MicroRNAs (miRNA) are a type of non-coding small RNA (smRNA) that regulate gene expression at the post-transcriptional level by base pairing with complementary sites in messenger RNA (mRNA), causing either mRNA degradation or translational inhibition. The overall goal of this project is to understand the relationship between miRNAs and abiotic stresses in plants. How plants employ miRNAs to alter gene expression when they encounter various stresses, like drought, cold, submergence, and salinity, is of great agricultural importance. Plants must develop sophisticated ways to cope with these stresses since they are unable to evade them. We first utilized small RNA biogenesis mutants such as dcl1, dcl2/3/4, or rdr2 to enrich specific small RNAs, and then used these mutants to make smRNA libraries. We treated Arabidopsis rdr2 seedlings with submergence, salt, and both submergence and salt, while treating Arabidopsis rdr2 flowers with drought, salt, and cold. Their low molecular weight RNA were isolated, and smRNA libraries were constructed. We checked the quality of the libraries by traditional cloning and sequencing. From these smRNA libraries, miRNA expression will be analyzed by high-throughput sequencing by SBS (sequencing by synthesis) and sent to a company (Illumina) or a facility at the Delaware Biotechnology Institute to sequence, and then for data mining by computational analysis. Potential miRNA candidates will be identified and undergo validation. R.S.H.B was supported by Delaware EPSCoR, through National Science Foundation Grant EPS-0447610 and the State of Delaware, and NSF grant MCB#0548569 to P.J.G. provided research support.

Vibrio parahaemolyticus is a Gram-negative halophilic marine bacterium found in estuarine and marine coastal ecosystems worldwide. The ability of the organism to survive and proliferate under conditions of fluctuating salinity is well known and can be contributed in part to the uptake of compatible solutes, organic molecules which allow the bacterium to avoid plasmolysis due to excess water loss in a hyper osmotic habitat. We identified a number of putative compatible solute transporters belonging to the betaine/carnitine/choline (BCCT) family of transporters using bioinformatic analysis on the V. parahaemolyticus RIMD2210633 genome sequence. VP1723, a putative BCCT family transporter was found as part of a unique cluster of systems on chromosome I of V. parahaemolyticus. Using TMHMM, SOSUI and hydropathy profiling we identified the transmembrane helices of this transporter and compared our results with those of previously characterized BCCT transporters from other organisms. We amplified VP1723 from V. parahaemolyticus and cloned it into the broad host range vector pBBR1MCS. The vector was then transformed into a transporter deficient E. coli strain MKH13 in order to functionally characterize the specificity of compatible solute uptake by this transporter. Growth of the E. coli strain under conditions of elevated salinity will confirm a fully functional transport system. Assays using radiolabelled compatible solutes will determine the affinity of the transporter for betaine, carnitine or choline. NMR analysis will allow us to determine whether uptake of these solutes facilitates the synthesis of glycine betaine using choline as a precursor molecule. Supported by the Charles Peter White Undergraduate Science Education Program.

The placenta is a highly organized and complex organ in mammals. Its primary function is to transport nutrients from the mother to the fetus during gestation. The trophoblast cells that are characteristic to the placenta are in direct contact with the maternal tissue and consequently these cells are likely to be the key regulators of placental function. Here the focus is on Cathepsins 1 and 2, both are members of a family of placental cysteine proteases found on chromosome 13 in rats, and are expressed early in gestation. Both cathepsins are localized to invasive trophoblast giant cells, and are strong candidates as more proteolytic cathepsins involved in embryonic nutrition or extensive remodeling during invasive implantation. The primary objective of this 10 week study is to express cathepsin 1 respectively through such methods as RT-PCR, TOPO-cloning, RE Digestion, and Insertion into Expression Host. Supported by Grant Number 2 P20 RR016472-08 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).

Prostate Cancer (PCa) is the second most common type of cancer among men and results from the uncontrolled growth of epithelial cells that line the ducts of the prostate gland. The cause of death in these men is rarely attributed to the original tumor, but rather the metastasis of the cancer to its primary targets, bone and lymph. Our lab has shown that bone cells release ATP in response to a number of stimuli. ATP then signals surrounding cells through purinergic receptor activation to induce bone formation. We postulate that this release may play a role in the affinity of PCa cells to bone and that more metastatic cells release greater amounts of ATP. To test this hypothesis, we examined levels of ATP released from cells of the LNCaP model; LNCaP, C4-2 and C4-2B cells. These cells were developed to become increasingly metastatic. LNCaP cells representing the least metastatic and C4-2B cells being the most aggressive. Our lab has demonstrated that the release of ATP is mediated through entry of calcium into the cell. We treated the three cell lines with ionomycin, a calcium ionophore. We found that the C4-2B cells released the greatest amount of ATP compared to untreated control cells and that LNCaP cells released the least. These studies suggest that more metastatic cells release large amounts of ATP to stimulate cell migration and invasion of PCa cells into bone. Future studies will focus on the mechanism of ATP release from the metastatic cells and the effector response of these cells to ATP. (Funded by Department of Defense PCRP-W81XWH-06-1-0244 and NIH/NIAMS R01 AR051901)

RhoC is a small signaling protein, more specifically a monomeric GTPase, and is a member of the Ras sub family. Rho proteins are involved in multiple cellular processes, such as cell division, intracellular trafficking, and the organization of cytoskeletal components. The RhoC protein has been shown to be directly involved in cancer cell motility and invasion. Breast cancer, inflammatory breast cancer, and pancreatic cancer are among a few of the cancers in which RhoC is known to play an active role. Insulin-like Growth Factor (IGF-1) plays a significant role in cell growth regulation and development, as well as cellular DNA synthesis. This experiment uses three different cell lines to determine their ability to adhere to Bone marrow endothelial cells (BMEC) in the presence of IGF-1. The three types of PC-3 cell lines used were parental PC-3, PC-3’s containing a dominant negative RhoC (dnRhoC), and a control PC-3 vector Lac Z. These cells were treated with IGF-1 for varying time points ranging from 5 to 30 minutes, and their adhesion to BMEC cells was measured by performing an adhesion assay. The hypothesis of this experiment is that IGF-1 regulates PC-3 adhesion through RhoC GTPase activity. Prostate cancer cell adhesion to Bone marrow endothelial cells is a critical step in invasion and metastasis to bone, which is a major clinical concern. Funding for this project has been provided by the Department of Defense.

Salmonella spp. are gram-negative pathogens that are estimated to be responsible for 1.4 million cases of food poisoning annually in the United States. With the ever increasing use of anti-microbial products, multi-drug resistant strains have appeared in natural, clinical, and laboratory environments. This resistance is thought to be at least partially the product of the up-regulation of membrane efflux pumps. In this study Salmonella enterica 4931 strains were developed which were able to grow in the presence of 500-600 ppm Dodecyltrimethylammonium chloride (DTAC), a quaternary ammonium compound. A microarray analysis of mRNA from a reduced susceptibility (SRS) versus a parental strain both growing in DTAC was done. Results indicated an increase in the amount of transcription of acrB, an efflux pump, in the SRS strain which has been linked to multi-drug resistance. Penicillin G is another known substrate of acrB and these new findings supported earlier results that SRS strains with a reduced susceptibility to DTAC also showed reduced susceptibility to Penicillin G. In addition, this study also showed that SRS strains in the presence of the efflux pump inhibitor Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) showed a greater sensitivity to DTAC than the parental strain. This is possibly another physiological indication of a greater reliance of the SRS strains on efflux pumps for defense against anti-microbial compounds. The discovery of efflux pump inhibitors such as CCCP could lead to advances in combating SRS strains in clinical and natural environments. This research was generously funded by the Science and Engineering Scholars Program.

Calcium and Integrin Binding Protein (CIB1) is primarily important in the processes of angiogenesis, hemostatis, thrombosis and spermatogenesis. More specifically, CIB 1-/- mice are unable to produce sperm and exhibit defects in ischemia-induced angiogenesis. CIB1 is a specific binding partner for Integrin aIIb β3, the platelet fibrinogen receptor. CIB1 is a member of a family which includes three other closely related proteins (CIB 2, 3 and 4). This study seeks to determine if there is a member of the CIB protein family that compensates for the absence of CIB1 and functions in way that parallels CIB1. To test this hypothesis, RNA will be isolated from platelets, endothelial cells, lungs and testis from wild type and CIB1-/- mice. RNA purity will be determined by analysis of 260/280 and 260/230 absorbance ratios using a nanodrop spectrophotometer. Pure RNA samples will be used for One Step Reverse-Transcriptase PCR utilizing specific primers for CIB 1, 2, 3, and 4 to detect transcription of these proteins. For each sample, a β-actin control using reverse transcriptase and DNA polymerase will allow for the evaluation of RNA integrity and another β-actin control without reverse transcriptase will detect any genomic DNA contamination. In a certain tissue, if there is a CIB family member (CIB 2, 3 or 4) that is expressed in the CIB1-/- mouse but not in the wild type mouse, a compensatory molecule can be identified and researched further. We have found that CIB3 is not expressed in wild type and CIB1-/- mouse testis, whereas CIB2 and CIB4 are both expressed equally amongst the wild type and CIB1-/- testis. Therefore, we can conclude that CIB2 and CIB4 are not compensating for the absence of CIB1 since no sperm are produced in their presence. If the compensatory molecule that we identify proves to restore function to cells that are defective in the absence of CIB1, a new avenue of research into another CIB family member will open. This project is supported by the Howard Hughes Medical Institute and National Institutes of Health grant UPN.

The mammalian eye lens consists of fiber cells covered on the anterior surface by monolayer of epithelial cells. The most abundant protein in adult lens fibers is BetaB2-crystallin. Previously, our laboratory demonstrated that mice heterozygous for a 12 nucleotide deletion in the BetaB2-crystallin gene (Crybb2Phil) develop cataracts. However, in homozygous mutants, the lens epithelium undergoes an epithelial mesenchymal transition (EMT) leading to severe lens abnormalities. In order to understand the molecular basis of this phenotype, a SuperArray real time RT-PCR gene expression panel was used to test the hypothesis that TGFbeta signaling is activated in the Crybb2Phil  homozygous lens. We discovered that of the 84 genes represented on the panel, 18 were significantly upregulated in the Crybb2Phil lens indicating that some aspects of TGFbeta signaling are induced in lenses exhibiting EMT.  One of these genes (IGFBP3) is upregulated 160 fold in the Crybb2Phil lens epithelium as compared to wildtype, and this protein is upregulated in the Crybb2Phil  lens as assayed by immunofluorescence, although the most notable upregulation was in the fibers.  Since IGFBP3 induces EMT in other systems by binding to its receptor TGFbetaRV, we investigated the expression of TGFbetaRV in the lens by RT-PCR and confirmed that it is expressed.  Neither IGFBP3 nor TGFbetaRV have ever been previously reported in the lens, so these data suggest that this receptor ligand pair may be a novel inducer of EMT in the lens.  This work was supported by the Howard Hughes Medical Institute and the National Institutes for Health.

The molecular events associated with prostate development are being elucidated slowly. This study sought to validate the gene expression profile from previous microarray (MA) data that showed significant enrichment of hundreds of candidate genes in specific domains of the urogenital sinus (UGS) namely: urogenital epithelium (UGE), urogenital mesenchyme (UGM), urogenital dorsal (UGD), urogenital ventral (UGV) halves. Of these candidate genes, twelve were chosen with significant differences in both UGM/UGE and UGD/UGV, while another eleven were chosen with significant differences in either UGE/UGM or UGD/UGV. We collected samples of 16.5 days p.c. UGS and separated them either into UGE and UGM, or bisected them into UGD and UGV. RNA was extracted and used to synthesize cDNA. Oligonucleotide primers suitable for quantitative-polymerase chain reaction were designed for each of the candidate genes. Reaction conditions for each primer set were determined empirically by gradient RT-PCR. Immunofluorescence (IF) was then used to confirm protein localization for one candidate gene in each of the eight compartments or domains of the UGS. The first round of Q-PCR confirmed the predicted localization for 20 of 23 genes tested, though not always at the same fold change as predicted by the MA. The validation using IF is still ongoing, but so far has localized clearly: Tpm in UGM, E-cad in UGE, Pax2 in UGD, Myh3 in UGM, Msc in ventral UGM, and Fgfr2 in dorsal UGE. These techniques should result in validation of the developmental prostate microarray results and add critical knowledge about gene segregation, regional expression and early morphogenetic determination in the UGS.

Colon carcinomas arise from the normal epithelium of the colon. This epithelium contains three types of differentiated cells: enterocytes, goblet cells & neuroendocrine (NE) cells and is structurally organized in subunits called colonic crypts. The role of NE cells in regulation colonic crypt homeostasis is largely unknown, but they are postulated to have a regulatory role on maintaining the stem cell (SC) niche located at the crypt base. Since a growing body of scientific evidence supports the concept that cancers, including colon cancer, arise from stem cells, this suggests that dysregulation of NE cells may contribute to colon cancer development. We qualitatively and quantitatively compared the expression of several NE and SC markers in normal and malignant colonic tissues using immunohistochemical analysis. A double-labelling immunofluorescence technique was also used to quantitatively assess co-expression of the NE marker CgA and the putative stem cell marker ALDH1A1 in cells in normal and malignant colon tissue. The results revealed that most, if not all, normal crypts and colon cancers contain cells that stain positive for both NE and SC markers. CgA and ALDH1A1 positivity was more frequently co-localized in cells in normal crypts (90%) compared to cells in colon cancers (30%; p<0.001). These findings suggest that NE cells frequently display a SC-like phenotype in normal crypts but this phenotype occurs less frequently in NE cells in colon carcinomas. This study suggests that alterations occur in the NE cell population during colon tumorigenesis and this alteration might lead to changes in the crypt stem cell niche and contribute to colon cancer development. This project was funded by Imperial College Undergraduate Research Opportunity Programme, Christiana Care and Department of Chemistry at Hamline University.

Head and neck cancer patients who undergo radiation therapy suffer from salivary gland hypofunction. To help this problem, a current project is underway that involves tissue engineering to engineer an artificial salivary gland. As a part of this project, the purpose of this investigation is to explore the expression pattern of extracellular matrix genes in salivary glands. This study focuses on two of the major salivary glands, the submandibular and the parotid. Finding the optimal matrix will assist dissociated salivary cells to self-assemble then differentiate into their glandular phenotype. In order to analyze ECM gene expression, a SuperArray-based analysis of extracellular matrix (ECM) molecules was conducted. RNA was isolated individually from submandibular and parotid tissue and reverse transcribed to cDNA to use in the SuperArray. The ECM gene expression of both glands was compared and it was found that collagens, selectins, laminins, certain integrins, MMPs, and hyluronan synthase 1 were highly expressed in the submandibular gland relative to the parotid. Primers were designed for these highly expressed genes and a Q-PCR-based assay was used to validate the SuperArray results. It was found that the genes were in fact highly expressed in the submandibular versus the parotid. Further work will involve validating the expression of the ECM molecules at the protein level. This research was funded by the Center for Translational Cancer Research, the Howard Hughes Medical Institute, and by private philanthropic contribution.

Prox1 is a homeodomain transcription factor that is important for regulation of lens, liver, pancreatic, and lymphatic system development. Although Prox1 function is understood, it is not known how this protein is controlled. Previously, Arid2 (also called BAF200 and zipzap) was isolated from a yeast-two-hybrid assay as a potential Prox1 interacting protein. Arid2 belongs to the ARID family of proteins that are important for cell development, gene expression, and cell growth regulation. It is also a vital component of SWI/SNF complexes that function in chromatin remodeling. This work seeks to test the hypothesis that Prox1 and Arid2 interact and to determine the biological relevance of this interaction. A GAL4 yeast two-hybrid assay was used to detect an interaction between proteins by the activation of reporter genes that are transcribed if the two proteins are able to join. Plasmids Prox1-pGBKT7 and Arid2-pACT2 were transformed into two different yeast strains of two different mating types. These were mated and plated on selectable media lacking nutrients that the reporter genes are responsible for making to screen for interactions. Yeast colonies grew on selectable media, suggesting that Prox1 and Arid2 do interact. Fluorescent immunohistochemical analysis was also performed on four-week C57B6 mouse lens, showing that Arid2 and Prox1 are colocalized in the lens fiber cells. This work currently seeks to determine if Arid2 could affect Prox1 activity within transfection tests using the chloramphenical acetyltransferase reporter gene and our study will continue to establish whether the interaction between Prox1 and Arid2 occurs in vivo. This research is funded by the Howard Hughes Medical Institute.

Based on previous research, the downregulation of the RhoC GTPase in PC-3 human prostate cancer cells derived from bone metastasis leads to increased and sustained levels of Rac GTPase activity. It has been shown that the Rac GTPases are involved in prostate cancer cell migration and invasion particularly through bone marrow endothelial cells. There are 3 isoforms of Rac and a homologous protein, RhoG, that has been implicated in the activation of Rac1.  In the current study, we examine the levels of expression, activation, and phenotypic effects of Rac1, Rac3 and RhoG GTPases. The relative and quantitative levels of Rac1, Rac3, and RhoG were compared in PC-3 cells, C3 exotransferase (an inhibitor of RhoC GTPase) treated PC-3 cells and siRNA treated cells. A tumor cell diapedesis assay was done across a monolayer of bone marrow endothelial cells after the siRNA treatment of Rac1, Rac3, or RhoG to determine the individual contributions of each GTPase to a cell₼s invasive capability. RhoG, Rac1, and Rac3 ability to undergo diapedsis was tested. In the future a GLISA experiment will be done on siRNA treated PC-3 cells. We will determine the phenotypic and physiological effects of Rac1, Rac3, and RhoG more closely. As well, we plan to calculate changes in morphology by determining the contribution of each GTPase to the formation of lamelipodia.

Spinal Muscular Atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons and muscle wasting. SMA is caused by deletion or mutation(s) in the Survival Motor Neuron 1 gene (SMN1). The highly homologous gene, SMN2, is present in all patients but cannot compensate for the loss of SMN1. The functions of SMN have been implicated in the assembly of ribonucleoprotein complexes, pre-mRNA splicing, neurite outgrowth, and cell survival. However, it remains unclear how SMN deficiency results in selective loss of motor neurons in SMA. The long-term goal of this project is to functionally characterize the SMN protein. In this study, we expressed and purified glutathione S-transferase (GST)-fused SMN protein in baculovirus-based expression system. We cloned GST-SMN into pFastBac vector, generated recombinant bacmid, and transfected recombinant bacmid into Sf9 cells. We have also amplified the GST-SMN baculovirus stock and measured virus titers by immunofluorescence staining. Small amount of GST-SMN was purified by affinity chromatography. Western blotting analyses of the purified GST-SMN showed accurate expression. We are currently preparing large amount of the purified GST-SMN. We will use this recombinant protein for structural analyses of the SMN protein by partial protease digestion and Western blotting analyses. This research is supported by the Howard Hughes Medical Institute's Undergraduate Science Education Program and Dr. Wenlan Wang’s Research Group.

MicroRNAs recognize and bind to mRNAs of target genes and alter protein expression of those gene products.The goal of this project was to identify and examine microRNA sequences from prostate cancer (PCa) cell lines representing disease progression from lymph node to bone metastases. These sequences were generated by large scale sequencing of small RNAs found in LNCaP and its bone metastatic derivative, C4-2B.Various bioinformatics tools and gene databases were used to identify potential targets for both novel and known microRNAs expressed by PCa cells during disease progression from slow growing androgen sensitive status, represented by LNCaP, to androgen independent lethal disease, represented by C4-2B. Prostate cancer gene expression microarray data were used to further narrow the prospective target genes for known microRNAs. Biobase (http://bkl.biobase.de/cgi-bin/bkl/idb/1.0/searchengine/start.cgi) software was used to place these gene targets in relevant cellular pathways. Unknown potential microRNA sequences were also analyzed in an attempt to validate their status as genuine microRNAs. Future work on this project is designed to validate the action of microRNAs on target genes involved in prostate cancer progression using real-time PCR. (Supported by NCI P01 CA098912 and University of Delaware Science and Engineering Scholars Cancer and Genetics Fellowship.)

Obesity is currently the second leading cause of preventable death in the United States. This is due to the fact that obesity is often a risk-factor for many diseases that eventually can cause death, such as heart disease, diabetes, non-alcoholic fatty liver disease (NAFLD), and others. NAFLD is characterized by the presence of an abnormal amount of fat in the liver, which is defined as more than 10% fat by weight. My aim was to find what effect junctional adhesion molecule-A (JAM-A) has on weight gain and the eventual development of NAFLD. To analyze this relationship, groups of Jam-A (+/+) and Jam-A (-/-) mice were put on either a high fat or low fat diet for 20 weeks. During this time the mice were weighed every two weeks to track the effects of the diet on body weight. Every four weeks blood samples were taken with the aim of testing for plasma levels of cholesterol and triglyceride. At the end of the 20 weeks, the mice will be sacrificed and the livers and fat pads will be removed to find further effects of the diet, including any progression of NAFLD as examined through histological staining. At this point it has been found that Jam-A (-/-) mice on the high fat diet have gained significantly more weight than that of the other groups. This project was supported by Grant Number 2 P20 RR016472-08 under the INBRE Program of the National Center for Research Resources (NCRR), National Institutes of Health (NIH).