VTechWorks Repository :: Browsing by Author "Denbow, Cynthia J."

Browsing by Author "Denbow, Cynthia J."

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Altered Autonomic Nervous System Function in Chickens Divergently Selected for Body Weight
Kuo, Alice Yi-Wen (Virginia Tech, 2000-08-04)
Autonomic nervous system activity is related to body weight regulation. Based on the MONA LISA hypothesis it has been suggested that most obese subjects and animals have low sympathetic nervous system activity. The aim of this study was to investigate whether there are differences in autonomic nervous system activity between lines of chickens selected for either high (HWS) or low body weight (LWS). In Exp. 1, various pharmacological agents were injected intravenously, and the changes in blood pressure (BP) and heart rate (HR) of both HWS and LWS chickens were compared. The results showed that the HWS birds had a greater increase in BP and HR than the LWS following injection of atropine, a muscarinic receptor blocker, and LWS birds had a greater decrease in BP and HR to propranolol, a beta- adrenergic receptor blocker than the HWS birds. These results suggested that HWS chickens have higher parasympathetic tone, whereas LWS chickens have a higher sympathetic nervous system tone regulating the cardiovascular system. HWS and LWS chickens displayed a similar response in BP and HR following injection of the ganglion blocker tetraethylammonium chloride. These results suggest that there is no significant difference in the central autonomic nervous system in the cardiovascular regulation between HWS and LWS together. Since there does not appear to be any differences in the activity of the autonomic nervous system activity at the level of the central nervous system, these findings imply that the difference in response to atropine and propranolol could be caused by differences in adrenal activity. The ratio of heart rate and blood pressure after the injection of phenylephrine showed significant difference between these two lines of birds, but not when phenylephrine was injected following atropine. This result indicated that HWS are more dependent on the parasympathetic nervous system to regulate the baroreceptor reflex. The percentage of adrenal and sympathetic impact on the regulation of heart rate showed that LWS females required greater adrenal activity than the other birds. In Exp. 2, the body weight and food intake responses of HWS and LWS chickens to ip injections of reserpine were compared. Reserpine caused a transitory decrease in food intake and body weight in both lines of birds. However HWS chickens recovered more slowly from the depression caused by reserpine than the LWS chickens. This could be due to lower sympathetic nervous system activity. In conclusion, it appears that HWS may have lower sympathetic activity than LWS. Combining the results of both experiments, it appears that the HWS birds have lower sympathetic and higher parasympathetic activity. Furthermore central nervous system autonomic activity in BP and HR regulation is not different between HWS and LWS, but the activity of the adrenal gland may be different between these two lines of birds.
Antibacterial activity of jalapeño pepper (Capsicum annuum var. annuum) extract fractions against select foodborne pathogens
Bacon, Karleigh; Boyer, Renee R.; Denbow, Cynthia J.; O'Keefe, Sean F.; Neilson, Andrew P.; Williams, Robert (Wiley, 2017-01)
Capsicum annuum fruits have been investigated for antimicrobial activity in a number of studies. Capsaicin or other cinnamic acid pathway intermediates are often suggested to be the antimicrobial component, however there are conflicting results. No research has specifically fractionated jalapeño pepper (Capsicum annuum var. annuum) extract to isolate and identify compound(s) responsible for inhibition. In this study, fractions were collected from jalapeño pepper extracts using reverse-phase HPLC and tested for antibacterial activity using the disk diffusion method. Following initial fractionation, two fractions (E and F) displayed antibacterial activity against all three pathogens (p > .05). Commercial standards were screened to determine when they elude and it was found that capsaicin elutes at the same time as fraction E. Fractions E and F were subject to further HPLC fractionation and antibacterial analysis using two methods. The only fraction to display clear inhibition using both was fraction E1, inhibiting the growth of L. monocytogenes. Fraction E1 was analyzed using HPLC-MS. The resulting mass spectra revealed fraction E1 contained compounds belonging to a group of C. annuum-specific compounds known as capsianosides. Limited research is available on antibacterial activity of capsianosides, and a pure commercial standard is not available. In order to confirm the potential antimicrobial activity of the compound(s) isolated, methods need to be developed to isolate and purify capsianosides specifically from jalapeño peppers.
Association of foodborne pathogens with Capsicum annuum fruit and evaluation of the fruit for antimicrobial compounds
Huff, Karleigh Rose (Virginia Tech, 2011-09-07)
Hot peppers are gaining popularity in the United States as both a vegetable and a spice. In 2008, jalapeño peppers were involved in a multistate outbreak of Salmonella Saintpaul. This is the first outbreak implicating jalapeño as a vehicle for foodborne illness. Hot peppers contain many compounds thought to possess antimicrobial characteristics. This research was conducted to provide more information on the interactions of pathogenic bacteria and jalapeño peppers, as well as to identify properties of Capsicum annuum that affect bacterial survival, growth, and inhibition. Behavior of pathogens associated with jalapeños was investigated by inoculating jalapeño fruits with a cocktail of Listeria monocytogenes, Salmonella enterica, or Escherichia coli O157:H7 on the intact external surface, injured external surface, or intact internal cavity and storing the jalapeños at 7°C or 12°C. Intact external jalapeñosurfaces did not support the growth of the bacteria tested under storage conditions of 7°C. However, L. monocytogenes populations remained detectable throughout the 2 week study. At 7°C, pathogenic bacteria were able to survive but not grow on injured and internally inoculated jalapeño, but populations increased at 12°C (p=0.05). The most supportive growth environment for the pathogenic bacteria was the internal cavity of jalapeño held at 12°C. This study demonstrated the importance of intact uninjured produce and proper storage temperatures for food microbial safety. Inhibitory properties of jalapeños were studied by making extracts from fresh jalapeño peppers to test for antimicrobial activity. A disk diffusion assay determined that the extracts were capable of inhibiting the growth of the pathogenic bacteria tested. Listeria monocytogenes was especially sensitive to the extracts. jalapeño extracts were fractionated using HPLC and used for inhibition assays using disk diffusion and growth curve generation. Two fractions stimulated bacterial growth (p=0.05), while two other fractions inhibited bacterial growth. The inhibitory fractions were separated further using HPLC and tested for antimicrobial activity. Fraction E1 suppressed the growth of L. monocytogenes. HPLC-MS analysis revealed that Fraction E1 contained compounds known as capsianosides. To prove that inhibition is caused by capsianoside(s) and determine minimum inhibitory concentrations, a method to isolate the pure compound should be developed.
Biological, Nutritional, and Processing Factors Affecting Breast Meat Quality of Broilers
Santiago, Hector Luis (Virginia Tech, 2002-02-07)
A series of experiments were conducted to investigate the effects of certain biological, nutritional, and processing factors on breast meat quality of broilers. In the first experiment, the influence of genetic strain, plane of nutrition, and age at slaughter on breast meat quality was evaluated. Breast meat from a high yield line of broilers had lower pH at 24 h postmortem (PM), greater L*, a*, and b* values, lower water holding capacity (WHC), and higher expressible moisture (EM) than those of a line selected for rapid growth. Breast meat from birds processed at 42 d had lower WHC than those processed at 53 d, even though no differences in pH and L* values at 24 h were observed. No significant effects due to plane of nutrition on meat quality traits were observed. In Experiment two, the influence of strain and chilling methods (ice or air chilled) on breast meat quality was studied in broilers. Breast meat quality significantly differed among strains, with one of the strains evaluated having higher muscle pH, lower L* values, and higher WHC than the other strains. Ice-water chilling significantly reduced the rate and extent of PM pH decline, but had significantly lower WHC and higher EM than those from carcasses chilled by air. However, chilling conditions did not influence breast meat color. In Experiment three, the effects of strain and gender on breast muscle quality of broilers was studied. Meat quality traits were evaluated on both sexes of six genetic crosses of commercial strains. No significant differences in breast meat quality traits among strains were observed. However, differences between sexes were highly significant. The P. major muscles of females had lower pH values at all PM times, higher L*, a*, and b* values, and lower WHC than males. The P. minor of females had significantly lower pH, lower WHC, higher EM, but similar color L*, a*, and b* values than males. In Experiment four, the effects of strain, gender, and age at slaughter on breast meat quality were studied. Strain differences were observed in both sexes, but these differences did not show any specific relationship with the strain genotype. Breast muscles from a male pure line had superior meat quality, with higher muscle pH, and WHC, but higher L* values than the other strains. Significant differences in breast meat quality traits due to age at slaughter were also observed. Regardless of gender, breast muscle pH at 24 h PM and WHC decreased linearly with age, while breast muscle temperature and L* values increased in a linear fashion with advancing age at slaughter. No significant strain by age interactions were observed for any of the meat quality traits evaluated. The results of these studies indicate that commercial genotypes differ significantly in PM muscle metabolism and subsequent meat quality. The results also indicate that female broilers and older birds might be more susceptible to meat quality problems.
Bridging the CGMP Gap Between the U.S. Government, Academia, and the Food Industry
West, Joey (Virginia Tech, 2012-12)
In communication theory, one of the most important aspects is to always remember who the audience is at all times. In this case, the audience are members of the food industry who are expected to translate the knowledge of the CGMPs into practice. The goal of this research is twofold; to effectively communicate the need and nature of the CGMPs and to simply reconnect the regulations to the audience they were developed to assist. Further, a definition of perception is an individual’s capacity for comprehension. We find that in the real world, an individual’s comprehension does not always neatly fit inside the perimeters that the CGMPs provide. In some cases compliance with regulations does not allow for continuation of traditional processing methods, short-cuts, and individual bias when it comes to how firms may prepare, process, hold, and ship food products. That leaves it up to the government to find a means to bridge the gaps that exist between comprehension, perception, and reality. The main focus is to present information so that the ordinary person can understand and act upon the regulation on a consistent basis. The answer may lay not in the regulation itself, but Food Industry members’ ability to relate to and understand the need for compliance.
Characterization of Gene Expression During Adenosine 3':5'-Cyclic Monophosphate Induced Neuroendocrine Differentiation in Human Prostatic Adenocarcinoma
Goodin, Jeremy Lee (Virginia Tech, 2002-04-03)
The LNCaP cell line is a versatile and useful model that is suitable for the study of human prostate cancer in vitro. The elevation of LNCaP intracellular cAMP levels through the addition of membrane permeable cAMP analogues, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal transduction pathway can induce reversible neuroendocrine differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights for the molecular mechanisms governing neuroendocrine differentiation, early detection of prostate cancer, and/or potential targets for gene therapy. In this study, differential display PCR was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and cloned. DNA sequencing and database comparisons were performed. Among the differentially expressed genes, the human ribosomal S3a gene was identified as down regulated in response to LNCaP differentiation. In order to better ascertain the mechanism by which HRS3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site-directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the HRS3a gene. Two cyclic AMP response elements, a Sp1 element and a GA-binding protein element, were involved in the regulation of HRS3a gene expression. In order to ascertain the effect of HRS3a down regulation in LNCaP cells, antisense phosphorothioate oligonucleotides were designed to inhibit HRS3a gene expression. Treatment of LNCaP cells with antisense HRS3a oligonucleotides did not influence cAMP induced neuroendocrine differentiation but antisense treatment did result in a decrease in LNCaP cell growth. In addition, it was determined that morphological changes associated with cAMP induced differentiation of LNCaP cells from the epithelial to the neuroendocrine state may not require alterations in gene expression nor the expression of novel proteins.
Characterizing glucose, illumination, and nitrogen-deprivation phenotypes of Synechocystis PCC6803 with Raman spectroscopy
Tanniche, Imen; Collakova, Eva; Denbow, Cynthia J.; Senger, Ryan S. (2020-03-30)
Background. Synechocystis sp. PCC6803 is a model cyanobacterium that has been studied widely and is considered for metabolic engineering applications. Here, Raman spectroscopy and Raman chemometrics (Rametrix (TM)) were used to (i) study broad phenotypic changes in response to growth conditions, (ii) identify phenotypic changes associated with its circadian rhythm, and (iii) correlate individual Raman bands with biomolecules and verify these with more accepted analytical methods. Methods. Synechocystis cultures were grown under various conditions, exploring dependencies on light and/or external carbon and nitrogen sources. The Rametrix (TM) LITE Toolbox for MATLAB (R) was used to process Raman spectra and perform principal component analysis (PCA) and discriminant analysis of principal components (DAPC). The Rametrix (TM) PRO Toolbox was used to validate these models through leave-oneout routines that classified a Raman spectrum when growth conditions were withheld from the model. Performance was measured by classification accuracy, sensitivity, and specificity. Raman spectra were also subjected to statistical tests (ANOVA and pairwise comparisons) to identify statistically relevant changes in Synechocystis phenotypes. Finally, experimental methods, including widely used analytical and spectroscopic assays were used to quantify the levels of glycogen, fatty acids, amino acids, and chlorophyll a for correlations with Raman data. Results. PCA and DAPC models produced distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on (i) growth in the presence of 5 mM glucose, (ii) illumination (dark, light/dark [12 h/12 h], and continuous light at 20 mE), (iii) nitrogen deprivation (0-100%NaNO3 of native BG-11 medium in continuous light), and (iv) throughout a 24 h light/dark (12 h/12 h) circadian rhythm growth cycle. Rametrix (TM) PRO was successful in identifying glucose-induced phenotypes with 95.3% accuracy, 93.4% sensitivity, and 96.9% specificity. Prediction accuracy was above random chance values for all other studies. Circadian rhythm analysis showed a return to the initial phenotype after 24 hours for cultures grown in light/dark (12 h/12 h) cycles; this did not occur for cultures grown in the dark. Finally, correlation coefficients (R > 0.7) were found for glycogen, all amino acids, and chlorophyll a when comparing specific Raman bands to other experimental results.
Characterizing metabolic stress-induced phenotypes of Synechocystis PCC6803 with Raman spectroscopy
Tanniche, Imen; Collakova, Eva; Denbow, Cynthia J.; Senger, Ryan S. (2020-03-30)
Background. During their long evolution, Synechocystis sp. PCC6803 developed a remarkable capacity to acclimate to diverse environmental conditions. In this study, Raman spectroscopy and Raman chemometrics tools (Rametrix (TM)) were employed to investigate the phenotypic changes in response to external stressors and correlate specific Raman bands with their corresponding biomolecules determined with widely used analytical methods. Methods. Synechocystis cells were grown in the presence of (i) acetate (7.5-30 mM), (ii) NaCl (50-150 mM) and (iii) limiting levels of MgSO4 (0-62.5 mM) in BG-11 media. Principal component analysis (PCA) and discriminant analysis of PCs (DAPC) were performed with the Rametrix (TM) LITE Toolbox for MATLABR (R). Next, validation of these models was realized via Rametrix (TM) PRO Toolbox where prediction of accuracy, sensitivity, and specificity for an unknown Raman spectrum was calculated. These analyses were coupled with statistical tests (ANOVA and pairwise comparison) to determine statistically significant changes in the phenotypic responses. Finally, amino acid and fatty acid levels were measured with well-established analytical methods. The obtained data were correlated with previously established Raman bands assigned to these biomolecules. Results. Distinguishable clusters representative of phenotypic responses were observed based on the external stimuli (i.e., acetate, NaCl, MgSO4, and controls grown on BG-11 medium) or its concentration when analyzing separately. For all these cases, Rametrix (TM) PRO was able to predict efficiently the corresponding concentration in the culture media for an unknown Raman spectra with accuracy, sensitivity and specificity exceeding random chance. Finally, correlations (R > 0.7) were observed for all amino acids and fatty acids between well-established analytical methods and Raman bands.
Corticotrophin Releasing Hormone Modulation of Feed Intake, Gastric Motility, and Behavior in Low and High Body Weight Selected Lines of Chickens
Cline, Mark A. (Virginia Tech, 2005-05-04)
The effect of intracerebroventricular (ICV) injection of corticotrophin releasing hormone (CRH) and related compounds on appetite, behavior, and gastric motility in lines of chickens, one selected for low body weight (LWS) and the other high body weight (HWS), was determined. Nucleotide sequence and expression patterns of the CRHr2 receptor, involved in appetite regulation, were also determined. Some individuals of the LWS line are anorexics and many die simply from not eating while some individuals in the HWS line are compulsive eaters and exhibit obesity. CRH is a 41 residue peptide that initiates an organism's stress response and is a potent inhibitor of appetite. An ICV injection of CRH dose-dependently decreased feed intake in both lines but did not effect water intake. When CRH receptor antagonists were ICV injected an increase in feed intake in the LWS line but not in the HWS line was observed, however the appetite reducing effect of CRH was attenuated in the HWS line but not in the LWS line. The LWS line has higher concentration of corticosterone than does the HWS line. In both lines at all times treatment with CRH caused an increase in locomotion and no CRH-treated chicks from either line slept post injection. Chicks from the LWS line that were treated with CRH exhibited other anxiety related behaviors sooner than the HWS line. The LWS line showed a liner increase in crop emptying time as the dose of ICV CRH increased. The HWS line responded with a quadratic dose response to CRH treatment. Polymorphisms in the CRHr2 receptor were found in both lines in the same positions, thus we concluded these differences do not significantly contribute to body weight differences. However, differences detected in expression patterns between lines for the CRHr2 receptor may contribute to their different body weights. We conclude that differences in the CRH system, its concentrations and differential receptor action, of these two lines may be partly responsible for their altered body weight phenotype.
The Effects of Dietary Lipids on Bone Chemical, Mechanical and Histological Properties in Japanese Quail (Coturnix C. Japonica)
Liu, Dongmin (Virginia Tech, 2000-06-26)
Japanese quail were used as animal models in four experiments to evaluate the effects of supplementing diets with different lipids on bone chemical, mechanical, and histological properties. In Exp. 1, laying hens were fed a basal diet containing either 5% soybean oil (SBO), hydrogenated soybean oil (HSBO), chicken fat (CF), or menhaden fish oil (FO). The addition of SBO in the maternal diet increased the levels of total n-6 fatty acids and arachidonic acid (AA, 20:4n-6) in yolk and tibial bones of newly hatched progeny (P<0.01), whereas the maternal FO diet elevated the concentrations of total n-3 fatty acids, eicosapentaenoic acid (EPA, 22:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and total saturated acid, but greatly decreased the amount of AA in both egg and progeny tibiae (P<0.01). The maternal HSBO diet resulted in the accumulation of trans-18:1 fatty acid in egg yolks and tibiae at hatch. The addition of FO or HSBO to the maternal diet significantly lowered the ex vivo PGE2 production of tibiae in newly hatched quail compared to those from hens given the SBO or CF diets (P<0.01). In Exp. 2, the addition of different lipids in the maternal diets did not affect growth, tibial length, diameter or collagen content of the progeny. However, supplementing the maternal diet with 5 % FO or HSBO increased the percent bone ash , increased bone pyridinium crosslinks of collagen, enlarged the cartilaginous proliferative and hypertrophied zones, increased diaphyseal cortical thickness of the tibiae in embryos (P<0.05), and subsequently increased tibial shear force, stiffness (P<0.05) and improved cortical thickness, density and trabecular density in early growth and development of progeny compared to those from hens consuming the SBO or CF diets (P<0.05). In Exp. 3, male quail at one month of age were fed a basal diet containing either 5% SBO, HSBO, CF or FO for seven months. Long-term supplementation in the diets of different lipids did not affect body weight, food intake, tibial length or diameter, but the FO group had the highest tibial percent ash, and both FO and HSBO increased tibial mineral content in aged quail compared to those fed the SBO or CF diets (P<0.05). At 8 months of age, quail fed FO had the highest concentrations of (n-3) fatty acids (20:5n-3, 22:5n-3, 22:6n-3) but the lowest amounts of 20:4n-6 in lipids from tibial cortical bone, whereas the SBO and CF diets greatly elevated (n-6) fatty acids and 20:4n-6 levels. The HSBO diet which contains t18:1 fatty acid resulted in t18:1 accumulation in bone. Long-term supplementation with FO or HSBO increased tibial shear force, stiffness and shear stress, as well as improved cortical thickness and density compared with the SBO or CF diets ( P<.05). In Exp. 4, the addition of SBO or CF to the diet for seven months decreased tibial mineral content compared to the FO diet (P<0.05). Quail fed SBO increased collagen concentration in the tibiae (P<0.05), but the level of collagen crosslinks was higher in quail fed FO or HSBO compared to those given the SBO or CF diets (P<0.05). The PGE2 production in bone organ culture and marrow was greatly increased in quail maintained on the SBO or CF diets (P<0.05). PGE2 production in the bone microenvironment was negatively correlated with the tibial percent ash and collagen crosslinks but had a positive correlation with tibial collagen concentration. The results of these studies demonstrate that either supplementing the maternal diets with or long-term exposure to different lipids alters the chemical composition and metabolism of skeletal tissue in both embryos and aged quail. Maternal dietary SBO or CF had an adverse effect on bone growth and development in embryos. Likewise, long-term exposure to SBO or CF diet impaired bone metabolism and remodeling. In contrast, the FO or HSBO diet had beneficial effects on bone modeling in embryos and remodeling in adult quail.
Egg Safety: Control Factors of Salmonellosis
Wier, Deborah (Virginia Tech, 2013-05)
Salmonella enterica serotype Enteritidis (SE) continues to be one of the most commonly identified bacteria associated with outbreaks of human salmonellosis around the world, particularly in developed countries (CDC, 2011). In the United States, salmonellosis is a common infection with occurrences reaching up to 1.4 million in a year (Braden, 2006). From May to November of 2010, approximately 1,983 illnesses were reported and associated as a result of a single Salmonella spp outbreak detected in the United States (CDC, 2011). The source of the outbreak was traced to Wright County Egg in Galt, Iowa and subsequently led to the voluntary recall of 380 million eggs distributed throughout the country. Hillandale Farms, also in Iowa, voluntarily recalled another 170 million eggs (CDC, 2011). Identifying the source of the outbreak was the result of coordinated investigations by the Center for Disease Control (CDC), local public health authorities and the Food and Drug Association (FDA). According to the United Egg Producer’s (UEP) website, the chance of eggs containing SE is rare in the United States. Estimates are 1 in 20,000 eggs might be contaminated with SE (UEP, 2010; Guard-Petter, 2001). This would mean that a consumer would encounter an infected egg once in 84 years. The number is also claimed to be decreasing due to the increased protection measures adapted by egg producers over the past decade. Improved sanitary housing systems, vaccinations and cleaning processes have all led to the claim of decreased cases of SE throughout the nation (Braden, 2006; UEP, 2010; Patrick, et al., 2004). The use of average outbreaks can be misleading due to the nature of infection and their correlation to single producer outbreaks. Investigation into SE outbreaks between 1985 and 1999 by Patrick, et al., 2004 concluded that outbreaks of SE infections in human decreased by almost 50% between 1995 and 1999. These reports may have left people feeling like things were on the right track, then the Wright County Egg incident occurred. Without constant vigilance and willingness to incorporate new ideas, the next big outbreak is around the corner. This paper will look at the various methods available to increase the quality of eggs and integrating those ideas into a viable program to fortify public safety.
Evaluation of different solvents to extract antibacterial compounds from jalapeño peppers
Bacon, Karleigh; Boyer, Renee R.; Denbow, Cynthia J.; O'Keefe, Sean F.; Neilson, Andrew P.; Williams, Robert C. (Wiley, 2016-08-31)
Chili peppers (Capsicum spp.) may possess antibacterial properties and have potential to be used in foods as antimicrobial. The complete chili pepper extract should be evaluated to determine which compounds are responsible for the antimicrobial activity. Extraction of compounds from the pepper is completed using a solvent. The type of solvent used for extraction influences which compounds are isolated, therefore the best extraction method needs to be determined. The purpose of this study was to identify which solvent is most successful at extracting unknown antibacterial compounds from jalapeño peppers. Fresh jalapeño peppers were chopped, weighed, and blended with a solvent (sterilized hot water, 70% methanol, 95% methanol, 70% ethanol, or 95% ethanol) at a 1:1 ratio (g/g) until the mixture was homogenized, followed by shaking for 15 min. The slurry was centrifuged; supernatant was removed and used for antibacterial testing against Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica. The diameter of growth inhibition was measured and statistically evaluated using ANOVA to determine the extract with the greatest antimicrobial activity. Solvents were tested alone as a control. There was greater bacterial inhibition from extracts created with methanol and ethanol than hot water. Listeria monocytogenes was significantly more susceptible to the extracts than E. coli or Salmonella isolates. Each solvent extract was then analyzed using high-performance liquid chromatography (HPLC) and fractions (A–G) were collected and used for subsequent disk diffusion analysis against L. monocytogenes. Fractions E and F (eluded between 20 and 30 min) exhibited the most antibacterial activity. There were no differences between solvents used (p = .05). Further investigation into specific compounds within these extracts will be completed in the future.
The Expression and Characterization of Human Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Tobacco
Witt, William T. (Virginia Tech, 2003-05-29)
The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most studied membrane protein models because of its clear clinical significance. Mutations within the CFTR gene lead to cystic fibrosis, the most common autosomal recessive genetic disorder in the Caucasian population. CFTR, a large 160 kDa glycoprotein, is a chloride ion channel in the ABC superfamily of transporter proteins. Due to low natural abundance of CFTR and difficulties producing sufficient amounts in heterologous systems, the exact protein function/structure relationship is unknown. Expression of CFTR in E. coli is lethal and mammalian culture systems are expensive and low yielding. However, successful bioproduction of many complex human proteins has been shown in transgenic plants. Our research objective is to develop tobacco as a model system for expressing human CFTR. Constructs of full-length CFTR fused to the 35S double enhanced promoter could not be propagated in E. coli, suggesting that the CFTR product generated by "leaky" expression was detrimental to bacteria. Two strategies were undertaken to address the problem: 1) a plant intron was introduced into CFTR sequence and 2) a more tightly regulated wound-inducible promoter MeGATM was used. Tobacco was transformed with all constructs. CFTR presence was determined by polymerase chain reaction (PCR). Expression and intron splicing was analyzed by reverse transcriptase-PCR. Splicing did not occur presumably due to intron /exon contexts. In tobacco expressing MeGA:CFTR, however, novel high-molecular-weight membrane-associated proteins were immunodetected using anti-CFTR antibodies suggesting that tobacco may be capable of producing human CFTR.
Expression strategies for plant-based production of a vaccine adjuvant
Verbiest, Leen (Virginia Tech, 2000-04-20)
Today's development of novel vaccines stresses the need for edible vaccines that are inexpensive, easily administered and capable of being stored and transported without refrigeration. Without these characteristics, developing countries find it difficult to adopt vaccination as the central strategy for preventing their most devastating diseases. A promising approach is the production of vaccines in plants we commonly consume. Two major obstacles have been encountered in developing vaccines in plants. First, the expression level of foreign antigens tends to be low and second, co-expression of an adjuvant may be required to facilitate an appropriate immune response. Ricin, a plant toxin that survives the human digestive process, has been proven to stimulate an immune response and could therefore serve as a suitable adjuvant. The long-term goal is to produce a vaccine that protects against the disease entamoebic dysentery. The specific goal of this research was to produce ricin in tobacco as adjuvant for the vaccine. Vectors were constructed that fused the ricin coding sequence to different plant promoters and transgenic tobacco plants were generated by transformation with Agrobacterium tumefaciens. The levels of expression in these transgenic plants were tested using immunoblot assays. Southern blot analysis was performed for the highest expressors of each construct. The enzymatic activity of the tobacco-synthesized ricin was shown using a protein translation inhibition assay. Expression of ricin was also confirmed using transient transformation of hairy root cultures. Future experiments will address the practical use of the tobacco-synthesized ricin as adjuvant, as well as the expression of the ricin B subunit fused to a protective antigen of Entamoeba histolytica in tobacco as edible vaccine.
Fasting alters histone methylation in paraventricular nucleus of chick through regulating of polycomb repressive complex 2
Jiang, Ying (Virginia Tech, 2013-09-19)
The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting.
Genomic and Physiological Differences for Ghrelin and Leptin Receptor in Lines of Chickens Selected for High and Low Body Weight
Kuo, Alice Yi-Wen (Virginia Tech, 2003-12-08)
Autonomic nervous system (ANS) activity is related to body weight regulation. Based on the hypothesis that Most Obesities kNown Are Low In Sympathetic Activity (MONA LISA), it has been suggested that most obese subjects and animals have low sympathetic nervous system activity. Leptin, leptin receptor, and ghrelin genes influence the ANS regulation of body weight and food intake. The aim of this study was to investigate whether there are differences in leptin, the leptin receptor, or ghrelin regulation between lines of chickens selected for high (HWS) or low body weight (LWS). Intraperitoneal injections of reserpine were administrated to chickens from the HWS and LWS lines. Body weight and food intake were then compared to evaluate ANS regulation. While reserpine caused a transitory decrease in food intake and body weight in both lines, the magnitude of the change was greater in the HWS than in the LWS chickens. However, chickens from the LWS line exhibited greater catecholamine and indoleamine level changes in response to reserpine than those from the HWS line. Therefore, HWS chickens were more sensitive to the body weight-reducing effects of reserpine than LWS lines, while LWS chickens appeared to have greater sympathetic nervous system activity. Food and water intakes were differentially affected in HWS and LWS chickens in response to intracerebroventricular administration of human recombinant leptin. Leptin caused a linear decrease in food intake in the LWS line, but no effect on food intake in the HWS lines. The HWS chickens tended to have reduced water intake following leptin administration. These results suggest that the leptin receptor, or the down-stream neuropeptide regulation pathway mediating the effect of leptin; may be different between chickens from the HWS and LWS lines. Leptin, insulin like growth factor (IGF)-1, and IGF-2 concentrations in the plasma of HWS and LWS lines of chickens were evaluated. Leptin, IGF-1 and IGF-2 levels were significantly higher in the LWS than HWS chickens. The HWS female leptin concentrations were significantly lower than in HWS males or LWS females. Male chickens had greater IGF-1 concentrations in the plasma than female chickens. However, the concentration of IGF-2 did not differ between sexes. The difference in leptin concentrations in these lines and sexes may explain the differences in age of sexual maturity. Different IGF-1 and IGF-2 concentrations may be involved in the obese and anorexic conditions, fast and slow growth, and high and low food consumption found in these two lines of chickens. Differences in the gene sequence of the leptin receptor were observed in HWS and LWS lines of chickens. A single nucleotide polymorphism (SNP) in the intron between exon 8 and 9 introduced a restriction site for the enzyme Sel I in the HWS, but not the LWS line. Two SNP were detected in the leptin receptor cDNA region at nucleotides 189 and 234. At nucleotide 189, the LWS line has both a homozygous (T-T) and heterozygous (C-T), whereas the HWS line has only homozygous (T-T) form. The SNP found in nucleotide 234 introduces a restriction site Mse I in the HWS, but not the LWS line. These specific changes may be directly involved or closely linked to differences between the two lines in either the coding or regulatory domains of the leptin receptor. Differences in the leptin receptor gene expression between HWS and LWS lines of chickens in various organs and ages were observed. Leptin receptor expression in the whole brain was significantly different between sexes at 28 days-of-age in the HWS and LWS lines. The LWS line had higher leptin receptor gene expression in the liver at 2 days-of-age than at 56 and 363 days-of-age, but no differences were observed in the HWS line. In addition, at 2 days-of age, liver leptin receptor gene expression was higher in LWS than HWS chickens, but the reverse was observed at 363 days-of age. In adipose tissue, leptin receptor expression was higher in the LWS than HWS line. Leptin receptor expression in adipose tissue was greater at 363, than 28 and 56 days-of-ages. Our results showed that changes in the regulation of leptin and the leptin receptor were associated with sex, age, and growth. Differences in the ghrelin gene in the HWS and LWS lines under different feeding conditions were investigated. Both HWS and LWS chickens have six extra base pairs in the 5'-untranslated region. The LWS male ghrelin gene expression was significantly lower than in the LWS female and HWS male. The 84 day-old males had lower gene expression than 84 day-old females and 363 day-old males. When comparing different feeding methods, females allowed ad libitum feed consumption had a lower cycle threshold cycle number (CT) ratio than males allowed ad libitum feeding or fasted females. However, the inflection point cycle number of ad libitum fed females was lower than that of the ad libitum fed males, but greater than the fasted females. Ghrelin gene expression was different between the two lines of chickens, and the expression of ghrelin in chickens was influenced by body weight selection, sex, age, and feeding condition.
An H5N1 Pandemic: The Virginia Tech Campus as a Model of State, National, and Global Emergency Preparedness
Cornett, Rose (Virginia Tech, 2013-05)
Experts suggest the likelihood of another human influenza pandemic to be high, or maybe even inevitable. There’s a good chance that it could be H5N1, and the possible effects are worrisome. No one can know for sure when the next pandemic will occur. A pandemic has no regard for borders; once it begins, it has serious economic implications all levels. Organizations at all levels have put programs in place to prepare for such an event, and models allow them to better prepare by estimating illnesses, hospitalizations, deaths, and other factors pertinent to public health. Virginia Tech, a community in itself, will not be except from the fallout, and must ensure preparedness with regard to resources, expertise, and services in order to mitigate the effects of a full-blown pandemic.
Identification of the Lyme Disease Vectorin Southwest Virginia
Sheppard, James (Virginia Tech, 2009-05)
In the mid-1970s, fifty-one residents from the quaint little Atlantic seaside town of Lyme, Connecticut came down with something that resembled rheumatoid arthritis (Garrett, 1994). The ailment was dubbed Lyme disease and quickly spread to all fifty states. Once physicians became aware of the symptoms, the disease was diagnosed in many states of the Northeast, but there was a clear upward trend in Lyme disease found throughout the United States. By 1992, Lyme disease was the most reported vector-borne disease in the country (Steere, et al.). In fact, from 1995 to 2009, more than 300,000 cases of Lyme disease were reported to the Centers of Disease Control (CDC) by state health departments and the District of Columbia (http://www.cdc.gov/nc) (Appendix A). During this 14-year period, most of the cases were reported from northeastern states including Connecticut, Delaware, Massachusetts, Maryland, Minnesota, New Jersey, New York, Pennsylvania, and Rhode Island. In 1982, Lyme disease was first seen in Virginia and, following the nationwide trend, has steadily increased since that time (Lyme Disease, 2010b). The cases of Lyme disease in Virginia included 29,959 confirmed cases and 8,509 probable cases being reported just in 2009 (http://www.cdc.gov/nc).
Involvement of AMP-activated protein kinase in differential regulation of appetite between lines of chickens selected for low or high juvenile body weight
Xu, Pingwen (Virginia Tech, 2011-04-21)
This study was to determine (1) if genetic selection for high (HWS) or low (LWS) body weight in chickens has altered the hypothalamic AMP-activated protein kinase (AMPK) system and (2) if this alteration contributes to the dissimilar feeding response to various appetite modulators between HWS and LWS lines. Compared to HWS, LWS chickens had higher levels of AMPK α and acetyl-CoA carboxylase (ACC) phosphorylation, which was caused by upregulation of the upstream factor calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK β). There was greater mRNA expression of carnitine palmitoyltransferase I (CPT1), leptin receptor (LEPR) and neuropeptide Y (NPY) and less mRNA expression of ACC α, fatty acid synthase (FAS), fat mass and obesity associated gene (FTO), pro-opiomelanocortin (POMC) and orexin in LWS than HWS chickens. At 5 days of age, intracerebroventricular (ICV) injection of AICAR, 5-amino- 4-imidazolecarboxamide riboside, caused a quadratic dose-dependent decrease in food intake in LWS but not HWS chicks. Compound C, (6-(4-(2-piperidin-1-yl-ethoxy)-phenyl))-3-pyridin-4-yl-pyrazolo(1,5-a)-pyrimidine, caused a quadratic dose-dependent increase in food intake in HWS but not LWS chicks. The anorexigenic effect of AICAR in LWS chicks and orexigenic effect of Compound C in HWS chicks resulted from either activation or inhibition of other kinase pathways separate from AMPK. There is a lower threshold for the anorexigenic effect of ghrelin in LWS than HWS chicks, which was associated with differential hypothalamic AMPK signaling. ICV injection of ghrelin inhibited corticotrophin-releasing hormone (CRH), 20-hydroxysteroid dehydrogenase (20HSD), glucocorticoid receptor (GR), CPT1 and FTO expression in LWS but not HWS chicks. Additionally, the hypothalamic mRNA level of ghrelin was significantly higher in LWS than HWS chicks, which may also contribute to the differential threshold response to ghrelin in these two lines. Obestatin caused a linear dose-dependent increase in food intake in HWS but not LWS chicks. The orexigenic effect of obestatin in HWS chicks was not associated with altered AMPK. Obestatin inhibited LEPR and FTO expression in HWS but not LWS chicks. Thus, selection for body weight may alter the hypothalamic response to ghrelin by the AMPK pathway, CRH pathway, CPT1 and FTO, and to obestatin by LEPR and FTO.
John H. Kerr Dam and Reservoir: Ingredient in a Recipe for Disaster
Hood, Debra Gaye (Virginia Tech, 2010-04-30)
In today's world, dams have become possible bioterrorist tools to cause harm, destruction, and death. One such dam is the John H. Kerr Dam and Reservoir that was created to control flooding and produce hydroelectric power for parts of Virginia and North Carolina. A history of its development as well as its many benefits to the areas surrounding the facility in terms of wildlife habitats and its recreational uses will be discussed in addition to those involved with flood control, hydroelectric power, and drinking water supply. This paper will present scenarios of a possible threat from bioterrorists as well as the possibility of an accidental compromise to the John H. Kerr Reservoir. Responses by local, state, and federal agencies to both the accidental release of the herbicide atrazine and to the purposeful contamination with the radiological material, tritium, will be presented. Because these scenarios will eventually be published online as teaching tools, questions and answers based on the materials presented have been provided for that purpose.

Browsing by Author "Denbow, Cynthia J."

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