LBRN 12th Annual Meeting Program - Louisiana State University
December 22, 2017 | Author: Anonymous | Category: N/A
Short Description
Jan 31, 2014 Louisiana Tech University and LSUHSC-New Orleans In this study, BSA was allowed ......
Description
Louisiana Biomedical Research Network
12th Annual Meeting Marriott Convention Center New Orleans, LA January 31—February 2, 2014 LBRN 12th Annual Meeting
Administrative Structure Administrative Core Thomas Klei (PI) Bill Wischusen (PC)
Bioinformatics, Biostatistics, & Computational Biology Core
Molecular Cell Biology Core K. Gus Kousoulas
Brygg Ullmer Hilary Thompson
Steering Committee External Advisory Committee Vincent McKoy (Chair) Richard Hart Jessica Kissinger Edward Mocarski John Quackenbush Harold Silverman
Thomas Klei (Chair) Mark Batzer Gene D’Amour Rachel Cruthirds Prescott Deininger Ann Findley Thomas Gettys Wesley Gray K. Gus Kousoulas Stan Napper Mario Philipp Robert Rhoads Paul Sisson Michael Stubblefield Hilary Thompson E. William Wischusen LBRN 12th Annual Meeting 2
Table of Contents
LBRN Administrative Structure
2
Table of Contents
3
Agenda
4
Meeting Floor Plan
6
Poster Session Abstracts
8
Oral Presentation Abstracts
25
Meeting Attendees
38
42
Oral Presentation Abstracts by Author
44
45
Index Poster Session Abstracts by Author
Upcoming Events
LBRN 12th Annual Meeting 3
Agenda Mee ng Room
FRIDAY, JANUARY 31, 2014
Riverbend, 2nd 4:30 ‐ 5:30 pm Mee ng Registra on / Poster Set‐up / Hotel Check‐in Fl
6:00 ‐ 7:00 pm
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Welcome Dinner
7:00 ‐ 9:00 pm 2nd Fl ‐ Atrium Poster Session (with dessert and coffee)
Mee ng Room
SATURDAY, FEBRUARY 1, 2014
Research Presenta on
Mee ng with EAC, Julia Room, 2nd Floor
Blaine Kern, Breakfast 1st Fl Riverbend, 2nd 8:00 ‐ 8:15 am Introduc on Fl 7:00 ‐ 8:00 am
Concurrent Session 1
Concurrent Session 2
8:15 ‐ 8:35 am
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Thomas Huckaba4
8:35 ‐ 8:55 am
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Shuja Bai4
8:55 ‐ 9:15 am
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Prerna Dua4
9:15 ‐ 9:30 am
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Bashir A e a2
Huckaba and Mentors
9:30 ‐ 9:45 am
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Steven Adelmund1
Bai and Mentors
9:45 ‐ 10:00 am
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Thomas Bishop2
Dua and Mentors
10:15 ‐ 10:35 am
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Break
10:35 ‐ 10:55 am
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Rebecca Giorno‐McDonnell3
10:55‐ 11:15 am
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Tara Williams‐Hart4
11:15 ‐ 11:30 am
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Amal Kaddoumi4
11:30 ‐ 11:45 am
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Kui Chen2
Giorno and Mentors
11:45 ‐ 12:00 pm
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Derek Jones1
Williams‐Hart and Mentors
12:00 ‐ 12:15 pm
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Agasthya Kas bola1
Blaine Kern, Lunch 1st Fl Riverbend, 2nd 1:30 ‐ 1:50 pm Eduardo Mar nez‐Ceballos4 Fl 1:50 ‐ 2:10 pm " Elahe Mahdavian4
Kaddoumi and Mentor
12:15‐1:30pm
Agasthya Kasibotla1
1
Summer Graduate Student, 2Summer Faculty, 3Pilot Project PI, 4Project PI
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Agenda cont. Mee ng Room
SATURDAY, FEBRUARY 1, 2014
Research Presenta on
Mee ng with EAC, Julia Room, 2nd Floor
2:10 ‐ 2:30 pm
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Harris McFerrin4
Mar nez‐Ceballos and Mentor
2:30 ‐ 2:45 pm
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Sachin Khiste1
Mahdavian and Mentors
2:45 ‐ 3:00 pm
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Dominique Washington1
McFerrin and Mentor
3:00 ‐ 3:15 pm
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Break
3:30 ‐ 3:50 pm
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Adarsh Radadia3
3:50 ‐ 4:10 pm
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Seetharama Satyanarayanajois4 Satyanarayanajois and Mentors
4:10 ‐ 4:25 pm
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Cecily Benne ‐DeFleece3
Benne and Mentors
4:25 ‐ 4:40 pm
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Niharika Mente1
Concurrent Session 4
4:40 ‐ 5:00 pm
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2 William Yu
Concurrent Session 3
Radadia and Mentors
6:00 ‐ 7:00 pm Riverbend 2nd Fl Informal Recep on 7:00 ‐ 9:00 pm Blaine Kerne, 1st Fl Dinner Presenta on: "Transla onal Research: The Na on's Impera ve" Guest Speaker: Sidney A. McNairy, Jr., Ph.D. D.Sc. Re red Member of the Senior Execu ve Service Former Associate Director, NCRR and Director, Capacity Building Branch, NIGMS Na onal Ins tutes of Health Bethesda, Maryland
SUNDAY, FEBRUARY 2, 2014
7:00 ‐ 9:00 am Blaine Kerne, 1st Fl Breakfast Hotel: Marrio Conven on Center 859 Conven on Center Blvd New Orleans, LA 70130 (504) 613‐2859 (Desk) (504) 613‐2860 (Fax)
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Meeting Floor Plan Marriott Convention Center New Orleans, LA
Marriott Convention Center 859 Convention Center Blvd New Orleans, LA 70130 (504) 613-2859 (Desk) (504) 613-2860 (Fax) LBRN 12th Annual Meeting 6
Notes
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Poster Session Abstracts
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Poster Session Abstracts 1
Iden fica on of biological markers in NF‐kB pathway of AD using text analy cs
Harikrishna Adigoppula, Pradeep Chowriappa, Prerna Sethi‐Dua and Walter J. Lukiw Louisiana Tech University and LSUHSC‐New Orleans Alzheimerʹs disease (AD) and age related macular degeneration (AMD) are two significant health care concerns in the US. Both AD and AMD represent the leading causes of progressive, inflammatory degeneration of the brain and retina leading to irreversible cognitive decline and visual dysfunction. It is known that these diseases share a significant number of amy‐ loidogenic (inflammatory) disease markers, and common disease mechanisms. However, there is no significant evidence to support this claim. We hypothesize that the elucidation of potential metabolic patterns between markers of both AD and AMD can help reveal insights into this degenerative pathology. To this end, we propose a text mining framework to help identify patterns of genes, proteins, and miRNAʹs (targets) of both AD and AMD. In this work we focus on the NF‐kB pathway and its associated genes, proteins, and miRNAʹs. We employ text analytics (TA) to identify and select reliable patterns from fifty published articles selected from the Neuroscience Information Framework (NIF). Text Analytics is car‐ ried out in two stages namely (a) Information Retrieval (IR) and (b) Information Extraction (IE). Using the gene ontology (GO) resource template we extracted prominent categories and concepts using sentiment analysis. We then cluster extract‐ ed concepts to identify clusters of related terms. As a proof of principle, this work will demonstrate the efficiency of using text analysis to curate patterns extracted from scientific documents that help identify genes, proteins, and miRNAʹs associ‐ ated with NF‐kB. As part of our future work we intend to utilize the extracted patterns of these genes, proteins, and miRNAʹs in the creation of an interactome for AD and AMD. This interactome will help us explore comorbidities between both AD and AMD.
2
The Use of Ultra Filtra on and Fluorescence Spectroscopy for the Study of Phenol Red Binding to Serum Albumin
Tracy Ash, Joshua Lutz, Phillip Paylok and Dr. Elahe Mahdavian Louisiana State University—Shreveport The purpose of this project was to study the binding affinity of phenol red to bovine serum albumin (BSA) through the methods of ultrafiltration and fluorescence spectroscopy. In this study, BSA was allowed to react to equilibrium with phe‐ nol red dye at various concentration ratios in a series of samples. These mixtures were then centrifuged through ultrafil‐ tration filter tubes. Using Uv‐Vis spectrophotometry, the concentration of phenol red dye in the filtrate was determined. This allowed the concentrations of free dye in equilibrium with protein bound dye to be established for each sample. With the concentration of protein and the total amount of dye in each sample, the number of bound phenol red molecules per protein molecule was estimated. From the collected data, a saturation curve and Scatchard plot were created to show the total number of phenol red molecules bound to serum albumin by one or two interacting sites with different binding con‐ stants. A double reciprocal plot of the saturation curve was created to determine the dissociation constant, Kd. The inter‐ actions between phenol red and BSA were further investigated using fluorescence spectroscopy. It was observed that phe‐ nol red has a strong ability to quench the intrinsic fluorescence of BSA through a static quenching process. A Stern‐ Volmer plot was created to determine the association constant, Ksv.
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Poster Session Abstracts 3
Finding Posi vely and Nega vely Co‐expressed miRNA Profiles in miRNA Datasets
Sphoorthy Asuri Maringan , Prerna Sethi‐Dua and Walter J. Lukiw Louisiana Tech University and LSUHSC‐New Orleans Alzheimerʹs is a neurodegenerative disease whose cause is still unknown. In Alzheimerʹs disease, the brain cells get degen‐ erated and die, causing a steady decline in memory and mental function. MicroRNAs (miRNAs) are a class of short non‐ coding RNAʹs, which show tissue‐specific regulatory activity on genes. Expression profiling of miRNAs is an important step for understanding the pathology of Alzheimerʹs disease (AD). It has been shown in a number of studies that coexpres‐ sion is correlated with functional relationships, though coexpression does not necessarily imply a causal relationship among transcript levels. In this approach, miRNAs that have similar expression patterns across a set of samples are hy‐ pothesized to have a functional relationship. We analyzed two miRNA datasets from human brain, of which the first da‐ taset consists of 5 samples each from control, temporal and hippocampus and the second dataset consists of 27 control and 54 diseased samples collected from patients over a period of 10 years. We performed correlational analysis on the filtered dataset using the Pearson correlation with a specified threshold and focused on the miRNAs having high correlation val‐ ues as the most significantly co‐expressed miRNAs. In this study, we scrutinize five miRNAs: hsa‐miR‐125b‐5p, hsa‐miR‐9‐ 5p, hsa‐miR‐34a, hsa‐miR‐146a‐5p and hsa‐miR‐155‐5p which have previously been stated to be cooperatively or redun‐ dantly regulating an inflammation pathway for Alzheimerʹs and thus playing a subtle role in the disease. Later, we com‐ pared the commonly co‐expressed miRNAs in all the experiments with the biological modules to find the biological signifi‐ cance and confirm the co‐expression linkages.
4
The Interac ve Chroma n Modeling Webserver: ICM
Dr. Thomas Bishop, James Liman, William Johnston and David Donze Louisiana Tech University and Louisiana State University Chromatin is a complex composed of DNA and histones that folds DNA so that it can fit in a cell nucleus. Experimental studies of chromatin reveal topologies ranging from regular, ribbon‐like structures with various topologies to irregular beads‐on‐a‐string structures. Models describing these topologies range from a simple two angle model to coarse‐grain models. Since chromatin contains only a single thread of DNA, it can be used as a scaffold to assemble a 3D structure of chromatin. Our Interactive Chromatin Modeling webserver (ICM http://latech.edu/~bishop) employs this approach. ICM rapidly generates a putative chromatin fold given only a sequence of DNA. The nucleosome positions in the proposed chromatin fold can be determined automatically by ICM or the nucleosome positions can be specified. The goal of this effort was to interface ICM with LAMMPS as a means of optimizing and assessing the models generated by ICM. We im‐ plemented the coarse‐grained model developed by Korolev et al in LAMMPS. Chromatin folds were generated by ICM for the CHA1, PHO5 and HIS3 promoter sequences of S. cerevisiae using known nucleosome positions, as well as, posi‐ tions automatically determined by ICM. Some but not all of the reported nucleosome positions are compatible with the sequence specific geometric properties of DNA and x‐ray structures of the nucleosome. In all cases there were unoccupied segments of DNA resulting in an extended conformation for the chromatin. In some instances nucleosome clusters were formed. In no instance could a regular or fiber‐like structure be identified. Chromatin folds obtained from nucleosome positions associated with different experiments differed significantly. The organization of chromatin described by nucleo‐ some positions is not sufficient to describe the spatial organization of chromatin. The question remains: To what extent do the DNA and nucleosomes in chromatin differ from their canonical structures?
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Poster Session Abstracts 5
Finding Alzheimer's disease related genes from GO/pathway term networks
Naveen Kumar Chalasani, Prerna Sethi‐Dua and Walter J. Lukiw Louisiana Tech University and LSUHSC– New Orleans Uncovering the underlying genetic components of Alzheimerʹs (AD) disease is key to understanding its pathophysiology. Although AD research is intensive, underlying genetic information is still unclear. In our approach, we investigate gene expression dataset of 31 AD patients at different levels of severity. The data pre‐processing follows the steps of standardi‐ zation and normalization followed by feature ranking using minimum Redundancy Maximum Relevance (mRMR) criteria to obtain top 100 and 250 gene expressions. With the obtained data subsets we create functionally organized GO/pathway term networks using GO term and KEGG/BioCarta pathway information in Cytoscape. After the formation of GO/pathway term networks, we perform biomedical literature search with obtained significant GO terms to find AD relat‐ ed genes. We compare the two data subsets and observe the GO terms related to AD. By this approach, we found regula‐ tion of protein deacetylation, Visual behavior, Spliceosomal complex assembly were related to AD and the associated genes found were ATXN1, HMGCR, CELF2, and PSIP1.
6
Construc on of a Chimeric BK and Squirrel Monkey Polyomavirus
Lara Crawford, Gloria McClure, Donna Rogers and John A. Vanchiere University of Louisiana at Monroe, LSUHSC ‐ Shreveport Human polyomaviruses have been present in humans for thousands of years but were identified less than 60 years ago. Typically harmless, these viruses can cause significant disease in severely immune compromised individuals such as pa‐ tients with AIDS and organ transplant recipients. However, in the past decade, the increased use of immunosuppressive drugs for the treatment of rheumatoid arthritis and other autoimmune diseases has led to increased polyomavirus‐ associated disease, thereby prompting more intensive study of these viruses. Unfortunately, the lack of sufficient model systems has hampered the study of human polyomaviruses. By amplifying selected DNA sequences of the virus, recombi‐ nant DNA technology can be used to aid in the development of new experimental models. These new models will broad‐ en our ability to study these viruses and develop new treatment and prevention strategies.
7
Compara ve Analysis of A3, A4, and A10 Mycobacteriophages: Sequence Homology at the Repressor Binding Site Results in Homoim‐munity of Related Mycobacteriophages
Dr. Ann Findley, Erin Rizzo, Stephen Jackson, Jobi Arceneaux, Rebecca Baudin, Rudolf Beutner, MaryBeth Borque, Lara Crawford, Amy Fontenot, Brice Gillikin, Mallorie Hayes, Rachel Johnston, Meghan Kurz, Maroutcha Mouawad, Abigayle Reed, Catherine Schilling, Zachary Streeter, John Vu, Tyriana Wilson, Amanda Sco , Meghan Richters, Dus n Lovas, Grant Jernigen, Thai Nguyen, Swapan Bhuiyan, J. Derek Jones, Bri any Miller, Jeremy Harmson, Christopher R. Gissendanner and Allison M.D. Wiedemeier University of Louisiana—Monroe Restriction digestion analyses for preliminary cluster assignment were conducted with the customary BamHI, ClaI, EcoRI, HaeIII, and HindIII enzyme panel. As in the past, many ULM isolates cut only with HaeIII, which indicates that these iso‐ lates are likely members of the ʹA miscellaneousʹ group of Mycobacteriophages. In an effort to gain further insight into their true assignment, the NEB Cutter 2.0 virtual restriction digestion utility was used to identify additional enzymes whose restriction patterns could distinguish between members of subclusters A2‐A10. Restriction enzymes NaeI, PflFI, PpuMI, SacI, SacII, SbfI, ScaI, and StuI were chosen for the subsequent digestion of targeted isolates. Additionally, we pre‐
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Poster Session Abstracts viously developed (and confirmed by PCR) a lysogen culture of Mycobacterium smegmatis containing the prophage Peaches (A4). This culture was utilized to screen novel phage isolates to determine their homo/heteroimmunity with My‐ cobacteriophage Peaches. Two phage isolates, Rockstar and Trike, were unable to infect the Peaches lysogen culture, and therefore, were initially assumed to be probable members of the A4 cluster of Mycobacteriophages. However, data from the additional restriction enzyme digestion panel screen and subsequent full genome sequencing of Rockstar and Trike indicated that they were members of the A3 and A10 clusters, respectively. A functional annotation of Trike and its com‐ parison to other A10 and related A3 and A4 cluster members will be presented. We also provide a comparative analysis of repressor binding site sequence homology for selected A3, and all known A4 and A10 Mycobacteriophages which furnish‐ es additional evidence for the observed homoimmunity between members of these related, but not identical, cluster repre‐ sentatives. Additionally, we discuss the unique opportunity that the small genomes of Mycobacteriophages Rockstar and Trike present as a screening mechanism for gene knockout experiments and the implications of their lack of the immunity repressor gene shared by fellow cluster members.
8
Rescuing a neurodegenera ve disease‐causing muta on with Mn2+
Sco Jennings, Davon J. Carter, David J. Nathan, Madeline Chenevert and Thomas M. Huckaba Xavier University of Louisiana Hereditary Spastic Paraplegia (HSP) is a clinically and genetically heterogeneous group of inherited diseases characterized by progressive stiffness (spasticity) in the lower limbs, caused by the degeneration of corticospinal tract axons. Patients with the pure form of the disease exhibit only this limb spasticity, while patients with the complex form also exhibit vari‐ ous neurological disorders. 10% of the complex forms of human HSP are due to mutations in the kinesin molecular motor Kif5A, which transports cargoes to the pre‐synapse region of the axon by traveling along microtubules (MTs). One of these mutations is S203C: a mutation of a conserved serine, which coordinates a magnesium ion (Mg2+) in the nucleotide binding site, to a cysteine. We hypothesize that this mutation causes HSP because of cysteineʹs decreased affinity for Mg2+, and that by substituting an alternate divalent cation ‐ such as manganese (Mn2+) ‐ we can partially rescue the S203C mutantʹs function. We recombinantly expressed Kif5A WT and S203C motor domain constructs and performed MT glid‐ ing assays in buffers containing varying ratios of Mg2+ and Mn2+ ions, ranging from 100% Mg2+ to 100% Mn2+. We found that S203C glides MTs at a rate approximately 3% that of WT in 100% Mg2+, but that S203C gliding velocity increas‐ es proportionally with increasing Mn2+ concentration, ultimately gliding MTs four to five times faster in 100% Mn2+. We also performed ATPase assays on these constructs in buffers containing 100% Mg2+, 50% Mg2+ / 50% Mn2+, and 100% Mn2+, and found that while S203C exhibits significantly lower ATPase activity than WT in the presence of Mg2+ alone, the addition of Mn2+ significantly increases the S203C ATPase rate. These results support the hypothesis that substituting Mn2+ for Mg2+ has a restorative effect on the performance of S203C‐mutant Kif5A.
9
Design and structure‐ac vity rela on of D‐aminoacid containing pep domime cs for inhibi on of EGFR heterodimeriza on
Shanthi Kanthala, Ted Gauthier and Seetharama D. Satyanarayanajois University of Louisiana—Monroe and Louisiana State University EGFR (Epidermal Growth Factor Receptor) family is normally involved in the signal transduction pathways leading to cell growth and differentiation. Overexpression of HER2 and deregulation of its signaling has implications in breast, ovarian and lung cancer. We have designed several peptidomimetics to block the HER2 mediated dimerization resulting in anti‐ proliferative activity on cancer cells. Our previous work has shown that a peptidomimetic compound 5, designed based on the crystal structure of HER‐2 and trastuzumab has antiproliferative activity in nanomolar range. The objective of this pro‐ ject is to investigate the structure‐activity relationship of peptidomimetic analogs of compound 5. Compound 5 was con‐ formationally constrained by N‐ and C‐termini modification and cyclization as well as substitution with D‐amino acids at N‐and C‐termini. Among the compounds studied in this work, a peptidomimetic compound 21 with D‐amino acid substi‐ tution with its N‐ and C‐termini capped with acetyl and amide functional group and reversed sequence in comparison
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Poster Session Abstracts with compound 5 exhibited better antiproliferative activity and selectivity for HER2 overexpressed breast, ovarian and lung cancer cell lines. The compound 21 was further evaluated for its ability to inhibit protein‐protein interaction using pathhunter assay, proximity ligation assay and western blot analysis. Results suggested that compound 21 is able to block HER2: HER3 interaction and inhibit phosphorylation of kinase domain of HER2. To evaluate the binding of compound to domain IV of HER2 protein, we have expressed domain IV of HER2 protein in S2 cell lines. Binding of compound 21 was also studied by docking the compound to HER2 protein domain IV. Based on these results, a model for binding of com‐ pound 21 to HER2 protein and inhibition of heterodimerization was proposed.
10
Uptake, Internaliza on and Quan fica on of LHRH tagged gold coated Superparamagne c Iron Oxide nanopar cles in Cancerous MCF‐7 cells
Sachin Khiste, S. Batra, S. Jeyaseelan, C.S. Kumar, S.N. Murthy and R.M. Uppu Southern University and A&M College—Baton Rouge and Louisiana State University Previous studies from our laboratory have shown that gold coated superpara‐magnetic iron oxide nanoparticles (SPIONs@Au) linked to LHRH can selectively kill cancer cells expressing receptors for LHRH. In these studies, we used cysteamine to cross link SPIONs@Au to the carboxy terminal of LHRH. In the present study, we synthesized SPIONs@Au linked to LHRH through the amino terminal using 12‐mecaptododecanoic acid (MDDA) as spacer. The resulting conju‐ gates, SPIONs@Au‐MDDA‐LHRH were studied for their uptake and internalization by LHRH receptor over expressing MCF‐7 cancer cells. When incubated SPIONs@Au‐MDDA‐LHRH (0.2 mg/mL) for 24 h, we found substantial uptake (180 pg/mL; measured by ICP) of the nanoparticles by MCF‐7 cells. TEM studies show evidence of clumps of SPIONs@Au‐ MDDA‐LHRH indicating their uptake. The nanoparticulate clumps were found located inside the membranous compart‐ ment together with cell organelles like mitochondria and ended up in late endosomes (autophagosomes), confirming the endocytic pathway of uptake. Further studies are underway to examine the selective tumorocidal activities of SPI‐ ONs@Au‐MDDA‐LHRH under the influence of low strength magnetic field. These studies would pave way for designing nanomaterials with tumorocidal properties and better management of cancer patients.
11
Mo fBrowser: Robust High‐Throughput Genome‐Wide Mining and Explora on of Mo fs
Phillip Kilgore, Marjan Trutschl, Urska Cvek and Rona S. Sco Louisiana State University—Shreveport and LSUHSC‐Shreveport Modern sequencing technology has permitted the collection of extensive sequence data. Complete genomes for over 2700 species exist right now, and will grow through initiatives such as the 10K Genome Project. This is a boon for life scientists who are interested in motif extraction to identify novel phylogenetic relationships, to locate regulatory elements that facili‐ tate transcription factor binding, or to predict secondary structure. However, it also presents computational and pro‐ cessing challenges. We present a method of genome‐wide mining of sequence motifs. Our pipeline begins by extracting sequences in the [‐1000, 4000] interval of each geneʹs sequence. This region was chosen to capture cis elements in the core and proximal promoters in the 1kb upstream sequences, while the 4kb downstream sequence would include regulatory sequences residing in the 5ʹ end of genes. We then supply these data to Dispom, a tool for the de‐novo discovery of differ‐ entially abundant transcription factor sites, to construct a database of candidate motifs. We partition the genome into ran‐ domly‐assigned bins to serve as input for independent Dispom processes executed on a 312‐core computational cluster, and then use a high‐pass frequency filter to remove outliers. We then construct a greedy prefix tree from the motif data‐ base in order to find the exact location (hit) of each motif within the sequence. We also introduce the concept of an ʹimplied hitʹ to allow for the possibility that some motifs may be super‐sequences of simpler, known motifs. We provide a data‐ base‐driven interface to allow for multiple views of the data. Users may supply a list of various identifiers in order to ob‐ tain a report restricted to the genes of interest, may browse a motif for genes associated with it, or may obtain all of the motifs for a single gene. Initially, we used the GRC37h reference assembly and identified several candidate motifs of high variation, length, and frequency.
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Poster Session Abstracts 12
Cyclin‐dependent kinase inhibitors reduce HSV‐1‐associated ocular neovasculariza on in a mouse model
Elise LeMelle, Tatyana T. Santoke, Bria Carmichael, Eric Stewart, Heba A. Sarhan, Thomas Vu, Sydney Turner, Briana M. Jarre , Partha S, Bha acharjee, Deborah E. Sullivan, Konstan n G. Kousoulas and Harris McFerrin Xavier University of Louisiana, Emory University, Tulane University and Louisiana State University Herpes simplex virus type 1 (HSV‐1) infects greater than 90% of humans worldwide and during ocular infection produces inflammation and angiogenesis that can lead to blindness. In the United States, HSV infection is the leading cause of infec‐ tion‐induced blindness; nearly 40,000 new cases are reported and 300,000 cases are treated yearly. Cyclin‐dependent kinas‐ es, mostly known for their involvement in the cell cycle and transcription, are involved in HSV transcription and replica‐ tion. Cyclin‐dependent kinase 9 (CDK9) and its downstream target, serine 2‐phosphorylated RNA polymerase II, are es‐ sential in HSV‐1 transcription and replication however, to date there is little literature on the role of CDKs in HSV infection of the eye or on the efficacy of CDK inhibitors in preventing HSV‐1‐associated ocular neovascularization and its conse‐ quences. We are testing the hypothesis that cyclin‐dependent kinase inhibitors and shRNA against CDK9 decrease angio‐ genesis and angiogenic signaling pathways up‐regulated by angiogenic factors in vitro and in vivo. To date, we have demonstrated that these inhibitors decrease vascular endothelial cell migration, invasion, tubule formation in vitro and angiogenesis in chick embryo and mouse Matrigel angiogenesis models. We have determined that the compounds tested are non‐toxic in rabbit and mouse eyes, and both drugs decrease mouse corneal neovascularization due to HSV‐1 corneal infection to levels observed in mice treated with an anti‐herpetic control drug. Additionally, the drugs block HSV‐1 repli‐ cation in culture. We have begun to develop novel inhibitors of CDKs and will determine whether these compounds in‐ hibit CDK9 in vitro and reduce corneal neovascularization due to recurrent HSV‐1 infection in vivo.
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The Use of Ultra Filtra on to Study Kynurenine Binding to Bovine Serum Albumin
Vijay Letchuman, Joshua Lutz and Dr. Elahe Mahdavian Louisiana State University—Shreveport Fusarochromanone has been proposed as an angiogenic inhibitor, which can be used to prevent the growth of new cancer cells. Fusarochromanone has been shown to inhibit the growth of pre‐malignant skin and bladder lesions and malignant skin, breast, bladder, and prostate cancers. However, its mechanism of action has not been determined. Using its structur‐ al analog, Kynurenine, its binding affinity to Bovine Serum Albumin (BSA) was studied. The kynurenine ligand was mixed at different concentrations with BSA and its absorbance, using a UV‐VIS Spectrum, was measured in order to deter‐ mine the number of molecules of kynurenine that had bound with the BSA molecule. The peak wavelength (λmax) was determined to be 359 nm. The dissociation constant, Kd, was found to be 3.00 x 101 µM. The fluorescence spectrum was obtained in order to reinforce the results of the UV‐VIS Spectrum. The results showed that kynurenine does in fact bind to BSA with a high affinity and this was shown in one assay, at certain concentrations of kynurenine and BSA. This finding was confirmed using the fluorescence spectrum of BSA and kynurenine.
14
RNA‐seq Analysis Reveals the Differen al Expression of Hoxa1 Target Genes in Mouse ES Cells in Response to Re noic Acid
Dr. Eduardo Mar nez‐Ceballos Southern University and A&M College—BatonRouge The Hox genes are members of the homeobox superfamily and are central in regulating embryonic development. They are a family of transcription factors and, in mammals, they are separated into four clusters (A, B, C and D) located on four different chromosomes. The expression of these genes occurs sequentially (3ʹ to 5ʹ) a along the anterior posterior axis acting
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Poster Session Abstracts to regulate the activation or repression of genes located downstream. Hoxa1, the most 3ʹ member of the A cluster, in chro‐ mosome 7, has been found to be essential for different aspects of morphogenesis and organogenesis during embryonic development. Sequential activation of Hox genes is induced by all‐trans retinoic acid (RA), a derivative of Vitamin A, through the action of its nuclear RA receptors (RAR alpha, beta, and gamma). Although targets of Hoxa1 have been iden‐ tified using microarray analyses, very few direct Hoxa1 target genes are known. For the present project, we employed the ChIP‐on‐chip technology to identify novel Hoxa1 direct target genes. Next, we examined the expression profile of putative Hoxa1 direct targets by performing RNA‐ sequencing (RNA‐seq) assays. For these experiments, we employed Wild type (Wt) versus Hoxa1 knockout (K.O.) ES cells treated for 48 hours with 1 µM RA. Control cells were treated with vehicle only. Integrative analyses of ChIP‐on‐chip and RNA‐seq were performed to examine the response of direct Hoxa1 target genes to RA treatment.
15
Disrup on of Neuronal Networks During Epileptogenesis
Daniel McBride, Dr. Alberto Musto Louisiana State University—Shreveport and LSUHSC‐New Orleans Epilepsy is a brain disorder characterized by abnormal brain activity and repeated spontaneous seizures. Epilepsy in the U.S. affects nearly 3 million people and costs more than $15 billion per year, according to the CDC. Yet the vast majority of people with epilepsy are found in developing nations, according to the WHO. There is no known cure. Epileptogenesis, the process by which a normal brain changes into an epileptic one, is not well understood. This means patients may go untreated until their symptoms (i.e. seizures) are chronic. A better understanding of epileptogenesis may allow us to (1) diagnose earlier and begin therapy sooner, (2) develop better therapies or a cure, and (3) prevent epilepsy from develop‐ ing. My research involved analyzing electrical signals recorded in the brains of living mice. Many of the mice were given treatments to cause seizures, a process which models epileptogenesis. Because mice brains are in many ways similar to human brains, learning what occurs in these mice may translate into a better understanding of epileptogenesis in humans. In particular, my research focused on patterns in hippocampal activity and relationships between different frequencies of that activity.
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Amyloid Beta Hepa c Clearance in Sandwich‐Cultured Primary Rat Hepatocytes
Loqman Mohamed and Dr. Amal Kaddoumi University of Louisiana—Monroe Failure in amyloid‐β (Aβ) systemic clearance across the liver has been suggested to play a role in Aβ brain accumulation and thus to contribute largely to the pathology of Alzheimerʹs disease (AD). The purpose of this study was to characterize in vitro the transport mechanisms of Aβ40 across the liver using sandwich‐cultured primary rat hepatocytes (SCHs) and to determine its biliary clearance (CLbile) and biliary excretion index (BEI%). 125I‐Aβ40 BEI% was time dependent and reached steady state at 30 minutes, with an average value of 29.8% and a CLbile of 1.47 ml/min per kilogram of body weight. The role of low‐density lipoprotein receptor‐related protein‐ 1 (LRP1) in mediating the basolateral uptake of 125I‐ Aβ40 in SCHs was assessed using receptor‐associated protein (RAP, 2 µM). A significant reduction in 125I‐Aβ40 BEI% and CLbile with RAP was observed, demonstrating a major contribution of LRP1 in mediating hepatic uptake of intact 125I‐ Aβ40 via transcytosis. Furthermore, activity studies suggested a lower role of receptor for advanced glycation end prod‐ ucts (RAGE) in 125I‐Aβ40 hepatic uptake. Verapamil (50 µM) and valspodar (20 µM) significantly reduced 125I‐Aβ40 BEI%, indicating a role for P‐glycoprotein (P‐gp) in the biliary excretion of 125I‐Aβ40 in SCHs. LRP1‐ and P‐gp‐mediated 125I‐Aβ40 biliary excretion was inducible and increased BEI% by 26% after rifampicin pretreatment. In conclusion, our findings demonstrated that besides LRP1, P‐gp and, to a lesser extent, RAGE are involved in 125I‐Aβ40 hepatobiliary dis‐ position and support the use of enhancement of Aβ hepatic clearance via LRP1 and P‐gp induction as a novel therapeutic approach for the prevention and treatment of AD.
LBRN 12th Annual Meeting 15
Poster Session Abstracts
17
Characteriza on of Kif5A Switch I Muta ons that cause Hereditary Spas c Paraplegia
David Nathan, Davon Carter, Sco Jennings, Madeline Chenevert and Thomas Huckaba
Xavier University of Louisiana
Hereditary Spastic Paraplegias (HSPs) are a group of neurodegenerative disorders that arise from the progressive degen‐ eration of corticospinal tract axons, causing lower limb spasticity and weakness. An autosomal dominant form of HSP (AD‐HSP) is caused by mutations in Kif5A, a neuronally enriched form of the kinesin‐1 family of transport motors. We have mutated the wild type Kif5A gene with each of the separate AD‐HSP‐causing mutations and have begun to test the mechanical properties of recombinantly‐expressed mutant motors in biochemical and biophysical assays. Here we report the results of five separate mutations in Switch I and one mutation in the L11 loop predicted to be at the microtubule inter‐ face. Microtubule‐stimulated ATPase assays show that each mutation causes an alteration in ATPase rate, but to varying degrees. Performing microtubule pelleting assays with saturating levels of ATP, we found that M198T, S202N and S203C had a significantly higher microtubule affinity than wild type Kif5A or the other mutants, suggesting a new rate‐limiting step in the ATPase cycle that follows a high affinity intermediate. Stopped‐flow experiments are being performed to deter‐ mine which of the steps in the ATPase cycle are being affected. Conversely, in the presence of a non‐hydrolyzable ATP analog, R204W, R204Q, and E251K had a significantly lower microtubule affinity than wild type and the other mutants, suggesting a defect in communication of the ATPase state with the microtubule‐binding site. In microtubule gliding as‐ says, wild type Kif5A moved microtubules at a rate of 486±35 nm/s. The M198T and S203C mutants moved microtubules at significantly lower rates of 54±6 and 15.4±5.7 nm/s, respectively. The remainder of the mutants bound microtubules to the glass surface in rigor. Our studies provide insight into the mechanistic changes in Kif5A activity as a result of HSP‐ causing mutations, as well as information about the roles of separate amino acids in Switch I.
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Eosinophil Recruitment into the Airways Following Intravenous Injec on of Spleen Cells Isolated From Sensi zed Mice
Samuel Okpechi, Mohamed Ghonim, Bashir Rezk A eia, Amarjit S. Naura, Jimena Trillo‐Tinoco and Hamid A. Boulares Southern University—New Orleans, Stanley Sco Cancer Center, and LSUHSC‐New Orleans Allergic asthma ranks among the most common chronic diseases in the United States, affecting approximately twenty mil‐ lion individuals and more than hundred million people worldwide. It is characterized by inflammation of the lung, re‐ versible airway obstruction and airway hyper‐responsiveness. Allergic airway inflammation is characterized by peribron‐ chial eosinophil accumulation within the submucosa of the airway of the lung. The aim of this project is to investigate the effect of IV injection of the total population of spleen cells isolated from sensitized mice in the infiltration of inflammatory cells including but not limited to eosinophils into the lungs of wild type mice. Total population of spleen cells were isolat‐ ed from a group of sensitized wild type mice (spleen cell donor) using standard method. Four million spleen cells were injected intravenously into each of the wild type mice (experimental samples). Seventy two hours after the IV injection, the wild type mice were then challenged with 3% aerosolized OVA (ovalbumin) for 30 minutes each day for three days. Mice were euthanized 48 hours after the last challenge. The lungs were lavaged with cold PBS and the cell suspension was cyto‐ spinned onto coated Superfrost Plus microscope slides. The cells on the slides were stained with Different Quick staining kit. Differential cell counting was performed by a pathologist blinded to the treatment groups. Our results showed a sig‐ nificant elevation in the number of eosinophillic cells in the bronchoalveolar lavage fluid from the OVA‐sensitized and challenged mice, 2.3 ± 0.3 cell/mm2 vs 1.8 ± 0.6 cell/mm2 in the control group (P
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