UC San Diego SearchMenu
2016 Research Showcase
GAMB Abstracts
Abstract Title : M13 Phage Display of Thermus aquaticus Variable Protein
Abstract : Bio-macromolecular crystallography requires samples (e.g., proteins or RNA) that form large, well-defined crystals in order to determine molecular structures through X-ray diffraction. However, there are still many proteins that remain non-crystallizable, which can be due to their unstable structure or unfavorable entropy. Thermus aquaticus variable protein (TaqVP) can potentially aid in crystallizing these difficult proteins by acting as a crystallization chaperone. Thermostable proteins tend to crystallize better, and TaqVP has been crystallized and is thermostable up to 90C. TaqVP can also potentially accommodate ~1014 different sequences, with a potential to bind many proteins. TaqVP has been cloned into a phagemid vector for M13 phage display via the pIII coat protein, and its expression on the phage coat is being determined. If expression on the phage is successful, a phage library will be generated to select a TaqVP mutant that possesses high affinity and specificity of a crystallization candidate protein.
Abstract Title : Analysis of the IceWarrior phage
Abstract : Streptomyces phage are important to research because phages can manipulate the host genome and the host can produce antibiotics. We extracted IceWarrior, a lytic phage, from a soil sample and isolated it with host Streptomyces platensis. Electron microscopy revealed that IceWarrior is a siphovirus with a hexagonal capsid. The phage genome was annotated in DNA Master to identify open reading frames (ORFs) representing 85 potential gene products. Proteins predicted included generic structural proteins as well as proteins involved in genome replication. More than 50% of the gene products have unknown functions, and one gene in particular, product 47, might be a member of a novel protein family. Further genomic analysis using Phamerator to compare IceWarrior to other phage genomes in the same cluster showed that certain regions of the IceWarrior genome are conserved. This data could aid in understanding the evolutionary origins and history of phages. Although more research is required to characterize the unknown proteins, this work could yield important findings that facilitate the development and discovery of novel antibiotics. ADDITIONAL PRESENTERS: Alicia Ho, Shivani Lakkaraju, Aishwarya Vuppala, Caroline Ren, and Jennifer Doan
Advisor : DR. GERRY BOSS
Abstract Title : Generation of Transgenic Mice for the Investigation of the Role of Protein Kinase G II in Bone
Abstract : Osteoporosis affects millions of Americans and costs related to the disease amount to billions of dollars annually. It is characterized by progressive loss of bone mass and strength, which increases risk of fractures and causes significant morbidity and mortality. Teriparatide, the only FDA-approved drug to stimulate bone formation, can only be used for a limited period since its anabolic effects wane over time. Thus, novel therapies to promote bone formation are needed. Our laboratory has shown that the nitric oxide/cGMP/protein kinase G pathway mediates the pro-survival and pro-proliferative effects of mechanical stimulation and estrogens in osteoblasts. Protein kinase G II was shown to be critical for the anabolic effects of mechanical stimulation. In order to further investigate the role of protein kinase G II in bone, a PRKG2 R242Q transgenic mouse model was developed to study the effects of osteoblast-specific expression of constitutively active protein kinase G II. Analysis of their static and dynamic bone formation parameters will allow insight to protein kinase G II's role in bone formation. In vitro growth and differentiation of primary osteoblasts derived from PRKG2 R242Q transgenic mice will contribute to elucidating protein kinase G II's specific role in osteoblast development. Based on previous studies, these findings will contribute to the implication of protein kinase G II's potential as a therapeutic target for osteoporosis.
Abstract Title : Identification of Plasmodium falciparum gametocytogenesis genes
Abstract : An important component of the malaria eradication campaign is eliminating transmission from the human to the mosquito. This process is governed by gametocytes, the sexual stages of Plasmodium falciparum. However, we know little about gametocyte biology and especially how the induction of gametocytogenesis is controlled at the molecular level. In order to investigate the cellular processes which control gametocytogenesis in P. falciparum parasites, 28 clonal parasite strains were isolated by limiting dilution and screened using high-content imaging. Three of those clones produced fewer gametocytes, and one of those clones possessed a nonsynonymous mutation in a transcription factor subunit by whole-genome sequence analysis. The other two clones had no detectable changes at the genome level and we hypothesize that they possess differences at the transcriptional level. Identification of genes involved in gametocytogenesis will advance our current understanding of P. falciparum sexual stages and provide new targets for transmission-blocking vaccines or drugs.
Abstract Title : Isolating antibiotic-producing strains of Streptomyces
Abstract : The number of antibiotic-resistant bacterial strains has been increasing exponentially over the last several decades, resulting in a dire need for new antibiotics. Since most clinically useful antibiotics are derived from soil bacteria, we isolated 36 strains of bacteria from soil samples around San Diego County, fourteen of which belong to the antibiotic producing genus Streptomyces. We then established the phylogenetic relationships between these Streptomyces strains based on similarities in their 16s rRNA to determine if there are correlations between related strains and antibiotic production. To discover the amount and spectrum of antibiotic productivity in these strains, we tested their ability to kill three different strains of bacteria: a wild-type strain of E. coli, an E.coli tolC mutant that is more sensitive to antibiotics due to its defective efflux pumps, and a strain of Bacillus subtilis to find compounds effective against gram-positive organisms. We identified strains that killed the E.coli tolC mutant but not the wild-type, suggesting they produce antibiotics that are substrates for the efflux pumps. We also found some Streptomyces strains that killed E. coli but not Bacillus, which indicates that they produce compounds specific to killing gram-negative bacteria. Finally, we discovered Streptomyces that killed all three strains, suggesting that they produce broad-spectrum antibiotics. Based on these results we will attempt to isolate new varieties of antibiotics in the near future. ADDITIONAL PRESENTERS: Vipula Racha, Carleen Villarreal, Elaine Luterstein, Quinn Fujii, Elena Estrada, Sriya Panchagnula
Abstract Title : CRISPR/Cas9 Mediated Double Mutant Generation of Tandem Genes VICTR and VICTL1 in Arabidopsis thaliana
Abstract : The advent of CRISPR/Cas9 genomic editing has allowed for the creation of precise, targeted changes in the genome, and our goal is to utilize CRISPR/Cas9 genomic editing to characterize highly homologous tandem genes VICTR and VICTL1 in Arabidopsis thaliana plants. VICTR is a member of the Nucleotide Binding Leucine Rich Repeat (NB-LRR) immune receptor family and was reported for its role in crosstalk between ABA phytohormone signaling and immune responses. Since NB-LRR genes are often functionally redundant and clustered in the genome, generating a victr victl double mutant could allow us to fully characterize this immune receptor gene. We transfected victl1 mutant plants with a CRISPR/Cas9 construct targeting the VICTR gene. The generated victr victl1 double mutants were confirmed by comparing them to victr mutant phenotypes, demonstrating the efficacy of CRISPR/Cas9 constructs in targeted mutagenesis in plants. However, eliminating the CRISPR/Cas9 consruct is in progress.
Abstract : DNA is wrapped around histone proteins to form chromatin. Histones can be post-translationally modified by enzymes, such as acetyltransferases and kinases, which add acetyl and phosphate groups respectively to target residues. Addition of these chemical groups to histones alters chromatin structure and accessibility of DNA. One such histone-modifying acetyltransferase, Gcn5, interacts with Swe1, a kinase that regulates cell cycle checkpoint pathways. Specifically, deletion of SWE1 alleviates stress sensitivities of gcn5 null cells. The nature of this interaction has remained unknown. We identified specific mutations in two kinases downstream of Swe1, Cdc28, the main cyclin dependent kinase in budding yeast, and Ipl1, which promotes chromosome bi-orientation, that also alleviate gcn5 null phenotypes. However, the gcn5 null phenotypes alleviated by deletion of SWE1 only partially overlap with those rescued by cdc28 and ipl1 mutants. This array of genetic interactions between GCN5 and SWE1, CDC28, and IPL1 suggests functional interactions between Gcn5 and multiple, non-overlapping pathways controlled by Swe1. Levels of relevant histone modifications are a crucial parameter for determining the mechanism by which mutants of Cdc28 and Ipl1 rescue gcn5 null phenotypes, and preliminary results showing levels of particular histone modifications in these mutants will be presented here.
Abstract Title : Senataxin, a potential modulator of mutant C9ORF72 mediated cytotoxicity in an in-vitro model study of ALS pathogenesis
Abstract : Two autosomal dominant, gain of function mutations: L389S and R2136H in the Senataxin (SETX) gene lead to ALS4, a juvenile onset form of amyotrophic lateral sclerosis. To examine the effects of these mutations, we used a L389S knock-in, a transgenic R2136H, and a C9ORF72 (G4C2)450 BAC repeat expansion mouse model. Cytotoxicity of mutations was measured using LDH-Cytotoxicity Colorimetric analysis and Propidium Iodide Staining. These assays revealed that both mutations in SetX and repeat expansions in C9ORF72 leads to considerable cell death in primary cortical neurons (upper motor neurons). Knockdown of Senataxin further exacerbates C9ORF72 pathophysiology.
Abstract Title : Sequences complementary to the miRNA 3' end are necessary for specific miRNA targeting
Abstract : MicroRNAs (miRNAs), small RNAs 20-22 nucleotides long, post-transcriptionally regulate messenger RNAs (mRNAs) via imperfect base pairing. MiRNA families, such as the let-7 family in C. elegans, have identical seed sequences (nucleotides 2-8 from the 5' end), which are known to be critical in miRNA targeting of mRNAs. Thus, families are thought to have overlapping targets. However, lin-41 has been established to be regulated only by let-7, suggesting that there may be non-seed sequences that are important for specific miRNA-mRNA interaction. Using CRISPR/Cas9, we created a strain in which let-7 binding sites on the lin-41 3'UTR are modified at the region where the 3' end of let-7 binds. Using a survival assay, we showed that this modification changes the specificity of lin-41. This work furthers our understanding of miRNA targeting by providing insight into how differences on the 3' end of the miRNA can affect miRNA-mRNA interaction.
Abstract Title : The usefulness of novel enteroid-derived monolayers in understanding diarrhea caused by Salmonella infection
Abstract : Non-typhoidal Salmonella is the second leading cause of foodborne illness worldwide. Despite its prevalence, the mechanism through which Salmonella elicits diarrhea isnot fully understood. We have previously shown that infection with Salmonella causes decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Downregulated in Adenoma) in the colon of mice, likely contributing to luminal fluid accumulation. In humans, Salmonella primarily colonizes the small intestine. We therefore studied DRA expression in murine ileum following Salmonella infection and found two-fold decreases in DRA expression accompanied by four and almost three-fold increases in Atoh1 and Muc2, respectively, which are markers of the secretory epithelial lineage. Staining of tissue segments also revealed increased goblet cell numbers and overall proliferation of the intestinal epithelium. These findings suggests a bias toward secretory over absorptive lineages following infection. To further investigate these changes we developed a 3D enteroid model that recapitulates the various epithelial lineages present in the small intestine, as well as monolayers derived from these enteroids. Infection of enteroid-derived monolayers was associated with similar decreases in DRA expression accompanied by increases in Atoh1 and Muc2, showing the utility of this in vitro model. Currently, we are using our enteroid model to delineate changes in the composition of the intestinal epithelium following infection, focusing on the Wnt/NOTCH signaling pathways responsible for proliferation and differentiation of the intestinal epithelium. In addition, we are working to delineate the transcriptional pathways through which DRA repression could occur. ADDITIONAL PRESENTER: Eileen Kim
Abstract Title : Rare Variants of Vinculin and Tropomyosin 1 in Dilated Cardiomyopathy
Abstract : Dilated Cardiomyopathy (DCM) is a condition in which the heart's ability to pump blood is decreased. It is a major public health problem, one of the major causes of heart failure, and can result in rhythm disturbances and death. It is also a major reason for cardiac transplantation given that other effective treatments for advanced disease are not available. Therefore, discovering the basic mechanisms of DCM could contribute to better and more accurate ways to diagnose and treat heart failure. Recently, with advances in sequencing technology, over 1000 variations in more than 40 genes have been associated with cardiomyopathies, including DCM. We identified novel variants in a large family in the genes encoding the costameric protein Vinculin (VCL) and the sarcomeric regulatory protein Tropomyosin 1 (TPM1). Sequencing these loci in 31 family members showed that the combination of VCL and TPM1 variants cosegregated with all family members diagnosed with cardiomyopathy across 3 generations. Analysis revealed that the combination of these variants is significantly associated with cardiomyopathy. Given this we hypothesized that these novel variants in VCL and TPM1 act combinatorially to predispose individuals to develop DCM. To testify this hypothesis, we used CRISPR-mediated genomic editing to introduce the VCL and TPM1 variants into the mouse genome, so that we could study the effect of these variants in combination and in isolation. There is no significant differences in vital signs or cardiac function at baseline between groups. When challenged with cardiac stress by thoracic aortic banding, mice carrying both variants exhibited stigmata of DCM including significant reductions in cardiac contractility and increased cardiac chamber dimensions and heart weight, confirming that these variants combinatorially predispose to the development of cardiomyopathy. The aim of my project is to investigate the mechanism of the novel VCL and TPM1 variants in causing DCM. First, we tested the VCL and TPM1 protein expression in myocardium of the mice harboring the variants. VCL protein expression was reduced 50% in VCL heterozygous variant mouse hearts, compared to littermate controls. Mice homozygous for the VCL variant showed embryonic lethality since VCL expression was ablated completed. Thus the VCL homozygous variant mouse were identical to the global VCL knockout mice, suggesting the VCL variant is functionally equivalent to a null allele. TPM1 expression was unchanged in the TPM1 variant mice compared to controls.Next we investigated the organization, expression and localization of intercalated disk (ICD), sarcomeric, costameric and Z- band related proteins, as well as the overall structure of myocardial tissue and isolated CMs from the single variant, doubly-heterozygous variant mice and littermate controls. We found that ICDs were disorganized(evidenced by widened connexin-43 immunostaining) in the ICD of variant mice vs. controls. The Z-band protein (α -actinin-1), costameric proteins (VCL and paxillin), and ICD protein (ZO-1) were reduced in double heterozygous variant mice, vs. controls. Finally we investigated the contractility and calcium handling properties of cardiac myocytes (CMs) isolated from the mice, using an IonOptix MMSYS (Myocyte Calcium & Contractility Recording System system). Preliminary resultsfound contractility of single CMs reduced and calcium handling abnormal in double heterozygous variant mice vs. controls. In summary, we found two novel variants in VCL and TPM1 that appear to act combinatorially to provoke DCM in patients, a phenotype partly recapitulated in mice where mice harboring both the VCL and TPM1 variants showed disorganization of ICD , along with abnormal calcium handling and CM contractility.
Abstract Title : Emricasan Improves Survival, Portal Hypertension, and Fibrosis in a Bile Duct Ligation Model of Liver Disease
Abstract : Portal hypertension is an important factor in the prognosis of liver cirrhosis, and novel pharmacological interventions are greatly needed. This study aims to determine whether Emricasan, an oral pan-caspase inhibitor, ameliorates portal hypertension (PHT) and fibrosis in an experimental model of short and long term cholestasis. To test this, mice underwent either a disease-inducing bile duct ligation (BDL) or a sham procedure, and then received either Emricasan or a placebo. The mice were euthanized after 10 days or 3 weeks, and liver histopathology and molecular assays were performed. The 10 day BDL mice receiving the pan-caspase inhibitor had significantly reduced levels of fibrosis (p<0.05) and apoptosis (p<0.05) compared to the placebo BDL mice. Additionally, the 3 week BDL mice receiving the pan-caspase inhibitor had significantly improved survival (75% versus 40%) and significantly reduced portal pressure (p<0.05) compared to those receiving the placebo. Accordingly, Emricasan is a potential therapy for PHT.
Abstract Title : The Role of Heparan Sulfate in Brown Adipose Tissue Activation
Abstract : Activation of brown adipose tissue (BAT) is particular interesting as it is considered a therapeutic strategy to increase energy expenditure in obese patients and to lower plasma triglyceride levels in hypertriglyceridemia patients. BMP7 is one of the key regulators of cold-induced BAT thermogenesis, but also a heparan sulfate (HS) binding protein that requires heparan sulfate proteoglycans (HSPGs) at the cell surface to exert its biological activity. We showed that activation of BAT leads to a reduction in atherosclerosis development and that LPL fueling of brown adipose tissue upon cold activation involves an initial heparin-sensitive binding. Based on these observations we postulate that brown adipose HS regulates BAT activation. To address this hypothesis groups of 6-9 male and female HSPG-deficient mice (Ndst1f/fAdipoqCre+) and wild-type control mice (Ndst1f/fAdipoqCre-) on a chow diet will be exposed to subthermoneutral temperature (21C) and cold (4C) to study the impact of HS composition on BAT activation. During acute exposures we will monitor BAT activation by measuring body temperature (using rectal probe), body weight and plasma triglyceride levels. At the end of the exposure mice will undergo necropsy to assess expression of specific BAT signature genes in BAT via qPCR, Western blotting and immunohistochemistry. We anticipate that BAT activation should be impaired in HSPG-deficient mice.
Abstract Title : Genome mining soil Streptomyces for antibiotic-producing genes
Abstract : In trying to identify novel antibiotics, we isolated 36 bacterial strains from soil samples, 14 of which were identified as Streptomyces. With Streptomyces being major producers of antibiotics, we further characterized these strains by testing for antibiotic production. Strains DF, JS, and QF were able to kill E. coli, and their genomes were subsequently sequenced. We used antiSMASH, a program that analyzes antibiotics and secondary metabolites in bacteria, to identify which biosynthetic clusters in the genome may be responsible for producing antibiotics. By comparing the gene clusters of DF, JS, and QF to known natural product biosynthetic genes, we can determine potential gene clusters that produce similar products. Several clusters could potentially have significant roles in strain DF's antibiotic production that are related to known antibiotics such as Friulimicin and Daptomycin. The differences between known biosynthetic pathways and the clusters identified are significant enough to indicate novel antibiotic production. This data, taken together with antibiotic mode-of-action analysis using BCP (Bacterial Cytological Profiling) may lead to the classification of a novel antibiotic. ADDITIONAL PRESENTERS: Sean Kim, Jem Soriano, Thomas Dawson, John Paul Davis, Danielle Le
Abstract Title : Quick and cost effective CRISPR guide RNA validation in vitro
Abstract : CRISPR/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable sgRNA. We have developed a cost effective and straight forward method for screening sgRNA function in vitro. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative PAM site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.
Abstract Title : The Role of Kisspeptin Signaling in Brown Adipose Tissue in Mice
Abstract : The peptide kisspeptin and its receptor, Kiss1r, regulate reproduction by stimulating reproductive hormone secretion from the brain. Besides controlling reproduction, Kiss1r was also recently shown to regulate body weight (BW), metabolism, and glucose tolerance. Outside the brain, Kiss1r is found in the gonads, liver, pancreas, white adipose tissue, and brown adipose tissue (BAT). BAT is known for its high density of mitochondria, making it an important tissue for metabolic regulation. Here, we tested the hypothesis that kisspeptin signaling in BAT influences BW, metabolism, and/or glucose tolerance. Comparing wild-type mice with conditional Kiss1r knockout in BAT, we found that absent Kiss1r in BAT does not alter reproduction, BW, metabolism, or glucose tolerance. This suggests that Kiss1r in the BAT does not have a requisite role in metabolic or glucose regulation, and the previously-observed obesity and metabolic dysfunction in global Kiss1r KOs are due to impaired kisspeptin signaling in another tissue(s).
Abstract Title : Elucidating the Mechanism of Circadian Clock Protein KaiC Localization in Synechococcus Elongatus PCC 7942
Abstract : Circadian clocks are endogenous, self-sustained, 24-hour oscillations that are present in a wide variety of organisms. Cyanobacteria are the only known bacteria to possess a circadian clock. In my research, I focus on the clock of the cyanobacterial model organism Synechococcus elongatus PCC 7942. The cyanobacterial circadian clock revolves around three main proteins: KaiA, KaiB, and KaiC. KaiC is the central component that undergoes a 24-hr phosphorylation rhythm; KaiA enhances the auto-phosphorylation of KaiC, while KaiB enhances the auto-dephosphorylation of KaiC. KaiC has been shown to localize to the cell poles; this localization is enhanced at night and is independent of other clock-specific factors. Furthermore, KaiC localization is conserved in E. coli. This suggests that there is a conserved mechanism by which KaiC localizes to the cell poles. I screened for essential genes that may be responsible for KaiC localization in E. coli. I transformed a plasmid expressing YFP-KaiC into a library consisting of 238 essential genes of E. coli, that are tagged in a manner that allows for inducible depletion.This tool allows me to decrease the expression of essential proteins so that I can observe the effects on KaiC localization. Out of the 238 tagged proteins, 11 candidates expressed a phenotype of KaiC delocalization. Out of these 11 candidates, 9 have homologs in cyanobacteria, all of which are essential in S. elongatus. I will observe the effects of depleting these 9 essential proteins on KaiC localization. If any of these candidates are found to affect KaiC localization, I will observe how it affects the circadian clock as a whole to understand how KaiC localization is important to the function of the circadian clock.
Abstract : Saccharomyces cerevisiae is a species of yeast, one of the most intensively studied eukaryotic model organisms in biological research. S. cerevisiae has two haploid modes, A and Alpha, which allow for the mating of yeast. These two modes have seen to act differently in colony formation on different medias. The difference in colony formation is hypothesized to be due to cell to cell contact. This cellular contact can be linked to cancer, a devastating group of diseases that continues to ravage victims by the millions, is marked by uncontrolled cell division sans contact inhibition. The result of this experiment, in turn, can lead to a further understanding of metastasis, which is the spread of a cancer from one part of the body to another not directly connected with it. The primary investigation of this research involved focusing on S. cerevisiae Mat-A's growth being dependent upon cell contact. To achieve this, an experiment consisting of various plating techniques were utilized that placed Mat-A S. cerevisiae onto SD-Met media, with Mat-alpha as a positive control. The techniques incorporated were pinning, streaking, spreading, and dropping. In addition, pinning on SD-Met with +2 lead was completed. These techniques were selected due to pinning and streaking keeping cells in contact with each other as they were plated, while spreading and dropping require the use of liquid media to plate, which spreads cells apart from each other. The results from these techniques showed that Mat-A yeast cells were able to grow using pinning and streaking, but spreading and dropping the cells did not cause growth, most of the time. Mat-alpha cells grew with all the techniques done. The pining on the lead plate lead to an unexpected color change of the media with the Mat-A cells but not the Mat-alpha cells. This shows that S. cerevisiae requires cell contact in order to grow on deficient medias. ADDITIONAL PRESENTER: Michael Herron
Abstract Title : Visualization of the interaction between Wnt9a and its specific Fzd receptors
Abstract : Wnts are secreted signaling molecules involved in many developmental processes and in maintaining adult tissue homeostasis. Previous work in our laboratory has shown that one particular Wnt ligand is important in hematopoiesis, or the development of blood stem cells. Using a Wnt transcriptional reporter cell line, we have identified two of the fourteen zebrafish Frizzled (Fzd) receptors that our candidate Wnt protein can interact with. Using expression constructs of the Fzd receptors fused to a fluorescent protein, we have developed a strategy to visualize the interaction between Wnt and its Fzd receptors. By exploiting one mechanism by which cells negatively regulate their own signal, cells can internalize the entire ligand-receptor complex. We can then visualize this internatlization by following the Fzd fusion proteins, allowing us to confirm interacting proteins. We can also use this system to interrogate other Wnt pathway members and how they affect Wnt-Fzd interactions.
Abstract Title : Six3: Role in Circadian Rhythms, Fertility, and Metabolism
Abstract : The suprachiasmatic nucleus (SCN) is a small structure of the hypothalamus that coordinates circadian rhythms in the body. SIX3 is a homeodomain transcription factor and is required for normal brain and SCN neuron development. However, its role in adulthood is unknown. It has been observed that deletion of Six3 results in an abnormal metabolic phenotype, which has been known to affect body weight and fertility. Here, we examine the origin of the differing phenotypes in the Six3Flox:SynapsinCre mice, a mouse model allowing specific deletion of Six3 in mature neurons. We first performed lineage tracing of SynapsinCre expression by tracing cells expressing RosaLacZ. We show that SynapsinCre is expressed only in certain structures of the brain, including the SCN, where it deletes Six3. We next investigated the effects on circadian rhythms of Six3Flox:SynapsinCre by observing running-wheel activity in male mice. We show that the loss of Six3 in neurons leads to abnormal circadian rhythms in male mice, indicating Six3 is critical for maintaining SCN output.
Advisor : Dr. JOE POGLIANO
Abstract Title : A Novel Approach for Identifying Molecules with Activity Against New Targets in Bacteria
Abstract : The emergence of multi-drug resistant bacteria and the decline in the number of new antibiotics coming to market poses a major global health threat. There is an urgent need to discover antibiotics that act by novel mechanisms of action (MOA). We have developed a rapid and versatile platform for identifying drugs that act by novel MOAs called Bacterial Cytological Profiling (BCP). BCP utilizes fluorescence microscopy to observe changes in cytological parameters of bacteria exposed to lethal concentrations of antibiotics. Antibiotics that hit targets in different pathways generate different cytological profiles. For novel drug targets, those for which there are currently no antibiotics, we have developed Rapid Inhibition Profiling (RIP), in which we generate cytological profiles by degrading essential proteins, providing a visual screen for antibiotics inhibiting novel targets. We have utilized RIP to obtain profiles of targets within a number of essential pathways in Bacillus subtilis and we have screened a library of 25,000 antitumor drugs from the National Cancer Institute for molecules that inhibit bacteria via specific mechanisms of action. We demonstrate that the screen is capable of identifying the MOA of previously characterized antibacterial molecules, that it identifies anticancer drugs that target the DNA, and that it is capable of identifying new antibacterial molecules with specific MOAs. In the future we will combine BCP with virtual screening to first identify molecules that can interact with target proteins via molecular dynamic simulations, and then to determine if they inhibit these drug targets in vivo.
Abstract Title : Factor Analysis of Temperament and Personality Traits in Bipolar Patients: Correlates with Comorbidity and Disorder Severity
Abstract : Temperament and personality traits have been suggested as endophenotypes for bipolar disorder based on several lines of evidence. Previous work suggested an anxious-reactive factor identified across temperament and personality inventories that produced significant group discrimination. We have further characterized this factor structure in a sample of 1195 bipolar patients. Dimension reduction identified 18 factors explaining 39% of the variance. The two largest factors reflected affective instability and general anxiety. All factors revealed specificity for associated clinical features in a predictable pattern. Subsequent cluster analysis identified one group with a strong lack of general anxiety and low affective instability represented. The remaining subjects could be distinguished into two groups by the presence of either more positive characteristics, including persistence, spirituality, expressivity, and humor, or more negative characteristics of depression and anxiety. These results suggest that cross-analyses of temperament and personality inventories may have utility for identifying clinical profiles in bipolar patients.
Abstract Title : alin 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 non-myocyte 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. Electron microscopy (EM) and Evan's blue dye (EBD) uptake were performed to determine costamere integrity and membrane stability respectively. 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 (Tln2KO/Tln1cKO). Tln2KO/Tln1cKO 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 beta1D integrin and displacement of vinculin from the costameres. Increased EBD uptake in the Tln2KO/Tln1cKO vs. control showed that membrane stability was reduced with loss of CM talin. Conclusions: These data suggest that Tln2 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 : Identification and characterization of phage infecting Streptomyces platensis
Abstract : Through studying phage genomics, we can better understand their evolutionary history, their known biological functions, and discover new phage properties and functional roles. Because phage can manipulate host genomes, understanding phage properties and their host range could lead to the application of phage as vectors for therapy to treat bacterial infections. Twenty-eight phage were isolated and purified from Streptomyces soil bacteria. After conducting gel electrophoresis and electron microscopy, we sequenced the genomes of phages: IceWarrior, BartholomewSD, TrvxScott, and Shawty. Clustering the sequencing data of these four phage revealed BartholomewSD and TrvxScott clustered together while IceWarrior and Shawty clustered uniquely. These four phage were tested on twelve different Streptomyces hosts to determine their host range. Differences in the phages' hosts range allowed us to examine relationships between cluster and host range. ADDITIONAL PRESENTERS: Yuya Fujita, Pranathi Gutala, Prianka Kumar, Samuel Shing, Sharon Tran