2016 Trainees

		Marianna Barnhart Mentor: David Wright Home Institution: Emmanuel College Research Abstract: Nucleic acids are used as biomarkers in diagnostic tests, as they are highly sensitive and specific. However, amplification and detection of nucleic acids for disease diagnosis is often done using high cost, specialized techniques, such as loop-mediated isothermal amplification (LAMP) and PCR, limiting its functionality in low-resource settings. Here, we report the use of a sensitive bio-barcode assay for the detection of malarial DNA to be read on a lateral flow assay (LFA). The 18S rRNA gene, which is responsible for coding a component of the small eukaryotic ribosomal subunit, is conserved in every species of Plasmodium making it a valuable sequence for detection. Target DNA was extracted from the sample using a magnetic microparticle and bound to labeling bio-barcode gold nanoparticles (AuNP) resulting in the target sequence being sandwiched between a AuNP and a magnetic microparticle. To detect the target sequence, barcode DNA was then released from the conjugated AuNPs and visualized using an LFA. Because the AuNPs carry large numbers of barcode DNA per target DNA binding event, substantial amplification is achieved. We hypothesize this will allow for the sensitive lateral flow detection of DNA from all species of malaria without the use of expensive, time consuming techniques.
		Janna Berman Mentor: Nathan Schley Home Institution: University of Puget Sound Research Abstract: Pincer complexes are common in synthetic laboratories as organometallic catalysts, however, they are rarely observed in enzymes. A pincer ligand framework is present in the nickel cofactor of lactate racemase, an enzyme that converts lactic acid between its D- and L- isomers, thus suggesting catalytic potential. We plan to investigate the hydride transfer capability of a lactate racemase model because this property activates inert C-H bonds to impart new reactivity to a substrate. Given pincer complexes are used to facilitate the generation and transfer of hydrides, observing similar activity in this enzyme may suggest that a pincer ligand framework plays a large role in its biological activity. Herein, we complete the synthesis of a model complex of lactate racemase. The proposed synthetic route takes advantage of simple, commercial starting materials via a Hantzsch ester synthesis. In addition, synthesized compounds are thus far air-stable and product yields range from fair to excellent. Future plans include metalation of the pincer ligand with nickel and experiments to probe the catalytic hydride transfer capability. We expect hydride transfer on the model compound of lactate racemase to occur directly at nickel or at C4 of the pyridinium ring on the ligand. Provided catalytic hydride transfer occurs with our synthesized model compound of lactate racemase, we will demonstrate the utility of the pincer ligand framework in influencing enzymatic activity associated with racemization of lactic acid.
		Danielle Boley Mentor: Jeff Johnston Home Institution: University of Notre Dame Research Abstract: Arginylglycylaspartic acid (RGD) is a tripeptide cellular recognition sequence that interacts selectively with cell surface receptors, called integrins. These interactions are known to induce cell signaling pathways that are important in several pathological processes. The current understanding of protein-integrin interactions has influenced the development of macrocyclic peptide drugs with RGD motifs. Although synthetic methods have been developed to produce these molecules, using them to create RGD cyclic peptides and depsipeptides still poses a significant challenge. We propose a strategy to produce cyclic RGD depsipeptides that uses a combination of enantioselective synthesis and Umpolung Amide Synthesis (UmAS). This approach achieves the broadest possible scope while overcoming synthetic challenges, such as racemization. This synthesis includes an enantioselective Henry addition of bromonitromethane into an aliphatic aldehyde, followed by coupling of the bromonitroalkane product with a dipeptide, L-Orn(Z)-Gly-OMe, using UmAS. Preliminary results suggest successful synthesis of the necessary catalyst, production of amide product, and coupling of amino acids. Future directions include saponification and subsequent coupling with an Asp-containing tridepsipeptide. The resulting hexadepsipeptide will then be used in the synthesis of large RGD macrocycles, which could be medically important in the future since integrin-related drugs have the potential to treat heart disease, cancer, and autoimmune diseases.
		Mattie Braselton Mentor: Eva Harth Home Institution: Georgia Southern University Research Abstract: Hydrogels are formed by crosslinked polymer chains that form networks, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are superabsorbent natural or synthetic polymers. Hydrogels also possess a degree of flexibility similar to natural tissue due to their significant water content which allows them to be injectable into the human body. Oxime click reactions, the reaction between an amino-oxy functionality with a ketone or aldehyde, are optimal for the synthesis of hydrogels. The efficiency of oxime formation provides an opportunity to synthesize functional polymeric materials where the formation of water as the only side-product make it attractive for the synthesis of polymers that can be bioorthogonal. The Harth lab has established a process to synthesize semibranched polyglycidols that display properties to polyethylene glycol (PEG), are hydrophilic, and deliver a platform for further functionality. For small hydrophobic drugs, a common way to enable delivery is through nanoparticles of controlled size. The nanoparticles create an environment for encapsulating hydrophobic small molecules which are otherwise incapable of being dispersed in the body. In this research, an amino-oxy functionalized polyglycidol and a linear polyester containing ketone functionalities were used to create a hydrogel that contained drug-loaded nanoparticles entrapped within the system. Since the polyester component of the nanoparticle and hydrogel can degrade either enzymatically or hydrolytically, the molecule drugs encapsulated inside the nanoparticle are released during degradation of both nanoparticle and gel. This dual delivery system has shown efficacy for the release of two different classes of therapeutics.
		Heidi Carpenter Mentor: Steve Townsend Home Institution: University of South Carolina Research Abstract: Carbohydrate-based vaccines are promising targets for cancer immunotherapy.Aberrant glycosylation patterns, called tumor associated carbohydrate antigens (TACAs) appear on the surface of malignant cells. The syntheses of TACAs have led to promising steps in the development of cancer treatments. If properly presented to the immune system, a TACA should elicit an immunological response resulting in the eradication of malignant cells expressing the TACA. Although a useful tool for therapies, carbohydrates are poorly immunogenic and thus require conjugation to immunoenhancing elements in order to stimulate an effective immune response. Zwitterionic polysaccharides (ZPSs) isolated from bacteria have a promising future in vaccinology because they have immunoenhancing properties. In theory, vaccine constructs can be developed by conjugating clinically important TACAs to ZPSs to invoke an effective immune response. Chemical synthesis of these ZPSs would allow for the manipulation of the compounds involved. The goal of this project is to chemically synthesize building blocks needed for the total synthesis of a ZPS isolated from Morganella morganii.
		Sarah Hahn Mentor: David Wright Home Institution: Louisiana State University Research Abstract: Global infectious disease burdens often fall on low-to-middle income countries, with Lateral flow assays (LFAs) being a widely used format for diagnostics in these low-resource settings. In these tests, antibodies are immobilized on nitrocellulose, and the analyte is captured at the antibody line. Antibody-conjugated gold nanoparticles are used to detect the presence of the analyte. LFAs are fast, cheap, durable, and disposable, allowing for use in more rugged environments while returning same-day results. Due to the positive/negative result format, the sensitivity and complexity of the assay are limited. To improve both, an enzymatic turnover reaction could correlate the formation of a colored product at the test line to the quantity of analyte. In a lateral flow format, this colored product would not remain stationary due to capillary flow on the test strip, thus removing evidence of the positive test. Our current project is to polymerize barriers in order to hold 4 μL of colored solution. This was done by printing a photoinitiator (eosin conjugated to bovine serum albumin) onto nitrocellulose, wicking up the polymer precursor mixture of PEGDA, TEA, VP, and free eosin, and illuminating with green LEDs. The concentration of striped EITC, PEGDA, TEA, VP, free eosin, wicking time, and illumination time were all optimized in order to obtain controlled polymerization at the initiator site. This novel assay format will be applied to the malarial biomarker pLDH, which has natural enzymatic activity producing a colored product, allowing for the development of a rapid, simple, quantitative detection method for pLDH.
		Zasmine Hymes-Green Mentor: Gary Sulikowski Home Institution: Alabama State University Research Abstract: Protein interaction are responsible for many internal and external responses within living systems. To determine the exact cause and effect relationship that small molecules have on their targets, these proteins must be isolated and identified. One popular method used to do so is the biotin-streptavidin complex. When small molecules attach to biotin, this acts a probe to enter the cell where the binding of target proteins can occur. Streptavidin, a bacterial protein, then binds to biotin. The biotin-streptavidin complex is then able to be used in a variety of pull down assays to obtain the target protein for analysis. However, streptavidin has such a high affinity for biotin that releasing the target protein from the complex requires harsh conditions that can denature or further damage the protein. Therefore, this project seeks to synthesize a UV-cleavable linkage to be inserted between the biotin-streptavidin complex and the target protein. These mild conditions circumvent the complications that arise from traditional methods. Utilizing a readily available amino-protected phenylglycine, we have demonstrated rapid access to an unprecedented photocleavable core in only 2 synthetic steps. With our new synthetic strategy validated, we can now turn our attention to derivatization of our photocleavable core. We plan to covalently attach both biotin and bicylononyne moieties. This new linker will be used to study the protein targets of various biologically active compounds.
		Adam Perez Mentor: David Cliffel Home Institution: San Diego State University Research Abstract: In order to more rapidly develop disease treatments, research is currently being conducted to form a "milli-human," which is a combination of artificial organs used to accurately simulate a human without the need for an actual test subject. The development of these "organs-on-chips" requires a method to monitor health of the organs. Therefore, we are developing an electrochemical sensor that utilizes platinum screen-printed electrodes to perform multianalyte detection of pH, glucose, lactate, and glutamate levels of the organs. With a device that can detect these compounds and conditions, we can simulate response of human neurovascular cells to certain health stresses. The advantage of this device is that it allows simultaneous multianayte detection with high specificity, high sensitivity, and low limits of detection. The real-time detection utilizes oxidase enzyme films and iridium oxide sensors in order to accurately monitor cell metabolism and is the first time glutamate, a crucial neurotransmitter, has been measured in this system. We use a CH 1440 Electrochemical Analyzer to measure the open circuit potential (OCP) of our electrodes to determine pH and current to determine analyte concentrations. While our experimentation with iridium-oxide plated electrodes produce replicable signals that can be calibrated upon addition of each analyte, its complete functionality remains still to be explored as to whether it can detect glucose, lactate, and glutamate. Our research examines the effects of iridium oxide plated electrodes as an alternative or interdependent component to measuring pH, glucose, glutamate, and lactate with enzyme films.
		Christian Ramsoomair Mentor: Walter Chazin Home Institution: College of the Holy Cross Research Abstract: Pathogenic bacteria pose a significant threat to global public health due to emerging antibiotic-resistant strains. Eukaryotic organisms have evolved a response known as “nutritional immunity” which slows the growth of the bacteria by limiting access to key metal nutrients. Understanding this response could lead to better therapeutic strategies to combat these pathogens.Calprotectin (CP), has been identified as a key member of nutritional immunity by utilizing high affinity transition metal-binding sites. Previous studies have illustrated its ability to sequester manganese and zinc to deprive bacteria of these key nutrients. Observations of CP protection against copper-dependent bacteria and the Irving-Williams series suggest that CP should not only bind copper, but have a higher affinity than the other transition metals. Using assays such as isothermal-titration calorimetry, X-ray crystallography, and fluorescence spectroscopy, we aim to establish the affinity of the CP-Copper interaction and its structural characterization.Beyond its metal sequestration activity, CP interacts with the cell surface receptor RAGE (the receptor for advanced glycation end products). This interaction activates a pro-inflammatory signaling cascade when large amounts of RAGE ligands are present. Here a positive feedback cycle is established, which can cause chronic inflammation and several adverse effects of diabetes. The Chazin lab has begun fragment-based discovery for inhibitors of RAGE as a promising therapeutic target in diabetes. We are focusing on identifying a RAGE mutant that is conducive for structural studies needed to develop a multivalent RAGE inhibitor. This could lead to drugs that specifically target RAGE and its adverse signaling during diabetes.
		Aron Sulovari Mentor: Brian Bachmann Home Institution: Vassar College Research Abstract: Global increases in antibiotic resistance necessitate innovative natural product development. Genetic manipulation of underdeveloped antibiotic molecular scaffolds is a promising area of research for addressing the shortage of new antibiotics. The complex oligosaccharide everninomicin is one such scaffold that proceeded to Phase III clinical trials, but was withdrawn from development due to toxicity concerns. The organic synthetic route for everninomicin, however, is too complex (> 130 steps) for feasible generation of analogues to reevaluate its toxicity and clinical efficacy. The natural biosynthesis of everninomicin is hypothesized to be significantly more efficient and potentially capable of generating analogues. Therefore, we aim to express and purify the acyltransferase (EvdD1), orsellinic acid halogenase (EvdD2), polyketide synthase (EvdD3) and aromatic O-methyltransferase (EvdM5) involved in the biosynthesis of the dichloroisoeverninic acid (DCE) moiety of everninomicin for future characterization of enzyme function and substrate selectivity. Protein cloning, expression and purification procedures will be used to obtain pure enzymes and enzymatic assays will be performed to evaluate biotransformation activity. EvdD1, EvdD2 and EvdM5 have already been expressed. Biochemical assays using synthesized substrates will confirm the enzymes' role in the biosynthesis of DCE, and permit detailed mechanistic dissection of DCE pathway enzymes. Elucidation of the biosynthetic mechanism of everninomicin will enable harnessing of the biosynthetic pathway for the generation of semisynthetic analogues to further explore its clinical application.
		Danielle Worrell Mentor: Ray Blind Home Institution: Tuskegee University Research Abstract: Nuclear receptors are ligand activated transcription factors whose activity can be regulated both by a ligand as well as a variety of co-regulatory proteins. Previous research has shown that phosphoinositide phospholipids (PIPs) are important in the regulation of the transcriptional activities of steroidogenic factor 1 (SF-1), a subgroup 5 nuclear receptor. The mechanism of liver homolog receptor 1 (LRH-1) activation is currently uncharacterized; but, due to high sequence homology with SF-1, we predict that the two will share a common mode of activation. To assess this, we will treat human embryonic kidney (HEK293) cells expressing tetracycline-inducible LRH-1 with aurintricarboxylic acid (ATA). ATA is a known inhibitor of inositol polyphosphate multikinase (IPMK), a kinase that phosphorylates PIP2 to PIP3 when bound to SF-1. We isolated RNA from untreated and ATA treated cells, and then ran real time reverse transcriptase polymerase chain reaction (qRT-PCR) using primers for LRH-1 target genes. Upon analysis of these data, we will be able to determine the effect of ATA on LRH-1 dependent transcription. We expect to see the expression of LRH-1 target genes decrease upon ATA treatment: this would suggest that IPMK activates LRH-1 in the same way as it does SF-1. Abnormal hormone signaling is a strong driver of cancer development; thus, drugs targeting IPMK could be used to inhibit tumors.