While the current, classical, and experimental treatment for ovarian cancer focuses on destroying malignant cells, these cells are actually moving targets for therapies. The tumor “stroma” contains dominantly tumor-associated macrophages (TAMs). TAMs produce factors that support tumor growth and block immune responses against the tumor. Our strategy to eliminate TAMs is based on a new adenovirus vector that expresses suicide gene specifically in TAMs. Our adenovirus vector will transduce TAMs in cell culture and mouse tumor models. We will use a microRNA based system to avoid suicide gene expression in myeloid cells other than TAMs (such as white blood cells). To generate that system, we used mouse tumor models and isolated macrophages from blood and tumors and delineated their micoRNA expression profile using microarrays with >1,000 known microRNAs. We selected the microRNA with the highest expression level white blood cells and a low level in TAMs (mmu-miR-106a). As a negative control, we chose a microRNA that was high in TAMs (mmu-miR-210). My task was to insert 4 target sites with 100% homology to the selected microRNAs into the 3’UTR of a GFP gene, which was under the control of an EF1a promoter. The GFP expression cassette, together with a cassette containing the mCherry gene under the control of the PGK promoter, were inserted into adenovirus (Ad5/11) vectors. We demonstrated in vitro and in vivo, after intravenous injection into mice, that transduction efficiency of white blood cells and TAMs was comparable for the Ad5/11-miR106 and control vector (based on mCherry expression). However GFP expression from Ad5/11-miR106 was absent in white blood cells due to miRNA-mediated degradation of GFP mRNA. My future plans are to substitute the GFP gene with a suicide gene and to demonstrate that corresponding Ad5/11 vectors selectively kill TAMs in vivo, which in turn should slow down tumor progression.

Chagas Disease is a tropical disease caused by the parasite Trypanosoma cruzi. An estimated 100 million people are at risk in 21 endemic countries and ~8 million persons are currently infected. Left untreated, Chagas disease can cause fatal heart rhythm abnormalities, a dilated heart, a dilated esophagus, and/or a dilated colon. The efficacy of current treatments decreases the longer the person is infected, and 40% of treated patients experience adverse reactions, demonstrating the need for new and improved drugs. A drug target has been identified in T.cruzi: the Methionyl-tRNA synthetase (MetRS). Like tRNA synthetase enzymes in other species, the T.cruzi MetRS is crucial for protein production and survival. To exploit this target in T.cruzi we developed a robust assay (Z' factor ≥ 0.5) to identify hits against the MetRS enzyme using a luminescence ATP detection method (Promega's Kinase-Glo™). Compounds that inhibit the T.cruzi MetRS enzyme will be tested for specificity by assaying the compound with the human MetRS homolog. Specific T.cruzi MetRS inhibitors will be tested for potency against intracellular T.cruzi amastigotes and cytotoxicity against mammalian cells. Compounds that are toxic to T. cruzi, not cytotoxic to mammalian cells, and have good pharmacological properties will be selected for hit--to--lead drug development. 

Adenoviruses (Ads) are the most exhaustively researched and well-understood viral platforms for gene therapy and oncolytic virotherapy, with potential applications including the treatment of numerous inborn and acquired genetic diseases, including cancer. However, when injected into the bloodstream, Ad vectors can bind several blood coagulation factors that facilitate the transduction of hepatocytes, sequestering the virus in the liver. Although both coagulation factors VII and X bind the Ad5 hexon with very high affinity, only FX appears to play a role in mediating Ad hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to the virus and its poor capacity at supporting cell entry, we conducted structural analyses on the Ad5-FVII complex. Using high-resolution cryo-electron microscopy followed by molecular dynamics flexible fitting (MDFF) simulations, we have determined that the FVII GLA-domain sits within a surface-exposed hexon trimer depression in a different orientation than found for FX, althouth several of the same hexon residues that mediate FX binding also mediate FVII binding to hexon. Furthermore, we found that two proximal FVII molecules on the viral surface may interact via their serine protease (SP) domains, burying potential heparin sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. Several viruses with point mutations in critical residues of the hexon protein were engineered and grown. Infection assays revealed cell transduction efficacy that varied independently of the strength of the interaction between the hexon and FVII. MDFF simulations revealed an altered interface between hexon and FVII that could affect the orientation of FVII in the pocket and, thus, the efficacy of dimer formation. Since the occluded HSPG domains are crucial for cell entry, our results suggest that differing binding orientations for FX and FVII GLA domains lead to disparate quaternary structures, which ultimately determine the cell-transduction efficacy of the complex.

Prostate cancer is the most common type of male malignancy in the Western world. Patient death often occurs when treatments which decrease circulating levels of male reproductive hormones are no longer effective. This lethal stage is termed castration-resistant prostate cancer (CRPC). Under normal conditions, androgens function through androgen receptors (ARs) to cause AR activation. The recently identified androgen receptor splice variants (AR-Vs), which lack part or all exons coding for the AR-ligand binding domain, are believed to cause the continuous AR activation found in androgen-independent conditions such as CRPC. Ubiquitin-conjugating enzyme E2 C (UBE2C) is an oncogene expressed in many types of solid tumors and has a function of marking proteins for degradation. Uncontrolled cell growth can be caused by overexpression of UBE2C which targets proteins that serve as cell-cycle check points to be degraded. Overexpression of UBE2C is reported to be positively correlated with AR-V expression. The transcription activation of UBE2C requires chromatin-looping through a phosphorylated transcription factor, mediator of RNA polymerase II transcription subunit 1 (p-MED1). My project investigates the relationship between AR-V and p-MED1 in mediating UBE2C gene expression. We used LnCaP and M12 human prostate cancer cell lines and transfected them with AR-Vs. Expression assays were carried out to measure the UBE2C gene expression levels and the presence of p-MED1 in response to AR-V expressions. We also performed functional assays such as co-immunoprecipitation to determine the functional interplay between AR-V and p-MED1 in mediating UBE2C transcription. We hypothesized that p-MED1 is required in AR-V-directed UBE2C expression. Our data suggest that, through a distinct transcriptome, the presence of p-MED1 as a mediator is required in AR-V-directed UBE2C expression and cancer cell proliferation. The importance of AR-V, p-MED1 and their mechanisms underlying CRPC progression might provide a new therapeutic approach for targeting AR-independent CRPC.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to lead to improved drugs. Most eukaryotic organisms synthesize sterols for essential biological functions that when manufactured incorrectly can lead to cell death. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediates of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T. cruzi in vitro and are now the center of research regarding anti T. cruzi drug discovery. We are developing novel azole compounds that target the sterol 14-demethylase homolog of T. cruzi to deprive the cell of its necessary sterols. However, further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of this research project is to analyze the ERG3 homologs of T. cruzi. The putative homologs have been cloned and sequenced. No significant mutations were found. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

The pancreatic islet β-cell maintains blood glucose homeostasis by releasing insulin, a process that becomes dysfunctional in type 2 diabetes mellitus. Thus, maintaining normal β-cell function is critically important and occurs through many mechanisms including interactions with the surrounding extracellular matrix (ECM). We have recently shown that the polysaccharide hyaluronan is a normal component of the islet ECM. Under normal conditions, hyaluronan exists in a high molecular weight (HMW) form (>500 kDa) which exerts anti-inflammatory effects on surrounding cells. One study has shown that HMW hyaluronan can potentiate β-cell glucose-stimulated insulin release (GSIS). Our preliminary data suggest, in diabetic islets, hyaluronan can become fragmented. Low molecular weight (LMW) hyaluronan (<10 kDa) is pro-inflammatory. Furthermore, agents that can activate the same intracellular signaling pathways as LMW hyaluronan (e.g. free fatty acids and lipopolysaccharide) have been shown to impair GSIS, suggesting LMW hyaluronan may have a similar effect. I hypothesize that HMW hyaluronan potentiates GSIS, whereas LMW hyaluronan inhibits GSIS in vitro. Rat pancreatic islet cells were cultured on plates coated with HMW hyaluronan, LMW hyaluronan, or L-ornithine (control). After 48 hours GSIS was determined by comparing insulin release under basal (low glucose) and stimulated (high glucose) conditions. Our preliminary data show that as expected HMW hyaluronan potentiates GSIS 2 fold while LMW hyaluronan decreases GSIS by 30% compared to control. Thus it appears HMW hyaluronan is beneficial for β-cell function whereas LMW hyaluronan lacks this effect. Hence, hyaluronan fragmentation in the islet under diabetic conditions may contribute to impaired β-cell function.

Neprilysin (NEP) is a plasma membrane metalloendopeptidase expressed in pancreatic islet β cells. In non-islet tissues, it has been shown to modulate ion flux. Whether NEP modulates potassium and/or calcium flux in β cells that are necessary for glucose-stimulated insulin secretion (GSIS) is unknown. Thus, the purpose of this study was to determine whether NEP acutely regulates GSIS and if so, where NEP acts in the secretion pathway. To do this, islets were isolated from 8-10 week old C57BL/6 mice. Following overnight recovery, islets were pre-incubated for 30 minutes in 2.8 mM glucose and then stimulated for 60 minutes with either 2.8 mM (basal) or 20 mM (stimulated) glucose in the absence or presence of the NEP inhibitor thiorphan (50 uM). To measure the effects of NEP inhibition on GSIS mediated by Ca2+ channel activity independent of K+ATP channel-induced depolarization, in some conditions a Ca2+ channel agonist (BayK-8644, 1 uM) was used in combination with a K+ATP channel opener (diazoxide, 500 uM). The results showed that acute inhibition of NEP significantly reduced 20 mM glucose-stimulated insulin secretion (128.1±16.0 vs. 80.5±11.4 pM/5 islets; n=11, p=0.03) without affecting basal insulin secretion (13.9±3.8 vs. 8.7±2.3 pM/5 islets; n=11, p=0.26). In addition, NEP inhibition did not alter insulin secretion in response to BayK-8644 plus diazoxide (33.0±6.1 vs. 38.2±8.1 pM/5 islets; n=6, p=0.62), suggesting NEP may be acting to modulate GSIS at a step that is proximal to the Ca2+ channel. It can be extrapolated from this data that NEP may play an important role in the insulin-secretion pathway and thus, acutely upregulating NEP may prove effective in treating type 2 diabetes. 

Dystonia Chorea is a very rare disease that includes the recurrent, aimless, uncontrollable, and sometimes painful movements of chorea, as well as the characteristics of dystonia, such as twisting, contorted movements or abnormal postures caused by sustained muscle contractions. The objective of the Raskind Lab is to identify the genes that are responsible for rare forms of neurogenetic diseases using exome sequencing and linkage analysis. For the Dystonia Chorea family, chromosome X was selected for initial analysis because the pedigree suggested possible X linkage. Chromosome 4 and 9 held candidate genes with some evidence for linkage. Within these regions, the variants were filtered for sequence-read quality and sharing of variants by affected people. Only variants for which the affected people were heterozygous for the alternate and reference bases were further considered. We did literature searches for protein functions and disease similarities with Dystonia Chorea, however none were identified. We will assess a more complete linkage analysis to identify new regions of interest on other chromosomes for analysis of exome variants.

Mycobacterium tuberculosis infection is a leading cause of death worldwide, but the full details of how the human immune system responds to these bacteria are not known. Our lab studies CD1 proteins, which allow T-cells to recognize and respond to lipids which are the major constituents of the mycobacterial cell wall. We recently discovered that low expression of CD1A on dendritic cells is present in ~10% of the population and is associated with increased susceptibility to tuberculosis in Vietnam. In addition to low surface protein expression, CD1A transcription is also impaired in CD1A-deficient individuals. The molecular mechanism of CD1A-deficiency is unknown. I hypothesized that CD1A expression is regulated by a single nucleotide polymorphism (SNP) that alters transcription factor binding. I cloned and analyzed the CD1A 5’ untranslated region (UTR) from four CD1A-deficient and five control individuals. There was one SNP, rs366316, that showed an association with CD1A-deficiency. I genotyped 163 individuals and examined the association of the rs366316 genotypes with CD1A expression. I found that the C/C genotype (N=9) was associated with CD1A-deficiency in comparison to the C/T and T/T genotypes (N=154, p<0.01). Based on this association, I examined whether the C/C genotype regulated transcriptional activity. I isolated 5’UTR sequences in normal and deficient individuals that were identical except for rs366316. I used site-directed mutagenesis to generate C to T mutants and T to C mutants. I made separate luciferase expression vectors for each of the four 5’UTR constructs and found that the normal and mutated 5’UTRs with a C allele showed 44% less expression than the normal and mutated 5’UTRs with a T allele (p<0.001). These data show that a SNP in the 5’UTR of CD1a is causally associated with human CD1A-deficiency. Together, these data suggest that CD1A-deficiency is common, regulated by a SNP, and important in MTB pathogenesis.

Malaria is a prominent parasitic disease that afflicts 300 to 500 million people and results in 1 to 2 million deaths each year, approximately 90% of which are young children under the age of 5 and pregnant women in sub-Saharan Africa. New therapeutic compounds for treating malaria are urgently needed due to the emergence of multidrug-resistant parasites including Plasmodium falciparum, the most fatal Malaria causing species that infects humans. A mass spectrometry-based screening of a natural product library yielded seven securinine-based compounds that were determined to bind to Plasmodium falciparum 2'-deoxyuridine 5'-triphosphate nucleotidohydrolase (PfdUTPase). This enzyme catalyzes the formation of deoxyuridine monophosphate (dUMP) from deoxyuridine triphosphate (dUTP) which has important implications on DNA replication and thus makes dUTPase a good target enzyme for drug discovery. These seven compounds were tested using a standard biochemical assay and enzyme activity was measured through the detection of pyrophosphate, a product of the catalyzed reaction. Detection was done with two independent detection kits: indirectly through the detection of inorganic phosphate (pyrophosphatase and Malachite Green Kit) or directly through the detection of pyrophosphate (PPiLight Kit). In most drug screens, the goal is to discover possible drug-like molecules that inhibit the target enzyme, however, these securinine compounds were actually found to increase PfdUTPase activity. While it is unusual to pursue compounds that do not inhibit target enzymes, these compounds might still prove to be possible drug candidates by over activating PfdUTPase and killing the parasite in this manner. Whether this is a viable method of utilizing these compounds remains to be tested.

 In 2010 an estimated 1.3 million people in the U.S. were living with type 1 diabetes mellitus, a disease that leads to elevated blood glucose levels and has been shown to accelerate atherosclerosis, which causes cardiovascular disease. As inflammatory macrophages are key to the etiology of atherosclerosis, we are interested in how diabetes leads to inflammatory status in macrophages. We have shown that the enzyme acyl-CoA thioesterase 7 (ACOT7) in macrophages increases production of inflammatory mediators that cause the accumulation of these cells in artery walls. ACOT7 is up-regulated in macrophages in a murine model of type 1 diabetes. Since glucose levels and inflammatory mediators are altered in diabetes, the goal of this project was to investigate the effects of these alterations on ACOT7 expression. Through the use of real time quantitative PCR and Western blotting, ACOT7 mRNA and protein levels, respectively, were measured in response to the above conditions. ACOT7 was up-regulated in response to Tumor Necrosis Factor-alpha (TNF-α, a pro-inflammatory chemical secreted by macrophages) and high glucose levels, as well as the inflammatory stimulus lipopolysaccharide (LPS). This increased understanding of ACOT7’s role in the production of inflammatory mediators offers a helpful framework for designing treatments to prevent or retard the formation of atherosclerosis in type 1 diabetics.

Pseudomonas aeruginosa can cause chronic and latent infections within many human diseases especially those related to immunocompromised individuals such as cystic fibrosis. This species also engages in a density dependent communication known as quorum sensing, which can often increase the species' virulence through the production of extracellular products such as proteases and components for the production of biofilms. Quorum sensing is a highly conserved process in nature yet quite interestingly we find that cystic fibrosis lungs provide a unique environment for the profileration of quorum sensing mutants (LasR mutants) as opposed to free living forms of the species elsewhere in the general environment. I am working to characterize 100 environmental isolates of Pseudomonas aeruginosa to test the hypothesis that quorum sensing mutants are infrequent within free living populations of the bacteria and to this point, I have not conclusively found an environmental mutant. Any information will in turn help us understand more about the selective pressures leading to the emergence of LasR mutants in the cystic fibrosis lung and consequently the therapeutic significance or insignificance of targeting quorum sensing in cystic fibrosis chronic infections.

Recent studies demonstrate that certain reproductive factors such as parity (defined as the number of completed pregnancies) are disparately associated with various pathologic subtypes of breast cancer. One factor, fetal microchimerism - the term for persistent child-origin cells in a woman’s circulation and tissues long after delivery - is generally deficient in women with unselected breast cancer and suggests a protective role. Here, we are testing the hypothesis that high-grade triple negative breast cancer (positive association with parity) will be distinctly associated with fetal microchimerism compared to low-grade luminal breast cancer (inversely associated with parity). Buffy coat specimens (n=450) were obtained from breast cancer patients and control participants aged 18-44 from the REACH project (Fred Hutchinson Cancer Research Center, PI Christopher Li). Using quantitative polymerase chain reaction, DNA extracts from these specimens were tested for the presence and concentration of Y chromosome sequence DYS14, a marker of male fetal microchimersim. In an interim analysis from the first 87 specimens, 33% (9/27) of women who developed luminal breast cancer tested positively for the presence of DYS14, as did 33% (10/30) of healthy controls. 47% (14/30) of women who developed triple negative breast cancer tested positively for the presence of DYS14. Preliminary results demonstrate a higher prevalence of fetal microchimerism in patients with triple negative breast cancer compared to control or luminal participants consistent with the original hypothesis. Additional samples will be tested. Fetal microchimerism testing may represent a novel non-invasive method to identify women with poor prognosis breast cancers.

 Human aortic diseases such as aneurysms (abnormal dilation of the aorta) dissections (tears in the wall of the aorta) are often life-threatening. These diseases present significant challenges because: 1) their pathogenesis is not well understood; 2) they are difficult to diagnose, often presenting at an advanced, life-threatening stage; 3) they have limited treatment options; and 4) the treatments are all associated with major complications. Our work focuses on achieving a better understanding of the pathogenesis of aortic aneurysms and dissections, with a long-term goal of preventing or stabilizing these diseases. Signaling pathways initiated by transforming growth factor beta (TGF-β) in smooth muscle cells (SMC) of the aorta are essential for normal vascular development. Mouse embryos lacking SMC TGF-β signaling exhibit abnormal aortic structure, including dilation of the distal aorta. However, the role of SMC TGF-β signaling in maintaining the structural and functional integrity of the adult aorta is unclear. We use a transgenic mouse model to test the hypothesis that TGF-β signaling in adult aortic SMC preserves aortic health and prevents aortic aneurysms and dissections. With this model we can delete a gene that is critical for TGF-β signaling specifically in SMC of adult mice, and thereby discover the role of SMC TGF-β signaling in the regulation of SMC gene expression and aortic structure. We expect to find abnormal gene expression and aortic structure in adult mice with loss of SMC TGF-β signaling. Our study may uncover signaling pathways and gene expression patterns that maintain aortic health. These pathways may eventually be exploited to develop therapies that prevent aortic aneurysms and dissections.

Expression of glycans or carbohydrate antigens on mucosal surfaces is known to influence interactions between the host and bacteria. Variation in the expression of a single glycosyltransferase B4galnt2 is associated with significant shifts in the composition of the intestinal microbiota of mice (Staubach F, ISME, 2012). Glycosyltransferases are a class of enzymes that transport and assemble glycans. Preliminary studies of a mouse Salmonella model has found a significant association between Salmonellosis and expression of B4galnt2. In this project we will test the hypothesis that Salmonella spp. show preferential binding to the B4galnt2 β 1-4 GalNac sugar, which is commonly expressed by human intestinal epithelial cells (Mohlke et al., Cell 1999). We have adapted an in vitro method from Ruiz-Palacios et al. (JBC, 2003) to study this question, which utilizes Chinese hamster ovary (CHO) cell genetically engineered to express specific glycans to assay bacterial binding. Cells will be grown toconfluency in chamber slides then they are they are fixed and blocked. Clinical Salmonella enterica isolates will be grown in culture tubes overnight. Diluted bacteria will then be directly applied to the CHO Cells in the chamber slides and incubated together. After incubation the slides will be fixed and stained with a fluorescein isothiocyanate (FITC) conjugated anti Salmonella antibody. Stained slides are examined under a fluorescent microscope to quantify the number of bacteria bound to CHO-B4galnt2 cells vs. control CHO cells. Increased numbers of bacteria binding to the CHO-B4galnt2 cells compared to the control CHO cells would support a role for the B4galnt2 β 1-4 GalNac in host-Salmonella interactions. If no significant difference in binding is seen by this method, we will also test the hypothesis by doing introducing shear in a modified dot blot binding assay using mucus from B4galnt2 (+/+) and (-/-) mice to investigate Salmonella spp. differential adherence.

Proper patient-provider communication (PPC) regarding choices available to patients and management of diabetes-specific care can lead to better patient outcomes. Our pilot study describes the specific demographic and clinical characteristics associated with PPC in families with children who have type 1 diabetes (T1D). Thirty-four caregivers of youth with T1D ages 2-18 were recruited for a study describing quality of care (QOC) measures, specifically PPC and quality of diabetes-specific education. Parent-report of patient quality of life (QOL), via a standardized assessment, was also collected. Clinical measures including insulin regimen and current glycosylated hemoglobin (A1c) was recorded from the medical record. Descriptive statistics, including Pearson and Spearman correlation coefficients, chi-squared tests and one-way analyses of variance tests were used to explore differences between demographic and clinical variables with QOC. Thus far, 34 caregivers have completed enrollment. On a scale of 4-16, with higher scores reflecting better PPC, the mean was 13.8 +/- 2.6 (range 8-16), indicating relatively high PPC overall. When asked to indicate whether 7 diabetes-specific topics (i.e., what to do for a low or high blood sugar) have been addressed, 23/34 (67.6%) reported all 7 as being discussed (mean = 6.6 +/- 0.7; range 5-7). Among clinical variables (QOL, A1c, and insulin regimen), QOL associated with PPC (r=.42, p=.01). For quality of diabetes education, income was the only demographic variable associated (families with higher income were more likely to report maximum education scores; chi-squared = 9.48, p=.05). No clinical variables were associated with diabetes education scores. Initial results from this pilot study indicate high levels of quality of care in this population. Certain demographic indicators, such as income, did affect quality of care indicators. Quality of life was also an important correlate with PPC, which suggests adequate communication plays a role in patient outcomes.

Acute myeloid leukemia (AML) is a cancer characterized by the accumulation of abnormal immature white blood cells in the bone marrow. This proliferation impairs normal blood production and leads to suppression of normal blood counts, increasing susceptibility to infection and bleeding. Adhesion of leukemia cells within the bone marrow confers resistance to chemotherapy drugs. E-selectin is a molecule on the surface of endothelial cells lining blood vessels; it mediates the adhesion of white blood cells to vessels within bone, and may play a role in the residence of leukemia in the bone marrow “vascular niche.” Interaction of cancer cells with E selectin has been shown to activate survival pathways such as PI3K-NFκB, making cells less susceptible to apoptosis (programmed cell death). Using an assay for E-selectin and analysis via RT-PCR, we have preliminary data suggesting that binding of cells from a specific patient sample to E-selectin up-regulated genes in the TGF-beta, Wnt and Hedgehog signaling pathways, the latter of which may play a role in drug resistance in CD34+ leukemia cells. Further work aims to demonstrate that the activation of these survival pathways is consistent across a range of cell samples, and to investigate whether up-regulation extends to other pathways known to inhibit apoptosis in cancer cells. Knowledge of the mechanisms by which leukemia cells increase their survival upon binding ligands in their environment such as E-selectin could aid the design of therapies aimed at treating AML, and potentially help overcome the persistent problem of resistance to chemotherapeutic agents.

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous species of pathogenic bacteria and a major cause of hospital-acquired pneumonia. Immune responses to P. aeruginosa infection in mice are mediated by a cytosolic multiprotein complex called the NLRC4 inflammasome. The NLRC4 inflammasome assembles upon ligand recognition leading to the release of the pro-inflammatory cytokine interleukin (IL)-1β and culminating in cell death. P. aeruginosa elicits a flagellin-dependent release of IL-1β and cell death in mouse macrophages, but research has not found human macrophages to recognize flagellin. This immune response is evoked in human macrophages by needle protein, an element of the type three secretion system (T3SS) infection apparatus used by P. aeruginosa. The mechanisms human macrophages use to regulate immune response to P. aeruginosa have not been fully established. We hypothesize that NLRC4 regulates immune response to P. aeruginosa in humans and therefore, NLRC4 deficient macrophages infected with P. aeruginosa will have impaired cell death and IL-1β production compared to normal NLRC4 expressing cells. Small interfering RNA (siRNA) will be used to knock down mRNA and protein expression of NLRC4. Macrophages treated with non-specific siRNA and NLRC4 targeted siRNA will be infected with live P. aeruginosa. We will then assay for IL-1β with ELISA and cell death using a lactate dehydrogenase (LDH) release assay. We expect NLRC4 knockdown cells to release less IL-1β and LDH compared to cells treated with non-specific siRNA thus indicating that in humans, the NLRC4 inflammasome is associated with defense against P. aeruginosa infection. Upon establishing the function of human NLRC4 during P. aeruginosa infection, we will proceed to investigate the roles of NLRC4 inflammasome subunits in ligand recognition and initiation of an immune response. Defining these roles could further our understanding of mechanisms behind immunity or susceptibility to P. aeruginosa and potentially lead to novel treatments for infection.

Author: Amanda Tanadinata, Karol Bomsztyk, Daniel Mar. Background: Apart from DNA mutations, alterations in chromatin structure, also referred to as epigenetic changes, have been shown to be among principal causes of tumor genesis that leads to cancer. However, unlike mutations, the usage of these chromatin alterations in gene expression is reversible. This feature, in fact, is what has nurtured the idea of creating an epigenetic-based treatment for cancer. One such treatment uses the compound 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltrasferase inhibitor. DNA methylation can be crucial for transcriptional repression and activation, by altering recruitment of enzymes and transcription factors to chromatin . The goal of this study was to profile a set of pathways through the analysis of 5-aza-dC -mediated changes in DNA methylation, mRNA expression , RNA Polymerase II, histone marks, and enzymes. Method: Colorectal cancer cell line (HCT116) cells treated with or without 5-aza-dC were probed with different antibodies to see chromatin density differences at specific sites of the genome by Matrix Chromatin Immunoprecipitation (Matrix ChIP). Methylated DNA Immunoprecipitation (MeDIP), was used to assay DNA methylation sites at selective gene sites. The results were then quantified with real time PCR. In this study, SPARC promoter, H19, and UBE2b were examined specifically. Results: Treatment with 5-aza-dC increased SPARC mRNA levels, and reduced DNA methylation This was associated with changes in RNA polymerase II levels ,histone acetylation and methylation, and chromatin modifying enzymes at the SPARC but not the control H19 and UBE2b genes. Conclusion: These results imply that 5-aza-dC induced de-methylation of selective genomic sites alters enzyme binding and chromatin structure which then causes changes in rates of transcription.

Dysregulation of the Epidermal Growth Factor Receptor (EGFR) pathway has been implicated in 50-60% of glioblastoma multiforme (GBM) tumors. The EGFR signaling network regulates cell proliferation, differentiation, growth, and apoptosis. The binding of an extracellular ligand activates the EGFR molecule, initiating a cascade in which proteins are serially activated and deactivated through phosphorylation and dephosphorylation. This process is time-sensitive: proper function depends upon each protein remaining active for the proper interval. Wild-type astrocyte proteins respond to phosphorylation by tyrosine kinase, dephosphorylating within five to ten minutes. A dysfunction of EGFR resulting in sustained low-intensity activity or over-activity of the EGFR pathway causes distal abnormalities, including the aplastic over-proliferation and aggressive invasion of astrocytes endemic to GBM. A detailed temporal map of signal transduction through various components of EGFR pathway is needed to find the proteins contributing to the deviant cell behavior. To that end, we will create a 7-time point model of the EGFR pathway in GBM. The GBM cell line will be exposed to EGF for various lengths of time starting from 5 minutes through 24 hours. At each time point the phosphorylation status of pathway proteins will be profiled and quantified.  This assay is anticipated to yield approximately 500 phosphorylated proteins. We will use high throughput phosphoproteomics strategy, in which quantitative mass spectrometry will be utilized to measure phosphorylation levels of each component of the pathway. Information generated through phosphoproteomics will be overlaid on the computational EGFR network for each time point resulting in seven different EGFR pathway models. We will compare the network at each time point with the other to track the signal transduction through different proteins in the pathway. At the end of this project, we will have seven detailed EGFR networks, which can elucidate the dynamics of signal transduction from ligand exposure to the cell’s response through gene activation.

In adults, acute myeloid leukemia (AML) generally remains difficult-to-treat, and most patients are currently expected to die either of their disease or treatment-related toxicities. The need for novel therapies is thus unquestioned. With increasing availability of specific small molecule inhibitors, one line of research has focused on targeting signaling pathways that are pivotal for the in vivo growth, survival, and propagation of AML cells as a means to improve therapeutic outcomes in this disease. Emerging evidence suggests that the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway, a key physiologic regulator of transcription, translation, cell cycle progression, differentiation, metabolism, and suppression of apoptosis, could provide a rational drug target in AML. Constitutive activation of AKT signaling is indeed observed in many patients with AML, and several groups have reported inferior relapse-free and overall survival in AML cases with AKT activation. Given these data, we recently conducted an early phase clinical trial testing MK-2206, a highly specific allosteric AKT inhibitor, in patients with relapsed AML. However, MK-2206 has insufficient anti-AML activity at tolerated doses. Therefore, the goal of my project in the preclinical studies that I am currently carrying out is to identify signaling pathways that are critically important for AML cell survival in the presence of MK-2206 with the goal of developing combination therapies that optimize chemosensitization that can be achieved with AKT inhibitors. This is being done by mixing the MK-2206 inhibitor with various conventional chemotherapic drugs and measuring their toxicity on different AML cell lines by use of flow cytometry.

Pancreatic cancer is the fourth most common cause of cancer-related deaths in the United States. The overall survival rate is low because current therapies are ineffective. However there is growing research that High-Intensity Focused Ultrasound (HIFU) can be used to significantly improve current therapies for treating pancreatic cancer. HIFU works by accumulating ultrasound waves (i.e. energy), which generates heat causing tissue damage (i.e. hyperthermia). The thermal damage caused is measured by using thermal dose. Thermal dose describes the temperature the tissue is treated at and how long that treatment occurs. The theory of thermal dose has been tied to hyperthermia of cancer cells since at least the 1980s and there is a lot of strong mathematical background behind the time-temperature relationship. However with the rapid development of clinical hyperthermia for cancer therapy there is a need for accuracy when it comes to predicting thermal dose. My research focuses on whether or not equivalent thermal doses actually mean equivalent cell death (with cell types Panc-1 and MIA PaCa) and if this can be translated into a clinical setting where higher acoustic power can be used (i.e. using higher energy with HIFU to get lower time of treatment). I simulate HIFU by treating pancreatic cancer cells in water baths at equivalent thermal doses and measure cell survival with spectroscopy. My initial results suggest that the thermal dose relationship is accurate in relating amounts of cell death. An early conclusion from this is that thermal dose can be used to provide better calculations in a clinical setting and help evaluate data. Patients may even be able to experience shorter treatment times through using higher acoustic power with HIFU resulting in a more comfortable experience.

According to the American Cancer Society, pancreatic cancer has a five-year survival rate of less than 15 percent and is the fourth leading cause of death from cancer in the United States. Present-day chemotherapy treatments attempt to lengthen the lives of patients but are often unsuccessful due to the nature of the disease. Doxorubicin and Gemcitabine are already commonly prescribed as pancreatic cancer treatments while Thermodox®, a lysolipid thermally-sensitive liposome (LTSL) technology, is still undergoing clinical trials. LTSLs encapsulate Doxorubicin releasing the drug when the agent is exposed to temperatures greater than 40 degrees C. The goal of this study is to compare the in vitro cytotoxicity of four chemotherapeutic conditions: unheated Thermodox®, heated Thermodox®, Doxorubicin, and Gemcitabine. Varying concentrations of each drug condition are applied to LMP, PANC-1, and Mia-PaCa cell lines, which originate from mouse and human pancreas carcinoma. The remaining post-treatment viable cells and relevent controls are then stained with a fluorescent dye (Hoechst 33342) and analyzed by a fluorescent plate reader, from which cell death can be determined. Preliminary results show that that unheated Thermodox is not toxic to cells, and heated Thermodox®, Doxorubicin, and Gemcitabine is toxic to cells. The results of this experiment can lead to greater knowledge of the cytotoxicity of current chemotherapy agents as well as new technologies in cancer treatment.