Symposium Poster Session

Csaba Horvath Memorial Award Symposium 2018

Poster Session

 SPECIFICITY OF PEPTIDE SUBSTRATE REPORTORS FOR PROTEIN KINASE B FROM DICTYOSTELIUM DISCOIDEUM

Misha A. Mehra ‘21, Michelle L. Kovarik

Dictyostelium discoideumhas a relatively short social cycle, which makes it an ideal species for timely viewing of each of its life stages. Nutrient deprivation marks the beginning of the Dictyosteliumsocial cycle. Starvation of the amoebae leads to secretionof cAMP, which attracts other amoebae to move towards the signal. Protein kinase B (PKB) is an enzyme found in Dictyosteliumthat is activated in response to the release of cAMP and signals information about social development within the cell. The aim of the experiment was to investigate the specificity of three peptide substrate reporters (VI-B, AP-I or Crosstide) and conclude which is most specific for the enzyme PKB from Dictyostelium. Each of the peptides was developed in human cells and has not previously been applied outside of mammalian systems. Cells were treated with combinations of cAMP and the PI3K inhibitor LY294002. Capillary electrophoresis coupled with laser-induced fluorescence detection was used to obtain the percent phosphorylation in each of the peptide substrate reporters. As expected it was found that the percent phosphorylation was highest for the (+) cAMP (-) LY294002 treatment, and Crosstide and AP-I had higher phosphorylation than VI-B. Further work will be done to compare the two isoforms of PKB in Dictyostelium. PKBA is an isoform of PKB that is active early in development and is susceptible to LY294002, but the isoform PKBR-1 expressed later in development is not. We will therefore compare time points at 1 h and 6 h of social development.

Misha Mehra is currently a sophomore at Trinity College. She is originally from Nagpur, India, and she plans to major in chemistry or biology. Her research project is on the adaptation of fluorescently-labeled peptides for use as reporters on kinase activity in the social amoeba, Dictyostelium discoideum.

IN VITRO MEASUREMENT OF PROTEIN KINASE B ACTIVITY ON PEPTIDE SUBSTRATE REPORTERS 

Sababa Anber ’20 , Michelle L. Kovarik

 Protein kinase B (PKB) is an enzyme involved in regulation of metabolism, cell survival and apoptosis in many cell types. The activity of this enzyme in intact cells and cell lysates can be measured using fluorescently-labeled peptide substrate reporters. However, it is necessary to understand the kinetics of the reaction to interpret the resulting data. The purpose of this experiment was to investigate the rate of enzyme activity invitro, using three different peptide substrate reporters (VI-B, Crosstide and AP-I) with purified PKB from human andDictyostelium discoideum cells. Seven concentrations (2 - 50 µM) of the three peptides were prepared, and the human enzyme and substrate were allowed to react for 0 to 45 min. Enzyme activity was measured by the rate at which the substrate was phosphorylated using capillary electrophoresis with laser-induced fluorescence. As expected based on Michaelis-Menten kinetics, preliminary results revealed that with increased peptide substrate concentration, the phosphorylation rate increased until it reached a maximum velocity, Vmax (Crosstide) = 6 × 10-12mol/min, Vmax (AP-I) = 3 × 10-12mol/min, Vmax (VI-B) = 8 × 10-13mol/min. The KMvalue, which corresponds to the substrate concentration at half Vmax, was ~10 µM for Crosstide, ~30 µM for AP-I and ~20 µM for VI-B. However recent results revealed that at higher concentrations, the velocity curve decreases. This could be due to substrate inhibition, in which the substrate itself acts as an inhibitor and impedes the activity of its enzyme. Future work on this project will determine whether substrate inhibition is affecting the velocity curve by looking at whether the results are reproducible and will compare theVmaxand KMvalues of (Crosstide, AP-I, VI-B) using the amoeba version of the enzyme.

Ababa Anber is a third year undergraduate student at Trinity College majoring in Neuroscience. She is on the pre-med track and aspires to go to medical school after college.

Accelerated Solvent Extraction of Polycyclic Aromatic Hydrocarbons from Avian Bill Horns and Subsequent Analysis by UPLC/UV 

Son T. C. Nguyen, John T. Ciurylo, Eric Noi Jr., Anthony A. Provatas, Alexander V. Yevdokimov, James D. Stuart, and Christopher R. Perkins

Polycyclic aromatic hydrocarbons (PAHs) are toxic persistent organic compounds that are comprised of multiple unsubstituted, aromatic carbon rings. PAHs are commonly produced as a result of the combustion of petroleum fuels and can be found in large concentrated amounts in crude oil. Due to their carcinogenic and mutagenic properties, PAHs pose a serious environmental concern and have become increasingly relevant with the occurrence of the Deepwater Horizon oil spill in the Gulf of Mexico in April 2010 and more recently with the Santa Barbara oil spill in May 2015. 

     The American white pelican is a large aquatic soaring bird which breeds in interior North America, moving south and to the coasts, as far as Central America and South America. The bill horns of male pelicans shed after mating and are typically composed of beta-keratin, a porous and absorbent material. PAHs and other persisting organics are absorbed and for that reason the ability to analyze the bill horns is an important measure of the pelican’s total exposure to PAHs. A method has been developed and validated for the detection and quantification of 16 PAHs in avian bill horns. Method detection limits and precision and accuracy studies were performed utilizing a Waters Acquity UPLC/UV system. The method detection limits for PAHs ranged from 2.0 ng/mL to 10.6 ng/mL while analyte concentration recoveries from a 1000-ng/mL spike ranged from 72.2% to 105.4% with a relative standard deviation of 0.6% - 3.2%.

Son Nguyen is a 5th year student majoring in Chemistry at the University of Connecticut. He is working at UConn’s Center for Environmental Sciences and Engineering as a senior laboratory assistant since 2017. He has presented his posters at the Eastern Analytical Symposium and Exposition and the Frontiers Undergraduate Research Poster Exhibition

Dr. Anthony Provatas

As a Project Scientist since 2009 at the Center for Environmental Sciences & Engineering (CESE), I have been extensively involved and led various research projects including Biofuels Research and Environmental Organic Analysis. Prior to joining CESE, I worked at Pfizer Inc. in Groton, CT. for over eight years. I have extensive experience in laboratory automation and various analytical instruments. 

UPLC-MS/MS Analysis of Cortisol in Hair and Saliva Collected from Human Subjects

Jacob E. Cortigiano , Sarah D. Anderson, Patrick T. Kaplita, James D. Stuart, Anthony A. Provatas, Christopher R. Perkins

Cortisol, a well-known organic compound belonging to the steroid family, is called the “stress hormone” as humans under significant stress produce cortisol from the adrenal cortex. The analysis of cortisol in hair gives a measure of chronic stress while its analysis in saliva measures a short-term stress level.  In the past, to test for cortisol levels field researchers would collect human saliva in test tubes and submit them to a laboratory for analysis.   In this study, we used a novel approach of blotting saliva onto Whatman FTA blood spotcards. The saliva spots were cut out and then extracted with methanol. Hair samples were mechanically minced and pulverized prior to the addition of methanol as the extraction solvent. Extensive sample sonication and vortexing followed and the extracts were evaporated to dryness in an automated evaporator. For both extracts, a sensitive method utilizing ultra-high performance liquid chromatography/tandem mass spectrometry was developed to measure cortisol levels in both hair and saliva. Method detection limit, accuracy and precision studies were performed for method validation followed by sample screening of hair and saliva collected human subjects.

Jacob Cortigiano is a senior undergraduate at the University of Connecticut majoring in Chemistry. His permanent residence is in Stamford, Connecticut, where he has lived for a majority of his life. After completing his undergraduate career, he hopes to pursue further education in the fields of Biology and Chemistry. His main interests lie in medicinal chemistry, with a heavy focus on organic synthesis. Because of this he will most likely enroll in a Biochemistry or Chemical Biology doctorate program. 

Dr. Anthony Provatas

As a Project Scientist since 2009 at the Center for Environmental Sciences & Engineering (CESE), I have been extensively involved and led various research projects including Biofuels Research and Environmental Organic Analysis. Prior to joining CESE, I worked at Pfizer Inc. in Groton, CT. for over eight years. I have extensive experience in laboratory automation and various analytical instruments. 

 Analyzing Nitrosamines in Drinking Water by Solid Phase Extraction Followed by Ultra High Performance Liquid Chromatography/Tandem Mass Spectrometry

Trevor D. McBrine, Julia M. Lineweber, Benjamin S. Reale, Abdurrahmaan Sallam, James D. Stuart, Anthony A. Provatas, Christopher R. Perkins

 Nitrosamines are a group of organic compounds consisting of a nitroso group bonded to an amine. Nitrosamines are commonly found in drinking water as by-products due to the use of chlorine, chloramine, and anion-exchange processes to treat surface water sources. The presence of nitrogen precursors from upstream waste water disposal allows for the formation of nitrosamines through the reaction of dimethylamine with dichloramine. N-nitrosodimethylamine (NDMA) is the most frequently detected nitrosamine in water. The presence of these compounds in drinking water poses environmental and public health concerns because they have been shown to cause high incidences of cancer in both animals and humans. Unfortunately, the low molecular weight of nitrosamines prevents their removal by reverse osmosis, and instead water must be treated with UV exposure. The purpose of this project was to determine the concentrations of nitrosamines in drinking water samples using UPLC-MS/MS after a solid phase extraction (SPE) sample preparation step. Coconut charcoal SPE cartridges were used to capture the nitrosamines and were eluted with methylene chloride. The eluent volume was reduced on a nitrogen blow-down unit, with the residue taken up in methanol, while ensuring the sample was not brought to dryness. The methodology used was validated by method detection limit, precision and accuracy studies, which displayed excellent analyte sensitivity and recovery. 

Trevor McBrine is a senior chemistry major at the University of Connecticut. He began working at the Center for Environmental Sciences and Engineering (CESE) in May and will continue to do undergraduate research for his last two semesters. His goal after graduating is to work in industry where he can apply the skills he has learned at CESE and continue to gain experience in the field of analytical chemistry.

Dr. Anthony Provatas

As a Project Scientist since 2009 at the Center for Environmental Sciences & Engineering (CESE), I have been extensively involved and led various research projects including Biofuels Research and Environmental Organic Analysis. Prior to joining CESE, I worked at Pfizer Inc. in Groton, CT. for over eight years. I have extensive experience in laboratory automation and various analytical instruments. 

Mass Spectrometry Quantitation of Metabolites in Bacterial Vitamin B2 Biosynthesis Pathway for Study of Human Microbiota-Immune System Interaction

Lei Wang1 and Xudong Yao*1

In collaboration with:

Cihan Tastan2,3, Ece Karhan2, Wei Zhou2, Elizabeth Fleming2, Anita Y. Voigt2, Meghan Horne2, Lindsey Placek2, Lina Kozhaya2, Julia Oh*2, and Derya Unutmaz*2

1Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA

2Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA

3Department of Microbiology, NYU School of Medicine, New York, NY 10016, USA

 Correspondence: xudong.yao@uconn.edu

Vitamin B2 or riboflavin is synthesized by bacteria but not human cells. Therefore, human mucosal-associated invariant T cells (MAIT) subtly detect invasive bacteria via T cell receptor (TCR) recognition of metabolites, which is captured and presented by the MR1 protein, from bacterial riboflavin biosynthesis pathway. However, it is still not clear whether and how MAIT cells can distinguish different strains of bacteria in the human microbiota. One hypothesis would be that the level of riboflavin and its precursors secreted by bacteria plays an important role here. To justify this hypothesis, as a complementary study in parallel to an MAIT-based in vitro functional assay, we developed analytical methods for quantitation of these compounds in various phosphate-buffered saline-based bacterial supernatant samples from distinct strains, using liquid chromatography-stable isotope dilution multiple reaction monitoring mass spectrometry (LC-SID-MRM MS). Although it was challenging to extract hydrophilic analytes from salty samples, we successfully quantified riboflavin with a limit of detection (LOD) of 72.4 femtomole, a limit of quantitation (LOQ) of 217.3 femtomole at 95% level of confidence and an average coefficient of variation (CV) of 4.9%. As consistent with the bioassay, the quantitation result suggested only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Moreover, the activation of MAIT cells also correlated with the level of riboflavin bacteria secreted. Currently, the analytical method development is still ongoing for two notoriously unstable precursors of riboflavin, 5-amino-6-ribitylamino uracil (5-ARU) and 6 7-dimethyl-8-ribityllumazine (lumazine), whose quantitation would require meticulous sample handling, perfectly-tuned chemical derivatization and separation.