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2017 Research Showcase
CADB Abstracts
Advisor : LARS BODE
Abstract Title : The Effects of Human Milk Oligosaccharides on Parasitic Organisms
Abstract : Human milk contains a reservoir of unique, structurally diverse, and highly abundant oligosaccharides. These human milk oligosaccharides (HMOs) play important roles in brain development, cognition, immune health and protection against bacterial and parasitic infections. Previous studies have shown that HMOs to be anti-adhesive antimicrobials that can lower the risk of pathogenic diseases, for example Entameba histolytica. Further, this project looked at the response of parasites, including the protozoa (Naegleria fowleri, Giardia lamblia, Trypanosoma cruzi, Trypanosoma brucei) and the flatworm (Schistosoma mansoni), to structurally different HMOs. Through whole organism screenings, the experimental results showed that HMOs incubation enhanced the T. brucei growth, inhabited the T. cruzi and Giardia, and had no effect on Naegleria fowleri and Schistosoma mansoni. The growth enhancement/inhibition results of the parasites were confirmed across different runs and will be followed by a dose response experiment to identify the lowest concentration that will produce similar effects.
Abstract Title : Nanoparticle Decoys for Treatment of Infection with Vibrio Cholerae
Abstract : Each year, enteric infections are associated with millions of deaths. The emergence of multidrug resistant pathogens is a matter of growing clinical concern, threatening the utility of existing antibiotics. The enterotoxins produced by these pathogens are central in disease pathogenesis, since mutation of the toxins or their host receptors markedly attenuates or abolishes disease. Because of their critical role, the respective host receptors constitute attractive targets for non-traditional antimicrobial strategies that do not rely on conventional antibiotics, because the receptors, contrary to any bacterial factors, are constant and do not mutate over therapeutically relevant time frames, eliminating concerns about antibiotic resistance development. One major approach to leverage host receptor specificity is the development of pharmacological decoys that are identical to and compete with the receptors, and thus act as harmless sinks or sponges for the toxins until they are removed or destroyed by the host. In this study, we developed GM1 ganglioside-conjugated nanoparticles (GM1 NP, GM1-polymer hybrid nanoparticle decoys) that can neutralize cholera toxin in a cell culture model and in mice infection that we tested in the current work. We also used intact vibrio cholerae for cell-based cAMP assay and mice ligated loop where GM1 NPs were incubated with the live bacteria. Interestingly, GM1 NP decreases cAMP1 following infection compared to the control particle. Together, the proposed project will develop a novel nanotechnology-based platform as a non-traditional antimicrobial strategy for the management of intestinal infections with enterotoxin-producing pathogens that cannot be treated with conventional antibiotics due to resistance or other biological reasons.
Abstract Title : Natural muscle loss in the Lesser Egyptian Jerboa as a model for muscular degeneration
Abstract : The lesser Egyptian Jerboa (Jaculus jaculus) is a small bipedal rodent that is closely related to a house mouse (Mus musculus). Its hindlimbs have three toes and elongated metatarsals, the long bones of the foot, that fuse together in postnatal development. Jerboas are born with muscles in the foot that appear normal, but then degrade rapidly within its first week. Muscle degeneration has parallels to human muscular disease but also differs in striking ways that may give us a deeper understanding of the fundamental aspects of muscular development and dysfunction. Therefore, we sought to characterize the natural loss of muscles in the jerboa foot. It is important to understand the dynamics of muscle loss so that we can identify the timing of the earliest events and the mechanisms of cell death or transdetermination. By quantifying the number of muscle fibers in a comparable region in the foot across stages in the first week, I found that muscle fiber loss is initiated between four and six days after birth. By transmission election microscopy, we found that the contractile sarcomere apparatus disassembles prior to myofiber loss. In order to test the hypothesis that sarcomere proteins are disassembled in an orderly fashion, similar to muscle atrophy, we performed immunohistochemistry of many of the major sarcomere proteins. We found that the striated pattern of desmin is lost prior to any of the other structural proteins similar to mouse models of human atrophy. This provides context to develop and test hypotheses to identify the initiating event(s) that lead to developmental loss of muscle in an evolutionary context.
Abstract Title : The Effects of Kappa-Opioid Stimulation Post-Cardiac Pressure Overload on Cardiac Function
Abstract : The stimulation of kappa-opioid receptors has been shown to protect the heart from injuries related to ischemia, or a shortage of oxygen. In addition, the increase in caveolae formation has been shown to play an important role in cardiac protection as well. In this study, we set out to see if the constant stimulation of kappa-opioid receptors would reduce pressure overload-induced heart failure caused by a transverse aortic constriction (TAC) surgery. Ten-week old mice were given either a sham or a TAC surgery. Two weeks after the surgery, the mice were given daily intraperitoneal injections of either a kappa-opioid receptor agonist U50488H or a vehicle (V). Their heart function was measured via echocardiography before the surgeries, and then at two-week intervals for ten weeks post-surgery. The hearts were also analyzed by transmission electron microscopy and immunohistochemistry at 11 weeks post-surgery. Analysis showed preserved mitochondrial structure and dense intercalated discs in TAC+U50 animals, while TAC+V animals showed bleached discs and swollen mitochondria. Accumulation of connexin-43, a gap-junction protein, was found intracellularly in TAC+V hearts but was found in the membranes of TAC+U50 hearts. Thus, we believe that U50448H administration could potentially become a treatment option for patients that are susceptible to heart failure.
Abstract Title : Interactions between the Basement Membrane Proteins PXN-2 and SPON-1 in C. elegans Embryogenesis
Abstract : Basement membranes are extracellular matrix (ECM) protein networks associated with tissue boundaries in all animals. They are essential for embryogenesis as well as post-embryonic tissue organization and structural integrity. Defects in basement membranes or ECM underlie numerous genetic diseases, including Alport Syndrome, benign familial hematuria, and epithelial basement membrane corneal dystrophy. We are studying basement membrane function using a genetic model organism, the nematode C. elegans. We focus on two basement membrane proteins, F-spondin SPON-1 and peroxidasin PXN-2, both of which promote muscle-epidermal attachment during embryogenesis. We performed a suppressor screen for a temperature sensitive mutant of spon-1, which is lethal at 25˚C, and found several new mutations that rescue spon-1 lethality. Using whole genome sequencing and two-point linkage mapping, we located one suppressor-associated missense mutation to be within the catalytic domain of pxn-2. The resulting change of a leucine to phenylalanine residue within this critical region of the enzyme may cause structural and functional alterations that change its interaction with nearby molecules, ultimately leading to rescue of spon-1 mutant defects. To further assess the effects of this suppressor mutation we crossed it with the null mutant of spon-1, which is 100% lethal at all temperatures, upon which there was no significant rescuing effect. The suppressor mutation was back-crossed into the original spon-1 temperature sensitive mutant to confirm the original suppression. This putative suppressor allele of pxn-2 appears phenotypically wild type, unlike loss of function in pxn-2 which exhibits muscle-epidermal detachment and abnormal morphology. This observation suggests that a gain-of-function allele of pxn-2 specifically suppresses partial loss of SPON-1 function either by direct interaction or by activating alternative signaling pathways. Elucidating how basement membrane proteins function will increase our understanding of this essential structural network’s role in fundamental biological processes such as embryonic development and wound healing.
Abstract Title : CRISPR/Cas9 disection of heparan sulfate
Abstract : Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with a large number of matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively in cells or model organisms, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. In this study, a genome-wide CRISPR/Cas9-mediated screen was developed to uncover and characterize novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, a high-throughput screening assay was adapted to identify lentiviral-encoded sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs. Parallel screens using alternative HS-dependent toxins or plant lectins that cause cytotoxicity dependent on other types of glycosylation were performed in order to sort genes that selectively affect HS biosynthesis. In preliminary library screens, we identified previously studied genes essential for HS formation and factors involved in the intoxication of cells by diphtheria toxin, an HS-dependent exotoxin. Furthermore, we uncovered potential candidate genes whose function is unknown relative to HS biosynthesis. Top hits from the screens were characterized and categorized based on their predicted gene functions and are currently being individually validated and examined for their potential involvement in the regulation of HS biosynthesis. Overall, these studies will give us a better understanding of the genetic regulatory factors involved in HS biogenesis.
Abstract Title : Enhanced wound repair in heparan sulfate biosynthetic mutant mice
Abstract : Mammals have a limited ability to repair wounds from traumatic injury. As heparan sulfate regulates many of the cell signaling pathways important for wound repair and cell growth, we hypothesized that heparan sulfate biosynthetic mutant mice may have altered wound repair. To analyze wound repair, full-thickness dorsal skin excisional wounds were performed on two mouse models of heparan sulfate deficiency, genetic and pharmacological. In the genetic model of heparan sulfate deficiency, the mice lack copies of the EXT genes that encode for heparan sulfate biosynthetic copolymerase complex. In the pharmacological model, mice are treated with a competitive substrate for the heparan sulfate biosynthetic copolymerase complex. Both the pharmacological model and genetic model that had altered heparan sulfate biosynthesis closed their dorsal skin wounds faster than the wild-type mice. Biochemical analyses were performed to compare the variation of heparan sulfate biosynthesis. Understanding how heparan sulfate functions in wound repair and regeneration can help enhance wound repair in patients.
Abstract Title : Discovery of Drugs against Deadly Amoebic Encephalitis caused by Naegleria Fowleri
Abstract : Primary amoebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living amoeba Naegleria fowleri (N. fowleri). To date, the treatment for PAM relies on the drugs Amphotericin B and Miltefosine. However, these drugs are not optimum or uniform – Amphotericin B causes renal toxicity with only about a dozen survivors worldwide. Considering the severity of the disease and the current treatments do not prevent lethal outcomes for the majority of the PAM patients, a better treatment option is urgently needed. Therefore, we conducted a screening of eighty-six FDA-approved compounds (repurposing of drugs: drugs already on the market for the treatment of other diseases) against Naegleria fowleri. To enable this screening an automated, high-throughput screening methodology was developed. A primary screening of eighty-six (blood brain-barrier crossing) drugs had been tested at 50uM concentration, in vitro for hits—killing activity of N. Fowleri. From this screening, we obtained six hits from drugs RAP1, RAP2, RAP3, RAP4, RAP5, and RAP6. Therefore, a secondary screening was performed to determine the EC50 of each compound. Through this screening we have identified 6 drug candidates with the EC50 values ranging from 3 to 6uM. This level of activity is within therapeutically achievable concentration range. Based on theses results a future in vivo experiments will be conducted in the mouse model of PAM to measure the efficacy of the compounds for the treatment of PAM
Abstract Title : Identifying the Mechanism of Action of New Antimicrobial Compounds Using Bacterial Cytological Profiling
Abstract : The emergence of multi-drug resistant bacteria and the decline in the number of new antibiotics creates 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 inhibit targets in different pathways generate different cytological profiles. We screened a library of over 2,000 compounds against E. coli ΔtolC, determined the MOA of the hits, and identified five previously uncharacterized compounds active against both E. coli ΔtolC and Staphylococcus aureus. We also obtained compounds with unknown MOAs from collaborators, and identified three that inhibit peptidoglycan biosynthesis in B. subtilis. These molecules can be further developed as antibiotics for the treatment of infections caused by drug resistant bacteria.
Abstract Title : Identification and Validation of Small Molecule Targets in Plasmodium Falciparum
Abstract : Today there is a significant need for new drugs to combat malaria due to resistance found in a number of current treatments. To create new antimalarial drugs, novel drug-able targets must be identified to reduce the risk of cross-resistance in Plasmodium falciparum. Small molecules can be used to select for parasite resistance, which can then elucidate the target through SNVs and CNVs found through whole genome sequencing. MMV011903, MMV032967, MMV023949, and MMV676459 are small molecules discovered through phenotypic screening to have antimalarial activity. These compounds were used to evolve parent strains of Plasmodium falciparum at sub-inhibitory concentrations. MMV085203 resistant lines have been found to have mutations in a putative transporter, dihydrouridine synthase, and protein kinase on the second chromosome through whole genome sequencing. These targets were validated through CRISPR-cas9 genome editing. By identifying novel targets in Plasmodium falciparum, more effective antimalarials can be produced and brought to the community at large.
Abstract Title : A naturally occurring homeotic transformation: Differential Hox10 expression causes a vertebral identity shift in the Lesser Egyptian Jerboa
Abstract : The Lesser Egyptian Jerboa (Jaculus jaculus) is a small bipedal desert rodent with a number of skeletal modifications that correlate with an upright stance. Despite this unusual rodent bipedality, jerboas are phylogenetically closely related to the mouse, and are therefore an ideal comparative developmental model to study how molecular mechanisms can result in divergent phenotypes. One such point of comparison is the single posterior shift in vertebral identity observed in the jerboa— while mice have 13 thoracic and 6 lumbar vertebrae, jerboas have 12 thoracic and 7 lumbar vertebrae. We hypothesize that this shift in vertebral identity is due to differential expression of Hox genes, which determine vertebral identity in a variety of species. Specifically, we chose to examine the expression of Hox10 genes (HoxA10, HoxC10, and HoxD10), which suppress rib identity in transgenic mice. We predicted that the expression of at least one Hox10 gene would be extended more anteriorly in jerboa embryos than in mouse embryos, correlating with the suppression of rib formation in the transformation of the thirteenth thoracic vertebra to an extra lumbar vertebra. We compared the expression of HoxA10, C10 and D10 mRNA in somites, the embryonic precursors of vertebrae in mouse and jerboa and found that both HoxA10 and HoxD10 are expanded anteriorly in the jerboa while HoxC10 is unchanged, suggesting that a trans regulatory factor mediates this shift.
Abstract Title : Identification of the Targets of a GRAS Family Transcription Factor of Maize via Transposon Insertional Mutagenesis and Virus Induced Gene Silencing
Abstract : A maize transcription factor (TF) called GRAS3 may affect hormone signaling involved in plant growth and immunity. It is predicted to regulate salicylic acid hormone metabolism, cell signaling, and protein degradation. We aim to validate the true targets of GRAS3. To investigate the importance of GRAS3 to maize plants, we screened plants with putative functionally disruptive transposon insertions in the gras3 gene. We genotyped the plants through a series of PCR reactions using gene specific primers and transposon specific primers to determine whether they contained the insertion and if they were homozygous. In the next generation, we will identify homozygous plants and then will check for transcript abundance of GRAS3 and its targets since the transposon should interrupt the coding sequence. We identified and self-pollinated plants which are heterozygous for our insertions of interest. In addition, we are using particle bombardment to infect plants with Foxtail Mosaic Virus that has been genetically modified to contain a silencing construct for GRAS3. This may manifest in phenotypic differences at the plant level (visual differences such as smaller size or less vibrant color as compared to the wild type) and at the molecular level (a change in gene expression of the targets). ADDITIONAL PRESENTERS: Bonny Pham, Kevin Celniker.
Abstract Title : Persistent expression of developmental genes in digits of heparan sulfate deficient mice: proposed mechanism for Multiple Hereditary Exotoses (MHE) and enhanced regeneration.
Abstract : Heparan sulfate can regulate cell proliferation and differentiation by mediating growth factor signaling. In humans, heparan sulfate deficiency causes Multiple Hereditary Exotoses (MHE), a disease characterized by abnormal growth of bone spurs and lumps. Preliminary experiments in heparan sulfate deficient mice suggests an enhanced ability to wound heal and regenerate, however the mechanism behind enhanced regeneration and MHE is not fully understood. We hypothesize that there are common mechanisms between enhanced regeneration and the development of MHE. To investigate the mechanism of enhanced regeneration and MHE, we performed qRT-PCR in neonatal digits of heparan sulfate deficient mice. We found that compared to wildtype mice, heparan sulfate deficient mice have persistent expression of a number of developmental genes including: Prrx1, Msx2, Tbx2, Tbx4 and shh pathway genes (shh, Ptch1, Gli1, HHIP). Based on these results, we propose a mechanism for MHE and enhanced regeneration through the persistent expression of developmental programs resulting in aberrant (MHE) and enhanced growth.