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.

The function of voltage-gated sodium channels has been well characterized, however its potential role in producing a more metastatic phenotype of prostate cancer (PCa) is not well known. beta-subunits of sodium channels have immunoglobulin (Ig) loops of the V-set family that are known to interact homotypically and heterotypically as cellular adhesion molecules. We hypothesize that the beta subunits contribute to metastasis to the spine through perineural invasion by interacting with other V-set Ig-molecules. Western Blot for the SCN2beta subunit was performed on cells of the isogenic human PCa progression model: LNCaP, C4-2, and C4-2B4. Imunofluorescence also was performed on the C4-2B4 cell line. Digitonin-derived whole cell extracts from C4-2 cells were subjected to a wheat germ agglutinin (lectin) affinity column to enrich SCN2beta in the samples for western blot analysis. Western Blot analysis of the cells of the LNCaP PCa progression series showed an increasing expression of SCN2beta that corresponded to increasing tumorigenicity, invasiveness and metastatic potential. The protein was found predominantly in triton soluble fractions. Immunofluorescence showed little specific interaction. The C4-2 lysate subjected to lectin chromatography showed little SCN2beta. Although the results of the immunoblot suggest that the expression of SCN2beta increases as PCa progresses to a more metastatic phenotype, the functional significance of its location in triton soluble fractions remains to be determined. Additional troubleshooting or affinity purification of the antibody may be for specificity. Preliminary results with lectin chromatography were disappointing but this method may still prove efficient for enriching beta-subunits. Funded by the INBRE Program- P20RR016472.

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).

Previous studies have shown that the effects of solvent variation of the available specific rates of solvolysis of frequently used pharmaceutical precursors can be analyzed using the extended Grunwald-Winstein equation. There are also many instances for solvents rich in fluoroalcohols where simple G-W plots lead to points lying above the correlation line. Here it is shown that this behavior can be treated by the addition of a new term called the aromatic ring parameter, to the extended G-W equation. By measuring the rate of solvolysis of cinnamyl chloride and applying the extended G-W equation with the aromatic ring parameter, this research will show that our studies of solvolytic acylation mechanisms could lead to better understanding of the pathways involved in the acylation of the hydroxyl groups of potential pharmaceuticals to increase their water solubility. It also helps us to understand mechanisms of reaction of cinnamyl halides, which are common precursors in the synthesis of useful haloenol lactones that are often used in treatment of drug resistant cancer and prevention of herbicide resistance in weeds. 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).

White rot fungi, specifically Phanerochaete chrysoporium, are able to produce non-stereo selective enzymes that oxidize compounds present in pollutants and, therefore, may be used for environmental remediation. Past research has investigated the ability of the fungal enzymes, lignin peroxidases (LiP) and manganese-dependent peroxidases (MnP), to degrade pesticides and other compounds present in wastewater and the herbicides, atrazine and alachlor, in contaminated soil. Although past studies have successfully measured the percentage of compound degradation, there still remain difficulties with measuring the enzyme activity after application to the soil due to interacting compounds. The goal of this research is to develop an assay and method to monitor the activity of LiP and MnP within the soil over time. The fungi have been grown within a packed-bed bioreactor, first supplied with growth media and then starvation media which cause the fungi to begin the ligninolytic activity. Once the fungi begin producing enzymes, the effluent will be collected and applied to soil. The enzymes will be extracted from the soil using the developed assay. The soil extract will be analyzed for enzyme activity. LiP activity will be monitored by using a continuous spectrophotometric assay that measures the oxidation of veratryl alcohol to veratraldehyde at 310 nm. MnP activity will be monitored by using a continuous spectrophotometric assay to measure the oxidation of o-dianisidine to a colored-product at 460 nm.  Future work will evaluate the efficiency of this proposed method and continue examining literature for other enzyme extraction methods from soil. Research funded by Delaware EPSCoR, through National Science Foundation Grant EPS-0447610 and the State of Delaware.

In the recent decades obesity has become an epidemic. Due to the overwhelming effects of obesity on mortality and morbidity more people are turning to bariatric surgery. Recent studies have shown that patients who consistently attend follow-up visits with their surgeon and health care team were more successful with long-term weight loss. It has also been shown that telephone consultations can be used to increase compliance and decrease post-operative complications. Our goal was to determine if regular telephone consultations would increase compliance with follow-up appointments and therefore lead to improved bariatric surgery outcomes. Patients undergoing pre-operative counseling were consented and then randomly assigned to the experimental or control group. The experimental group would receive weekly telephone consultations encouraging them to attend follow-up appointments and addressing any questions. The control group would receive standard care post bariatric surgery. A power analysis determined that the appropriate sample size is 64 patients for both the control and experimental group. To date only 39 out of the required 128 patients are 6 months post-operation. Therefore, the preliminary results are inconclusive and research is continuing to be conducted. This project described 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 chicken was domesticated approximately 7,000-10,000 years ago in Asia from the red jungle fowl. The advent of industrial scale agriculture in the early 20th century triggered intensive genetic selection to improve the chicken’s meat (broiler) production traits. This selection has yielded birds with a 2 fold increase in breast muscle mass and a significantly reduced time to market. Along with increased muscle, undesirable traits have arisen including cardiovascular failure leading to sudden death. We are comparing a heritage line (IL50) that grows as broilers did in the 1950s with a modern line (Ross708) to gain insight into the changes resulting from the selective pressure. A morphometric analysis revealed significant growth differences between the heart, breast muscle and liver of the heritage and modern lines. Current effort focuses on determining the size and number of cells in the heart and breast muscle of the two lines. This will allow evaluation of the relative contributions of hypertrophy and hyperplasia to the overall size of the organs. This research was funded by INBRE.

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).

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).

Aliphatic chlorothioformate esters are used to prepare low molecular weight thiocarbamate inhibitors of the enzyme elastase in mammals. Such small molecules are therapeutically effective in treating chronic inflammatory diseases such arthritis, asthma, colitis, and emphysema. Because of convenient rates of reaction at temperatures close to ambient, the specific rates of solvolysis of Isopropyl chlorothioformate are analyzed using the extended Grunwald-Winstein equation. Previously, in a comparison of chloroformate ester and chlorothioformate ester solvolyses by the ionization mechanism, it was assumed that sulfur would have a smaller tendency to use its electrons in a stabilizing interaction with the adjacent electron deficient carbon of an incipient carbocation. Kevill and D’Souza have shown that in ethyl chloroformate solvolysis, an addition-elimination channel dominates and only in the more ionizing and least nucleophilic solvents does the principal reaction channel involve ionization. For ethyl chlorothioformate, the relative importance of the two reaction channels is reversed, and for the majority of solvents, the ionization pathway is dominant. Thus it will be of great interest to observe the characteristics introduced by a bulkier isopropyl group from the isopropyl thioester.

The Science, Ethics, and Public Policy (SEPP) program at Delaware Biotechnology Institute serves as the major fountainhead for discourse concerning scientific research and its implications to human conduct.   The SEPP web page serves as the Science, Ethics, and Public Policy program’s ‘cyber-infrastructure.’  During the summer of 2008, Andrew Martin made a number of changes and additions to the SEPP website.  Cosmetic Changes included touching up images and adding new pictures and logos for projects and events, e.g, a November 2008 conference, Environmental Nanoparticles: Science, Ethics, and Policy and the Public Communication of Life Sciences project.  Formatting changes included rewriting all of the article links for uniformity.  The Content of the website was also improved with many new articles having been added.   Beyond his work in the information technologies, Andrew was able to aid in the research of two talks given by Dr. Thomas Powers in July.  They are titled "The Ethics of Biofuels" at Plants and Soils Montreal 08, and "Neuroimaging and Philosophical Moral Psychology" at the Institute of Clinical Research in Montreal.  Delaware EPSCOR (NSF) funded this work.

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.

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 /

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).

Increasing areas of residential development combined with the loss of forested land is an environmental concern in Delaware. There has been a 35% increase in urban land use throughout Delaware from 1992 to 2002. These land use changes throughout the state can contribute to impaired water quality issues. Proper land use balance is essential in order to protect the health of nearby bodies of water. The objective of this research was to determine the effect of current and potential future land use changes on the fate and transport of runoff, sediment erosion, and nitrate-nitrogen and phosphate-phosphorus deposition within the Mispillion watershed using the Loading Simulation Program C++ (LSPC). Land use type was altered for the Mispillion watershed to represent a present day calibration simulation (2001-2003), a future residential build-up, and a historic pre-colonial era where forest was the dominant land use. Results indicate that the large presence of agricultural land within the watershed combined with rainfall events are the defining factors for the output values of runoff, sediment loss, and nutrient deposition. These findings as well as the continued use of the LSPC model can provide land use planners quantitative information that is essential for making sound environmental decisions. Future simulation analyses will include growth projection estimates for the Mispillion watershed from the Delaware State Office of Planning. This project described was supported by Delaware EPSCoR, through National Science Foundation Grant EPS-0447610 and the State of Delaware.

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%.

Isopropyl Chloroformate decomposes on heating or on contact with acid producing toxic and corrosive fumes including chlorine and phosgene. Yet this highly flammable explosive compound has found pharmaceutical use as a precursor in the synthesis of molecules useful in treating prophylaxis, diabetes, atherosclerosis, cardiovascular diseases, hyperuricemia, inflammatory bowel diseases, or skin disorders related to an anomaly of differentiation of epidermic cells. Hence, understanding its mechanism of reaction in a variety of solvents will give real insight for the production of synthetically useful pharmaceutical products. Here the specific rates of solvolysis Isopropyl Chloroformate are analyzed using Grunwald-Winstein equations. Previous studies found that Alkyl or Alkenyl Chloroformates solvolyze in most of the commonly studied solvents by an addition-elimination mechanism with the addition step being rate-determining. In fluoroalcohols for Ethyl, and n-Propyl chloroformate, an ionization mechanism was indicated with up to 50% water content in HFIP-H2O and with up to 10% water content in TFE-H2O mixtures. Therefore in this project we provide kinetic studies of Isopropyl Chloroformate including 1,1,1,3,3,3- Hexafluoro-2- Propanol (HFIP) and 2,2,2-Trifluoroethanol (TFE). 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 suburban sprawl in the northeastern states has created refuge areas for white-tailed deer (Odocoileus virginianus) populations to drastically increase. These refuges prevent deer population control by limiting hunting access because of mandated safety zones. The balance between public safety and deer population control is a challenge for wildlife managers. Northern Delaware is a prime example of a densely human populated area with a large deer population. We examined possible impacts of altering safety zone size on the amount of land accessible to hunting. Using an aerial photograph of New Castle County, Delaware north of I-95 and I-495, a 183m (200yrds) buffer was established around each inhabited building, which represents the size of the current safety zone. Using this buffer we were able to quantify the current land available for hunting. We altered the buffer size around each building from 183m (200yrds) to 91m (100yrds) and 46m (50yrds) to determine the total area of deer habitat accessible for harvest under smaller safety zone sizes. By analyzing this data it was established that by reducing the safety zone to a reasonable 91m (100yrds), 71% of the land considered deer habitat would be available for harvest. By reducing the safety zone distance wildlife managers could have a more effective tool for controlling deer population and thus deer tick related diseases and environmental damages due to deer browsing.

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.]

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.

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)

The application of heat to skin tissue has many advantages in the wound healing process. Raising the tissue temperature promotes a variety of inflammatory processes, including leukocytosis, drainage, vasodilation, suppuration, and overall healing. Other studies have demonstrated that preoperative warming did decrease the incidence of wound infections following clean surgery. This knowledge laid the foundation for this research study in which the application of a heating pad prior to a Cesarean section (C-section) delivery was predicted to decrease the wound complication rate post procedure. Mothers scheduled for non-emergent C-sections were invited to participate. Once informed consent was obtained, subjects were randomized to one of two groups: routine care which involved no heating pad, or the application of a heating pad for 30 minutes within two hours of surgery. Subjects were called two weeks following their surgery to determine any incisional problems. To date, 161 subjects have been enrolled. Interim data analysis has shown no significant difference in wound complication between those subjects who received the heating pad and those who received standard care. However, the target sample size (N=250) has not been reached leaving the study underpowered and at risk for a type two error at this point in time.

This study was conducted in an attempt to determine whether a higher quality of life is prevalent among those patients who have had pulmonary lobectomies using a video-assisted thoracic surgery (VATS) approach rather than a thoracotomy (THOR) approach. The research was done by evaluating information available in the Helen F. Graham Cancer Center thoracic surgery database and by surveying both THOR and VATS patients on aspects of quality of life. Once the two groups were finalized, the length of stay was analyzed using the information provided by the database. The VATS patients had a lower average stay than the THOR patients, a difference that showed to be statistically significant (p=0.007). Other collected data that also indicated a higher quality of life among the VATS patients included the length of time on pain medications, pain level one week after surgery, and length of time until able to return to work. The conclusions drawn from this study indicate that a higher quality of life during the initial post-operative period exists for patients who have pulmonary lobectomies via VATS rather than by thoracotomy.

In the poultry industry, table egg laying hens are used to produce the eggs that are bought in the grocery store. Multiple layers are kept in one cage with 10,000 to 18,000 cages and 75,000-125,000 or more birds in a typical layer house.  In the poultry industry, the primary approach to highly infectious diseases is surveillance, quarantine, depopulation, disposal, and disinfection.  When birds are infected or suspected of infection, with a highly infectious disease, such as avian influenza, the birds must be depopulated in order to prevent the spread of the disease.  Depopulation methods have been developed for floor reared meat type birds, but caged layers remain a problem.  One of the critical questions is whether to depopulate the birds inside the cages or to remove the birds prior to depopulation.  After death, during rigor mortis, it becomes difficult to remove the birds from the cages. The purpose of this experiment was to determine the average rigor mortis onset and relaxation time for depopulated caged layer carcasses.  This will help determine when are the most efficient times for removing the carcasses from the cages, so that disposal of the carcasses and disinfection of the facilities can begin.

The National Center on Elder Abuse estimated 1-2 million Americans age 65+ were victims of abuse by a caregiver (2005). Elders are projected to comprise 20% of the US population in 2030. Frail elders with health care needs are at greatest risk for mistreatment by their caregivers. This study aims to elicit perceptions of elders about physical abuse as a kind of mistreatment. The first phase of the project, presented here, is the scale development. Kane’s (1991) family social support theory grounds construction of the scale and proposes that the more stable a family the more substantial the reciprocity, advice and feedback, and emotional involvement. This proposition will guide data interpretation with an assumption that the less stable the family the more likely they are to have destructive behaviors. A vignette design with Likert scaling method was chosen for the construction of the scale because it allows examination of context (Martin, 2004) while also presenting a sensitive topic in a non-threatening manner. A 30-item, 4-point Likert scale with three scenarios, each depicting a living situation is presented. In each scenario three different abusive themes are presented and three different barriers to reporting. Respondents rate the abusiveness of a situation and their likelihood to reporting it. Perceived definitions and likelihood of reporting are important because whether or not elder victims would report “abuse” is meaningless without knowing how elders perceive abuse. Since it is unlikely elders would self-report (Fulmer et al, 2004), these data are essential to health care professionals in screening for abuse. Funding: NIH grant 2 P20 RR016472-08 from NCRR & Buxbaum Scholarship.