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ATTENUATION OF ACQUIRED APLASTIC ANEMIA THROUGH TREATMENT WITH LEUKADHERINSJessica Gonzalez1, Roland Jurecic2.
University of Miami, Coral Gables, FL, 2University of Miami Miller School of Medicine, Coral Gables, FL.
1 Aplastic anemia (AA) is an autoimmune form of bone marrow (BM) failure, characterized by the development of selfreactive T cells. These T cells release inflammatory cytokines and trigger the apoptosis of hematopoietic stem cells (HSCs) and progenitors and mature blood cells, resulting in severe BM aplasia and pancytopenia. We hypothesized that by targeting the cells of the innate immune system with novel immunosuppressive drugs called Leukadherins (LAs), we could reduce their abilities to function as antigen presenting cells and prevent them from activating selfreactive T cells, attenuating AA. To test the efficacy of LA treatment we used a mouse model of immune-mediated BM failure, which is induced by infusing allogeneic lymph node (LN) cells. The mouse AA model is characterized by the expansion of allo-reactive donor T cells. The AA was induced in 2 groups of mice by tail vein injection of LN cells.
The AA group of mice was not treated whereas the AA/LA mice were treated with 1 mg/kg of LA/mouse/day for 7 days after LN cell infusion. The control group of mice did not receive LN cells and was not treated with LA. The peripheral blood cells and the status of the hematopoiesis in control, AA and AA/LA mice were analyzed 14 days after LN cell infusion. The treatment with LA partially protected the hematopoietic progenitors and completely protected the HSCs in the BM. These results suggest that treatment with LA could attenuate AA by diminishing the damage to HSCs and hematopoietic progenitors.
ANALYSIS OF IMMUNE SYSTEM CELLS IN SCLEROSTIN-DEFICIENT MICEArthur Chow, Larrisha Coney, Yvette Pellman, Jennifer O. Manilay.
University of California, Merced, Merced, CA.
When sclerostin (SOST), an essential glycoprotein for proper bone formation, is absent, hyperactive bone growth occurs along with a decrease in bone marrow cavity size. Recent studies have shown that Sost has significant influence over developing bone marrow environments that sustain B cells, however, not much is known of its role in B cell function. We are investigating the effects of age on B cell function in Sost-knockout (KO) mice. Peripheral blood samples will be analyzed at set time points to observe changes in mature and immature B cell populations in Sost-KO and control wild-type B6 mice. We hypothesize that B cell populations will be altered in the absence of Sost. If this is the case, it will indicate that anti-SOST antibodies that are currently in clinical trials for treatment of osteoporosis may have unintended effects on B cell immunity. Additionally, we are testing the efficacy of zinc based fixation for flow cytometric analysis of immune cells in Sost-KO mice. Zinc based fixation can reduce research costs considerably if epitopes are not altered by the fixation. To study this, splenic cells were freshly obtained from a wild-type B6 mouse and quantitatively compared with previously fixed splenic cells in Z7 zinc buffer. We hypothesize that if the zincfixed cells express similar fluorescence intensity, then we can verify its practicality for those particular antigenic determinants. If successful, we will test antibody cocktails and apply this method to cells in the Sost-KO mice.
BIOPROSPECTING FOR BIOENERGY: BACTERIAL DEGRADATION OF LIGNOCELLULOSIC COMPOUNDSMyrna Rios1, Jean Gárcia-Díaz1, José R. Pérez-Jiménez, PhD2.
Universidad del Turabo, Gurabo, PR, 2Puerto Rico Institute for Microbial Ecology Research, Universidad del Turabo, 1 Gurabo, PR.
Oil has now become a global concern due to its high demand and political control. One of the solutions to this problem is to opt for an alternate energy source. An effective and promising proposal would be biofuel. Fuels of biological origin can replace part of the consumption in traditional fuels, such as oil or coal fossil fuels. In search of a biomass that can produce biofuel, we focus on degradation of plant residues. Our objective is to isolate bacteria capable of degrading lignocellulosic compounds. Environmental samples containing plant material were screened for putative degraders. Liquid media separately containing xylan, cellulose, and lignin was inoculated and examined daily for changes in turbidity or color. Samples of grasses and leaves reacted favorably to cellulose and lignin. Wood samples reacted positively in xylan media. Recently, these samples were inoculated in media supplemented with essential trace elements to stimulate degradation. Purification and characterization of degraders is in progress.
HOST MICRORNA PROFILES FOLLOWING CHLAMYDIA TRACHOMATIS INFECTION AND THEIR
CONTRIBUTION TO GENITAL TRACT PATHOLOGYMilytza Carrasquillo-Medina1, Tanvi Arkatkar2, Jieh-Juen Yu2, Neal Guentzel2, Rishein Gupta2, Bernard Arulanandam2.
Universidad de Puerto Rico en Humacao, Humacao, PR, 2South Texas Center for Emerging Infectious Disease and 1 Center of Excellence in Infection Genomics, University of Texas at San Antonio, San Antonio, TX.
Chlamydia trachomatis (CT) is the leading cause of sexually transmitted bacterial infections in humans and is associated with upper reproductive tract pathologies. There is increasing evidence on the role of microRNAs (miRs) in host immune regulation. Antichlamydial protective innate and adaptive immune responses lead to collateral damage in upper genital tracts with resultant inflammation, tubal occlusion, and hydrosalpinx development. Our hypothesis in this current study is that miRs involved in early genital CT infection may contribute to development of upper genital pathologies. C. muridarum (murine strain of CT) elementary bodies (5 X 104 IFU) were inoculated intravaginally in wild-type (WT) C57BL/6 mice followed by analyses at early (day 30), established (day 55), and late (day 80) stages of pathology. Total RNA was extracted and SyBr-Green based miR specific PCRs (miScript miRNA PCR, Qiagen) were performed to determine modulation of miRs upon infection. We have shown previously that immunopathologyinflammation related miRs (miRs 125b-5p, 214, 16, 135a, 183 and 30e) are modulated at days 6 and 12 post C.
muridarum challenge in WT mice and are associated with the generation of immune responses. We expect these down-selected miRs may be modulated at later stages of C. muridarum infection and lead to development of C.
muridarum associated upper genital tract pathology. Host miRs associated with early C. muridarum genital tract infection may contribute to later development of upper genital pathologies. Results from this study could form the basis for future in-depth investigations into novel host markers for CT infection and reproductive tract pathology.
MECHANISMS USED BY HELICOBACTER PYLORI TO GENERATE HELICAL CELL SHAPEMax Ruben1, Nina Salama2.
New Mexico State University, Las Cruces, NM, 2Fred Hutchinson Cancer Research Center, Seattle, WA.
1 Helicobacter pylori is a Gram-negative, helical-shaped bacterial pathogen that chronically infects the stomachs of approximately 50% of the human population and causes gastritis, peptic ulcer disease, and stomach cancer. It is thought that H. pylori’s helical cell shape aids colonization. We have characterized several cell shape determining (Csd) proteins that are essential for normal morphology, which is dictated by cell wall structure. The cell wall is composed of peptidogylcan (PG), a polymer of glycan strands crosslinked by peptide chains. While many of the Csd proteins directly modify PG, our studies focus on determining the role of two Csd proteins without predicted enzymatic activity: Csd5 and CcmA. Csd5, a predicted trans-membrane periplasmic protein, possesses an SH3 domain, which can bind another Csd protein, Csd4, in vitro. We hypothesize that Csd5 helps localize Csd4. Thus, introducing mutations in the SH3 domain via site directed mutagenesis might disrupt the Csd4-Csd5 interaction and result in changes to H. pylori’s cell morphology. We also hypothesize that CcmA, a putative cytoskeletal protein, may assist localization of other Csd proteins. We will generate a fluorescent protein fusion, CcmA-Wasabi, joined by a flexible linker peptide, to determine the subcellular localization of CcmA in relation to morphological features of the
INHIBITORY CONCENTRATION CURVES OF SILVER NANOPARTICLE SOLUTIONSJoshua Davidson, Dulce Romero-Urbina, Miguel Yacaman.
University of Texas at San Antonio, San Antonio, TX.
Silver metal has been used for its antimicrobial effects for thousands of years. Its use declined as antibiotics developed as the primary method to target microbes. Due to the rise in antibiotic resistance, the use of silver as an antimicrobial agent has reemerged. Silver nanoparticles have been a focus for new antimicrobial treatments because of the properties that distinguish them from silver ions, particularly the capacity of silver nanoparticles to damage cellular membranes. In this study several solutions containing silver nanoparticles were tested to determine their inhibitory concentrations. Several solutions with various silver nanoparticle concentrations were made and their results were compared to control solutions containing silver ions. The inhibitory concentrations were determined for cultures of Bacilus cereus, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Staphylococcus epidermis, and Candida albicans. The cultures were under treatment for 24 hours then the absorbance values of the culture were determined using an OD600 method. The measured values were used to plot an inhibition curve.
The curve was used to form a minimal inhibitory concentration and half-maximal inhibitory concentration table which outlines the concentration of silver necessary to kill 10% and 50% of the microbes, respectively. These results can be used to evaluate the use of silver nanoparticle solutions for antimicrobial treatment. (Research supported by NIGMS MBRS-RISE GM 60655.) FRI-583
MUTATIONAL ANALYSIS OF H5N1 INFLUENZA HEMAGGLUTININ: EFFECTS ON VIRAL ENTRY AND PROTEINAna Cintron, Emily Rumschlag-Booms.
Northeastern Illinois University, Chicago, IL.
Influenza A is a virus that has a broad species tropism, ranging from humans to water fowl to horses. Avian influenza subtype H5N1 (also known as bird flu) jumped the species barrier from birds to humans in 1997, resulting in the culling of millions of birds and thousands of human deaths worldwide. H5N1 avian influenza continues to reemerge and is thought to have the potential to cause the next major global influenza pandemic in humans. Though there are many factors implicated in the spread of influenza, viral entry is thought to be one of the most important. Influenza entry is mediated via binding of the viral surface glycoprotein, hemagglutinin (HA), to its receptor, sialic acid. Amino acid changes within HA have the potential to alter the receptor tropism of HA and thus, the species tropism. The purpose of our research is to generate mutations within HA and observe how each mutation affects protein expression and viral entry using a pseudotyped virus carrying a luciferase reporter gene. We used site-directed mutagenesis targeting the residues Q226L and T318I within HA. These 2 mutations were chosen based on their implications in protein stability and infection. We hypothesize that Q226L will alter the conformation of the receptor binding site, increasing entry levels in human cells by changing the species tropism from birds to humans. In addition, we hypothesize that T318I will decrease entry levels in human cells but will increase HA protein expression and stability.
UNFOLDING THE STRUCTURE OF CLOSTRIDIUM PERFRINGENSLindsey Berger, Gilbert John.
Oklahoma State University, Stillwater, OK.
Clostridium perfringens is an anaerobic, Gram-positive bacteria typically found in the human intestinal tract. C.
perfringens contains an enzyme called azoreductase (AzoC) which cleaves azo dyes. Azo dyes are widely used in many consumer applications, including the food and textile industries. Certain metabolic azo dye products can cause cancer, creating concern among the medical and environmental community. However, it is difficult to predict how the enzyme will react with a particular azo dye given that the actual structure of the enzyme is unknown. Determining the structure would allow for a better understanding of the physiological role of the enzyme, which could aid in medicinal practices and solving environmental problems. This study used crystallography methods to further optimize the protein crystal conditions previously determined in order to generate a 3D image of the protein. The pure protein AzoC sample was obtained from an E.coli expression system. The experimental approach used to optimize the protein crystallization conditions involved minor changes associated with the pH, salt concentration, substrate dyes, and
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buffers using a 96 well additive screen or 24 custom-well screen. The screen plates created were observed each day for crystal growth. Sufficiently formed crystals were collected, frozen, and transported to the National Syncrotron Light Source for X-ray defraction. Computer software used the defraction data to obtain the final structure of the enzyme.
The results will provide important structure and function information for AzoC, which will impact the medical and environmental community.
PROBING THE NATURAL PRODUCT BIOSYNTHETIC POTENTIAL OF THE ACTINOMYCETE ACTINOSYNNEMAMIRUM Joseph Villanueva, Charles Melancon.
University of New Mexico, Albuquerque, NM.