxygen (O2) to tissues. These characteristics render LtEc a potent candidate for an Hb-based oxygen carrier (HBOC). The purpose of this study is to determine the systemic and microvascular hemodynamic changes and pharmacokinetics parameters of LtEc coated with polyethylene-glycol (PEG). We hypothesize that PEG conjugation to LtEc will have a longer circulation time preserving all the other functional aspects of the LtEc. Tangential flow filtration was used to purify the LtEc, which was then infused consecutively to increase the plasma protein concentration level in hamsters from 0.5 to 1.5 g/dL. The hematocrit percentage, plasma LtEc and PEG-LtEc concentration level, heart rate, mean atrial pressure, microvascular hemodynamics and blood flow, and noncompartmental variables of plasma LtEc and PEG-LtEc were all quantified. Preliminary data analysis showed that PEG-LtEc circulates 4 times as long as LtEc while preserving all other functional aspects of LtEc.

    FRI-708 ROOT MIMETIC SYSTEM

    • Christopher Capitan ;
    • Darcie Christensen ;
    • David Britt. Anne Anderson ;

    FRI-708

    ROOT MIMETIC SYSTEM

    Christopher Capitan, Darcie Christensen, David Britt. Anne Anderson.

    Utah State University, Logan, UT.

    Microorganisms are everywhere, including the plant’s rhizosphere zone. The goal of our project was to understand more completely the relationship between plants and bacteria, by building a device that can transport nutrients though fibers to grow biofilm. We used hollow membrane filters that would act like a root, releasing small compounds (i.e., nutrients) through their pores. We used a strong polymer to hold 6 fibers to a microscope slide and built-up compartments to create inlet and outlet chambers into which tubes were cemented, allowing nutrient flux. The system was sealed by gluing a cover glass over the tubes and fibers. The inlet tube was attached to a syringe pump containing Luria broth medium as a source of diffusible nutrients. The fibers were inoculated with cells of the beneficial root-colonizer, Pseudomonas chlororaphis O6. The device was wrapped in a paper towel saturated with distilled water to maintain moisture. After 48 h, growth of bacterial biofilm was observed on the outer surface of the fibers. The orange color of the biofilm cells was characteristic of production of antibiotic phenazines from the PcO6. Culturing from the cells on the outside of the fibers showed no contamination of the biofilm with any other microbes. Our experiment successfully demonstrated a working prototype for the root mimetic. Future use will increase the understanding of the interaction of the bacterium with compounds exuded by the root, a process vital to their beneficial response in overcoming plant stress.

    FRI-715 NON-INVASIVE CRICOTHYROID ELECTROMYOGRAPHY: AN ALTERNATIVE METHOD FOR DIAGNOSING PARKINSON'S DISEASE THROUGH SPEECH

    • Gladys Ornelas ;
    • Todd Coleman ;

    FRI-715

    NON-INVASIVE CRICOTHYROID ELECTROMYOGRAPHY: AN ALTERNATIVE METHOD FOR DIAGNOSING PARKINSON'S DISEASE THROUGH SPEECH

    Gladys Ornelas, Todd Coleman.

    University of California, San Diego, La Jolla, CA.

    Parkinson’s disease (PD) is a neuromuscular disorder characterized by a sudden death of dopamine-generating cells in the brain. The cricothyroid (CT) muscle in the larynx is affected by PD and becomes hyperactive leading to speech impediments in patients. The presence of CT hyperactivity has been observed at every stage in PD using invasive needle-based electromyography (EMG). These results suggest that monitoring the EMG of the CT muscle may provide an early diagnosis of PD. The purpose of this study is to determine if a noninvasive surface electromyography recording of the CT muscle could be used to define differences between a normal functioning CT muscle and the hyper-functioning CT muscle found in PD patients. The experimental procedure will test both healthy subjects and patients diagnosed with PD. EMG frequencies during phonation of a high /i/ sound will be compared to those from a low /i/ phonation. Spectrogram analysis will be used to extract the various frequency components of the EMG signal across time, which is effective in demonstrating differences in signal frequencies. Preliminary results have demonstrated that CT muscle hyperactivity can be detected noninvasively in both healthy and afflicted patients. Further testing is in progress to validate previous results and this EMG method as a new diagnostic tool for PD patients. If confirmed, noninvasive early CT muscle EMG detection will enable better treatment and an improved quality of life for PD patients.

    THU-707 QUARTZ NANOPIPETTE FOR INTRACELLULAR SUPEROXIDE SENSING

    • Joanna Perez ;
    • Rifat Ozel ;
    • Nader Pourmand ;

    THU-707

    QUARTZ NANOPIPETTE FOR INTRACELLULAR SUPEROXIDE SENSING

    Joanna Perez, Rifat Ozel, Nader Pourmand.

    University of California, Santa Cruz, Santa Cruz, CA.

    It is well understood that reactive oxygen species (ROS), including superoxide anions, are generated as byproducts in a wide variety of physiological pathways and are controlled by cellular antioxidant mechanisms. However, their overproduction can inflict oxidative damage to proteins, cells, and tissues, responses that are all known to be associated with the pathogenesis of a range of neurodegenerative disorders. Understanding the relationship between these disorders and superoxide can hold the key to the development of innovative therapies for combating neural degeneration in the cell. Previously used methods have been indirect, ambiguous, and incapable of performing real-time measurements. We have developed a novel technique for sensing superoxide radical anions using functionalized nanopipettes fabricated from single-barrel quartz capillaries. The nanopipette undergoes a series of modifications on its inner surface, including the immobilization of cytochrome-c. Cytochrome-c allows for the direct measurement of superoxide levels in the cell based on the redox reaction between cytochrome-c and the generated superoxide-radical anions. These electrochemical biosensors not only enable the quantitative analysis of superoxide levels in a single cell but also the monitoring of these levels over a period of time.

    FRI-717 IMPROVING WOUND HEALING THROUGH THE USE OF TETRAZINE-MODIFIED ALGINATE

    • Esmarline J. De Leon-Peralta ;
    • Brian Kwee ;
    • Yevgeny Brudno ;
    • David Mooney ;

    FRI-717

    IMPROVING WOUND HEALING THROUGH THE USE OF TETRAZINE-MODIFIED ALGINATE

    Esmarline J. De Leon-Peralta1, Brian Kwee2, Yevgeny Brudno2, David Mooney2.

    1University of Puerto Rico, Mayaguez Campus, Mayaguez, PR, 2Harvard University, Cambridge, MA.

    Ischemic diseases, such as myocardial infarction and peripheral arterial disease, are a leading cause of death globally. There has been increasing interest in treating these diseases with factors that induce angiogenesis: the growth of new blood vessels from pre-existing blood vessels. We have recently shown that alginate linked to tetrazine helps induce a greater recovery in blood perfusion compared to unmodified alginate in a murine model of hindlimb ischemia. The objective of this project is to understand how this biomaterial induces recovery in blood perfusion. Our hypothesis is that tetrazine acts directly on endothelial cells to induce angiogenesis and also activates immune cells that are pro-angiogenic. To validate our hypothesis, we have performed in vitro angiogenesis assays to study if tetrazine-modified alginate induces endothelial cell sprouting. Additionally, we have performed histology and immunohistochemistry on ischemic muscles from our in vivo experiments to see if this biomaterial increases blood vessel density, blood vessel remodeling, and the presence of pro-angiogenic immune cells. Our findings may lead to a novel method for treating people with ischemic diseases.

    THU-705 DEVELOPMENT OF TARGETED MESOPOROUS SILICA SUPPORTED LIPID BILAYER NANOPARTICLES FOR DELIVERY OF NUCLEIC ACID CARGO

    • Ayse Muniz ;
    • Amanda Lokke ;
    • Paul Durfee ;
    • Kimberly Butler ;
    • Jason Townson ;
    • Yu-Shen Lin ;
    • Jeffrey Brinker ;

    THU-705

    DEVELOPMENT OF TARGETED MESOPOROUS SILICA SUPPORTED LIPID BILAYER NANOPARTICLES FOR DELIVERY OF NUCLEIC ACID CARGO

    Ayse Muniz1, Amanda Lokke1, Paul Durfee1, Kimberly Butler1, Jason Townson2, Yu-Shen Lin2, Jeffrey Brinker1,3.

    1The University of New Mexico, Albuquerque, NM, 2Oncothyreon Incorporated, Seattle, WA, 3Sandia National Laboratories, Albuquerque, NM.

    Gene therapy remains a promising approach for treatment of a diverse range of diseases. However, providing reliable, cell-specific delivery of nucleic acids to cells in vivo remains a significant challenge. Improvement of delivery efficacy likely requires greater control over cargo stability, cellular specificity, gene integration, and expression. Current non-viral methods of in vivo nucleic acid delivery in mice such as cationic liposomes, polymers, peptides, or other nanoparticle-based complexes, have emerged as safer and cheaper alternatives to their viral counterparts. However, maximizing efficiency and specificity while minimizing toxicity using these methods is currently limited. Mesoporous silica nanoparticles (MSNPs) serve as an attractive option for targeted interaction with cells due to their ability to undergo surface modification while maintaining stability. We have modified MSNPs with 8 nm pore sizes to efficiently encapsulate and deliver nucleic acid cargos including DNA, siRNA, and mRNA to HeLa cells in vitro and highly vascularized chicken embryo models in vivo. Variables including nanoparticle shape, charge and size, and various liposomal formulations were explored to understand their effects on nucleic acid delivery. We demonstrate in vitro plasmid transfection efficiencies greater than 80% after 48 hours, greater than 95% efficiency for mRNA delivery, and 50% gene knockdown with siRNA delivery using our MSNP platform. It remains the goal of this research to modify MSNPs to deliver nucleic acids to targeted cells with high efficiency in vivo. By developing a universal nucleic acid carrier, current delivery methods could be dramatically improved to achieve higher specificity and lower toxicity.

    THU-703 MAKING BIOLOGICAL SENSE OF IMPORTANT GENES IN BREAST CANCER AND THEIR COORDINATED BEHAVIOR: INITIAL RESULTS

    • Camille Marrero ;
    • Mauricio Cabrera ;

    THU-703

    MAKING BIOLOGICAL SENSE OF IMPORTANT GENES IN BREAST CANCER AND THEIR COORDINATED BEHAVIOR: INITIAL RESULTS

    Camille Marrero, Mauricio Cabrera.

    University of Puerto Rico, Mayaguez Campus, Mayaguez, PR.

    Breast cancer is the most commonly diagnosed cancer among women in the U.S. Abnormal patterns of genetic expression are among the multiple factors that contribute to the occurrence of breast cancer. This work aims to provide biological evidence of genes that are deemed important to the presence and development of this illness, as well as their coordinated behavior. In our research group, the selection of candidate genes and the determination of their global correlation are carried out through mathematical optimization techniques applied to microarray experiments. These techniques include modeling gene coexpression with the travelling salesman problem (TSP) and, alternatively, with the minimum spanning tree (MST). Biologically speaking, it is first important to establish whether the results obtained through these methods are viable, and then to uncover interactions that could potentially lead to cause-effect relationships. These 2 tasks are approached in our work in breast cancer.

    THU-704 ENGINEERING CELL-CELL COMMUNICATION USING SIGNAL SENSING WITH CHEMICALLY INDUCED PROXIMITY TECHNOLOGY

    • Maria Alvarez ;
    • Guihua Zeng ;
    • Roushu Zhang ;
    • Fu-sen Liang ;

    THU-704

    ENGINEERING CELL-CELL COMMUNICATION USING SIGNAL SENSING WITH CHEMICALLY INDUCED PROXIMITY TECHNOLOGY

    Maria Alvarez, Guihua Zeng, Roushu Zhang, Fu-sen Liang.

    The University of New Mexico, Albuquerque, NM.

    Non-specific drug development in present drug markets causes different unwanted side effects in the body. Engineering a therapeutic system with higher specificity addresses this off target problem. This approach combines reactivity based OH signal sensing with chemically induced protein proximity technology. By using rapamycin as a chemical inducer, we can change chemically modified abscisic acid from an inactive form to an active, pro-drug form. This active form will diffuse to another cell and act as an inducer leading to expression of GFP. This strategy can also be used to induce EGF, which acts as an inducer to generate hydrogen peroxide leading to nuclear export in a cell. Engineering different cellular pathway communication models can lead to future development of gene therapy.

    FRI-703 RECOGNITION OF SPECTRALLY AND AUDIBLY VARIED STIMULI IN THE PRESENCE OF COMPETING STIMULI

    • Moriah Garcia ;
    • Edward Bartlett ;
    • Aravindakshan Parthasarathy ;

    FRI-703

    RECOGNITION OF SPECTRALLY AND AUDIBLY VARIED STIMULI IN THE PRESENCE OF COMPETING STIMULI

    Moriah Garcia, Edward Bartlett, Aravindakshan Parthasarathy.

    Purdue University, West Lafayette, IN.

    Listening is a complex process in which one must discriminate specific sounds from competing stimuli in conditions where ambient noise is often louder than the target sound. In these conditions, temporal processing deficits due to age make it difficult for the listener to distinguish the target sound. The purpose of this study was to determine how changing the spectral content and signal to noise ratio (SNR) of a target sound in the presence of a masker would influence the recognition of this sound. Five subjects sought to recognize a target sound in the presence of a competing sinusoidally amplitude-modulated tone. SNRs of 60, 50, 40, 30, 20, 10, 5, 0, -5, and -10 were used to vary the sound levels of the tones, while carrier frequencies of 2,000 Hz, 2,500 Hz, and 3,143 Hz were used to vary the spectral content. Recognition of the target decreased with an increase in SNR, showing a sharper decrease when the tones had similar carrier frequencies. This indicates that as ambient noise increases, the ability of a listener to recognize their sound of interest decreases, especially for noises that are spectrally similar to their target. This suggests that for a listener, both increasing the sound level and differing the carrier frequency of the sound of interest are key to gaining recognition of that sound in the presence of competing stimuli. This knowledge is of great importance when designing hearing aids, showing that they should acknowledge the frequencies that the user struggles to distinguish in addition to increasing sound levels.

    FRI-704 THE IMPLICATIONS OF E-CIGARETTES ON PLATELET COMPLEMENT ACTIVATION: ARE THEY REALLY SAFE?

    • Sarah Georges ;
    • Wei Yin ;

    FRI-704

    THE IMPLICATIONS OF E-CIGARETTES ON PLATELET COMPLEMENT ACTIVATION: ARE THEY REALLY SAFE?

    Sarah Georges, Wei Yin.

    Stony Brook University, Stony Brook, NY.

    In the U.S., cigarette smoking is responsible for 1 in 5 deaths annually. Smoke from tobacco cigarettes contains acetone, acetic acid, arsenic, formaldehyde, nicotine, and tar among other things. In an attempt to create a product that is less harmful to the body, e-cigarettes were developed and have become popular recently. The major components of e-cigarette juice include propylene glycol, glycerin, water, and nicotine. However, the effects of e-cigarettes on public health, especially on the cardiovascular system, have not been thoroughly studied. To understand the effects of e-cigarettes on the cardiovascular system, we investigated how platelet complement activation was affected by e-cigarette smoke extract, and compared that to pure nicotine using an enzyme-linked immunosorbent assay (ELISA). Complement activation is an important contributor to the immune responses. We have reported earlier that platelets can support complement activation to completion, and tobacco smoke can enhance platelet complement activation in vitro. We hypothesized that e-cigarettes may enhance platelet complement activation depending on their nicotine content. Following a 2-hour treatment of e-cigarette extract, platelet surface C1q, C4d, and C3b deposition was measured using a solid-phase ELISA. Preliminary results demonstrated that e-cigarettes could increase complement activation, especially C1 activation. These results suggested that e-cigarettes may play a negative role in immune responses associated with the cardiovascular system.

    THU-708 MECHANISMS OF COLLAGEN TYPE-1 ASSEMBLY AND ALIGNMENT THROUGH INDUCED FLUID SHEAR IN LAMELLAR BONE

    • Hernan Paz ;
    • Mark Appleford ;

    THU-708

    MECHANISMS OF COLLAGEN TYPE-1 ASSEMBLY AND ALIGNMENT THROUGH INDUCED FLUID SHEAR IN LAMELLAR BONE

    Hernan Paz, Mark Appleford.

    The University of Texas at San Antonio, San Antonio, TX.

    Mechanically competent human bone contains collagen fibrils that are aligned in parallel layers with alternating directions. The restoration of this alignment is responsible for successful outcomes in bone fracture healing. The mechanism responsible for this alignment is poorly understood. We propose a study in which human osteoblasts cultured on an aligned collagen substrate will be exposed to variable environmental conditions in order to identify the mechanism underlying development of 3D collagen fibril structures in lamellar bone. A perfusion bioreactor will be used to induce variable media flow conditions during culture of the osteoblast construct and compared to static control. The tested variables will include fluid shear rate and direction of media flow relative to the direction of the aligned collagen platform. Measurement of the alignment and composition of subsequently secreted collagen matrix produced by the osteoblasts will be performed using atomic force microscopy, scanning electron microscopy, and immunofluorescence techniques. We anticipate shear rate and direction will influence subsequent collagen alignment based on correlation analysis. The cellular mechanisms for procollagen formation as tested by immunofluorescence may help identify the subcellular protein complex and organization of the secreted matrix. Variables such as fluid shear may influence directionality during osteoblast secretion of its collagen matrix. If highly aligned fibrils can be developed, bioreactor-cultured bone could be used for tissue engineered grafts. Successful culture of aligned bone is especially needed for large, segmental bone injuries that cannot be bridged by traditional metal plating.