Amyloid beta (Aβ) is a protein commonly found in the membranes of neurons. Because of its tendency to misfold and oligomerize, the transport of Aβ out of the brain is crucial to cognitive health. The apolipoprotein E (apoE) is responsible for the transport of Aβ; consequently, its gene is the sole genetic cause of Alzheimer’s that has been widely agreed upon. Of the three alleles E2, E3, and E4, the apoE4 protein is the most closely associated with increased Alzheimer’s risk. ApoE4 differs from ApoE3 at the 112th amino acid; in this study, the positively charged 112 arginine of ApoE4 was mutated into a 112 serine to resemble the uncharged 112 cysteine in ApoE3. Six drugs still under development were obtained, and as a result their manufacturers and details must remain confidential; they will be referred to as drugs A through F. To determine whether the effects of drugs A-F on the ApoE4 mutant were significant, the EPR spectra for the E3-like mutant and the E4 clone were scanned and compared. The greatest difference between the E3-like and E4 spectra was found with the drugs D and F, indicating that the drugs may have changed the protein’s structure. To further observe the behavior of apolipoproteins, the stability of lipid-bound nanodiscs of apolipoprotein A with diameters of 7.8 nm, 8.4 nm, and 9.6 nm were observed through comparison of samples in SDS-PAGE gels, EPR, and Coomassie Assays in the second phase of this project. In general, the SDS-PAGE gels showed that the lipid-bound samples retained shape better than their lipid-free counterparts.
Perennial crop plants like grapevine (Vitis vinifera L.) use stored carbohydrates (CHO) from the previous season to sustain early season growth and flower development. Water deficits inhibit current season growth and yield of the current season and following season, but the effects on stored CHO are not known. CHO assays were performed on two grapevine genotypes, Grenache and Syrah, across three different irrigation treatments. Specifically, CHO concentrations in basal nodes and internodes were assayed just after the onset of veraison, the onset of ripening, which has been shown to be the annual low point in storage CHO concentrations in grapevine. A series of hot ethanol baths were used to extract soluble sugars. After extraction, CHO samples were digested into glucose using an alpha-amylase and amyloglucosidase solution. A PGO enzyme o-dianisidine dihydrochloride solution was added to each sample for a color reaction. The plate was read using a spectrometer and optical density numbers were recorded and analyzed with a standard starch calibration curve. In general, Syrah had higher storage CHO concentration than Grenache, which suggested that it was more efficient with water. Across both varieties and all three irrigation treatments, internodes contained higher concentration of storage CHO. CHO distribution in internodes supported the hypothesized relationship: decreased water availability results in decreased concentrations of storage CHOs. These data suggest that water deficit and storage CHO concentrations are positively correlated in perennial crop plants like grapevine (Vitis vinifera L.). CHO in the nodes did not show the predicted distribution. Syrah vines had lower midday leaf water potential than Grenache vines which can be attributed to their anisohydric tendencies. This information on CHO distribution and genotypic variation in response to water deficits is important for identifying water-efficient grapevines and for water-conservation in agriculture.
Panelists were given blind samples of body washes to evaluate at home on a seven point scale. They received sufficient training to be able to distinguish between product differences for each attribute of the body washes. The goal of this study was to improve on the current methods of analytical product assessment with a home use descriptive analysis study to reflect more realistic home use situations of body wash products.
Evolution of 2-Nitrotoluene 2,3-dioxygenase and Gene Manipulation of Catechol 2,3-dioxygenase in Acidovorax sp. Strain JS42
Acidovorax (formerly Pseudomonas) sp. JS42 is able to use the compounds 2-nitrotoluene (2NT) and nitrobenzene as carbon, nitrogen, and energy sources. In the first step of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase (2NTDO) converts 2NT to 3-methylcatechol with concomitant nitrite release. 2NTDO has three component proteins, and although it can break down all three mononitrotoluene isomers, JS42 cannot grow on 3-nitrotoluene (3NT) or 4-nitrotoluene (4NT). However, it is possible to force JS42 to adapt to 3NT and 4NT with resulting changes to 2NTDO. In this study, we evolved 4NT+ JS42 strains to grow on 3NT using long-term selection. Sequencing of the ntdAc gene encoding the α subunit of 2NTDO oxygenase revealed substitutions at the amino acid position 204 on the active site. Mutations at positions 238 and 248 were conserved from the parent 4NT+ strain and no other mutations were present, supporting the hypothesis that the amino acid position 204 is crucial for the evolution of JS42 to utilize new substrates. The next step of the 2NT degradation pathway involves catechol 2,3-dioxygenase (CDO). Both ctdE1 and ctdE2 encode CDO, and each has an associated regulatory gene (ctdR1 and ctdR2). Based on the phenotype of a mutant strain with deactivated ctdE1, ctdE1 seems critical to the degradation pathway. We will complement the ctdE1 mutants to verify the role of the enzyme in 2NT degradation. It is not yet known whether ctdR1 encodes an inducer or repressor, so ctdR1 inactivation will be used to determine the function of the regulatory protein.
According to the Institutional Animal Care and Use Committee (IACUC), Public Health Services (PHS), and Office of Laboratory Animal Welfare (OLAW), institutions must follow guidelines set by the Guide for the Care and Use of Laboratory Animals in their animal care and use programs when establishing mice populations within a cage to maintain the health and welfare of the laboratory mice2. The effects of mouse-caging density were studied in this project to determine the maximum number of mice that could be housed in a cage and maximum time between cage changes while maintaining the health and welfare of the mice. The CO2 level, Ammonia level, change in air flow, temperature, humidity, and any social, behavioral, or physical changes of the mice were evaluated. Seventy cluster of differentiation 1 (CD-1) outbred female adult mice were divided into four groups, with a minimum of two mice and a maximum of five mice per cage. Each week measurements of the microenvironmental gases in the cage, observations of the behavior and physical changes in the mice, cage temperature, cage humidity, cage airflow, and weights of the mice were recorded, for three weeks.
Chemotaxis allows some organisms like Pseudomonas bacteria to move in response to chemical stimuli. Methyl-accepting chemotaxis proteins (MCPs) are membrane-bound chemoreceptors that facilitate this process. Understanding MCPs could lead to potential application of chemotaxis in fields like bioremediation, where it could improve efficiency since bacteria could not only break down pollutants but seek them out as well. In order to characterize the MCPs of species like Pseudomonas putida F1, hybrid sensor proteins comprised of the sensory domain of bacterial MCPs and the signaling domain of a 2-component regulator were used in the optimization of a screening method. By using a filter paper disk to diffuse potential attractant/repellants in a plate, bacteria with these hybrid sensor proteins will show an inhibition of growth if they sense the compound. This screening method was optimized by testing various concentrations and volumes of compounds as well as plate media content. This method will allow for quick screening of MCP functions in P. putida F1 and other bacteria.
Cytokinesis is an essential developmental process for plants. Currently, scientists have limited knowledge on the processes that occur within cytokinesis. John Almazan is researching the effects of Endosidin 7, a chemical that inhibits the cytokinesis phase of plant cell replication, on Arabidopsis Thaliana mutant seedlings. The primary goal of his research is to confirm whether the mutant seedlings are hypersensitive to Endosidin 7. Studying ES7 and its hypersensitivity effects on the mutant seedlings will confirm that there are additional proteins involved in callose deposition at the plant cell plate. Callose deposition is a significant process for the later stages of cytokinesis when a polysaccharide called callose is deposited at the cell plate, providing mechanical support to the membrane network before the plant cell splits into two daughter cells. Using a software called ImageJ and confocal microscopy, John will monitor the seedlings’ growth and observe ES7’s effects on the plant cells. This research can potentially lead to the identification of the specific proteins that assist the callose deposition process.
Small unilamellar vesicles (SUVs) aptly model cell membranes. In fact, vesicles are currently a major area of focus. They are being used to study biological functions through the incorporation of various molecules into the vesicle membrane . In this research, the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) 30 and 100 nanometer vesicles were studied in phosphate buffered saline (PBS) and water. The lipid samples were adsorbed on an oxidized gold surface on a germanium crystal and studied with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). In both PBS and water, 30 and 100 nm vesicles adsorbed intact. However, deformation of the SUVs varied with the size of the vesicles and solvent. This study shows that DMPC vesicles reacted differently based on changes to these two factors. These observations will help advance surface chemistry research and the development of better cell bilayer models.
The buffering capacity of foods plays a pivotal role in food breakdown during gastric digestion. The ability of foods to resist changes in pH alters the acidity of the stomach, influencing acid secretion, enzyme function, and chyme viscosity[SO1] . All of these properties directly influence the stomach’s ability to break down foods. Therefore, a comprehensive and replicable method to determine the buffering capacity of foods would prove beneficial for helping to predict and compare digestive properties [SO2] of different foods. We have developed a preliminary methodology for determining the buffering capacities of certain categories and subcategories of undigested foods, and used these to determine buffering properties of certain semisolid foods before digestion.
The atmospheric carbon sequestration abilities of plants, and in specific of grasses, are important on a local and global scale. However, little is known about the difference in carbon sequestration rates of perennial and annual grasses. The goal of this study is to discover if and to what extent grassland restoration, which is commonly achieved by seeding native perennial grasses, increases carbon sequestration through the analysis of soil organic carbon (SOC) stocks. Two sets of soil samples were taken, one set with samples from restored and nearby unrestored sites, and one with pairs of adjacent annual and perennial samples. The soils were processed and analyzed for carbon content in external laboratories. Statistical analysis using JMP revealed that the unrestored sites generally have higher amounts of SOC, although there is a positive relationship between perennial grasses and carbon presence. We conclude that the varying management practices and land use histories greatly shape SOC stocks in grasslands and that there is preliminary evidence that perennial grasses are more effective than annuals in storing carbon.
"Using a Dairy Relevant Bacterial Mock Community to Compare Quantitative PCR, High-Throughput Sequencing, and Culture-Based Cell Enumeration Methods "
A study that will contribute toward the improvement of quantification methods for bacterial profiling. This study is comparing high-throughput sequencing, plate counting, and Quantitative PCR using a bacterial community with dairy-associated bacterial species in order to assess whether current methods produce consistent and similar results. Using these three methods, this mock community is also testing the biases introduced by PCR and DNA extraction kits.
Research and application of fullerenes have been limited by the work and cost of separation and purification. This paper studies sulfur as a separation agent with C60 and C70 solutions. Crystal tubes were made of sulfur and 1:1 molar ratio mix of C60 and C70. Resulting crystals were tested with HPLC and single crystal X-Ray diffraction. It was found that sulfur cocrystallizes with both C60 and C70, but the two fullerenes crystallize separately. The results of this experiment can be used to develop new separation methods for fullerenes and adds to existing knowledge of sulfur as a cocrystallizing agent with fullerenes.
The outer membrane protein 85 (Omp85) family proteins found in the outer envelope of chloroplasts are remnants of ancestral cyanobacterium protein since their endosymbiosis by eukaryotes. Flowering plants in particular have an additional beta barrel protein in the outer membrane of their chloroplasts, OEP80tr, whose exact function is unknown. This study attempts to determine the function of OEP80tr by investigating the effects of null expression mutations of the gene. Through analysis of germination and plant growth (in roots, hypochotyl, and leaves), this study compares the phenotypes of wild type plants with the functioning gene to plants without the gene. The resulting observations could then be used to determine the role in development of the OEP80tr protein.
Changes in M1 and M2 Macrophage Phenotype Numbers following Long-term Smoking Cessation in Spontaneously Hypertensive Rats
Smoking leads to increased susceptibility to heart attack, stroke, lung cancer, and other respiratory complications, such as chronic obstructive pulmonary disease (COPD), emphysema, bronchitis and persistent cough. COPD is the third leading cause of death in the world today (1). Previous findings on smoker rats show increased epithelial volume, mucin content, and also consistent inflammation in the form of high numbers of macrophages. The purpose of this project was to determine whether macrophage numbers in rat tissues after a period of smoking cessation could be used as a unbiased marker of former smoking. In addition, this study was designed to determine the relative proportion of M1 or M2 phenotype macrophages (markers of active inflammation or lung repair, respectively) in the lungs of old rats as well as former smoker rats as a possible marker of continued lung inflammation or repair. The lungs of 12 male spontaneously hypertensive (SH) rats were examined using histological methods. There were significantly more M1 macrophages found in the tobacco smoke (TS) group compared to the filtered air (FA) group. In the TS group, large numbers of foamy macrophages were observed in the subpleural and terminal bronchial regions. Meanwhile in the FA group, the macrophages found were more dense and scattered throughout the tissue. Findings suggest certain changes in rat lungs due to tobacco smoke are still present after long-term cessation- some changes are irreversible and result in chronic damage. The cellular changes that occur in the cell due to TS exposure are important to consider when designing treatments for those who have stopped smoking.
Studying the effects of allergens and pollution on mice. Since asthma is actually the body attacking itself through an immune response of antibodies to allergens, Kelsy will be able to dose mice with allergens and pollution to see their effects on the lungs. She will look at slides of exposed mice lungs with a microscope, analyze mice for indications of inflammation, and analyze a specific protein using immunohistochemistry to determine the damaging effects of air pollution in the lungs and how this may exacerbate allergic asthma. The results will be to see how these two factors of allergens and particulate matter affect those with asthma and the mechanism of asthma.