Juvenile Idiopathic Arthritis is an uncommon disease characterized by inflammation of joints or connective tissue in children. The cause is unknown. This study was performed to determine whether cytokine profiles and cell counts in samples of synovial fluid (SF) obtained from pediatric patients correlates with a Good or Bad clinical outcome. Cell counts were determined for each SF sample and cytospin/pulled smear slide preparations were made for differential analysis (volume dependent). Slides were stained (Wright-Giemsa) and analyzed microscopically. One hundred consecutive nucleated cells were counted and classified according to their morphology as neutrophils (PMN), lymphocytes, and monocytes. Levels of selected cytokines (IL-6, IL-10, IL-8 and TNF-alpha) were determined by enzyme linked immunosorbent assay (ELISA) from the cell-free aliquots of each SF sample. Patients were assigned to 1 of 2 groups based on clinical outcome. Forty-five patients were assigned to the Good outcome group (no flair for > 6 months post treatment) and 23 patients to the Bad outcome group (patients experiencing a flair of disease activity < 6 months post treatment). All samples were obtained at the time of baseline treatment during arthrocentesis. Cell-free aliquots of each SF sample were stored at -80°C until assayed. This study was approved by the institutional review board (IRB). Results of differential cell counts, determinations of cytokine levels and clinical outcome determinations were analyzed statistically using Sigma Stat software (Mann Whitney-Rank Sum, Student’s t test, and Multiple Linear Regression). 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).

The proximal tubule (PT) is a major transport segment of nephrons in higher vertebrates. Our laboratory studies the role of parathyroid hormone (PTH) using chick PT cells in primary cell culture. PTH in mammals affects PT cells by inhibiting reabsorption (lumen to blood transport) of phosphate and bicarbonate ions, resulting in increased urine pH and excretion of these ions. PTH in birds is similar and also causes increased bicarbonate and phosphate excretion. Moreover, a novel phosphate secretory mechanism is stimulated. This study focused on a hypothetical parallel system for bicarbonate secretion in the chick PT. Previous work had shown that PTH stimulated chloride secretion in PT cells via a cystic fibrosis transmembrane regulator (CFTR)-like chloride channel. Based on a bicarbonate secretion model in the mammalian colon, we hypothesized that the ion exchanger down regulated in adenoma (DRA) is present in chick PT cells. Colon studies showed that DRA works with CFTR to reabsorb secreted chlorine from the lumen and simultaneously secrete bicarbonate into the lumen. PT cells were cultured on membrane filters and in culture flasks to investigate the presence of DRA by immunoblotting methods, RT-PCR, and pH measurements. Immunoblotting studies were unable to detect DRA protein in the PT cell lysates. However, RT-PCR studies indicate that chicken mRNA for the DRA gene is present. pH studies suggest that bicarbonate is transported across the PT cells, resulting in an increase in apical pH. Optimization of these processes and electrophysiological studies on monolayer PT cells can further elucidate the presence of the DRA transporter. Research support from University of Delaware HHMI Undergraduate Research Program, Office of the Dean of Arts and Sciences and UOG NIH RISE Grant # GM063682.

Inflammatory Breast Cancer (IBC) is one of the most fatal forms of breast cancer, claiming about 80% of its victims within 10 years. Studies conducted on IBC have found a strong relationship between RhoC GTPase expression and the highly metastatic qualities of IBC cells. We have evidence that RhoB, and AKT are involved in the activation of RhoC.  Post-translational modification of RhoB, an intracellular transportation protein, by farnesyl transferase leads to the transportation of Akt to the plasma membrane.  Farnesylated RhoB is found in cancer cells. We hypothesize that Akt phosphorylates RhoC, leading to activation.  When active, RhoC is capable of mediating the metastatic activities of IBC.  RhoB can also be geranylgeranylated (gg), a similar type of modification that leads to relocalization within the cell.  ggRhoB should lead to re-distribution of Akt in the cell where it cannot contact RhoC.  Farnesyl transferase inhibitors (FTI) interfere with the RhoB farnesylation, which leads to accumulation of ggRhoB and in turn alters the activation of RhoC.  Utilizing a scanning confocal laser microscope we can study the subcellular localization of RhoB, RhoC, and AKT at different time intervals after FTI treatment.  After 48 hours, we expect to find increased RhoB in the cell, and a decrease in activated RhoC.  If we can stop RhoC from being activated, we can greatly decrease the metastatic capability of IBC cells.  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).

Making new proteins is an important component of the mechanisms underlying learning and memory. Local protein synthesis is required for long term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element Binding protein (CPEB) regulates protein synthesis and has been found to be important for long term memory formation, possibly through regulating local protein synthesis in neurons. CPEB is a highly conserved RNA-binding protein that promotes the elongation of the polyadenosine tail of messenger RNA. The experimental design for this project is two-fold. First we need to isolate the CPEB gene from Helix aspersa, a snail that serves as our model for learning and second, we developed a method for introducing it, and other genes, into living snails to study the role in behaviors. In order to isolate the gene that codes for the CPEB protein, we extracted DNA from the snail that serves as a template for PCR amplification. The DNA from an organism rich in mucous materials like the snail was difficult to purify; however, using repeated extractions and treatment with RNase A, free of DNase we were able to successfully purify high quality DNA. Gel electrophoresis of the DNA sample was used to evaluate the quality and quantity of the extract. We then designed oligonucleotides that are homologous to the CPEB conserved sequence in other organisms to serve as CPEB primers for PCR of the snail DNA. Gel electrophoresis was used to determine if the primers identified could amplify the DNA region of interest. The UV photograph of the gel, after a number of experimental variations showed no bands; we are still working on trying to resolve this problem.  Using Green Fluorescent Protein (GFP) as a reporter molecule, we also developed a tool to perform future "knockout" experiments. This method involves incorporating a DNA sequence into a living organism that can be used to block or over-express a test gene of interest. We successfully expressed the GFP in bacteria, and then utilized the GFP gene and a plasmid to construct a eukaryotic expression vector to observe GFP in snails using CMV as the promoter. Finally, we "painted" the plasmid of interest on the surface of the snail, allowing the plasmid to diffuse into the snail to observe incorporation and possible expression. Histological analysis will be performed to identify cellular incorporation. Funded by EPSCoR.

Veterinary antibiotics are routinely administered to animals not only for therapeutical treatments but also for growth promotion. After administration, these substances are partially metabolized and are excreted in the urine and feces. It has been evidenced that sub-therapeutic feeding of food animals for growth promotion along with casual use of antibiotics in household products, such as soaps and creams, is contributing to increased antimicrobial resistance in the environment. If steps are not taken to minimize selective pressure on bacteria, the effectiveness of antibiotics (hailed as `magic bullets') may be marginalized. The excretion of feces and urine from medicated animals and subsequent application of contaminated manure as fertilizer into agricultural land is one of the major routes through which veterinary antibiotics enter the environment. This exposes of microorganisms in the soil to low levels of the antibiotics create perfect conditions for selectively proliferating resistant bacteria. This leads humans and animals susceptible to infection by resistant pathogens either though direct contact or by indirect means such as through food and water supply putting animal and human health at high risk. . The research project aims at better understanding the type and activities of microorganisms present in the poultry litter and bedding material. The presented representative strains of bacteria were isolated and physiologically characterized. The degradation of two widely used veterinary antibiotics, sulfadimethoxine and tylosin, by the isolates was investigated. This project described was supported by Delaware EPSCoR, through National Science Foundation Grant EPS-0447610 and the State of Delaware.

 Fertilization, or the fusion of gametes, is an essential process in the development of a new organism. In mammals, the sperm has to be able to penetrate the barriers surrounding the egg in order to effect fertilization. Sperm accomplish this task by using enzymes called hyaluronidases (hyase) to break down hyaluronan, the major component found in the cumulus cell matrix and the zona pellucida that surround the oocyte. Although Hyal2 been classified as a somatic hyase, three pieces of evidence suggest that it may be involved in reproduction: HYAL2 is abundantly expressed in the testes, HYAL3, a closely-related acid-active hyase, is present in sperm where another acid-active hyase is also present, and RT-PCR shows that Hyal2 transcripts are present in the uterus where the protein may be secreted and acquired by sperm during sperm transit. Based on all of the above, Ii is hypothesized that HYAL2 is found on sperm and plays a role in fertilization. Thus this project is to determine the presence and the role of HYAL2 in fertilization and in the female tract. A Western Blot analysis was conducted to detect the presence of HYAL2 in sperm proteins.. Results indicate the presence of a 54 kDa HYAL2 band in sperm in different subcellular locations. Thus zymography will be performed to determine of the protein is active. / Supported by INBRE/DBI /

One apolipoprotein, apoE, has already been shown to play a significant role in cholesterol efflux from adipoctyes. The Usher lab has discovered another protein, apolipoprotein C-I, which may also be involved in cholesterol efflux. Previous studies have shown that apoC-I mRNA is high in late-phase 3T3-L1 adipocyte differentiation and is regulated in a similar manner as apoE. This study examines the production and detection of apoC-I and apoE during late-phase human and mouse adipocyte differentiation. Human preadipocytes and mouse 3T3-L1 fibroblasts were grown to confluency and induced to differentiate 48 hours post-confluency. Following differentiation, to determine the time-course of apoC-I secretion, supernatant samples and cell lysates were collected on D0 (Day 0), D7, D14 for human adipocytes and D0, D3, D6, and D9 for 3T3-L1 adipocytes. Human supernatants were centrifuged to determine if secreted apoC-I associated with lipoproteins. The individual collected centrifuge fractions were analyzed on 5%-20% gradient gels and immunoblotted with goat anti-human apoC-I antibodies. Immunoblots demonstrated that human apoC-I is associated with high-density lipoprotein fractions and produced during late-phase differentiation. Results suggests apoC-I involvement in cholesterol efflux, though further analysis of apoC-I secretion from mouse adipocytes is underway. Future studies will include using ELISA assays to quantify apoC-I concentration in human and mouse supernatant samples and will also examine methylation patterns in the apoC-I promoter region, which may elucidate apoC-I transcription regulation. Funded by the Howard Hughes Medical Institute.

Osteoarthritis, a leading cause of disability in the United States, results in pain and swelling of the joints due to the loss of articular cartilage that protects the bones of the joint. This loss reflects an imbalance in the catabolic and anabolic activities of articular chondrocytes, the cells of the cartilage. Because mechanical loading of the joint can accelerate the degeneration of the articular cartilage, the effects of mechanical stimuli on chondrogenesis was studied using fluid shear and hypotonic swelling. We have shown that increased intracellular Ca2+ levels ([Ca2+]i) and ATP release are initial events in mechanical signaling in bone and postulate that similar signaling occurs in chondrocytes. [Ca2+]i levels and ATP release were measured in primary bovine articular chondrocytes (BAC) in response to hypotonic swelling or fluid shear. We found that the [Ca2+]i response in BACs is different from osteoblasts, exhibiting greater peak calcium levels and spontaneous oscillations in [Ca2+]i. We also found that ATP was rapidly released, but that this release was different when the cells were grown on top of type II collagen or encased in the collagen. These responses could be major factors in cartilage loss with prolonged usage incurred in an aging individual. A better understanding of these early signals will provide insight into the initial degradation of articular cartilage during high impact loading and will potentially lead to an understanding of cartilage repair. (supported by the Science and Engineering Scholars Program and NIH/NIAMS R01 AR043222)

Colorectal is the third most common cancer and the second leading cause of cancer-related death. Most colorectal cancers occur sporadically, but 5-10% are hereditary due to a genetic predisposition. One of these syndromes, hereditary non-polyposis colorectal cancer (HNPCC) is characterized by an increased risk of colon cancer and many other types of cancer. The pedigrees and test results for eleven families found in the Familial Cancer Risk Assessment Program at the Helen F. Graham Cancer Center who tested positive for HNPCC were collected and analyzed for genotype phenotype correlations. Those positive for a mutation in MLH1 (MutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli)) were found to have early onset colon cancer, an average diagnostic age for endometrial cancer and absence of ovarian cancer. In contrast, those with a nonsense alteration type typically presented with gastric cancer. Those who tested positive for a mutation in MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)) were found to have the same early onset of colon cancer found in MLH1, but all families also had either breast or ovarian. Those testing positive for MSH6 (mutS homolog 6 (E. coli)) had an average age for colon cancer diagnosis meaning that it was consistent with the reported age of 62 in HNPCC individuals. This is a very small subsection of HNPCC families, so it may be that these results cannot be extrapolated to general population. Additional research will include more families with specific mutation types to make statistically significant conclusions.

Neuroblastoma is the most common soild tumor in children and is particularly difficult to treat in patients older than two years of age. The overall hypothesis is that the inhibition of cathepsins B and L will result in the death of neuroblastoma tumors without harming differentiated nervous tissue and non-neuronal cells. To meet this objective, Some of the SK-N-SH were used as control (non FYAD treated) and the others cells were treated with FYAD. FYAD induces cell death and then the purification of lysosomes was confirmed by western blot analysis. The objective of this study is to determine the mechanism by which an inhibitor of two lysosomal proteases cause cell death in neuroblastoma cells. This grant was supported by 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).

The purpose of this research was to determine the rate of non-therapeutic surgeries for solitary pulmonary nodules. Researching and viewing power charts, data bases, and medical records of the patients provided information to evaluate the trends of the negative surgeries for the past nine years. The sensitivity of CT scanning made it possible to detect small pulmonary nodules. However, it is not capable to accurately differentiate between benign and malignant lesions, which expose patients to a risk of negative surgical interventions. After the patients who underwent thoracic surgery were identified, their operative and pathology reports were reviewed, leaving patients who underwent resection for indeterminate solitary pulmonary nodule(s) as the subject of the study. Non-therapeutic surgeries were defined as a pathology reports demonstrating no malignancy, fungal infection or other anomaly that would jeopardize the patient’s health. The accessibility of PET scanning and the launch of I-ELCAP study decreased the rate of non-therapeutic thoractomies from 23% (before 2002) to 9% (2003 till present). The average prevalence of negative thoracotomies for the past five years at CCHS was determined to be 7.4%. This grant was supported by 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).

Background: Purinergic signaling stimulates many biological processes such as cell proliferation, differentiation, and apoptosis. Two classes of purinergic receptors, GPCR and S/T kinase, have been identified that bind ATP and other nucleotides as ligands. ATP has an antitumor effect on cancer in vivo. Thus clinical trials are being carried out to determine that ATP can be used as a therapeutic agent for cancer. Here we looked at the effect of ATP on an isogenic progression series of prostate cancer (PCa) cell lines (LNCaP, C4 2, and C4 2B4). We hypothesize that ATP stimulates neuroendocrine differentiation (NED) and growth in PCa cells. NED occurs with relatively high frequency in PCa and is correlated directly with poor prognosis. Methods: ATP, 0.001-1000μM, was added in log10 increments to PCa cell lines in vitro. Morphology was examined by photomicroscopy; cell number was determined using crystal violet staining; and, migration was examined using scratch assays. Results: ATP increased the growth of PCa cells over vehicle alone (LNCaP=2.7X, C4-2= 1.7X, C4-2B4= 1.4X) with peak stimulation occurring at 1nM ATP. Morphology was largely unchanged in response to ATP. Scratch assays are currently in progress. Conclusions: Growth response to exogenous ATP decreases as PCa cells become more androgen insensitive and metastatic. NED is not apparent morphologically but biochemical analysis is required to confirm this result. Funding by DoD PCRP-W81XWH-06-1-0244

Colon cancer arises from tissue changes in normal colonic epithelium, the structure of which consists of histologic subunits termed colonic crypts that maintain an incredibly high level of organization during the human lifespan. Slowly proliferating colonic stem cells normally reside at the crypt base and are progenitors of all other cell types in the crypt. Enteroendocrine cells — specialized endocrine cells of the gastrointestinal tract that also have neuronal properties —are thought to: i) be responsible for normally controlling the stem cell population and ii) contribute to the development of tumors in the colon. We hypothesize that tumorigenesis is coupled to abnormal cell cycle proliferation and migration of enteroendocrine cells in the crypt. A system of reaction-diffusion equations was designed to mathematically model the crypt’s dynamic spatial-temporal organization involving eight different cell types. The state of the system is predicted converge to a steady-state distribution of cells, as found in a normal crypt. Divergence from the normal steady state, in particular from the proportion of different cell types and their distribution in normal crypts, may predict development of a colon tumor. To obtain biologic data for validation of model output, we did immunostaining of normal and tumor tissue using antibodies against several different neuroendocrine markers. Our results indicated that neuroendocrine cells are abnormally distributed in colon tumor because positively-stained cells were located in the upper level of tumor crypts, as compared to being restricted to the lower level of normal crypt This finding suggests that communication between neuroendocrine cells and stem cells could be a key factor in tumor development. This project was funded by the Howard Hughes Medical Institute.

Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells.  It is ubiquitously expressed in vascularized tissue and the reactive stroma surrounding prostate cancer cell lines produces high levels of the protein.  Additional studies suggested that perlecan plays a role in delivery of growth and angiogenic factors, which aids survival and growth of metastatic tumors.  The overall goal of this project was to study the promoter in order to understand the up-regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis.  Our first step was to analyze the promoter to find conserved elements.  The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a sequence for the mouse perlecan promoter also found in online databases.  Several transcription factor binding sites of interest were identified for further study including NFkappaB [-2410 to -2398], CREB ([-1797 to -1777] and [-709 to -689]), Smad3 ([-1301 to -1293] and [-187 to -179]), Elk-1 [-1699 to -1679], c-Jun ([-2453 to -2441] and [-2496 to -2476]) and TCF/LEF-1 ([-1521 to -1505] and [-1247 to -1231]).  Currently we are working to build a promoter-reporter construct by isolating the promoter region using PCR amplification.  Our current strategy seeks to optimize the PCR reaction with different sets of primers.  The next step will be creating a luciferase or RFP reporter construct in order to test the effects of the identified pathways. This project was funded by the Howard Hughes Medical Institute.

Osteoarthritis (OA), a degenerative joint disease, affects many people over the age of 65 and a large portion of competitive athletes. Age, mechanical stress, and genetics contribute to the disease. Currently there are few effective therapeutic treatments. We are using use a hyaluronic acid (HA) based scaffold delivery system in combination with recombinant perlecan domain 1 (PlnDI) to permit the slow release of heparin binding growth factors (HBGFs) into osteoarthritic knees. We hypothesized that Synvisc®, a commercially available HA gel, would decrease diffusion of injected PlnDI from the knee cavity. To test this, we fluorescently labeled and injected PlnDI intra-articularly into murine knees. The retention time of PlnDI was compared using in-vivo imaging when injected alone or co-injected with Synvisc®. In both cases, PlnDI was undetectable in the knee cavity after 24 hrs, therefore the Synvisc® failed to prevent PlnDI loss from the injection site. We next investigated PlnDI’s ability to diffuse into articular cartilage of isolated femurs, tibias, and patellar explants by incubating them with labeled PlnDI and nuclear dye. The PlnDI signal did not overlap with the nuclear signal, but localized to a peripheral region near the cartilage matrix. Current effort will develop a papain-induced mouse model of OA for use in future studies to screen the efficiency of different PlnDI-based repair complexes including their ability to increase retention of HBGFs and for proper targeting to OA articular surfaces. Funding for this project has been provided by Charles Peter White Scholarship.

A family of Junctional Adhesion Molecules (JAMs) consisting of JAM-A, JAM-B, and JAM-C has recently been discovered.  These transmembrane proteins are involved in tight junctions, are often associated with PDZ domain-containing proteins, help anchor transmembrane proteins to the cytoskeleton, and hold together signaling complexes.  JAM-A and JAM-B are known to contain PDZ domain-binding motifs, and it has been shown that JAM-A uses this motif to interact with cytoplasmic PDZ proteins.  However, interactions between JAM-B and PDZ domain-containing proteins have not yet been identified.  To study this, protein microarrays can be used to identify protein-protein interactions between JAM-B and PDZ domain-containing proteins. A cDNA construct encoding for a JAM-B GST fusion protein with an HA-tag was first created using polymerase chain reaction.  The JAM-B fusion protein was expressed in IPTG induced BL21 E. coli cell cultures, which were lysed using a French press. Expression levels in induced cultures were observed with Coomassie staining and Western blot analysis.  The lysate was incubated with glutathione sepharose beads to bind the fusion protein, which was subsequently cleaved with thrombin, allowing the HA-tagged protein to be purified.  The purified HA-tagged protein will be used to probe a protein microarray spotted with PDZ domain-containing proteins and JAM-B – PDZ domain interactions will be detected by chemiluminescence using an anti-HA primary antibody and an HRP-conjugated secondary antibody.  This identification of JAM-B and its PDZ-domain containing binding partners will further our understanding of signaling pathways and molecular interactions, particularly pertaining to the formation and regulation of intercellular pathways and tight junction integrity. Funding was provided by the Beckman Foundation, and the NIH for Dr. Naik.

A viable route in tissue engineering and regenerative medical treatments for arterial diseases is to create synthetic, transplantable stretches of arterial tissue that have the ability to grow and interact with surrounding tissue in the patient. However, a serum free medium must first be developed that is capable of growing and sustaining the various cell types found in arterial tissue until transplantation is possible. In my evaluation, the three major human arterial cell types (endothelial cells, vascular smooth muscle cells, and fibroblasts) were cultured in vitro in both their recommended serum-containing medium and a defined serum free medium (SM3+) with and with out growth factors in order to determine the extent of cell death, proliferation, and viability. This SM3+ medium has been successful in sustaining cell life in analogous cell types found in neonatal rat heart cultures for extended periods of time, and theoretically should be able sustain cell life in a human model. However, it was found that the overall cell viability decreased substantially for the three cell types, causing the cells to detach from the polystyrene plates they were seeded on. Additionally, there was little or no proliferation and considerable cell death observed in the SM3+ medium. This project was funded by the Howard Hughes Medical Institute.

Study of microRNA expression in normal colonic epithelium and colon cancers Sepehr Sedigh Haghighat1, Greg Gonye2, Tao Zhang1, Bruce Boman1 1Department of Biologic Sciences, University of Delaware, Newark DE, 2Thomas Jefferson University, Philadelphia PA Dysregulation of crypt cell proliferation and differentiation has been implicated in the development of colorectal cancer (CRC). As part of our investigation of regulatory factors involved in the stem cell origin of CRC, we investigated the role that microRNAs (miRNAs) might play in colon carcinogenesis. miRNAs are RNAs 20-24 nucleotides long that were recently shown to modulate many cellular signaling pathways through post-transcriptional regulation of messenger RNA (mRNA) levels and thus protein synthesis. Our previous micorarray analysis (368 gene chip) showed increased expression of 37 miRNAs in CRCs compared to normal colonic epithelium. Here we further evaluated miRNA expression in CRC versus purified colonic epithelium by quantitative PCR (QPCR). Total RNA was immediately isolated from tissue by the TrizolTM method. RNA was transcribed into first-strand complementary DNA (cDNA) through reverse transcription (RT). cDNA was then amplified through QPCR. Statistical analysis and plots of expression data for miRNAs were done to find miRNA genes that are differentially expressed. We found, using QPCR, five miRNAs to be differentially expressed in CRCs versus normal colonic epithelium including mir-25 and mir-198. Identification of miRNAs specifically expressed in normal colonic crypts and changes in their expression in CRCs will provide important information to help understand mechanisms of colon tumorigenesis.

Selenium exists in the environment in several ox-red states and one of the unresolved features is the rate at which colloidal, elemental selenium is produced from selenium oxy -anions. While few bacteria have the capability of coupling the reduction of selenate or selenite to elemental selenium, many aerobic bacteria have a glutathione-based process of forming elemental selenium and several anaerobic bacteria use metal reducing systems to produce elemental selenium. From this study we have developed a novel colorimetric method for the measurement of elemental selenium under several conditions including sulfidogenic environments. Using environmental bacterial isolates, we have followed the reduction of selenite to colloidal elemental selenium by pure cultures and the method detects elemental selenium even if elemental selenium is formed inside the cells. Our research addresses a method for measuring colloidal red selenium in the presence of chemicals typically associated with soil or water containing toxic compounds. Using environmental bacterial isolates, we have followed the reduction of selenite to colloidal elemental selenium by environmental isolates and the method detects elemental selenium. [This project described was supported by Delaware EPSCoR, through National Science Foundation Grant EPS-0447610 and the State of Delaware.]

Ribosomal proteins (RPs) play an important function in the maintenance of a normal protein synthetic rate. Our group generated the first viable mouse mutant model lacking an individual ribosomal protein (RPL29). In these mutants, decreased rates of protein synthesis and cell proliferation resulted in skeletal growth defects leading to short adult stature. Recently, we demonstrated that the absence of RPL29 increases bone fragility due to poor tissue and extracellular matrix (ECM) quality, suggesting an important link between efficient protein production and skeletal tissue growth. We hypothesized that because of impaired ability to synthesize large volumes of ECM proteins, RPL29 null bones will exhibit a reduced response to mechanical load as compared to wild type controls. To establish a correlation between defects in ECM protein biosynthesis and an impaired response to mechanical stimuli, cyclic axial loading was performed in vivo on the tibia of adult RPL29 null mice and age matched wild type controls of the C57BL/6J (B6) background. The cortical and trabecular bone microstructure of both the loaded and unloaded tibiae from each sample was analyzed using micro-computed tomography (microCT). Three-point bending tests on the loaded and unloaded tibiae will assess the extent that cyclic loading improves the mechanical properties and fragility of RPL29-deficient bones compared to controls. Studies of dynamic histomorphometry will also be used to quantify the effect of mechanical loading on bone formation capacity between the null mice and controls. Altogether, these studies will establish the importance of high volume protein synthesis for the regulation of bone formation during adulthood. In the future, this information may be particularly useful in treating diseases such as osteoporosis, in which bone microarchitecture is altered due to an imbalance between mineral and organic (collagen/non-collagenous proteins) phases. This project was funded by the Howard Hughes Medical Institute.

Titanium dioxide (TiO₂) nanoparticles are widely used and can be found in sunscreens, paint and food coloring. These metallic compounds readily donate electrons to create reactive oxygen species (ROS) and induce oxidative stress. High levels of oxidative stress are toxic to cells, particularly sperm which are devoid of repair enzymes and thus may undergo deleterious effects. The objective of this project was to examine the effects of TiO₂ nanoparticle exposure during spermiogenesis. Sexually mature male mice were injected intraperitoneally with 100% anatase nano-TiO₂ suspension at 0 mg/ml (control), 0.625 mg/ml (low), and 1.25 mg/ml (high) for three consecutive days. The mice were sacrificed after 24, 48 and 120 hours from the final injection and caudal epididymal sperm were removed and testis weights recorded. Sperm were analyzed for motility, hyperactivated motility, retention of residual cytoplasm and the ability to acrosome react. Testes were prepared for histological examination, TEM microscopy and DNA analysis. The results showed a significant (p<0.005) increase in the number of sperm retaining excess cytoplasm, as well as a decrease in acrosome reaction rates and motility with nano-TiO₂ exposure. TEM microscopy revealed TiO₂ nano aggregates in fat tissue surrounding the testes. Testis histology was abnormal and testicular DNA was tested for fragmentation. These results indicate that TiO₂ nanoparticles induce retention of excess cytoplasm on sperm by disrupting spermiogenesis and may lead to male subfertility/infertility. Funding was provided by EPSCoR and the Charles Peter White fellowship.

Fungal infections are becoming increasingly common, with the most frequent fungal organism being Candida albicans. The molecular mechanisms for this increase in pathogenicity of C. albicans, has yet to be fully understood. For example, it still remains unknown as to why certain microorganisms present a significant pathogenic risk to humans. It has been previously hypothesized that fungal pathogens are able to mask their -glucans within their cell wall to prevent recognition by immune system cells; therefore, we hypothesize that the opportunistic pathogen, C. albicans, conceals -glucans within its cell wall, hindering recognition by pattern recognitions receptors (PRRs), such as Dectin-1 and toll-like receptors (TLRs) expressed by cells of the innate immune system. In this research we examined fungal cell wall structure by Transmission Electron Microscopy (TEM) and macrophage recognition of fungal pathogens by Atomic Force Microscopy (AFM). This research revealed that C. albicans was found to have an average cell wall thickness of 96nm, whilst Saccharomyces cerevisiae, a non-pathogenic control, was found to have an average cell wall thickness of 130nm. Immunolabeled sections imaged by TEM show that the -glucans of C. albicans are clustered within the cell wall, rarely being displayed on the surface. The importance of the -glucan-Dectin-1 interaction is further corroborated by AFM data showing a 28% reduction in unbinding force when the β-glucans are blocked, and a 20% reduction when Dectin-1 is blocked. In conclusion, the interaction between Dectin-1 and β-glucan is important in innate recognition of pathogens, and C. albicans is able to evade recognition by concealing β-glucans within the cell wall.

The Unfolded Protein Response (UPR) has been hypothesized to be a cause of cataracts. UPR is a stress-induced autoregulatory mechanism that arises from the accumulation of unfolded proteins in the endoplasmic reticulum. It restores homeostasis in stressed cells by modifying gene expression at both the transcriptional and translational levels or by inducing apoptotis. UPR signaling pathways may be initiated by three ER transmembrane proteins: IRE1, PERK, and ATF6. Our lab previously confirmed IRE1 activation in mouse lenses that accumulate unfolded collagen IV protein chains in the ER lumen. Therefore, we chose to investigate another UPR pathway, the PERK pathway, in the collagen IV transgenic mice. In response to ER stress, PERK phosphorylates eIF2α, halting global ER protein translation but increasing the translation of CREB2/ATF4. CREB2/ATF4 can then upregulate GADD153/CHOP, a pro-apoptotic transcription factor. The mechanism behind cell death occurring through CHOP-mediated apoptosis is not completely clear, but is connected to the abnormal lens phenotypes seen in collagen IV transgenic mice. Immunostaining of 16.5dpc lenses with ATF4 and CHOP antibodies showed considerable upregulation in the transgenic lens cells. Furthermore, ATF4 and CHOP protein levels in newborn lenses were increased in transgenic mice, whereas adult transgenic lenses exhibited normal levels of ATF4 and CHOP. This was expected, as the PERK pathway is the first to be activated but then must be deactivated for stress-specific gene induction. Collectively these data suggest that the PERK pathway of the Unfolded Protein Response is active within embryonic lens cells experiencing ER stress. Supported by Howard Hughes Medical Institute and the National Eye Institute.

Previous experiments have shown that MDA-MB-231 human breast cancer cells could be injected into the extra-embryonic vasculature of chick embryos and then cells which had metastasized to the brain could be isolated.  These cells had been transfected with the neor and LacZ genes, enabling selection of drug resistant colonies of cancer cells which could then be quantified following visualization with X-Gal.  The sensitivity of the in vivo chick embryo system is being tested by initially injecting embryos with a large number of cells (50,000) and then decreasing the number of cells injected until tumors no longer form in the brain.  Injections of 50,000 cells produced an average of 199.5 colonies after treatment with G418, and injections of 5,000 cells produced an average of 32.2 colonies.  Detection of breast cancer cells in the brain after injection of only 5,000 cells into the extra-embryonic vasculature suggests that the in vivo chick embryo is a sensitive system.  The next step of this study is to investigate the effects of re-injecting cells which have been through the brain on the metastatic potential and specificity for the brain.  Experiments with nude mice labs have shown that re-injection produced sublines with enhanced selectivity for the brain.  The goal of this study is to determine whether the increased selectivity results from a higher percentage of injected cells metastasizing to the brain, or from fewer cells metastasizing to other organs.  Based on preliminary results, it is predicted that the increased selectivity is not due to a higher percentage of cells going to the brain, but fewer cells going elsewhere. This grant was supported by 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).

Osteoblasts respond to mechanical load with a rapid release of ATP that, in turn, binds to two classes of purinergic receptors (P2 X and P2Y). Our lab has reported that P2X7 receptor activation is essential to mechanotransduction in osteoblasts. We have recently observed that activation of this receptor results in a rapid change in osteoblast morphology and induces cellular contraction. We hypothesize that activation of P2X7 receptors during mechanical stimulation activates two distinct pathways, the RhoA GTPase and Protein Kinase C, that lead to the contraction of the osteoblast. Here, we examined the changes in MC3T3-E1  preosteoblast morphology and contraction using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor and how these changes were affected by inhibition of specific sites in the RhoA GTPase and PKC pathways.  BzATP, a known agonist of the P2X7 receptor, was added to MC3T3-E1 cells and changes in cell area following BzATP stimulation were quantitated using Differential Interphase Contrast (DIC) microscopy.  Addition of 0.5mM BzATP to MC3T3-E1 cells resulted in a 42% reduction in cell area.  Inhibition of PKC with the non-specific inhibitor, GF109203X, attenuated the BzATP-induced contraction with only an 11% reduction in cell area.  We predicted that activation of myosin light chain kinase, a modulator of contraction and a downstream affector of RhoA, would have significant effects on P2X7-induced contractions.  However, inhibition of this pathway failed to block BzATP-induced contraction. These studies suggest that PKC interacts with purinergic signaling pathways to increase the skeletal remodeling. (supported by INBRE2 P20)

When caught early prostate cancer (PCa) is treatable. Insulin-like growth factor-1 (IGF-1) and transforming growth factor-Beta (TGF-Beta) are cytokines that are both involved in the progression of cancer. TGF is a tumor growth suppressor and IGF is a cancer growth promoter. It is possible for IGF-1 to suppress the surface expression of TGF- Beta's major receptor TR-II. Based on published findings it appears that IGF-1 down regulates TGF-Beta signaling. By using a progressive lineage of PCa cells our objective is to explain the down regulation of TGF-Beta by IGF-1, through TGF-Beta receptor (TR-II). As the disease progresses TGF-Beta evolves from tumor suppressor to tumor promoter. TGF-Beta usually decreases the likelihood of cell proliferation and IGF-1 could be what causes TGF-Beta to mutate and possibly speed up the progression of PCa and increase the risk of malignancy. Using FACS we sorted the cells according to the intensity of fluorescence they acquired during the process. The level of TR-II on the lineage of cells used: LNCap, C4-2, and C4-2B4, was evaluated and compared to the controls which were not treated with TR-II. This project was funded by the HHMI NUCLEUS Program.