Erythritol is a naturally occurring, zero calorie sweetener produced by fermentation and is used as an additive in many foods and beverages. Erythritol is produced commercially through yeast fermentation using an expensive filtration process. The discovery of a microbe that could produce erythritol in comparable amounts could provide an alternative to this process, especially if it is a food grade microbe that could remain in the finished product. Lactobacillus florum is a novel, heterofermentative, lactic acid bacteria that has been found to produce erythritol making it a potential candidate. Two strains of Lactobacillus florum, 8D and 2F, previously isolated from an unripe king palm fruit and a Valencia orange leaf, respectively, were tested for their ability and efficiency in producing erythritol when given glucose, fructose, or a combination of the two. Other metabolites, such as mannitol, acetic acid, lactic acid, and ethanol were also measured for. Since the erythritol pathway is not known in L. florum, enzyme assays were conducted to measure for the activity of enzymes responsible for erythritol production in Oenococcus oeni and yeast, erythritol 4-phosphate dehydrogenase and erythrose reductase, respectively. The results described will increase our knowledge of Lactobacillus florum, the pathway by which it can produce erythritol, and lead to areas of study for an alternative means of erythritol production. Both 8D and 2F were found to be capable of erythritol production when provided glucose, fructose, and the glucose-fructose combination, with 8D producing more than 2F.
Tyrosine sulfation is a post-translation modification that has been proven to strengthen protein-protein interactions. In this study, we examine the potential for tyrosine sulfation in the human G-protein coupled receptor rhodopsin, the prototypic GPCR which plays a role in the visual phototransduction pathway in the rod cells of the eye. Through the position-specific scoring matrix, tyrosine sites Y26, Y29, Y30, and Y191 were found to have a high likelihood of sulfation in human rhodopsin. These sites were all well aligned in other forms of rhodopsin and cone opsins found throughout the animal kingdom. Y191, which is involved in a hydrogen bond network on extracellular loop 2 between E181 and Y192, was demonstrated through site-directed mutagenesis studies to function in the proper maintenance of Metarhodopsin II, the active state of photo-activated rhodopsin. Therefore, we find that sites Y26, Y29, Y30, and Y191 are consistent with sulfation.
The Effects of Anaerobic Soil Disinfestation Treatments With Different Carbon Sources on Agrobacterium tumefaciens
As the worldwide restrictions for soil chemical fumigants increase, growers need alternatives to eradicate soilborne plant pathogens. Anaerobic soil disinfestation (ASD), developed in Japan and the Netherlands in the 2000s, is one of these alternative techniques that can control certain soilborne plant pathogens, plant-parasitic nematodes, and weeds in specialty crop systems. To utilize the technique, the soil has to have a carbon source applied, covered with plastic, and mixed with water to generate anaerobic conditions in the soil. The point of this experiment was to see the effects of different carbon sources such as ethanol, almond hulls, almond shells, and rice bran on the soilborne plant pathogen Agrobacterium tumefaciens, which causes crown gall disease in walnuts. The results showed that ethanol was the most effective ASD treatment at suppressing A. tumefaciens. However, the most anaerobic treatment was the almond shells, but it was also the treatment with the greatest abundance of A. tumefaciens. The anaerobic environment created by ASD may not be the only driving mechanism behind suppression of A. tumefaciens. The ethanol treatment did not have anaerobic conditions, but volatile compounds produced through ASD in the ethanol treatment could have suppressed the A. tumefaciens instead.
Glycosylation is an important modification to cell surface proteins. The brush border of intestinal epithelial cells (IECs) contains many glycoproteins that are key to intestinal function. This study tested how changes to glycosylation affects the function of cell surface glycoproteins in IECs. Specifically, how removing N-glycans with exoglycosidases, such as those secreted by intestinal bacteria, affects the function of brush border enzymes, such as Intestinal Alkaline Phosphatase and Dipeptidyl Peptidase IV. This was tested in Caco-2 cells, a human cell line derived from colon adenocarcinomas that is used as a model for intestinal function. Caco-2 cells were cultured for 12 days, and their expression of IAP was tracked throughout the differentiation process. Once they were fully differentiated, the cells were then treated with glycosidases, and the enzymatic activity of IAP and DPP IV was measured. The results showed that changing the glycosylation caused enzymatic activity to decrease. Removing all the N-glycans with the glycosidase PNgase F decreased IAP activity to about 40 %, confirming that glycans are key to the function of the enzyme. Of the individual monosaccharides, removing mannose had the greatest effect, and removing sialic acid and fucose also affected the enzymatic activity. However, removing individual sugars did not cause any large decreases in activity, especially compared to removing the entire glycan.
A Mechanistic Insight on Chemical Enhancement of Gold Nanoparticles through Electron Paramagnetic Resonance
Nanoparticles are capable of enhancing reactions under X-ray irradiation. In order to utilize this enhancement and apply it to other reactions, further knowledge of the enhancement mechanism is required. The experiment was performed in two parts, the first involving the quantification of chemical enhancement using a fluorometer to test the fluorescence of the hydroxylation of coumarin carboxylic acid in the presence of various concentrations of gold nanoparticles. The second part of the experiment involved understanding the hydroxyl radical, reactive oxygen species (ROS), pathway of the reaction using electron paramagnetic resonance (EPR). It was found that the surface of gold nanoparticles catalytically facilitates conversion 3-OH radical adduct to 7-OH-CCA in the presence of oxygen. Also the amount of hydroxyl radicals found in solutions with varying concentrations of gold nanoparticles was constant, implying that ROS generation is not a part of the catalytic process.
Chaperone-mediated autophagy (CMA) is a process that maintains cellular homeostasis and degrades dysfunctional or damaged proteins. It functions by transporting proteins to the lysosome for degradation with the help of the HSC70 and LAMP-2A molecules. Research demonstrating a five residue amino acid sequence and several substrate proteins has resulted in many proteins being identified as potentially CMA regulated. We analyzed all proteins in the genome of several species including humans, and concluded that many proteins and signaling pathways are likely CMA regulated. Using a novel prediction program, we have also found that a greater percentage of proteins (46.5%) in the human protein sequences possess KFERQ-like motifs than previously thought (30%). Testing of lysosomes from starved H9c2 cells showed that the three proteins, which are predicted to have KFERQ-like motifs, are present in the lysosome.
The research work is based on finding the characterizations of different catalysts to determine the most effective and appropriate catalyst. This will be done through various methods, such as IR spectrometry with unreactive gases to observe how much the spectra of the catalysts change. This work can be applicable to industrially used catalysts, which accelerate production and creation of other chemical-based materials.
Due to the susceptibility of mitochondrial DNA to insertions, deletions, and point mutations and the existence of multiple copies of mitochondrial DNA in each organelle, a phenomenon called heteroplasmy arises. High levels of heteroplasmy are known to be a factor in the development of mitochondrial diseases. Sung Bin "Sean" is optimizing a procedure to detect low levels of heteroplasmy. Applications of this research include future studies into heteroplasmy as well as diagnosis of risk of mitochondrial diseases.
Understanding The Role Of Salicylic Acid Signaling Pathway In Stromule Induction During Plant Innate Immunity
Stromules are plant cell specific structures that extend out from the chloroplast. The role of the stromule is not known, however, a recent publication from the Dinesh-Kumar lab suggests that they are involved in the plant’s immune response to protect the plant from pathogens. More detailed studies regarding the regulation mechanism about the stromule induction is crucial to understand their contribution in the plant immune response. The Dinesh-Kumar lab studies have suggested that the stromule induction pathway might be regulated through the plant hormone Salicylic Acid (SA) pathway that is essential to the plant immune response. Key genes in this pathway are SID2/EDS16 and ATG5. To obtain a better understanding of the role of SA in stromule induction, the sid2/eds16 and atg5 mutants were crossed to make sid2/eds16 atg5 double mutant. sid2/eds16 atg5 double mutant was identified by genotyping using polymerase chain reaction (PCR). The results show that there was a double mutant and we will introduce a stromule marker later to observe stromule induction. Characterization of stromule induction in single and double mutants will provide insights on how stromule induction is regulated during the plant immune response.
Tiankun’s project uses animal models to simulate health effects of exposure to air pollution on human lungs and involves the quantification and comparison of cellular damage caused by certain particulate matter (PM) between animals with different ages and between different compartments of lungs. Based on the previous work of his professor and through his own research, he might be able to determine the harmful portion of air pollution and potential biomarkers for susceptibility to PM that could be used either to identify or to treat afflicted individuals. The findings might also serve as reference and guidance to the government in regard of making environmental regulations and health policies.
Tommy is currently working with Dr. Doug Gubler at the UC Davis Department of Plant Pathology, and his research involves several strains of fungi from the genus Botryosphaeria. His project tests several potential biocontrols, or methods of controlling pathogens through natural means, against the Bot fungi and seeing if any of them have inhibitory effects on the fungi. Fungi in the genus Botryosphaeria have been known to cause tree and shrub diebacks and diseases in grapes, apples, and pears. Each year, millions of dollars in revenue are lost in the agricultural industry because of damages by the fungi. Finding a cost-efficient and environmentally friendly method to control these organisms will greatly benefit the agricultural community.
Researching the role of microRNA during early development of embryos. MicroRNAs are small RNA molecules that regulate gene expression post-transcriptionally and play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Consequentially, altered microRNA expression is likely to contribute to human disease, including cancer and HIV. By studying the mechanisms that are caused by microRNA function, additional insight and a deeper understanding of related processes will allow for the pursuit of further research and possibly more efficient treatments or cures. Currently, the success rate of In Vitro Fertilization or fertilization outside the body is only at a 30% success rate for females age 35. By studying the regulatory roles of microRNA, processes such as In Vitro Fertilization could potentially benefit from increased success rates from a greater insight of correlated processes and techniques
Beta-glucosidase B is an enzyme found in every organism that breaks down the glycosidic bondages of polysaccharide molecules. Currently, there is a lack of understanding of how enzymes work, partly because databases on how point mutations affect this enzyme are incomplete. In this study, we investigated the effects of how three specific point mutation affects the enzyme’s effectiveness by using kinetic constants to characterize the enzyme’s effectiveness. One mutation has significantly improved enzymatic activity, but the other mutations have caused the enzyme to be less effective. Investigations such as these could lead to an understanding of how enzymes work, which has many applications, such as the biofuels industry.
Chlorpyrifos (CPF) is an organophosphate found in common pesticides that inhibits the enzyme acetylcholinesterase. Chronic exposure of CPF in humans results in neurological defects, developmental and autoimmune disorders, probably due to the generation of reactive oxygen species (ROS). Furthermore, CPF has been shown to have an effect on the dynamics and movement of mitochondria in rat cortical neurons and to decrease the activity of Complex I in PC12 cells. In this study, precursor neural cells from mouse brain were treated with 10 and 80 µM CPF for 24 hours. ATPase activity was found to be 20% lower on average compared to non-CPF-treated cells, with no changes in citrate synthase activity. Mitochondrial morphology and distribution were also analyzed through confocal microscopy. CPF-treated cells showed a significant decrease in the amount of tubular mitochondria (9% of untreated cells) accompanied by an 80% increase in the number of circular and fragmented mitochondria. Our results demonstrate that CPF exposure affects mitochondrial function suggesting its potential to produce oxidative stress and its involvement in oxidative stress-related neurodegenerative disease.
The Effect of Computationally Designed Point Mutations on Structure and Kinetic Characteristics of Glycosidase Hydrolase Enzymes
The importance of predicting an enzyme's function and rate of function based on its structure. Due to the inaccuracy of current predictive algorithms, there is a need for large and comprehensive databases from which more accurate predictions can be made. Three mutants of the beta glucosidase B enzyme were tested for expression and then kinetically characterized through Michaelis-Menten kinetics. The ultimate goal of this project is to be able accurately mutate an enzyme for a specific purpose (i.e increased or slowed activity) and even create novel enzymes from scratch with the intended chemical activity.