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2017 Research Showcase
GAMB Abstracts
Abstract Title : Genomic and Host Range Analysis of Novel Bacteriophage Dubu
Abstract : Bacteria are constantly evolving resistance to modern antibiotics; resistance often arises within a few years of the discovery of a new antibiotic. For this reason, there is a high demand for new antibiotics. By studying how bacteriophage are able to disrupt the bacterial life cycle, we can glean insight into viable tactics for developing alternative antibacterial treatments. In this project, we isolated phage Dubu from a soil sample taken from San Diego and enriched it against a strain of Streptomyces. Once Dubu was purified, its full genome was obtained, sequenced, and from there, positional and functional annotations were performed. Notably, Dubu is a myoviridae in cluster BJ, a relatively unstudied cluster containing just one other bacteriophage. We identified 49 putative proteins encoded as well as their functions. One of these proteins is nearly identical to a Streptococcus pneumoniae binding protein, raising questions about evolutionary history and host range. The effectiveness of the bacteriophage was tested on a variety of host bacteria. ADDITIONAL PRESENTERS: Maria Keisler, Reily Cannon, Jason Nideffer, Monica Jeung, Ashley Furukawa.
Abstract Title : Impaired Mitophagy in Sanfilippo A mice Causes Hypertriglyceridemia and Brown Adipose Tissue Activation
Abstract : Mucopolysacharidoses ( MPS IIIa) is a lysosomal storage diseases (LSD) that is caused by the deficiency of an enzyme called sulfamidase (SGSH) which breaks down glycosaminoglycans (GAGs), long chains of sugar carbohydrates found in each body cell, in the lysosome. MPS IIIa patients experience early development delays, extreme activity and difficult behavior, and massive amount of weight and muscle loss (Wasting Syndrome). This inherited metabolic diseases has currently no approved cure, but Enzyme Replacement Therapies (ERT) using recombinant SGSH are currently in tested in clinical trials. At a cellular level, GAGs accumulate inside the lysosomes of MPS IIIa patients this results in impaired autophagosome-lysosome fusion. Our study reports increased plasma triglycerides levels and less adipocytes in white and brown adipose tissue (BAT) in MPS IIIa mice. We present data supporting the fact that MPS IIIa mice have an abnormal energy homeostasis, large metabolic shift, increase in BAT hyperactivity and hyperthermia due to impaired autophagosome-lysosome fusion. Finally, when MPS IIIa mice were treated with Enzyme Replacement Therapy (ERT) using recombinant SGSH. This treatment reversed the phenotype as we observed reduced autophagosomes accumulation, decrease mitochondrial content, and normal body temperature in MPS IIIa mice. We hypothesize that increased BAT activity and persistent increase in energy demand in MPS IIIa mice might explain the cachexia observed in MPS IIIa patients.
Advisor : DR. KANG ZHANG
Abstract Title : Effects of Methylation on Cell Aging by Vitamin C treatment
Abstract : The purpose of this experiment is to study the effects of demethylation by vitamin c treatment on cell aging and proliferation rate. DNA methylation is an epigenetic modification in which cytosine is methylated at the 5 position by DNA methyltransferase (DNMT) to 5-methylcytosine. DNA methylation is relevant to the silencing in gene expression and X chromosome inactivation. This epigenetic modification can be reversed by Ten-Eleven (TET) 1 hydroxylase, a dioxygenase, of which vitamin c is a cofactor. Cells were treated with vitamin c as they were grown and the gene expression of gene x was analyzed. The results show that vitamin C is sufficient to increase the expression of gene x. It was also sufficient to increase proliferation rate and decrease aging phenotype of the cells. Deciphering the mechanism will open a new gateway of understanding aging and suggest possible ways to alter it.
Abstract Title : Streptomyces Phage Isolation and Host Range
Abstract : Genomic analysis of bacteriophage allows us to explore the evolutionary history and possibly discover new phage properties. Many phage integrate their own genome into the host’s genome, so we may be able to trace the genesis of certain evolutionarily conserved genes back to phage. Due to their ability to infect a limited range of bacterial species, we can then establish the evolutionary history of various bacterial strains. Within our class, we isolated more than 26 different phages from student soil samples in the San Diego area, and amplified the amount of phage that was isolated. Out of the 26, we sequenced the genomes of two phage: Alvy and Dubu. Afterwards, we tested the susceptibility of different Streptomyces strains, also isolated by students, to the two phage and compared the results to other phages sequenced by students in previous years. Through comparison of genomes and the host ranges, we can extrapolate similarities between different phages and draw connections regarding the relations between different phage. In this DNA analysis, we hope to provide insight on how the phage genome correlates to its ability to infect various species of bacteria. ADDITIONAL PRESENTERS: Sherwin Fazelpour, Daniel Fan Val Jackson-hundley, and Daniel Melnick.
Abstract Title : Interrogation of the Cryptic Biosynthetic Gene Cluster IPF38-51 in Microcystis aeruginosa PCC7806 using Improved Genetic Tools and Methods
Abstract : Microcystis aeruginosa, a bloom-forming and toxin-producing cyanobacterial species, has caused harmful algal blooms across the world. The most threatening feature of this species is its production of the potent hepatotoxin microcystin, which causes local ecosystem issues and safety concerns on drinking-water. Although M. aeruginosa has been extensively studied, two of its biosynthetic gene clusters are still enigmatic and their products have yet to be identified. The focus of this project is to study the cryptic gene cluster, IPF38-51, which was shown to be regulated by external microcystin levels in a microcystin-deficient mutant strain. To identify the secondary metabolites produced by this gene cluster and to understand its regulation, a strain with a knockout mutation of the IFP48 gene and a strain harboring the yellow fluorescent protein driven by the upstream region of the IPF51 gene are being produced. Methods and tools for the genetic manipulation of M. aeruginosa are also being further developed.
Abstract Title : Targeting DNA Topoisomerase II -- Developing New Drug Leads for the Treatment of Primary Amebic Meningoencephalitis
Abstract : Naegleria fowleri, commonly known as brain-eating amoeba, is a species of protozoan amoebae that is distributed in warm freshwater and has been reported to fatally infect humans of different ages but mainly children. They enter victims’ nasal cavities with the water, and infiltrate the cerebral cortex migrating along the olfactory nerve plexus, and incur fatal cerebrospinal tissue damage causing primary amebic meningoencephalitis (aka PAM). Despite the gruesome high fatality of PAM, many efforts to discover effective drug therapy have not been uniformly successful. Combination drug therapy, including miltefosine and amphotericin B, has proven to be successful treatment in clinic. However, the therapeutic effect varies due to many factors and full success rate cannot be ascertained. Recent study has found that RNA interference can inhibit important virulence factor of N. fowleri. This research project aims to reveal the essentiality of DNA topoisomerase II in maintenance of N. fowleri biological functions using the technique of RNA interference (aka RNAi), specifically siRNA. In addition, newly synthesized topoisomerase II inhibitors against N. fowleri will be tested out. Overall, this project attempts to develop new drug leads for the treatment of PAM. ADDITIONAL PRESENTERS: Lily Hahn, Abdolhakim Mohamed
Abstract Title : Using Genome Mining to Search Streptomyces Strains SFW and JS for Potential Antibiotic-Producing Gene Clusters
Abstract : Antibiotics have been the most effective way of fighting back against bacterial pathogens. However, in recent decades, bacterial resistance to antibiotics has been increasing, while the discovery of new antibiotics has been decreasing (CDC). With the goal of discovering previously unknown antibiotics, and as a way to combat antibiotic resistant bacteria, we chose to analyze two Streptomyces strains, SFW and JS, from the soils of San Diego. Streptomyces strains are well-known to produce antibiotics, and recent advances in genome mining technology enabled us to analyze their biosynthetic gene clusters, which are sequences that encode antibiotic producing proteins. Utilizing the software antiSMASH, a program that identifies similar clusters between different genomes, we hypothesized which ones are likely to produce antibiotics based on products of other similar gene clusters. "Antibiotic / Antimicrobial Resistance." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 27 Apr. 2017. Web. 18 May 2017. ADDITIONAL PRESENTERS: Andrew Jespersen, Jennifer Rothman, Victoria Rodriguez, Shaye Stalians.
Abstract Title : Elucidating the Mechanism of KaiC Localization in Synechococcus elongatus
Abstract : Circadian clocks are endogenous, self-sustained timekeepers that drive 24-hour rhythms of biological processes in a wide variety of organisms. Cyanobacterial circadian clocks revolve around three main proteins: KaiA, KaiB, and KaiC. KaiC is the central component. KaiC has been shown to localize to the cell poles; this localization is enhanced at night and independent of other clock-specific factors. The goal of this research is to find the mechanism(s) responsible for KaiC localization in the cyanobacterial strain Synechococcus elongatus PCC 7942. Previous work has shown that KaiC similarly localizes in Escherichia coli, which suggests there is a conserved mechanism that localizes KaiC to the cell poles. Previous work has suggested that this mechanism uses essential proteins in E. coli. We utilized an E. coli essential protein depletion library, which allows the inducible depletion of individual essential proteins. After introducing a plasmid expressing YFP-KaiC into 238 strains in which the essential genes are tagged with a degradation tag, we observed the effects on KaiC localization under depletion conditions. 12 candidates expressed a phenotype of KaiC delocalization under depletion conditions; out of these 12 candidates, 9 have homologs in cyanobacteria, which are also essential genes. We are currently testing the effects of conditional depletion of these homologs on KaiC localization and circadian clock function in S. elongatus.
Abstract Title : New Interaction Between Galectin-3 and Cystinosis Reveals Mechanism of Kidney Pathogenesis in Cystinosis
Abstract : Cystinosis is a severe autosomal recessive lysosomal storage disorder with implications from an early age. This disease is caused by a mutation on the gene CTNS coding for cystinosin, a lysosomal transporter of cystine. In cystinotic patients, the lack of cystinosin leads to cystine accumulation and formation of cystine crystals resulting in degradation of multiple bodily systems. Cystinosis affects multiple organs like the eyes, liver and pancreas, with preeminent renal complications. Indeed, patients develop Fanconi Syndrome before the age of one, characterized by loss of electrolytes and nutrients, progressing to end stage renal disease. Currently, cysteamine, a cystine depleting agent, is the only form of treatment for the disease, it does not prevent the progression of kidney disease and many patients still require kidney transplants. The implication of kidney disease in cystinosis is still not fully understood. While looking for potential protein partners for cystinosin, our collaborators in Paris (France) have proven that cystinosin interacts with galectin-3 (Gal-3), a lectin family protein which is involved in acute inflammatory response such as neutrophil activation and chemoattraction of monocytes and macrophages. Using mouse models created in our lab knocked out for CTNS, Gal-3 and double knockout for CTNS and Gal-3 we were able to demonstrate that galectin-3 is overexpressed in the absence of CTNS. To look into the localization of Gal-3, we used immunofluorescence and found that cystinosin co-localizes with Gal-3 in the lysosome. Following a biotin assay, we observed that this co-localization leads to the degradation of Gal-3. When comparing CTNS deficient mice with the double knockout mice, we found that there is significantly more macrophage infiltration in CTNS deficient mice than the double knockout model.Thus, we believe that gal-3 is responsible for the macrophage infiltration and consequent inflammation observed in cystinotic renal pathology. We have also found a particular cytokine to be overexpressed in CTNS deficient mice serum when compared to the double knockout model which could explain the elevated levels of macrophage recruitment and infiltration observed in the cystinotic kidney. By further understanding this interaction, there is potential to develop a treatment to halt the macrophage infiltration and reduce the debilitating affect of cystinosis on the kidney.
Abstract Title : Loss of Talin Protein from the Cardiac Myocyte Causes Early Onset Cardiomyopathy: Talin is Essential for the Structural Integrity of Costameres and Membrane Stability of Cardiomyocytes.
Abstract : Background and Hypothesis—Integrins mediate cell-extracellular matrix (ECM) adhesion. In muscle cells, integrins are core components of costameres, the structures that form the physical connections between sarcomeric Z-lines and the ECM. Integrins bind directly to the ECM proteins but require adaptor proteins to link with the actin cytoskeleton and sarcomeres. Talin is one protein that mediates that connection. There are two talin genes, tln1 and tln2, in adult heart, Tln2 is expressed exclusively in cardiomyocytes (CMs) while Tln1 is expressed predominantly in nonmyocyte cells with low levels in CMs. We hypothesized that Tln2 is essential for the preservation of CM costameric structure. Methods—Tln2 null (Tln2KO) and Tln1 CM-specific KO (Tln1cKO) mice were generated by Cre recombinase-loxP technology. Evan’s blue dye (EBD) uptake was performed to determine membrane stability. Molecular mechanisms were studied with neonatal mouse ventricular myocytes (NMVMs) derived from the mouse models and treated with adenoviral LacZ or Cre recombinase. Results—Tln2KO mice did not exhibit a basal cardiac phenotype when evaluated up to 12 months of age. Yet, loss of Tln2 resulted in increased CM Tln1 protein, while the costameric proteins - b1D integrin and integrin link kinase were decreased, indicating that increased expression of Tln1 can only partially compensate for loss of Tln2. Therefore, to analyze CM Tln2 function in the absence of Tln1, we deleted CM Tln1 using a Cre/LoxP system (Tln1- flox/flox X nuclear alpha-myosin heavy chain-Cre recombinase) in the Tln2KO background to produce mice with CM deletion of both Tln isoforms (Tln1cKO/Tln2KO). Tln1cKO/Tln2KO mice were normal at birth but developed cardiac dysfunction by 4 weeks, with 100% lethality due to heart failure by 6 months. Prior to onset of cardiac dysfunction, mutant hearts exhibited decreased expression of b1D integrin and displacement of vinculin from the costameres. Increased EBD uptake in the Tln1cKO/Tln2KO vs. control showed that membrane stability was reduced with loss of CM talin. Down-regulation of both Tln isoforms in NMVMs provoked a downregulation of b1D integrin levels together with a mislocalization of b1D, vinculin and focal adhesion kinase from the costameres. Conclusions—These data suggest that talin is a key component required for maintenance of cardiac costamere, CM and ultimately, myocardial structural integrity and highlight the importance of studying the role of talin in human cardiomyopathy.
Abstract Title : Phage Alvy: An Annotation and Analysis
Abstract : Phage can directly target and kill specific bacteria and therefore research in phage could present a potential solution to fighting bacterial diseases, especially those with antibiotic resistance. Phage Alvy was isolated from the soil using Streptomyces platensis, a domesticated and naturally antibiotic-producing bacteria. To isolate this phage we used enrichment and plating techniques to purify and determine the concentration of the phage lysate. With this lysate we were able to sequence the genome needed for later bioinformatic techniques and were able to image the phage through electron microscopy. We utilized a program called DNA Master to annotate Alvy’s genome and assign these genes possible functions. This lytic, siphoviridae contains 83 genes, which when compared to the genomes of other phage isolated during previous years of the Phage Genomics Research Initiative showed strong resemblance to the phages BartholomewSD and TrvxScott. Supplementary genetic analysis was done using Phamerator, to compare the Alvy genome to these highly similar genomes. Further research is needed to determine whether the phage has implications in the medical industry. ADDITIONAL PRESENTERS: Julia Asay, Jesse Kim, Ginny Wu, Alyssa Pearson.
Abstract Title : Nuclear Factor of Activated T Cells (NFAT) and its role in the Adult Drosophila Heart
Abstract : Heart disease is currently the leading cause of death in the U.S. Understanding the function of new genes and pathways in a simple model system may provide novel insights and lead to new therapeutic targets for heart disease in humans. In humans, calcineurin-NFAT signaling has been implicated in regulation of the cardiac hypertrophic response. However, how this gene interacts with other molecular pathways in the heart is unknown. In order to investigate the role of NFAT in context of other signaling pathways, the singular function of NFAT in the heart was investigated, using the animal model system Drosophila melanogaster. Loss-of-function studies were conducted and the resulting heart phenotype was assessed. NFAT-RNAi lines were used to conduct gene knockdowns (KD) in a tissue-specific manner using the GAL4/UAS system. The flies were dissected to expose the beating heart tube; hearts were filmed and analyzed using Semi-Automated Optical Heartbeat Analysis (SOHA). Key cardiac performance indicators, such as heart period, fractional shortening, heart rate, stroke volume, and cardiac output, were measured. Obvious differences between NFAT gene knockdown and respective controls using a single RNAi KD line were not observed. However, this may be due to insufficient KD or due to compensation by changes in expression of other genes. We will examine the effects of KD using other independent NFAT RNAi lines, as well as possible non-autonomous effects by KD in other tissues. In addition, we will study the effect of NFAT KD in sensitized backgrounds, such as NFAT heterozygous mutants.
Advisor : DR. MAHO NIWA
Abstract Title : Does age affect the unfolded protein response signal transduction pathway?
Abstract : Folding and maturation of cell surface receptors and secreted proteins have to occur in a cellular compartment called the endoplasmic reticulum (ER). In response to increased demands of ER functions (termed ER stress), a conserved signal transduction pathway called the unfolded protein response (or UPR) is activated. The UPR is conserved in a single cell eukaryote, yeast, S. cerevisiae, underscoring its importance. Thus far, most of UPR studies have focused around young yeast cells and limited information is available if /how aged cells respond to ER stress. To understand the effect of age, I used a protocol to enrich aged yeast cells and tested their ability to activate UPR using a fluorescently tagged UPR reporter. As the importance of the UPR has been recognized for human diseases, understanding the impact of age on the UPR will provide an important step towards age-exacerbated human diseases such as Alzheimer and Parkinson diseases.
Advisor : John Paul Shen
Abstract Title : Employing CRISPR/Cas9 Screens to Synthesize Novel Therapeutic Treatments and Construct de novo Gene Interaction Networks Across Multiple Cancer Cell Lines
Abstract : Combinatorial CRISPR-Cas9 screens reveal many genetic interactions that are private to cell type. Through these screens with three different cells lines—HeLa, A549, and 293T—we constructed de novo genetic interaction networks. There were three primary goals of this research : (1) Create comprehensive maps of physical and genetic interactions in cancer cells, (2) Use these cancer cell maps to enable hypothesis-driven investigation of targeted therapeutic interventions, (3) Enable precision oncology—matching the patient’s cancer profile to the correct drug. To transduce the aforementioned cell lines, a DNA array was harvested and integrated into a combinatorial guide RNA (gRNA) library, which was then made into virus to transduce the HeLa, A549, and 293T cell lines. In total, 73 genes with three unique gRNA per gene, 23,652 double knockout constructs with two replicates per cell line. After transduction, the combinatorial CRISPR-Cas9 screens sought to examine differential growth and survival. The results from these combinatorial CRISPR-Cas9 screens led to four distinct results (1) our combinatorial CRISPR-Cas9 knockout approach allows for the robust assay of thousands of genetic interactions in a single high-throughput experiment, identifying many therapeutically relevant synthetic lethal interactions, (2) sampling gRNA abundance at multiple time points improves fitness measurement, increasing power to detect genetic interaction, (3) most of the identified synthetic lethal interactions (89.5%) were private to one cell line, (4) it will be important to consider cellular context—lineage, expression profile, mutation profile—when designing synthetic lethal cancer therapies. Moving on from here, we will be examining and constructing, genetic interaction networks across many more cancer cell lines, such colon, breast, ovarian, and head/neck cancers. Using this initial research as a template, we will be pursuing new targets to examine.
Abstract Title : Isolating Streptomyces and Producing Antibiotics
Abstract : Each year in the U.S., at least 2 million people are infected with bacteria resistant to antibiotics. Due to the the overuse of broad spectrum antibiotic medications, there is substantial evidence that the increasing incidence of antibiotic resistant strain infection in hospitals around the world will continue in the coming years. This study investigates new strains of Streptomyces for their potential to produce new antibiotics to combat evolving strains of antibiotic resistant bacteria. We used Streptomyces strains because the majority of currently used antibiotics were derived from Streptomyces strains. First, we isolated and purified 40 Streptomyces from soil collected from different locations in the San Diego area. We tested 10 of these strains for their ability to produce antibiotics that were able to kill E. coli and Bacillus. We found that the potency of killing was dependent on particular strain and media. Further research should be done to investigate the properties of these produced antibiotics and their potential uses against resistant strains. ADDITIONAL PRESENTER: Daniella Vo, Shane Elder, Mina Botros, Michelle Holland, Emily Frink
Abstract : Age/diabetes-related atrophy or fibrosis in urogenital muscles represents a significant risk factor for the development of incontinence/erectile dysfunction in geriatric populations. While the loss of muscle mass is a clear indication of dysfunction, little is known about the underlying molecular mechanisms of muscle degeneration. Rabbit and mouse (diabetic) models were used to establish a time course of age/diabetes -related urethral/penile muscle dysfunction and to study fibrotic mechanisms. Young and old rabbits as well diabetic mice were scarified to harvest urogenital tissue samples. Protein expression was determined using immunostaining and western blot analysis for its quantification. Immunological studies demonstrated upregulation of fibrotic and atrophy pathways involving TGF-β and downregulation of caveolin proteins confirming increased fibrogenic pathology in these samples. Urogenital muscle atrophy/fibrosis can greatly impact continence as well as sexual functions. Understanding of such mechanisms is important for the development of potential interventions.
Abstract Title : Identifying novel functions of RAD53 and MEC1 in S. cerevisiae that mediate replication of duplex telomere DNA
Abstract : Telomeres are the essential structures at the ends of linear chromosomes. One essential role is to protect the ends from DNA damage machinery. Recently, the Lundblad lab has become interested in the possibility that DNA damage machinery may also play a positive role in promoting telomere homeostasis, potentially by promoting replication through the duplex telomeric tract. Two major players in this pathway, Rad53 and Mec1, are important for DNA replication checkpoints. Based on previous work, we suspect these two proteins to be involved with telomeres. To this end, I am pursuing extensive mutagenesis of each protein by employing a novel, phenotype-based protocol developed in the Lundblad lab designed to rapidly detect separation-of-function mutations. By screening this panel of mutations for novel telomere-related phenotypes, I have found mutations that show such phenotypes that are currently undergoing further testing. Through further analysis of these mutations, I hope to elucidate the complexities of telomere homeostasis. ADDITIONAL CONTRIBUTOR: Corinne Moeller
Abstract Title : Impact of APOE on Heart Disease
Abstract : Heart disease is the leading cause of death in the United States of America. One of the main risk factors of heart disease is hypertriglyceridemia, which is the accumulation of Triglyceride-rich lipoproteins (TRLs). One of leading causes of hypertriglyceridemia is the glycoprotein called apolipoprotein C-III (APOC-III). APOC-III inhibits the clearance of triglycerides by blocking the low density lipoprotein receptor (LDLR) and LDLR related protein 1 (LRP1) receptors that are responsible for TRL removal. Apolipoprotein E (APOE) reduces the likelihood of hypertriglyceridemia by helping lipoproteins bind to LDLR and LRP1 receptors. There are 3 isoforms of APOE (called 2, 3, and 4) that differ by only one or two nucleotides. The goal of this project is to learn about the importance of the APOE isoforms in the inhibition of APOC-III in TRL clearance. This goal will be achieved by injecting APOE knockout mice with viruses containing isoforms of APOE and observing the differences in triglyceride clearance and LDLR/LRP1 binding activity between APOE isoforms. As expected, injection of the APOE 3 isoform significantly decreased plasma triglyceride and cholesterol levels. Based on human patient statistics, we anticipate that APOE 2 will have little effect on the mice while APOE 4 will significantly decrease triglyceride levels. The results from this study will help us discover more treatment options for heart disease.