Dr. Dayle Daines -- MUSM (approved for funding, Macon Campus)

We are studying a set of gene pairs that regulate stress-induced reversible growth arrest in nontypeable Haemophilus influenzae (NTHi), bacteria that are one of the leading causes of otitis media in children.  This novel mechanism facilitates chronic infections because it allows these organisms to become tolerant to many antimicrobials, even when a particular drug normally kills the bacterium.  Since most antibiotics target essential biosynthetic pathways, an organism that is not replicating is not susceptible.  Once the clinical course of antibiotics is completed, the improved conditions trigger the dormant bacteria become metabolically active again and re-establish the infection.  Understanding how the protein products of these gene pairs, known as toxin-antitoxin (TA) loci, synergize to facilitate this growth regulation is important, since blocking this activity could increase the efficacy of existing antimicrobial therapies and reduce the number of recurrent infections.  The larger significance of these studies is that TA loci have been identified in the genomes of many different bacteria, which suggests that metabolic regulation following stress (antibiotic, nutrient, or oxidative, for example) is a conserved survival mechanism shared among numerous microorganisms.  Therefore, this project has implications not only for medicine, but also for other areas such as bioremediation, food safety, and environmental engineering, to name a few.

John Bauer -- MUPHS (aproved for funding, Atlanta Campus)

Discovery of Novel Antibiotics from Uncultured Soil Bacteria

Since the beginning of the twentieth century, antibiotics have been used successfully to neutralize infectious diseases that were previously widespread, untreatable, and frequently fatal. Infections now occur from pathogens that are resistant to all current antibiotic options, thus emphasizing an urgent need for agents with specific activity against panresistant organisms. While most clinically useful antibiotics are natural products, predominantly derived from microorganisms, reisolation of previously identified compounds from cultured organisms has become a serious impediment to the discovery of new bioactive natural products. Culture-dependent techniques have been used to access microorganisms in their natural environment for well over a century. Yet, current biodiversity estimates suggest that less than one percent of bacteria can be cultured in the laboratory. Given that the majority of clinically relevant antibiotics have been derived from less than one percent of bacteria, it seems likely that homologous metabolic pathways that generate novel antibiotics are present in the heretofore uncultured majority of bacterial species normally present in soil. Uncultured bacteria are therefore, an unexploited and potentially rich source of novel antibiotics. Since most bacteria do not grow well in the laboratory, new methods must be developed to access this biodiversity and in turn the chemical diversity encoded in the genomes of these, as yet, uncultured organisms. One approach to this problem is the construction of genomic DNA libraries from microbial DNA extracted directly from environmental samples (Figure 1). Heterologous expression of natural product biosynthetic gene clusters found on large fragments of environmental DNA (eDNA) can provide access to many natural products produced by these once inaccessible organisms. Identifying eDNA clones that are likely to produce clone-specific natural products can be accomplished by genetic screening. PCR with degenerate primers can be used to identify and recover clones that harbor homologous natural product-specific biosynthetic genes. Each recovered clone can be computationally and functionally examined for the production of novel antibiotics. Unlike traditional natural product isolations, where the metabolites of a single cultured organism are systematically characterized, eDNA libraries should allow us to simultaneously screen thousands of bacterial genomes. Previous studies, resulting in the isolation and characterization of novel secondary metabolites from eDNA libraries, strongly suggest that uncultured bacteria represent a plentiful source of novel natural products. The MUBS summer research project in my lab will focus on a genetics-based approach for the recovery and characterization of novel beta-lactam antibiotics from uncultured soil bacteria.

Chalet Tan -- MUPHS (approved for funding, Atlanta Campus)

Hsp90 is a highly conserved intracellular chaperon protein that is responsible for the conformational maturation and stability of numerous signaling proteins, such as HER2, BCR-ABL, estrogen receptors, androgen receptors, insulin-like growth factor 1 receptor, B-Raf, cdk4, HIF-1a and Akt among numerous others. Because these oncogenic client proteins are involved in multiple hallmark traits of malignancy, such as deregulated cell cycle progression, apoptosis, angiogenesis, invasion and metastasis, inhibition of Hsp90 confers a potent combinatorial blockade on the cancer phenotype. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a potent heat shock protein 90 (Hsp90) inhibitor that is currently undergoing Phases I and II clinical trials in patients with advanced cancers. The therapeutic outcomes of 17-AAG have been hampered owing to its severe hepatotoxicity as well as the large amount of toxic organic excipients used in the current intravenous formulations. Our preliminary studies have demonstrated that 17-AAG is efficiently loaded and retained within polyethylene glycol 2000-distearoylphosphatidylethanolamine (PEG2000-DSPE)/a-tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles without the inclusion of any organic solvents. We aim to further functionalize these nanocarriers to achieve cancer cell-targeted drug delivery, and evaluate their anticancer activity in drug- sensitive as well as drug-resistant tumor models.

Nader Moniri -- MUPHS (approved for funding, Atlanta Campus)

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors, transducing numerous intracellular signaling cascades which control, among other things, hormonal regulation, cell growth, secretory processes and neurotransmission. These receptors also comprise the largest gene family in the human genome and as a consequence are excellent drug targets, accounting for roughly 40-50% of drugs used clinically today. Our laboratory studies two distinct GPCRs: the beta-2-adrenergic receptor which facilitates “fight or flight” effects in response to epinephrine and norepinephrine, as well as GPR120, a recently discovered free fatty acid receptor which facilitates downstream insulin secretion. Currently, we are assessing the ability of these receptors to interact with partner-proteins in order to elicit a biological response. This summer internship, through the Mercer University Biomedical Scholars program, will afford the student the opportunity to characterize the nature of such partner protein interactions using techniques such as plasmid DNA transfection, protein expression, co-immunoprecipitation, immunoblotting, and reverse transcription-polymerase chain reaction.

David Goode -- CLA, Chemistry (approved for funding, Macon Campus)

Central to a successful mammalian host cell invasion by a virus is the ability to gain control of apoptosis, or programmed cell death. There are multiple systems by which viruses accomplish this, and most require the mimicking of host proteins in order to halt the death signal. The host Bcl-2 family of proteins creates a complex balance of both pro-apoptotic and anti-apoptotic signals—the scales tipping one way or the other based upon cellular conditions. Virus invasion usually tips this scale towards apoptosis, but viral mimics of anti-apoptotic Bcl-2 proteins (M11L and F1L) help to restore the balance and give the virus time to overtake cellular machinery—resulting in viral replication. These viral mimics have very different primary sequences from the native Bcl-2 proteins, and thus could be selectively targeted by small, organic molecules. Disruption of the viral proteins should allow apoptosis to proceed—killing the initially infected cell, but also halting viral replication and spread. Compounds have been previously discovered to bind the native anti-apoptotic Bcl-2 proteins and induce apoptosis. One such compound is ABT-737 and its derivatives (see the target structure below). Students are needed to synthesize ABT-737 derivatives, express and purify proteins, and/or synthesize BH3-domain peptides for biochemical assays. This work will lead to cellular culture and possibly animal studies with a graduate-level professor in the future.

Virginia Young -- CLA, Biology (approved for funding, Macon Campus)

Viral infections of the central nervous system (CNS) can have severe consequences, including progressive neurodegeneration, encephalitis, and meningitis. However, the manner by which neurotropic viruses, such as measles virus (MV), cause CNS disease is poorly understood. Interestingly, for many viruses such as Herpes simplex virus, polio virus, and MV the mechanism of viral spread in neurons is distinct from that in non-neuronal cells. Furthermore, this specialized mode of viral spread is associated with no neuronal loss. Thus, it appears that these otherwise lytic viruses have mechanisms to exist in a quiescent, non-lytic state within neurons, while nevertheless continuing to spread within the CNS. Therefore, the research goals of my lab are to understand these two processes by addressing the following questions.

How are neurotropic viruses, such as MV, transported within an infected neuron to the presynaptic membrane? I hypothesize that MV will use the cellular railroad—the microtubule machinery and its associated motor proteins of the dynein and kinesin families—to move within an infected neuron. These studies will be accomplished through basic tissue culture techniques and biochemical methods such as RNA immunoprecipitations to determine the cellular binding partners of viral genomes and viral proteins as they travel within infected cells. The overall objective of these studies is to determine the mechanism by which MV components are transported down the axon to the presynaptic membrane and to identify the viral and cellular proteins that facilitate this process. My work with MV infection in neurons will provide novel insight to this field as my lab examines the transport of MV, a small RNA virus in which the roles of each of the gene products is well-understood.

What are the downstream consequences of the interactions of cellular proteins (e.g., NK-1R) and viral proteins (e.g., MV-F) at the neuronal synapse? Previous research indicates that MV, like other neurotropic viruses, can spread through the CNS in the absence of detectable virus budding and in the absence of cell-cell fusion, the classical modes of MV spread in non-neuronal cells. Furthermore, MV ribonucleoprotein complexes (RNPs) localize predominantly at the presynaptic membrane and spread to neighboring neurons through a trans-synaptic process. Preliminary data suggests that viral transport across the synapse is largely dependent on an interaction of MV fusion protein (MV-F) with the neurotransmitter receptor neurokinin-1 (NK-1R). The normal role of NK-1R is to bind the neurotransmitter substance P and send signals to the neuron that often are associated with pro-survival pathways of the cell. Thus, this trans-synaptic mechanism of MV spread may serve several functions. My hypothesis is that these viral-cellular protein-protein interactions serve a larger role than merely facilitating viral transmission across the synapse. These interactions may be a mechanism by which the virus can hijack a cellular pro-survival signaling pathway to ultimately subvert the host antiviral response and establish a “friendly” environment for MV replication and spread. Activation of transcription factors associated with survival pathways in neurons, such as p42/44 ERK, AKT, and PKC, will be assessed by western blotting of infected neuron lysates.

Robert McKallip -- MUSM (approved for funding, Macon Campus)

It is estimated that approximately 50,000 new case of malignant melanoma are diagnosed each year in the United States. Due to the widespread growth of the metastatic lesions, surgical treatment is usually ineffective and many of these tumors are relatively resistant to current chemotherapeutic agents. Therefore, there is great need for finding new adjuvant therapies. IL-2 therapy of malignant melanoma has shown limited success. Unfortunately, high dose IL-2-therapy can have severe life-threatening side effects, which significantly hamper its usefulness. More recently investigators have been exploring the efficacy of adoptively transferring IL-2-stimulated lymphocytes into melanoma patients in conjunction with high dose IL-2 treatment. This approach initially showed significant promise, increasing the response rate from 24% in patients treated with high dose IL-2 alone to 33% in patients treated with the combination of adoptively transferred IL-2-stimulated lymphocytes and high dose IL-2 treatment. Although progress has been made in the treatment of malignant melanoma, improved strategies are needed to more successfully treat these diseases. Studies from our laboratory demonstrated that following exposure to IL-2 there is a significant increase in the expression of various CD44 isoforms and that lymphocytes expressing high levels of CD44 have potent cytolytic activity. Little is known about the significance of CD44 expressed by activated lymphocytes in the interactions with melanoma. In the current study we will examine the role of CD44 in the interaction between IL-2-activated lymphocytes and melanomas and we will test the hypothesis that lymphocytes expressing high levels of CD44 (CD44hi) are responsible for mediating lysis of melanoma tumor cells and that this increase in CD44 expression is directly due to increased expression of CD44 isoforms. Furthermore, we will examine the potential use of the CD44hi cells in the treatment of melanoma. Knowledge gained from this study will lead to a better understanding of the role of CD44 in lymphocyte-mediated lysis of melanoma tumor cells which may ultimately lead to significantly improved treatment for a number of cancers including melanoma. More specifically, it may be possible to stimulate lymphocyte with IL-2 and then selectively transfer only lymphocytes expressing high levels of CD44 or certain CD44 variant isoforms back into melanoma patients leading to a significantly more effective treatment of the cancer.